Ubolsree Leartsakulpanich - vtechworks.lib.vt.edu · Chapters 1 and 2 are intended to serve as an...

Biochemical characterization of a novel iron-sulfur

flavoprotein from Methanosarcina thermophila strain TM-1

Ubolsree Leartsakulpanich

Dissertation submitted to the Faculty of the

Virginia Polytechnic Institute and State University

in partial fulfillment of the requirement for the degree of

Doctor of philosophy

in

Biochemistry

Dr. James G. Ferry, Chair

Dr. John L. Hess

Dr. Eugene M. Gregory

Dr. Timothy J. Larson

Dr. Dennis R. Dean

Dr. Robert H. White

June 1999

Blacksburg, Virginia

Keywords: Iron-sulfur flavoprotein, Methanosarcina thermophila

Copyright 1999, Ubolsree Leartsakulpanich

ii

Ubolsree Leartsakulpanich

ABSTRACT

The iron-sulfur flavoprotein (Isf) from the acetate utilizing methanoarchaeon

Methanosarcina thermophila was heterologously produced in Escherichia coli, purified

to homogeneity, and characterized to determine the properties of the iron-sulfur cluster

and FMN. Chemical and spectroscopic analyses indicated that Isf contained one 4Fe-4S

cluster and one FMN per monomer. The midpoint potentials of the [4Fe-4S]2+/1+ center

and FMN/FMNH2 redox couple were -394 and -277 mV respectively.

The deduced amino acid sequence of Isf revealed high identity with Isf

homologues from the CO2 reducing methanoarchaea Methanococcus jannaschii and

Methanobacterium thermoautotrophicum. Extracts of H2-CO2-grown M.

thermoautotrophicum cells were able to reduce Isf from M. thermophila using either H2

or CO as the reductant. Addition of ferredoxin A to the reaction further stimulated the

rate of Isf reduction. These results suggest that Isf homologues are coupled to ferredoxin

in electron transfer chains in methanoarchaea with diverse metabolic pathways.

Reconstituted systems containing carbon monoxide dehydrogenase/acetyl-CoA

synthase complex (CODH/ACS), ferredoxin A, Isf, and the designated electron carriers

(NAD, NADP, F420, and 2-hydroxyphenazine) were used in an attempt to determine the

electron acceptor for Isf. Isf was unable to reduce any of these compounds. Furthermore,

2-hydroxyphenazine competed with Isf to accept electrons from ferredoxin A indicating

that ferredoxin A is a more favorable electron partner for 2-hydroxyphenazine. Thus, the

physiological electron acceptor for Isf is unknown.

Amino acid sequence alignment of Isf sequences revealed a conserved atypical

cysteine motif with the potential to ligate the 4Fe-4S cluster. Site-directed mutagenesis

of the cysteine residues in this motif, and the two additional cysteines in the sequence,

was used to investigate these cysteine residue as ligands for coordinating the 4Fe-4S

iii

center of Isf. Spectroscopic and biochemical analyses were consistent with the

conserved cysteine motif functioning as ligating the 4Fe-4S center. Redox properties of

the 4Fe-4S and FMN centers revealed a role for the 4Fe-4S center in the transfer of

electrons from ferredoxin A to FMN.

iv

FORWARD

This dissertation focuses on the characterization of the heterologously produced

iron-sulfur flavoprotein from Methanosarcina thermophila by different approaches.

Chapters 1 and 2 are intended to serve as an introduction to biological methanogenesis

and iron-sulfur proteins. Chapters 3 and 4 describe the research pertaining to the

characterization of iron-sulfur flavoprotein and a summary is presented in Chapter 5. The

studies described in Chapters 3 and 4 have or will be published as follows:

Becker D.F., Leartsakulpanich U., Surerus K.K., Ferry J.G., and Ragsdale S.W.

1998. Electrochemical and spectroscopic properties of the iron-sulfur

flavoprotein from Methanosarcina thermophila. J. Biol Chem. 273:26462-26469

Leartsakulpanich U, Antokine M.L., Golbeck J.H., and Ferry J.G. A novel [4Fe-

4S] iron-sulfur cluster binding motif in the iron-sulfur flavoprotein of

Methanosarcina thermophila (manuscript in preparation).

v

ACKNOWLEDGEMENT

I would like to express my gratitude to my thesis advisor, Dr. James G. Ferry, for

giving me the opportunity, financial support, encouragement and guidance in my

academic career. This dissertation would be impossible with out his guidance and insight

toward my study. I would like to thank my committee members, Dr. T.J. Larson, Dr.

D.R. Dean, Dr. E.M. Gregory, and Dr. J.L. Hess, for their interest in my research and

their flexibility and understanding of my situation as an out of state student. In addition, I

would like to thank Dr. R.H. White for being substituted for Dr. Dean as another thesis

examining committee. I thank Dr. D.F. Becker, Dr. S.W. Ragsdale, and Dr. K.K. Surerus

for their contributions to the characterization of the iron-sulfur flavoprotein (Isf). I thank

M.L. Antokine and Dr. J.H. Golbeck for their assist on EPR experiments with Isf

variants. I gratefully acknowledge the financial support from MOSTE, Thailand

throughout my 5 years in the US.

I also would like to thank all the postdocs and students in Dr. Ferry's lab. Cheryl

Ingram Smith (who helped me during my research rotation and has been very helpful

since, also for her compassion and caring), Birgit Alber (who was another Hoakie, special

soul-mate, my volleyball coach, and my driver when I wanted to head south), Kerry

Smith and Rob Barber (for their muscles and advice and thousands of suggestion),

Madeline Rasche, Kavita Singh-Wissman, and Julie Maupin-Furlow (for their kindness

and patience in teaching me techniques), Sean O'Hearn (for let me know that being a

biochemist is better than being a chef, Really?) and Mike Painter, Tong Zhao, Rebecca

Miles, Christie Brosius, Birthe Borup, Prabha Iyer, Brian Tripp, Laura Lierman, and

Mark Signs, for their friendship, English teaching, and support (especially when I cannot

smile). My appreciation goes to the secretary of the department at VA Tech, Mary Jo

Smart, and several secretaries of the Ferry lab, Vonni Kladde, and Carol DeArmitt, for

taking care of things promptly, efficiently, and conveniently for me.

I want to extend my appreciation to my ex-roommates at VATech, Kitsiri

Kaewpipat and Chanpen Chanchao, together with Sunitiya Thuannadee, Charaspim

vi

Boonyanan, and Matt Mamorino who made the years in Blacksburg an incredibly

wonderful experience for me. Also, Somboon Kiratiprayoon, Patcharin Poosanaas,

Amin Tanuminhadjo, Bill and Barb Saxton, Joanie Zhoa and HenSiong Tan, Ari and

Purwadi Purwasumato Venyi Hoa, Joy Wang, Maki Murata, Kerwin Foster, and all other

members of ICF, who supply the happiness at PSU.

Finally and the most important, I would like to express my deep gratitude to my

family, Khajon (for taking all the burden off my shoulders and letting me continue my

studies), Sirinthip (for her compassion, consideration, and love), Paramate ( for his

sharing, and prompt assistance with seemingly ceaseless energy), and particularly to my

Mom and Dad (for their unconditional and endless love, supporting guidance, generosity,

sweetness, and always always being there for me); without their inspiration,

encouragement, motivation, and optimism, I would not be able to come this far.

Thanks again to all.

vii

TABLE OF CONTENTS

Page

Title page i

Abstract ii

Forward iv

Acknowlegement v

Table of contents vii

List of tables ix

List of figures x

Introduction xiii

Chapter 1: Methanogenesis 1

I. Microbiology 1

Methanoarchaea 1

Growth substrates 1

Ecology 2

II. Biochemistry 6

Coenzymes 6

CO2-reduction pathway 8

Acetate fermentation pathway 10

III. Bioenergetics and electron transport 14

References 18

Chapter 2: Iron-sulfur proteins 32

I. Introduction 32

II. Structural and properties of clusters 32

1[Fe] cluster type 33

[2Fe-2S] cluster type 34



III. Ligation of iron-sulfur clusters 39

IV. Function of iron-sulfur centers 41

viii

TABLE OF CONTENTS (cont.)

Page

A. Electron transfers 41

B. Catalysis 42

C. Regulatory role 43

D. Iron storage role 44

E. Structural role 44

V. Iron-sulfur cluster assembly in proteins 45

References 46

Chapter 3: Objectives 55

Chapter 4: Electrochemical and spectroscopic properties of

the iron-sulfur flavoprotein from Methanosarcina thermophila 57

Abstract 57

Introduction 57

Materials and Methods 59

Results 63

Discussion 81

Acknowledgement 84

References 85

Chapter 5: A novel [4Fe-4S] iron-sulfur cluster binding motif in the iron-sulfur

flavoprotein of Methanosarcina thermophila 88

Abstract 88

Introduction 89

Experimental procedures 91

Results 93

Discussion 118

Acknowledgement 120

References 121

Chapter 6: Summary and future directions 124

Curriculum Vista 126

ix

LIST OF TABLES

Page

Chapter 1

Table 1. Substrates for methanogenesis 3

Chapter 5

Table 1. EPR properties of wild-type Isf and variants 115

Table 2. Rates for reduction of FMN in wild type Isf and variants 117

x

LIST OF FIGURES

Page

Chapter 1

Figure 1. Microbial food chain 5

Figure 2. Structure of the coenzyme of the coenzymes involved

in methanogenesis 7

Figure 3. Methanogenesis from CO2 reduction pathway 9

Figure 4. Proposed pathway for acetate conversion to CO2 and

CH4 in Methanosarcina thermophila 13

Chapter 2

Figure 1. Structures and properties of the structurally

characterized iron-sulfur centers that are

involved in biological system 36

Figure 2. Arrangement of residues involved in coordination of

[2Fe-2S] (A), [4Fe-4S] or 2[4Fe-4S] (B), [3Fe-4S] or

[4Fe-4S] (C), and 2[4Fe-4S] or [3Fe-4S] plus [4Fe-4S]

(D) clusters 38

Chapter 4

Figure 1. Corrected nucleic acid sequence and predicted amino

acid sequence of isf from M. thermophila 64

Figure 2. EPR spectroscopy of the [4Fe-4S] cluster in Isf poised at

various redox potentials in 50 mM potassium phosphate

buffer (pH 7.0) 66

Figure 3. Semilogarithmic plot of P1/2 versus 1/T, which shows a

linear relationship according to equation P1/2 = Aexp (∆-/kT) 67

Figure 4. Mössbauer spectra recorded at 100 K. A) oxidized Isf protein.

B) reduced Isf protein 69

Figure 5. Mössbauer spectra of reduced Isf protein recorded at 4.2 K

and 450 G applied parallel (A) or 450 G applied perpendicular

(B) to the γ beam 70

xi

Figure 6. Potentiometric titration of the FMN in Isf (3.2 µM) in

50 mM potassium phosphate buffer (pH 7.0) at 20o C

(curves 1-7, fully oxidized, -262, -272, -281, -290, -305,

and –342 mV respectively). Inset, Nernst plot of the

potentiometric data 72

Figure 7. EPR spectrum of Isf (170 µM dimer) was recorded at

10 K following incubation for 17 min with CO and CODH

(0.5 µM) at 25o C) 73

Figure 8. A fit of the Isf midpoint potential data to a theoretical

curve generated from the Nernst equation for two redox

centers with reduction potentials of –277 mV (n = 2)

and –394 mV (n = 1) 74

Figure 9. Time course for reduction of methanophenazine with

Isf from M. thermophila 76

Figure 10. Multiple amino acid sequence alignment of Isf from

M. thermophila with sequences deduced from open

reading frames identified in the genomic sequences of

M. jannashii and M. thermoautotrophicum 78

Figure 11. Time course for reduction of Isf with extract from M.

thermoautotrophicum 79

Figure 12. Time course for reduction of Isf with extract from M.

thermoautotrophicum 80

Figure 13. Proposed electron transport pathway for oxidation of CO

or the carbonyl group of acetyl-CoA 82

Chapter 5

Figure 1. Multiple amino acid sequence alignment of Isf from

M.thermophila (MST) with sequences deduced from open

reading frames identified in the genomic sequences of

M. jannaschii (MCJ), M. thermoautotrophicum (MBT),

Archaeoglobus fulgidus (AF), Chlorobium vibrioforme (CV),

Chlorobium tepidum (CT), and Clostridium difficile (CD). 94

xii

Figure 2. Coomassie blue stained native PAGE of wild type Isf and

variants 97

Figure 3. UV-visible absorption spectra of as-purified, denatured, and

reconstituted wild type Isf 99

Figure 4. UV-visible absorption spectra of wild type Isf and alanine

variants 100

Figure 5. UV-visible absorption spectra of wild type Isf and serine

variants 101

Figure 6. EPR spectra of reduced wild type Isf with different

processes to recover iron-sulfur center 102

Figure 7. EPR spectra of C16X (X = A or S) 104

Figure 8. EPR spectra of reduced C180A with different reconstitution

processes 105

Figure 9. EPR spectrum of reduced C180S 106

Figure 10. EPR spectrum of as-purified C50A 107

Figure 11. EPR spectra of as-purified C59A 108

Figure 12. EPR spectrum of reduced C47A 110

Figure 13. EPR spectrum of reduced C53A 111




xiii

INTRODUCTION

Methanogenesis is a prominent process in the biological world, in which it

represents the final step in the carbon cycle in anaerobic environment. Methanoarchaea

have a major impact on the environment and human activities. More than 109 tons of

methane have been released into the atmosphere. Methane is produced by two major

pathways. The first is the CO2 reduction pathway in which CO2 is reduced to methane

using electrons derived from either H2 or formate. Acetate is a key product in the

decomposition of organic compounds and is the primary substrate for methane

production with two-thirds of all biological methane derived the methyl group of acetate.

However, only species of the genera Methanosarcina and Methanothrix are known to

convert acetate to methane and CO2. The study of methanogenesis has made an

enormous impact in many areas of physiology, ecology, biochemistry, molecular biology,

and evolution.

Methanosarcina thermophila strain TM-1 is a moderate thermophile in the

Archaea domain. It can utilize acetate, methanol and methylamines as growth substrates.

In the past decades, one carbon metabolism in acetate catabolism has been well

established in Ms. thermophila, but the details of the path of electron flow and energy

conservation are less well understood. The carbon monoxide dehydrogenase/acetyl CoA

synthase (CODH/ACS) enzyme complex of M. thermophila is a key enzyme in acetate

metabolism and previous studies showed that ferredoxin A accepts electrons from

CODH/ACS. The electrons are then donated to iron-sulfur flavoprotein (Isf). Isf was

partially characterized and contains iron-sulfur cluster and FMN. As a result, the

properties of the Fe-S cluster and FMN were examined. Site-directed mutagenesis was

performed in an effort to identify the iron-sulfur cluster ligands.

1

CHAPTER 1

METHANOGENESIS

I. Microbiology.

Methanoarchaea. During the past three decades, the increasing interest in

methane-producing microorganisms has resulted in a rapid accumulation of knowledge.

The advent of 16S rRNA sequencing introduced a new classification scheme in which all

forms of life could be categorized into three "primary domains"; Eucarya, Bacteria, and

Archaea (42, 130). These three domains replaced the conventional classifications of

either the five kingdom system or the prokaryote/eukaryote dichotomy. The Eucarya

domain is comprised of plants, animals, and fungi while the Bacteria and Archaea

domains contain the prokaryotes. Methane producers, extreme halophiles, sulfate-

reducers, and extreme thermophiles are members of the Archaea (129). All

methanoarchaea belong to the Euryarcheota kingdom, and are classified into five orders,

ten families, and twenty-five genera (16).

The methanoarchaea represent the most diverse and extensively studied members

of the Archaea domain. Despite the fact that they share the common feature of methane

production, they are not closely related phylogenetically. They show diversity in: (1)

morphology (rod, coccus, spirillum, and aggregate forms) (30, 57, 61, 62, 106, 108); (2)

habitats with variable temperatures (2o to more than 100o C), pH (3 to 9.2), and salinity (1

mM to 3 M salt) (136); (3) cell wall components, such as pseudomurein (59), protein,

glycoprotein, and heteropolysaccharides (7, 68); (4) the appearance of novel cofactors

such as F430, coenzyme B, and coenzyme M in their metabolic pathways; and (5) the

ability to grow on one- and two-carbon substrates.

Growth Substrates. The sole means by which methanoarchaea obtain energy for

growth is through methanogenesis (123). They are extremely specialized in using only a

limited number of simple compounds as their growth substrates (Table 1) (136). They

require a minimum reduction potential of – 300 mV to achieve growth (52).

2

Most methanoarchaea are able to utilize H2 and CO2 as sources of energy and

carbon (eq. 1, Table 1). However, several methanoarchaea including Methanosarcina

thermophila strain TM1 lack this ability (134). Several methanoarchaea contain formate

dehydrogenase, which allows them to use formate as a reductant (eq. 2, Table 1) (64).

Methanobacterium thermoautotrophicum and some Methanosarcina sp. are able to

oxidize CO for their growth (eq. 3, Table 1) (23). Acetate is a catabolic product of many

fermentation processes; however, only Methanosarcina and Methanosaeta species can

ferment acetate to CO2 and methane (eq. 4, Table 1) (136). The methanoarchaea in the

genus Methanosarcina are able to catabolize methyl containing compounds such as

methanol and methylamine (eqs. 5-7, Table 1). Utilization of short chain alcohols such

as ethanol has been observed in some hydrogenotrophic methanoarchaea (eq. 8, Table 1)

(128, 133). A small number of methylotrophic species utilize di-methylated sulfide (eq.

9, Table 1) (91).

Ecology. Biological methane production is a strictly anaerobic process; thus,

methanoarchaea are exclusively found in anaerobic environments, although some can

tolerate a brief exposure to O2. Methanoarchaea can be found in diverse anaerobic

habitats such as marine and freshwater sediments, hot springs, sites of geothermal

activity, and in ruminant animals. They have also been found in association with human

activities such as rice paddy fields, sewage sludge digesters, and landfills.

Methanoarchaea are restricted to only a few substrates that in nature are provided

by other microbes (136). Many such metabolic interactions among microbes in different

communities can occur (109), some examples of which are: neutralism, mutualism,

symbiosis, or competitive interaction. Environments containing sulfate (SO42-)-reducing

microbes and methanoarchaea involve competitive interactions. Sulfate reducing

microbes have been reported to grow on H2 plus CO2 and acetate; in addition, SO42-

reducers have a higher affinity for these substrates than methanoarchaea (97, 103). In

nutrient limited environments, SO42- reducers out-compete methanoarchaea for these

substrates resulting in inhibited growth of the latter (103).

3

Table 1. Substrates for methanogenesis.

Reactants Products

1) 4H2 + HCO3- + H+ CH4 + 3H2O

2) 4HCO2- + H+ + H2O CH4 + 3HCO3

-

3) 4CO + 5H2O CH4 + 3HCO3- +3 H+

4) CH3COO- + H2O CH4 + HCO3-

5) 4CH3OH 3CH4 + HCO3- + H2O + H+

6) CH3OH + H2 CH4 + 3H2O

7) 4 (CH3) 3-NH+ + 9H2O 3CH4 + HCO3- + 4NH4

+ + 3H+

8) 2CH3CH2OH + HCO3- 2CH3COO- + CH4 + 3H2O

9) 2(CH3)2-S + 3H2O 3CH4 + HCO3- + 2H2S + H+

4

The methanoarchaea execute the terminal step in the degradation of complex

biomass to methane (Fig 1) (40), which is very important for the global carbon cycle. The

microbial degradation of biomass requires three inter-dependent metabolic groups of

microbes (37). The fermentative microorganisms degrade the large complex molecules

such as cellulose to the simple molecules H2, CO2, formate, and acetate, as well as

various fatty acids. Then, acetogens metabolize fatty acids into H2 and CO2, formate, and

acetate. Finally, methanoarchaea reduce CO2 with H2 or formate to methane, and ferment

acetate to methane and CO2. Hydrogen-producing acetogens also provide substrates for

methanoarchaea. The methanoarchaea in turn maintain a low H2 partial pressure that is

beneficial to acetogens because high concentrations of H2 inhibit the acetogens' metabolic

activity. Much of the released methane is utilized by methane oxidizing bacteria, called

methylotrophs, as their growth substrate (67). However, a large amount of methane

escapes and reaches the atmosphere where it is a major greenhouse gas (118). About 1%

annual increase of methane in the atmosphere has been observed (136), and is mainly due

to human activities.

Recent work has focused on using methanoarchaea in bioremediation. In sewage

sludge digesters, methanoarchaea and a mix of other anaerobic microorganisms degrade

organic waste into methane which can then be used as an alternative energy source (90).

In addition, studies are being performed on the ability to detoxify pollutants produced by

industry and agriculture (65, 66, 100).

5

Complex organics

CH3CH2COO-

CH3CH2CH2COO-Fermentative bacteria

H2 + HCOO-

H2-reducingacetogenicbacteria

CH3COO-

CO2

CO2-reducingacetogenicbacteria

H2

CO2

Methanogenic archaea

CH4 CH4

CO2 + CH4

Figure 1. Microbial food chain (40). Three different metabolic groups of

microbes are required to decompose complex molecules to methane and carbon

dioxide. The principle intermediates and the major route of carbon flow (solid

lines) are shown.

6

II. Biochemistry.

Methanoarchaea are diverse in physiology and phylogeny; however, they show

similarity in their metabolic pathways. These unique biochemical processes involve

several novel coenzymes (Fig 2) (125).

Coenzymes. Methanofuran (MF), a low molecular weight C1-intermediate

carrier, has been found in methanoarchaea and a SO42- reducing archeaeon, Archaeglobus

fulgidus (60, 126). MF binds CO2 and forms formyl methanofuran in the first step of the

CO2 reduction pathway (76). Tetrahydromethanopterin (H4MPT) has a similar structure

and function to tetrahydrofolate found in the Eucarya and Bacteria domains (123).

Tetrahydrosarcinapterin (H4SPT), isolated from Methanosarcina sp., has an additional

glutamyl group which differs from H4MPT (119). H4MPT has been isolated from

methanoarchaea, Archaeoglobus fulgidus, and the methylotroph Methylobacterium

extorquens AM1 from the Bacteria domain (19). Coenzyme M is first found in

methanoarchaea and is the smallest of all coenzymes known. The structure is a thiol

attached to sulfonic acid by two methylene groups (110). Coenzyme M is methylated

and CH3CoM is further reduced to methane by CH3CoM methylreductase. This reaction

is found in all methanogenesis pathways (29). Coenzyme B is a low molecular weight,

heat stable, oxygen-sensitive compound (29). It contains a reactive thiol group which

donates electrons in the methyl reductase reaction of CH3CoM (32). Factor F430, named

for its characteristic absorption at 430 nm (46), is a Ni-porphyrin cofactor that is tightly

associated with methylreductase (24-26, 28, 127). F430 is present exclusively in the

methanoarchaea (27). F420 has structural resemblance to FMN or FAD, but it is an

obligate 2-electron carrier equivalent to NAD(P) (123). The redox potential of F420 is in a

range of -340 to –350 mV. Due to the strong fluorescence of F420, it has been used to

determine the presence of methanoarchaea in mixed cultures. Factor III is a corrinoid-

containing compound. It is a component of methyl transferases and the carbon monoxide

dehydrogenase/acetyl CoA synthase complex (78).

7

Figure 2. Structure of the coenzymes involved in methanogenesis (125).

8

Methanoarchaea also contain cofactors that are commonly found in Eucarya and

Bacteria such as thiamin, riboflavin, pyridoxine, biotin, niacin, panthothenate, p-amino

benzoic acid, and molybdopterin (59, 75).

CO2 reduction pathway. Our knowledge of methanogenesis from the CO2

reduction pathway is mostly derived from studies of Methanobacterium

thermoautotrophicum strains ∆H and Marburg (37). Figure 3 illustrates the CO2

reduction pathway. The process is conducted by several one-carbon (C1) intermediate

carriers (14). Electrons for reductive steps in the pathway are derived from the oxidation

of H2 by hydrogenase or formate by formate dehydrogenase (FDH) (14). Hydrogenases

are classified into 3 groups based on their metal composition: NiFe-dehydrogenase (5, 9),

NiFeSe-hydrogenase (86, 131), and Fe-hydrogenase (4). When cells are grown in

formate, FDH oxidizes formate to CO2, which then enters the pathway (107).

Molybdenum- and tungsten-containing formate dehydrogenases have been identified (11-

13, 101, 102). Tungsten-FDH has higher O2 sensitivity than Mo-FDH. FDH contains

FAD, molybdopterin, nonheme iron and acid labile sulfur (8, 56, 116). The FDH of

Methanocoocus vannielii also contains Se (58).

The CO2 reduction pathway is initiated by transferring CO2 to MF followed by the

reduction of CO2 to formyl-MF (76). The formyl group is then transferred to the C1-

carrier H4MPT to produce 5-formyl-H4MPT. Reduction of the formyl moiety proceeds

via F420H2 and involves methenyl, methylene, and methyl redox states (104). Next, the

methyl group is transferred to coenzyme M by a corrinoid-containing methyltransferase

(115). The methyl group of CH3-CoM is finally reductively demethylated to CH4 by the

enzyme complex methyl-CoM methyl reductase (MCR) (32, 33). Electrons for methyl

CoM reduction are derived from coenzyme B which, after oxidation, bonds with

coenzyme M to form the heterodisulfide CoM-S-S-CoB as a byproduct (34). Two MCR

isoenzymes have been identified. Expression of the enzymes is growth phase dependent

(88, 93) and is correlated to H2 levels in the growth medium (15, 83, 120). Recently, the

crystal structure of MCR I was determined and has helped elucidate the active site and

9

Figure 3. The CO2 reduction pathway of methanogenesis (125). X, unknown electron

carrier; MFR, methanofuran; H4MPT, tetrahydromethanopterin; HS-CoM, coenzyme

M; HS-CoB, coenzyme B.

CO2XH2 + MFR

H2O + X

CHO-MFR

CHO-H4MPT

CH H4MPT

CH2 H4MPT

CH3-H4MPT

CH3-S-CoM

CH4

H4MPT

MFR

H2O

F420H2

F420

F420H2

F420

CoM-SH

H4MPT

CoB-SH

CoM-S-S-CoB

10

catalytic mechanism (35). The MCR enzymes contain the F430-Ni porphyrin coenzyme in

which Ni (I) is the catalytically active form (6, 54, 99). The CoM-S-S-CoB is

regenerated to active thiol compounds by a heterodisulfide reductase (49, 51, 104).

Electrons are derived from either H2 or formate.

Acetate fermentation pathway. The fermentation of acetate contributes two-

thirds of all biologically produced methane. Figure 4 summarizes the pathway of acetate

conversion to methane and CO2. In summary, the methyl group of acetate is reduced to

methane by electrons derived from the oxidation of the carbonyl group to CO2. Perhaps

the best characterized acetate fermentation pathway is from Methanosarcina thermophila

TM1. Methanosarcina and Methanosaeta are capable of growth on acetate; however,

they exhibit different affinities for acetate. At high acetate concentrations

Methanosarcina predominate, whereas in acetate-limited environments Methanosaeta

out-competes Methanosarcina (82, 89, 124, 135).

The first step of the acetate fermentation pathway requires activation of acetate.

In Methanosarcina, acetate kinase (3) and phosphotransacetylase (79) activate acetate to

acetyl CoA. In Methanosaeta this reaction is catalyzed by a single enzyme, acetyl CoA

synthetase (acetate thiokinase) (55). The carbon monoxide dehydrogenase/acetyl CoA

synthase (CODH/ACS) enzyme complex is a central enzyme in the pathway.

CODH/ACS catalyzes the cleavage of the C-C and C-S bonds of acetyl CoA, the

oxidation of CO to CO2, and transfer of the methyl group to H4SPT (95, 113).

CODH/ACS enzymes are widespread in procaryotes from both the Bacteria and Archaea

domains, and play roles in oxidation of CO, synthesis of acetyl CoA, or cleavage of

acetyl CoA (38). Enzymes from Methanosarcina sp. contain 5 different subunits which

can be divided into three components (1, 43, 69). Based on the studies of CODH/ACS

from M. thermophila and Clostridium thermoaceticum, the first component, a Ni/Fe-S

enzyme comprised of two subunits, cleaves acetyl CoA and transfers the methyl moiety

to the second component (53, 77). The Ni/Fe-S component also oxidizes CO and reduces

ferredoxin (111, 112, 114). The second component, the two-subunit Co/Fe-S enzyme,

contains factor III in which the active Co (I) is methylated (1, 53). The Co/Fe-S

11

component is a methyltransferase that transfers the methyl group from Co (III) to H4SPT

resulting in CH3-H4SPT (44, 45). The third component is very unstable and only the

truncated subunit from M. barkeri has been characterized. It appears to have acetyl

transferase activity that is responsible for binding of CoA and acetyl CoA (45). The

genes encoding the five subunits of CODH/ACS from M. thermophila cluster in an

operon (cdh) (80). In addition to the genes encoding the five subunits, a sixth open

reading frame (ORF) is co-transcribed with the cdh operon. It was suggested that this

ORF may encode a protein required for maturation of CODH/ACS (36, 39). The methyl

moiety on CH3-H4SPT is finally transferred to CoM by methyl transferase. Methyl

transferase from acetate grown cells has not been characterized. However, it is proposed

that a corrinoid-containing methyl transferase, as present in CO2-reducing

methanoarchaea, is likely to be involved (36, 37). CH3-SCoM is reductively

demethylated to methane in the same way as in the final step in the CO2 reduction

pathway.

Electrons derived from CO oxidation by the Ni/Fe-S component are used to

reduce ferredoxin (1, 114). Ferredoxin from M. thermophila contains 2 [4Fe-4S] centers

(20), which are potentially coordinated by two cysteine motifs of CXXCXXCXXXCP

(21). Electrons from ferredoxin are eventually transferred to heterodisulfide reductase

(HDR) to generate the active sulfhydryl forms of CoM and CoB, as described in the CO2-

reducing pathway. A reconstituted CO:CoM-S-S-CoB oxidoreductase system can be

established with the following purified components: ferredoxin, CODH/ACS,

membranes, and HDR (92). Electron carriers between ferredoxin and HDR have been

identified, including novel iron-sulfur flavoprotein (Isf) and membrane bound carriers.

Heterologously-produced Isf is a homodimer containing two FMN, 7-8 Fe and acid labile

S. Isf stimulates electron transfer from ferredoxin to the heterodisulfide reductase (74).

Further characterization of Isf is described in chapters 4 and 5. At present, what is known

about the electron transport chain is drawn from the CO:CoM-S-S-CoB oxidoreductase

system. The midpoint potentials (Em) determined for each component is consistent with

the electron flow as CODH →ferredoxin →Isf →cytochrome B→ heterodisulfide

reductase (36), but other components may be required. HDRs from M. thermophila and

12

M. barkeri have been purified (72, 105), and both contain b-type hemes and 4Fe-4S

clusters. They lack FAD, which is different from the HDR of M. thermoautotrophicum.

13

Figure 4. Proposed pathway for acetate conversion to CO2 and CH4 in Methanosarcina

thermophila. Ack, acetate kinase; Pta, phosphotransacetylase; CdhABCDE, CODH/ACS

CO dehydrogensae/acetyl-CoA synthase complex; THSpt, tetrahydrosarcinapterin; FdxA,

ferredoxin A; Isf, iron-sulfur flavoprotein; Cyt b, cytochrome b complex; Cam, carbonic

anhydrase; MTase, methyltransferase; Mcri,methyl CoM reductase (inactive); Mcra,

methyl CoM reductase (active); Hdr, heterodisulfide (CoM-S-SCoB) reductase. The

carbon atoms of acetate are marked with* and # to distinguish between the carboxyl and

methyl groups.

*CH3#COSCoA

CdhCCdhB

CdhA

CdhE

CdhD

*CH3-THSPt

THSPt

*CH3-S-CoM

*CH4

Mcra

Mcri

FdxA

FdxA

e-

e-

Cam

Isf

e- Hdr

HS-CoM

HS-CoB

CoM-S-S-CoB

MT-

ase

H+ + H#CO3-

H2O

H+

*CH3#CO2

-

+

CoA

CO

Pta

#CO2

Membrane

Membrane

Cyt b

Ack

14

III. Bioenergetics and electron transport carriers.

As in organisms belonging to the Bacteria and Eucarya domains, ATP is a

general currency in methanoarchaea. Substrate level phosphorylation and electron

transport phosphorylation are the two major mechanisms for ATP synthesis in all

procaryotes. So far, there is no evidence for ATP synthesis by substrate level

phosphorylation in methanoarchaea (85). It has been proposed that a chemiosmotic

mechanism with electron transport-driven phosphorylation is required for ATP synthesis

(117). Several experiments have been performed to test this hypothesis. A number of

thermodynamically favorable reactions associated with ion gradient formation have been

described. Methanoarchaea use both proton and sodium gradients for ATP synthesis and

endergonic reactions. The formation of ion gradients occurs during electron transfer

through a membrane-bound pathway that results in reduction of CoB-S-S-CoM. This

reduction is dependent on H2, F420, or ferredoxin in different methanogenic pathways

(94).

Knowledge of electron transfer pathways in methanogenesis is limited. Not all

electron carriers involved in this process are known; however several redox cofactors

from methanoarchaea have been identified, purified, and characterized. Although some

of these cofactors can serve as electron carriers, their physiological roles are uncertain.

Examples of known electron carriers involved in the electron transfer process are

ferredoxin, cytochromes b or c, and F420.

Ferredoxins are small, acidic redox proteins which contain clusters of non-heme

irons and acid labile sulfides. These iron-sulfur clusters are ligated to proteins by

cysteines. Clusters which serve as redox centers have been identified as [2Fe-2S], [3Fe-

4S], and [4Fe-4S] cluster types. The reduction potentials of ferredoxins range from –145

to +400 mV (81), which are suitable for a variety of redox reactions (18, 132). Unlike

several other iron-sulfur proteins, ferredoxin shows no enzyme activity. Ferredoxins

from the methanoarchaea M. thermophila strain TM1 (20, 112, 114), M. barkeri strains

MS (22, 84) and Fusaro (47), and Methanococcus thermolithotrophicus (48) have been

15

purified and characterized. These ferredoxins have been shown to be central electron

carriers in anabolic and catabolic pathways in methanoarchaea (3, 4, 41, 47, 48, 111)

Polyferredoxins are a class of proteins containing multiple iron-sulfur clusters.

Polyferredoxin from M.thermoautotrophicum contains 12[4Fe-4S] clusters (50, 96). The

genes encoding polyferredoxin (mvh B) from M. thermoautotrophicum, Methanothermus

fervidus, and Methanococcus voltae are located in the methyl viologen (MV)

hydrogenase (mvh) operon and are conserved (50). Therefore, polyferredoxin has been

proposed to function in association with the MV-hydrogenase system; however, the

reduction of polyferredoxin by MV-hydrogenase in the presence of H2 is very low. The

second possible role for polyferredoxin is iron-storage (50, 96).

Cytochromes are ubiquitous membrane-bound electron transfer components in

nature. Only the methanoarchaea that are able to grow on methyl-containing compounds,

such as acetate, methanol and methylamine, contain cytochromes (71). This indicates

that cytochromes are not involved in methane formation from CO2 and H2, or formate. It

has been proposed that cytochromes function in the methyl oxidation of these methyl-

containing compounds (71). Two b-type cytochromes and one c-type cytochrome were

detected in methanol- and methylamine-grown cells whereas the acetate-grown cells

contain an additional b-type cytochrome (70). The midpoint potentials for b-type

cytochromes found in methanol-, methylamine- and acetate-grown cells are –325, -183,

and –253 mV, respectively (70). Recently, the membrane fraction from methanol-grown

Methanosarcina mazei Gö1 revealed two b-type cytochromes and two c-type

cytochromes (63). The midpoint potentials for the b-type cytochromes are –135 and -240

mV and -140 and -230 mV for the c-type cytochromes (63). There has been evidence

suggesting that cytochromes are involved in electron transfer between F420H2 and CoM-

S-S-CoB (63).

F420 is another required electron carrier for methanogenesis. It is an obligate two-

electron carrier in Archaea which functions in analogy to NAD and NADP in the

Eucarya and Bacteria domains. The variable amounts of F420 among methanoarchaea

may be due to different requirements for this electron carrier in diverse metabolism (31).

16

Proteins known to interact with F420 include F420-reducing hydrogenase, NADP-F420

oxidoreductase, formate dehydrogenase, methylenetetrahydromethanopterin

dehydrogenase, methylenetetrahydromethanopterin reductase, secondary alcohol

dehydrogenase, puruvate synthase, and α-ketoglutarate synthase (29).

The FMN-containing flavoprotein, flavoprotein A, was recently purified, cloned,

and sequenced from M. thermoautotrophicum strain ∆H and Marburg (87, 121). It

copurified with the H2:heterodisulfide oxidoreductase complex. The flavoprotein A from

strain Marburg was purified as a homotetramer with a 43 kDa molecular mass per subunit

whereas the one from strain ∆H was a homodimer with a monomeric molecular mass of

45 kDa. The expression of either flavoprotein increased when cells were grown in iron

depleted media. The physiological role for flavoprotein A remains speculative, although

the FMN containing property suggests a function as an electron carrier. It may function

to substitute an essential iron-containing protein during iron starvation. Polyferredoxin

seems to be the most feasible protein for which flavoprotein A can substitute. With many

completed genome sequencing projects, a number of flavoprotein A related protein

sequences have been identified. The sequence comparisons reveal a conserved region for

FMN binding (122).

Methanophenazine is a recently discovered redox-active cofactor. It was first

isolated from the membranes of M. mazei Gö1 and shown to be very hydrophobic. The

structure is a 2-hydroxy phenazine derivative connected to a polyisoprenoid by an ether

bond. It has a molecular mass of 538 Da (2). The 2-hydroxy phenazine (a soluble

analogue of methanophenazine) is able to accept electrons from both F420-hydrogenase

and MV hydrogenase (2). Furthermore, reduced methanophenazine donates electrons to

heterodisulfide reductase from M. thermophila (10). Therefore, methanophenazine has

been proposed to play an important role in vivo in membrane-bound electron transport

systems of F420-hydrogenase: heterodisulfide oxidoreducatase and H2: heterodisulfide

oxidoreducatase. Diphenyleneiodonium chloride (DPI), a competitive inhibitor of

methanophenazine, inhibited both membrane-bound electron transport systems of M.

mazei Gö1 (17).

17

Preliminary investigation of M. thermoautotrophicum membranes is consistent

with the presence of low potential electron carriers (73). EPR studies suggest that iron-

sulfur clusters are membrane components. Upon the addition of either F420 or CH3-CoM

to membranes, the EPR spectra changes, results which are consistent with the

involvement of membrane components in the electron transfer process (98).

18

REFERENCES

1. Abbanat, D. R., and J.G. Ferry. 1991. Resolution of component proteins in an

enzyme complex from Methanosarcina thermophila catalyzing the synthesis or

cleavage of acetyl-CoA. Proc. Natl. Acad. Sci. USA. 88:3272-3276.

2. Abken, H.-J., M. Tietze, J. Brodersen, S. Bäumer, U. Beifuss, and U.

Deppenmeier. 1998. Isolation and characterization of methanophenazine and the

function of phenazines in membrane-bound electron transport of Methanosarcina

mazei Gö1. 180:2027-2032.

3. Aceti, D. J., and J.G. Ferry. 1988. Purification and characterization of acetate

kinase from acetate-grown Methanosarcina thermophila. J. Biol. Chem.

263:15444-15448.

4. Adams, W.W., L.E. Mortenson, J-S. Chen. 1981. Hydrogenase. Biochim.

Biophys. Acta. 594:105-176.

5. Albracht, S. P. J., E.G. Graf, and R. K.Thauer. 1982. The EPR properties of

nickel in hydrogenase from Methanobacterium thermoautotrophicum. FEBS Lett.

140:311-313.

6. Albracht, S. P. J., D. Ankel-Fuchs, R. Bocher, J. Ellermann, J. Moll, J. W.

Van der Zwaan, and R. K. Thauer. 1988. Five new EPR signals assigned to

nickel in methyl-coenzyme M reductase from Methanobacterium

thermoautotrophicum, strain Marburg. Biochim. Biophys. Acta. 941:86-102.

7. Aldrich, H. C., R.W. Robinson, and D.S. Williams. 1986. Ultrastructure of

Methanosarcina mazei. Syst. Appl. Microbiol. 7:314-319.

8. Barber, M. J., L. M. Siegel, N. L Schauer, H. D. May, and J. G. Ferry. 1983.

Formate dehydrogenase from Methanobacterium formicicum. Electron

paramagnetic resonance spectroscopy of the molybdenum and iron-sulfur centers.

J. Biol. Chem. 258:10839-10845.

9. Baron, S. F., and J. G. Ferry. 1989. Purification and properties of the

membrane-associated coenzyme F420-reducing hydrogenase from

Methanobacterium formicicum. J. Bacteriol. 171:3846-3853.

10. Baumer, S., E. Murakami, J. Brodersen, G. Gottschalk, S. W. Ragsdale, and

U. Deppenmeier. 1998. The F420H2:heterodisulfide oxidoreductase system from

19

Methanosarcina species. 2-Hydroxyphenazine mediates electron transfer from

F420H2 dehydrogenase to heterodisulfide reductase. FEBS Lett. 428(3):295-8.

11. Bertram, P. A., M. Karrasch, R. A. Schmitz, R. Bocher, S. P. J. Albracht,

and R. K. Thauer, 1994. Formylmethanofuran dehydrogenases from

methanogenic archaea - substrate specificity, EPR properties and reversible

inactivation by cyanide of the molybdenum or tungsten iron- sulfur proteins. Eur.

J. Biochem. 220:477-484.

12. Bertram, P. A., and R. K. Thauer. 1994. Thermodynamics of the

formylmethanofuran dehydrogenase reaction in Methanobacterium

thermoautotrophicum. Eur. J. Biochem. 226:811-818.

13. Bertram, P. A., R. A. Schmitz, D. Linder, and R.K. Thauer. 1994. Tungstate

can substitute for molybdate in sustaining growth of Methanobacterium

thermoautotrophicum - identification and characterization of a tungsten

isoenzyme of formylmethanofuran dehydrogenase. Arch. Microbiol. 161:220-

228.

14. Blaut, M., V. Muller, and G. Gottschalk. 1992. Energetics of methanogenesis

studied in vesicular systems. J. Bioener. Biomemb. 24:529-546.

15. Bonacker, L. G., S. Baudner, and R. K. Thauer. 1992. Differential expression

of the two methyl-coenzyme M reductases in Methanobacterium

thermoautotrophicum as determined immunochemically via isoenzyme-specific

antisera [published erratum appears in Eur. J. Biochem. 1992 Aug 1;207(3):1129].

Eur. J. Biochem. 206(1):87-92.

16. Boone, D. R., W.B. Whitman, and P. Rouviere. 1993. Diversity and Taxonomy

of Methanogens. Methanogenesis, J.G. Ferry, ed. Chapman and Hall, New York.

35-80.

17. Brodersen, J., S. Baumer, H. J. Abken, G. Gottschalk, and U. Deppenmeier.

1999. Inhibition of membrane-bound electron transport of the methanogenic

archaeon Methanosarcina mazei Gö1 by diphenyleneiodonium. Eur. J. Biochem.

259(1-2):218-24.

18. Bruschi, M. G., F. 1988. Structure, function and evolution of bacterial

ferredoxins. FEMS Microbiol. Rev. 54:155.

20

19. Chistoserdova, L., J. A. Vorholt, R. K. Thauer, and M. E. Lidstrom. 1998. C1

transfer enzymes and coenzymes linking methylotrophic bacteria and

methanogenic Archaea. Science. 281(5373):99-102.

20. Clements, A. P., L. Kilpatrick, W.-P. Lu, S.W. Ragsdale, and J.G. Ferry.

1994. Characterization of the iron-sulfur clusters in ferredoxin from acetate-

grown Methanosarcina thermophila. J. Bacteriol. 176:2689-2693.

21. Clements, A. P., and J.G. Ferry. 1992. Cloning, nucleotide sequence, and

transcriptional analyses of the gene encoding a ferredoxin from Methanosarcina

thermophila. J. Bacteriol. 174:5244-5250.

22. Daas, P. J. H., W. R. Hagen, J. T. Keltjens, and G. D. Vogels. 1994.

Characterization and determination of the redox properties of the 2[4Fe-4S]

ferredoxin from Methanosarcina barkeri strain MS. FEBS Lett. 356:342-344.

23. Daniels, L., G. Fuchs, R. K. Thauer, and J. G. Zeikus. 1977. Carbon monoxide

oxidation by methanogenic bacteria. J. Bacteriol. 132:118-126.

24. Diekert, G., R. Jaenchen, and R. K. Thauer. 1980. Biosynthetic evidence for a

nickel tetrapyrrole structure of factor F430 from Methanobacterium

thermoautotrophicum. FEBS Lett. 119:118-120.

25. Diekert, G., H-H. Gilles, R. Jaenchen, and R. K Thauer. 1980. Incorporation

of 8 succinate per mol nickel into factors F430 by Methanobacterium

thermoautotrophicum. Arch. Microbiol. 128:256-262.

26. Diekert, G., B. Weber, and R.K. Thauer. 1980. Nickel dependence of factor

F430 content in Methanobacterium thermoautotrophicum. Arch. Microbiol.

127:273-278.

27. Diekert, G., U. Konheiser, K. Piechulla, and R.K. Thauer. 1981. Nickel

requirement and factor F430 content of methanogenic bacteria. J. Bacteriol.

148:459-464.

28. Diekert, G., B. Klee, and R.K. Thauer. 1980. Nickel, a component of factor F430

from Methanobacterium thermoautotrophicum. Arch. Microbiol. 124:103-106.

29. DiMarco, A. A., , A. T. Bobik, and R. S. Wolfe. 1990. Unusual coenzymes of

methanogenesis. Annu. Rev. Biochem. 59:355-394.

21

30. Doddema, H. J., J. W. M. Derksen, and G.D. Vogels. 1979. Fimbriae and

flagella of methanogenic bacteria. FEMS Microbiol. Lett. 5:135-138.

31. Eirich, L. D., G. D.Vogels, and R. S. Wolfe. 1979. Distribution of coenzyme

F420 and properties of its hydrolytic fragments. J. Bacteriol. 140:1:20-27.

32. Ellermann, J., R. Hedderich, R. Bocher, and R. K. Thauer. 1988. The final

step in methane formation. Investigations with highly purified methyl-CoM

reductase (component C) from Methanobacterium thermoautotrophicum (strain

Marburg). Eur. J. Biochem. 172:669-677.

33. Ellermann, J., S. Rospert, R. K.Thauer, M. Bokranz, A. Klein, M. Voges, and

A. Berkessel. 1989. Methyl-coenzyme-M reductase from Methanobacterium

thermoautotrophicum (strain Marburg): purity, activity and novel inhibitors. Eur.

J. Biochem. 184:63-68.

34. Ellermann, J., A. Kobelt, A. Pfaltz, and R. K. Thauer. 1987. On the role of N-

7 mercaptoheptanoyl-O-phospho-L-threonine (componentB) in the enzymatic

reduction of methyl-coenzyme M to methane (FEB 05028). FEBS Lett. 220:358-

362.

35. Ermler, U., W. Grabarse, S. Shima, M. Goubeaud, and R. K. Thauer. 1997.

Crystal structure of methyl-coenzyme M reductase: the key enzyme of biological

methane formation. Science. 278:1457-62.

36. Ferry, J., G. 1997. Enzymology of the fermentation of acetate to methane by

Methanosarcina thermophila. BioFactors. 6:25-35.

37. Ferry, J. G. 1992. Biochemistry of methanogenesis. Crit. Rev. Biochem. Mol.

Biol. 27:473-503.

38. Ferry, J. G. 1995. CO dehydrogenase. Ann. Rev. Microbiol. 49:305-333.

39. Ferry, J. G. 1999. Enzymology of one-carbon metabolism in methanogenic

pathways. FEMS Microbiol. Rev. 23:13-38.

40. Ferry, J. G. 1997. Methane: small molecule, big impact [comment]. Science.

278:1413-4.

41. Fischer, R., and R.K. Thauer. 1990. Ferredoxin-dependent methane formation

from acetate in cell extracts of Methanosarcina barkeri (strain MS). FEBS Lett.

269:368-372.

22

42. Fox, G. E., L. J. Marrum, W. E. Balch, R. S. Wolfe, and C. R. Woese. 1977.

Classification of methanogenic bacteria by 16S ribosomal RNA characterization.

Proc. Natl. Acad. Sci. USA. 74:4537-4541.

43. Grahame, D. A., and T. C. Stadtman. 1987. Carbon monoxide dehydrogenase

from Methanosarcina barkeri. Disaggregation, purification, and physicochemical

properties of the enzyme. J. Biol. Chem. 262:3706-3712.

44. Grahame, D. A. 1991. Catalysis of Acetyl-CoA Cleavage and

tetrahydrosarcinapterin methylation by a carbon monoxide dehydrogenase-

corrinoid enzyme complex. J Biol Chem. 266:22227-22233.

45. Grahame, D. A., and E. Demoll. 1996. Partial reactions catalyzed by protein

components of the acetyl-CoA decarbonylase synthase enzyme complex from

Methanosarcina barkeri. J. Biol. Chem. 271:8352-8358.

46. Gunsalus, R. P., and R. S. Wolfe. 1978. Chromophoric factors F342 and F430 of

Methanobacterium thermoautotrophicum. FEMS Microbiol. Lett. 3:191-193.

47. Hatchikian, E. C., M. Bruschi, N. Forget, and M. Scandellari. 1982. Electron

transport components from methanogenic bacteria: the ferredoxin from

Methanosarcina barkeri (strain Fusaro). Biochem. Biophys. Res. Comm.

109:1316-1323.

48. Hatchikian, E. C., M. L. Fardeau, M. Bruschi, J.P. Belaich, A. Chapman, R.

Cammack. 1989. Isolation, characterization, and biological activity of the

Methanococcus thermolithotrophicus ferredoxin. J. Bacteriol. 171:2384-2390.

49. Hedderich, R., A. Berkessel, and R. K. Thauer. 1989. Catalytic properties of

the heterodisulfide reductase involved in the final step of methanogenesis. FEBS

Lett. 255:67-71.

50. Hedderich, R., S. P. J. Albracht, D. Linder, J. Koch, and R. K. Thauer. 1992.

Isolation and characterization of polyferredoxin from Methanobacterium-

thermoautotrophicum - The mvhB gene product of the methylviologen-reducing

hydrogenase operon. FEBS Letters. 298:65-68.

51. Hedderich, R., A. Berkessel, and R. K. Thauer. 1990. Purification and

properties of heterodisulfide reductase from Methanobacterium

thermoautotrophicum (strain Marburg). Eur. J. Biochem. 193:255-261.

23

52. Hungate, R. E. 1967. A roll tube method for cultivation of strict anaerobes.

Methods in Microbiology, Norris J.R., and Ribbons D.W. ed, Academic Press,

New York. 2B:117-132.

53. Jablonski, P. E., W. P. Lu, S. W. Ragsdale, and J. G. Ferry. 1993.

Characterization of the metal centers of the corrinoid;iron-sulfur component of the

CO dehydrogenase enzyme complex from Methanosarcina thermophila by EPR

spectroscopy and spectroelectrochemistry. J Biol Chem. 268:325-329.

54. Jaun, B., and A. Pfaltz. 1988. Coenzyme F430 from methanogenic bacteria:

methane formation by reductive carbon-sulfur bond cleavage of methyl

sulphonium ions catalysed by F430 pentamethyl ester. J. Chem. Soc. Chem.

Commun.:293-294.

55. Jetten, M. S. M., A. J. M. Stams, and A. J. B Zehnder. 1989. Isolation and

characterization of acetyl-coenzyme A synthetase from Methanothrix soehngenii.

J. Bacteriol. 171:5430-5435.

56. Johnson, J. L., N. R. Bastian, N. L. Schauer, J. G. Ferry, and K.V.

Rajagopalan. 1991. Identification of molybdopterin guanine dinucleotide in

formate dehydrogenase from Methanobacterium formicicum. FEMS Microbiol.

Lett. 77:2-3.

57. Jones, J. B., B. Bowers, and T. C. Stadtman. 1977. Methanococcus vannielii:

ultrastructure and sensitivity to detergents and antibiotics. J. Bacteriol. 130:1357-

1363.

58. Jones, J. B, and T. C. Stadtman. 1981. Selenium-dependent and selenium-

independent formate dehydrogenases of Methanococcus vannielii. J. Biol. Chem.

256;2:656-663.

59. Jones, W. J., D. P. Nagle, and W. B. Whitman. 1987. Methanogens and the

diversity of archaebacteria. Microbiol. Rev. 51:135-177.

60. Jones, W. J., M. I. Donnelly, and R. S. Wolfe. 1985. Evidence of a common

pathway of carbon dioxide reduction to methane in methanogens. J. Bacteriol.

163(1):126-31.

24

61. Kalmokoff, M. L., and K.F. Jarrell. 1991. Cloning and sequencing of a

multigene family encoding the flagellins of Methanococcus voltae. J. Bacteriol.

173:7113-7125.

62. Kalmokoff, M. L., K. F. Jarrell, and S. F. Koval. 1988. Isolation of flagella

from the archaebacterium Methanococcus voltae by phase separation with Triton

X-114. J. Bacteriol. 170:1752-8.

63. Kamlage, B., and M. Blaut. 1992. Characterization of cytochromes from

Methanosarcina strain Göl and their involvement in electron transport during

growth on methanol. J. Bacteriol. 174:3921-7.

64. Keltjens, J. T. 1984. Coenzymes of methanogenesis from hydrogen and carbon

dioxide. Antonie Van Leeuwenhoek. 50:383-96.

65. Kennes, C., M. C. Veiga, and L. Bhatnagar. 1998. Methanogenic and

perchloroethylene-dechlorinating activity of anaerobic granular sludge. Appl.

Microbiol. Biotechnol. 50:484-8.

66. Kennes, C., W. M. Wu, L. Bhatnagar, and J. G. Zeikus. 1996. Anaerobic

dechlorination and mineralization of pentachlorophenol and 2,4,6-trichlorophenol

by methanogenic pentachlorophenol-degrading granules. Appl. Microbiol.

Biotechnol. 44:801-6.

67. Kiene, R. P. 1991. Production and consumption of methane in aquatic systems.

Microbial production and consumption of greenhouse gases: methane, nitrogen

oxides, and halomethanes, J.E. Roger and W.B. Whitman, ed. American society

for microbiology, Washington, D.C. 111-146.

68. Kreisl, P., and O. Kandler. 1986. Chemical structure of the cell wall polymer

Methanosarcina. Syst. Appl. Microbiol. 7:293-299.

69. Krzycki, J. A., and J. G Zeikus. 1984. Characterization and purification of

carbon monoxide dehydrogenase from Methanosarcina barkeri. J. Bacteriol.

158:231-237.

70. Kuhn, W., and G. Gottschalk. 1983. Characterization of the cytochromes

occurring in Methanosarcina species. Eur. J. Biochem. 135:89-94.

25

71. Kuhn, W., K. Fiebig, H. Hippe, R. A. Mah, B. A. Huser, and G. Gottschalk.

1983. Distribution of cytochromes in methanogenic bacteria. FEMS Microbiol.

Lett. 20:407-410.

72. Kunkel, A., M. Vaupel, S. Heim, R. K. Thauer, and R. Hedderich. 1997.

Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri

is not a flavoenzyme. Eur. J. Biochem. 244:226-34.

73. Lancaster, J. J. R . 1986. A unified scheme for carbon and electron flow coupled

to ATP synthesis by substrate-level phosphorylation in the methanogenic bacteria.

FEBS. Lett. 199:12-18.

74. Latimer, M. T., M. H. Painter, and J. G. Ferry. 1996. Characterization of an

iron-sulfur flavoprotein from Methanosarcina thermophila. J. Biol. Chem.

271:24023-24028.

75. Leigh, J., A . 1983. Levels of water-soluble vitamins in methanogenic and non-

methanogenic bacteria. Appl. Environ. Microbiol. 45:800-803.

76. Leigh, J. A., K. L. Jr. Rinehart, and R. S. Wolfe. 1985. Methanofuran (carbon

dioxide reduction factor), a formyl carrier in methane production from carbon

dioxide in Methanobacterium. Biochemistry. 24:995-999.

77. Lu, W. P., P. E. Jablonski, M. Rasche, J. G. Ferry, and S. W. Ragsdale. 1994.

Characterization of the metal centers of the Ni-Fe-S component of the carbon-

monoxide dehydrogenase enzyme complex from Methanosarcina thermophila. J.

Biol. Chem. 269:9736-9742.

78. Ludwig, M. L., and R. G. Matthews. 1997. Structure-based perspectives on

B12-dependent enzymes. Annu. Rev. Biochem. 66:269-313.

79. Lundie, L. L., and J. G. Ferry. 1989. Activation of acetate by Methanosarcina

thermophila. purification and characterization of phosphotransacetylase. J. Biol.

Chem. 264:18392-18396.

80. Maupin-Furlow, J. A., and J. G. Ferry. 1996. Analysis of the CO

dehydrogenase/acetyl-coenzyme A synthase operon of Methanosarcina


81. Meyer, J. 1988. The evolution of ferredoxins. Trends Ecol. Evol.. 3:222-226.

26

82. Min, H., and S.H. Zinder. 1989. Kinetics of acetate utilization by 2 thermophilic

acetotrophic methanogens - Methanosarcina Sp Strain CALS-1 and Methanothrix

Sp Strain CALS-1. Appl. Env. Microbiol. 55:488-491.

83. Morgan, R. M., T. D. Pihl, J. Nolling, and J. N. Reeve. 1997. Hydrogen

regulation of growth, growth yields, and methane gene transcription in

Methanobacterium thermoautotrophicum deltaH. J. Bacteriol. 179(3):889-98.

84. Moura, I., J. G. Moura, B. Huynh, H. Santos, J. LeGall, and A. V. Xavier.

1982. Ferredoxin from Methanosarcina barkeri: evidence for the presence of a

three-iron center. Eur. J. Biochem. 126:95-98.

85. Muller, V., M. Blaut, and G. Gottschalk. 1993. Bioenergetics of

methanogenesis. Methanogenesis, J.G. Ferry, ed. Chapman and Hall, New York.

360-406.

86. Muth, E., E. Morschel, and A. Klein. 1987. Purification and characterization of

an 8-hyroxy-5-deazaflavin-reducing hydrogenase from the archaebacterium

Methanococcus voltae. Eur. J. Biochem. 169:571-577.

87. Nolling, J., M. Ishii, J. Koch, T. D. Pihl, J. N. Reeve, R.K. Thauer, and

Hedderich, R. 1995. Characterization of a 45-kda flavoprotein and evidence for a

rubredoxin, two proteins that could participate in electron transport from H2 to

CO2 in methanogenesis in Methanobacterium thermoautotrophicum. Eur. J.

Biochem. 231:628-638.

88. Nolling, J., T. D. Pihl, A. Vriesema, and J. N. Reeve. 1995. Organization and

growth phase-dependent transcription of methane genes in two regions of the

Methanobacterium thermoautotrophicum genome. J. Bacteriol. 177:2460-2468.

89. Ohtsubo, S., K. Demizu, S. Kohno, I. Miura, T. Ogawa, and H. Fukuda. 1992.

Comparison of acetate utilization among strains of an aceticlastic methanogen,

Methanothrix-Soehngenii. Appl. Env. Microbiol. 58:703-705.

90. Oremland, R. S. 1988. Biogeochemistry of methanogenic bacteria. Biology of

anaerobic microorganisms. Zehnder A.J.B. ed. John Wiley & Son, Inc. 641-705.

91. Oremland, R. S., R. P. Kiene, I. Mathrani, M. J. Whiticar, and D.R. Boone.

1989. Description of an estuarine methylotrophic methanogen which grows on

dimethyl sulfide. Appl. Environ. Microbiol. 55:994-1002.

27

92. Peer, C. W., M. H. Painter, M. E. Rasche, and J. G. Ferry. 1994.

Characterization of a CO:heterodisulfide oxidoreductase system from acetate-


93. Pihl, T. D., S. Sharma, , and J.N. Reeve. 1994. Growth phase-dependent

transcription of the genes that encode the two methyl coenzyme m reductase

isoenzymes and n-5- methyltetrahydromethanopterin:coenzyme M

methyltransferase in Methanobacterium thermoautotrophicum delta H. J.

Bacteriol. 176:6384-6391.

94. Rasche, M., E, and J. G. Ferry, 1996. Methanogens and Archaea, Molecular

biology of. Encyclopedia of Molecular Biology:66-78.

95. Raybuck, S. A., S. E. Ramer, D. R. Abbanat, J. W. Peters, W. H. Orme-

Johnson, J. G. Ferry, and C. T. Walsh. 1991. Demonstration of carbon-carbon

bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the

CO dehydrogenase complex from acetate-grown Methanosarcina thermophila. J.

Bacteriol. 173:929-932.

96. Reeve, J. N., G. S. Beckler, D. S. Cram, P. T. Hamilton, J. W. Brown, J. A.

Krzycki, A. F. Kolodziej, L. Alex, W. H. Ormejohnson, and C.T. Walsh.

1989. A hydrogenase-linked gene in Methanobacterium thermoautotrophicum

strain H encodes a polyferredoxin. Proc. Natl. Acad. Sci. USA. 86:3031-3035.

97. Robinson, J. A., and J. M. Tiedje. 1984. Competition between sulfate-reducing

and methanogenic bacteria for H2 under resting and growing conditions. Arch.

Microbiol. 137:26-32.

98. Rogers, K. R., K. Gillies, and J. R. Lancaster Jr. 1988. Iron-sulfur centers

involved in methanogenic electron transfer in Methanobacterium

thermoautotrophicum. Biochem. Biophys. Res. Commun. 153:87-95.

99. Rospert, S., M. Voges, A. Berkessel, S. P. J. Albracht, and R. K. Thauer.

1992. Substrate-analogue-induced changes in the nickel-EPR spectrum of active

methyl-coenzyme-M reductase from Methanobacterium- thermoautotrophicum.

Eur. J. Biochem. 210:101-107.

28

100. Schink, B. 1988. Principles and limits of anaerobic degradation: environmental

and technical aspects. Biology of anaerobic bacteria. Zehnder A.J.B. ed. Wiley

Interscience , New York. 771-846.

101. Schmitz, R. A., S. P. J. Albracht, and R. K. Thauer. 1992. A molybdenum and

a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic

archaeon Methanobacterium wolfei. Eur J Biochem. 209:1013-1018.

102. Schmitz, R. A., S. P. J. Albracht, and R. K. Thauer, 1992. Properties of the

tungsten-substituted molybdenum formylmethanofuran dehydrogenase from

Methanobacterium wolfei. FEBS Lett. 11479:78-81.

103. Schonheit, P. J., K. Kristjansson, and R. K. Thauer. 1982. Kinetic mechanism

for the ability of sulfate reducers to out-compete methanogens for acetate. Arch.

Microbiol. 132:285-288.

104. Schworer, B., and R. K. Thauer. 1991. Activities of formylmethanofuran

dehydrogenase, methylenetetrahydromethanopterin dehydrogenase,

methylenetetrahydromethanopterin reductase, and heterodisulfide reductase in

methanogenic bacteria. Arch. Microbiol. 155:459-465.

105. Simianu, M., E. Murakami, J. M. Brewer, and S.W. Ragsdale. 1998.

Purification and properties of the heme and iron-sulfur containing heterodisulfide

reductase from Methanosarcina thermophila. Biochemistry. 37:10027-39.

106. Sowers, K., R,Gunsalus,R,P. 1988. Adaptation for growth at various saline

concentrations by the archaebacterium Methanosarcina thermophila. J. Bacteriol.

170:998-1002.

107. Sparling, R., and L. Daniels. 1986. Source of carbon and hydrogen in methane

produced from formate by Methanococcus thermolithotrophicus. J. Bacteriol.

168:1402-1407.

108. Sprott, G. D., and T. J. Beveridge. 1993. Microscopy. Methanogenesis, J.G.

Ferry, ed. Chapman and Hall, New York. 81-127.

109. Stams, A. J. 1994. Metabolic interactions between anaerobic bacteria in

methanogenic environments. Antonie Van Leeuwenhoek. 66:271-94.

110. Taylor, C. D, and R. S. Wolfe. 1974. Structure and methylation of coenzyme M

(HSCH2CH2SO3). J. Biol. Chem. 249:4879-4885.

29

111. Terlesky, K. C., M. J. Barber, D. J. Aceti, and J. G. Ferry. 1987. EPR

properties of the Ni-Fe-C center in an enzyme complex with carbon monoxide

dehydrogenase activity from acetate-grown Methanosarcina thermophila.

Evidence that acetyl-CoA is a physiological substrate. J. Biol. Chem. 262:15392-

15395.

112. Terlesky, K. C., and J. G. Ferry. 1988. Ferredoxin requirement for electron

transport from the carbon monoxide dehydrogenase complex to a membrane-

bound hydrogenase in acetate-grown Methanosarcina thermophila. J. Biol. Chem.

263:4075-4079.

113. Terlesky, K. C., M. J. Nelson, and J. G. Ferry. 1986. Isolation of an enzyme

complex with carbon monoxide dehydrogenase activity containing a corrinoid and

nickel from acetate-grown Methanosarcina thermophila. J. Bacteriol. 168:1053-

1058.

114. Terlesky, K. C., and J. G. Ferry. 1988. Purification and characterization of a

ferredoxin from acetate-grown Methanosarcina thermophila. J. Biol. Chem.

263:4080-4082.

115. Thauer, R. K. 1990. Energy metabolism of methanogenic bacteria. Biochim.

Biophys. Acta. 1018:256-259.

116. Thauer, R. K., R. Hedderich, and R. Fischer. 1993. Reactions and enzymes

involved in methanogenesis from CO2 and H2. Methanogenesis, J.G. Ferry, ed.

Chapman and Hall, New York. 209-252.

117. Thauer, R. K., K. Jungermann, and K. Decker. 1977. Energy conservation in

chemotrophic anaerobic bacteria. Bacteriol. Rev. 41:100-80.

118. Tylor, S. C. 1991. The global methane budget. Microbial production and

consumption of greenhouse gases:, methane, nitrogen oxides, and halomethanes,

J.E. Roger and W.B. Whitman, ed. American society for microbiology,

Washington, D.C. 7-38.

119. Van Beelen, P., J. F. Labro, J. T. Keltjens, W. J. Geerts, G. D. Vogels, W. H.

Laarhoven, W. Guijt, and C. A. Haasnoot. 1984. Derivatives of methanopterin,

a coenzyme involved in methanogenesis. Eur. J. Biochem. 139:359-65.

30

120. Vermeij, P., J. L. Pennings, S. M. Maassen, J. T. Keltjens, and G. D. Vogels.

1997. Cellular levels of factor 390 and methanogenic enzymes during growth of

Methanobacterium thermoautotrophicum delta H. J. Bacteriol. 179(21):6640-8.

121. Wasserfallen, A., K. Huber, and T. Leisinger. 1995. Purification and structural

characterization of a flavoprotein induced by iron limitation in Methanobacterium

thermoautotrophicum marburg. J. Bacteriol. 177:2436-2441.

122. Wasserfallen, A., S. Ragettli, Y. Jouanneau, and T. Leisinger. 1998. A family

of flavoproteins in the domains Archaea and Bacteria. Eur J Biochem. 254:325-

32.

123. Weiss, D. S., and R. K. Thauer. 1993. Methanogenesis and the unity of

biochemistry. Cell. 72:819-22.

124. Westermann, P., B. K. Ahring, and R.A. Mah. 1989. Threshold acetate

concentrations for acetate catabolism by aceticlastic methanogenic bacteria. Appl.

Environ. Microbiol. 55:514-515.

125. White, R. H., and D. Zhou. 1993. Biosynthesis of the coenzymes in

methanogens. Methanogenesis, J.G. Ferry, ed. Chapman and Hall, New York.

409-444.

126. White, R. H. 1988. Structural diversity among methanofurans from different

methanogenic bacteria. J. Bacteriol. 170:4594-7.

127. Whitman, W. B., and R. S. Wolfe. 1980. Presence of nickel in factor F430 from

Methanobacterium bryantii. Biochem. Biophys. Res. Commun. 92:1196-1201.

128. Widdel, F. 1986. Growth of methanogenic bacteria in pure culture with 2-

propanol and other alcohols as hydrogen donors. Appl. Environ. Microbiol.

51:1056-1062.

129. Woese, C. R, and G. E. Fox. 1978. Methanogenic bacteria. Nature. 273:101.

130. Woese, C. R., Kandler, O., and M. L. Wheelis. 1990. Towards a natural system

of organisms. Proposal for the domains archaea, bacteria, and eucarya. Proc.

Natl. Acad. Sci. U.S.A. 87:4576-4579.

131. Yamazaki, S. 1982. A selenium-containing hydrogenase from Methanococcus

vannielii. J. Biol. Chem. 257:7926-7929.

31

132. Yoch, D. C., and R. C. Valentine. 1972. Ferredoxins and flavodoxins of

bacteria. Ann. Rev. Microbiol. 26:139-162.

133. Zellner, G., and J. Winter. 1987. Secondary alcohols as hydrogen donors for

C02-reduction by methanogens. FEMS Microbiol. Lett. 44:323-328.

134. Zinder, S. H., and R. A. Mah. 1979. Isolation and characterization of a

thermophilic strain of Methanosarcina unable to use H2-CO2 for methanogenesis.

Appl. Environ. Microbiol. 38:996-1008.

135. Zinder, S. H, S. C. Cardwell, T. Anguish, M. Lee, and M. Koch. 1984.

Methanogenesis in a thermophilic (58o C) anaerobic digestor: Methanothrix sp. as

an important aceticlastic methanogen. Appl. Environ. Microbiol. 47:796-807.

136. Zinder, S. H. 1993. Physiological ecology of methanogenesis. Methanogenesis,

J.G. Ferry, ed. Chapman and Hall, New York. 128-206.

32

CHAPTER 2

IRON-SULFUR PROTEINS

I. Introduction.

Since their discovery in the 1960s, a vast amount of data has been accumulated

regarding iron-sulfur proteins. These proteins are abundantly found in nature and are

regarded to be an ancient component of a chemoautotrophic process in which a Ni/Fe-S

center catalyzes carbon fixation (39). Although most iron-sulfur proteins are electron

carriers, they have also been shown to possess other functions such as providing substrate

binding sites, general structural roles, iron storage, and gene regulation. Developments in

the fields of biophysical techniques, molecular biology, chemical synthesis, and computer

modeling, in conjunction with site-directed mutagenesis methods, have resulted in a rapid

accumulation of data concerning iron-sulfur proteins. Insight regarding cluster assembly,

protein stability, electronic structure, functions, and ligand coordination has been

revealed.

II. Structure and properties of clusters.

Iron-sulfur proteins are broadly classified as either simple or complex. The first

group contains only iron-sulfur clusters, whereas the latter contains additional prosthetic

groups (42). Clusters in iron-sulfur proteins, rather than single-metal sites, provide the

diversity and versatility for these proteins to function properly (76). The clusters in iron-

sulfur proteins contain Fe with at least partial S coordination (42). Their structures are

Fe2+ or Fe3+ with approximately tetrahedral coordination to S atoms of cysteine residues

and inorganic sulfides. Inorganic sulfides are sometime referred to as "acid-labile

sulfides", because the sulfur atoms have a –2 valence state that are released as H2S with

acid treatment (85). Cysteine appears to be a major residue involved in cluster bridging to

the polypeptide; nonetheless, evidence of non-cysteinyl ligands has recently emerged.

The structure/function basis for non-cysteinyl versus cysteinyl ligands has not been

determined; however, it appears that when Fe is coordinated with non-cysteinyl ligands

the cluster is involved in substrate binding and there is a shift in reduction potential (42).

33

The properties of iron-sulfur clusters are dependent upon their electronic

configuration and capability of electron delocalization. Iron-sulfur proteins show a very

broad range of midpoint potentials. This can result from several factors including nature

of cluster ligands, hydrophobicity and charge of residues in the surrounding polypeptide

environment (61, 82). Electron paramagnetic resonance (EPR) and Mössbauer

spectroscopies are the techniques widely used to examine iron-sulfur clusters in proteins

and their properties. EPR spectroscopy is applicable to paramagnetic systems, and it is a

sensitive method which is ideal for studying many metalloproteins. Information obtained

from EPR analysis included electronic structure, metal coordination-sphere composition

and geometry (52). On the other hand, Mössbauer spectroscopy gives detailed pictures of

the chemical state of the iron atoms as well as the electron distribution in various redox

states of different iron-sulfur cluster types. Information derived from Mössbauer spectra

include the nature and arrangement of the ligand, spin of the iron atoms, spin coupling,

and the surrounding protein environment (41). To obtain an adequate Mössbauer

spectrum, the sample must contain a certain minimum quantity of the Mössbauer isotope,

which is 57Fe for iron-sulfur proteins. Due to the low natural abundance of 57Fe,

enrichment of the sample with 57Fe is always necessary (41).

[1Fe] cluster type. Basic structures of Fe-S clusters and their properties are

shown in Figure 1 (42). This cluster type, also referred to as rubredoxin-type cluster,

contains a single Fe atom bridged with four cysteines. The proteins show intense red

color when oxidized due to the charge transfer of S →Fe (17, 53). The structures of

rubredoxins from several microbial species have been solved. The EPR spectrum of the

ferric protein indicates the presence of high spin iron (S = 5/2) with g values of 4.3 and 9

(66). The oxidized rubredoxin with high spin Fe3+exhibits a small chemical shift at 0.25

mm/s at 77 oK and three quadrupole doublets in the Mössbauer spectrum (68, 71). A

large quadrupole split (3.16 mm/s at 77 oK) and small chemical shift (0.65 mm/s at 77 oK

has been observed for the Fe2+ form (68, 71). The coordinating cysteine motifs, CXXC,

at both the N- and C- terminal are conserved among these proteins (65). The midpoint

potential of the cluster is in the range of +20 to –60 mV.

34

[2Fe-2S] cluster type. This cluster type is composed of a Fe2S2 (S-cys)4 unit.

The oxidized form ([2Fe-2S]2+) contains two Fe3+ ions. When reduced by one electron, a

mixed valence of Fe3+- Fe2+ ([2Fe-2S]1+ cluster results. This type of cluster is always

described as a “plant-type ferredoxin”, since it is present in photosynthetic organisms.

However, this cluster type has also been characterized from ferredoxins of halophiles,

and species in the genera Rhodobacter, Clostridium, and Azotobacter from the Bacteria

domain. (31). A [2Fe-2S] subclass called the Rieskie cluster describes iron-sulfur

proteins where the [2Fe-2S] clusters are coordinated by non-cysteinyl ligation from two

histidines (55). This subclass has a higher reduction potential than other [2Fe-2S]

proteins with exclusively cysteine ligands. The sequence motif for this subclass is

CXHX15-17CXXH (7, 15). All of the sequences known for coordination are shown in

Figure 2. The two Fe3+ with S = 5/2 are antiferromagnetically coupled in the oxidized

state and, thus, are EPR silent. Conversely, with the reduced state of [2Fe-2S]+, the

antiferromagnetic couple results in S = _, and the typical gav value derived from EPR is

1.96 for plant type ferredoxin and 1.90 for Rieskie proteins (81). The Mössbauer

spectrum for the oxidized proteins shows a quadrupole split with a small chemical shift

value, while the reduced proteins give two quadrpole doublets. One doublet corresponds

to the Fe3+ ion in the cluster, and the other with larger quadrupole is derived from Fe2+

ion in the cluster (41).

[3Fe-4S] cluster type. This cluster type was first interpreted as an artifact of a

degradative product of a [4Fe-4S] center (6), because [4Fe-4S] centers can be

interconverted to [3Fe-4S] centers by removal of an iron unit. However, a considerable

amount of data has shown that the [3Fe-4S] cluster is indeed a true cluster in nature for

some proteins. The core structure is Fe3S4 (S-cys)3 which forms a cube-like structure due

to a missing Fe. The [3Fe-4S] center involves one electron transfers with 1+ and 0

reduction states. In the 1+ oxidation state, all Fe3+ are antiferromagnetically coupled

which results in S = _. The EPR signal for this [3Fe-4S]1+ cluster is either axial or

rhombic with the resonance at gav values of 2.02. The Mössbauer spectrum of oxidized

ferredoxin II from Desulfovibrio gigas is a single quadrupole doublet with chemical shift

at 0.27 mm/s characteristic of the Fe3+ in tetrahedral S coordination. The reduced cluster

35

produces a spectrum with two different intensities (2:1) of quadrupole doublet (40). The

less intense signal derived from Fe3+ is unchanged upon reduction, as indicated by the

Mössbauer parameter (40). Several sequence motifs coordinating [3Fe-4S] are shown in

Figure 2. The motifs contain two cysteines located closely to each other and another

distal cysteine. The cysteine motif coordinating the [4Fe-4S] cluster that is easily

converted to [3Fe-4S] cluster mostly contains aspartate in the place of the second

cysteine (9, 25, 42).

[4Fe-4S] cluster type. This cluster has a Fe4S4 (S-cys)4 cubane structure. It is

involved in one electron transfer reactions with three associated oxidation states, +3, +2,

and +1 (10, 11, 34). A cluster that undergoes three oxidation states in a biological system

has not been reported. These cluster types have a wide reduction potential in the range of

+450 to –700 mV. The [4Fe-4S] cluster with low reduction potential stays in the +2/+1

state, while the high reduction potential proteins (HIPIPs) stay in the +3/+2 state (31).

The low reduction potential of reduced [4Fe-4S]1+ and the oxidized HIPIPs with [4Fe-

4S]3+ are EPR active species. EPR analysis for [4Fe-4S]3+ of HIPIPs yields an axial

signal with g = 2.2 and 2.04 whereas the [4Fe-4S]1+ exhibits a rhombic type EPR with g =

20.6, 1.92, 1.88 with gav < 2. Mössbauer spectra of [4Fe-4S]2+ centers are generally

broad and also indicate almost equivalent iron atoms in the cluster. When the [4Fe-4S]2+

is oxidized or reduced, the spectra indicate an inequivalence of iron atoms in the clusters.

This feature is clearly evident from the spectra obtained in an applied magnetic field (41).

36

Cluster type oxidation EPR Mössbauer and spin number (g) ( δ)

Figure 1. Structures, and properties of the structurally characterized iron-sulfur centers

that are involved in biological systems (42, 52).

Fe2+ S = 2 - 0.65

Fe3+ S = 5/2 4.3, 9 0.25

[2Fe-2S]+ S = _ 1.89, 1.96, 2.05 0.25, 0.55

[2Fe-2S]2+ S = 0 - 0.26

[3Fe-4S]0 S =2 - 0.30, 0.46

[3Fe-4S]+ S =1/2 1.97, 2.00, 2.02 0.27

[4Fe-4S]+ S = 1/2 or 3/2 1.88, 1.92, 2.06 0.57

[4Fe-4S]2+ S = 0 - 0.42

[4Fe-4S]3+ S = _ 2.04, 2.04, 2.12 0.31

37

A.

N----CX4CX2CX29C----C Plant-type ferredoxins

N----CX5CX2CX36/37C----C Hydroxylase-type ferredoxins

N----CX3CX2CX36C30/31CX59C----C Biotin synthases

N----C-X2CX9CX31CX3C----C Clostridium pasteurianum ferredoxin

N----CXHX15-17CX2H----C Rieskie proteins

B.

N----CX6C----C (subunit 1) N----CX6C----C (subunit 2) [4Fe-4S] Fx in

photosystem I

N----CX34C----C (subunit 1) N----CX34C----C (subunit 2) [4Fe-4S] nitrogenase

N----C---------CX2C---------C [4Fe-4S] aconitase

N----CX2CX16CX13C----C [4Fe-4S] HIPIP

N----CX6CX2CX5C----C [4Fe-4S] Endonuclease

N----CXCX20CX20C----C [4Fe-4S] Leucine rich repeats

N----CX2CX11CX5C----C [4Fe-4S] Leucine rich variants

N----CX2CX2C---------C-P----C [4Fe-4S] ferredoxins

N----CX2CX2CX3C-P-------CX2CX2-8CX3CP----C 2[4Fe-4S] ferredoxins

C.

N----CX5C---------CP----C [3Fe-4S] ferredoxins

N----CX2DX2C---------CP----C [3Fe-4S] or [4Fe-4S]

ferredoxins

D.

N----CX2DX2CX3CP-------CX2CX2CX3CP----C 2[4Fe-4S] or [3Fe-4S] plus

[4Fe-4S] ferredoxin

N----CX7CX3CP--------- CX2CX2CX3CP----C [4Fe-4S][3Fe-4S] ferredoxins

38

Figure 2. Arrangement of residues involved in coordination of [2Fe-4S] (A), [4Fe-4S] or

2[4Fe-4S] (B), [3Fe-4S] or [4Fe-4S] (C), and 2[4Fe-4S] or [3Fe-4S] plus [4Fe-4S] (D)

clusters. "N" and "C" at the beginning and the end the motifs represent the N and C

terminal sequence of the proteins. Dashed lines indicate variable spacing between

residues. C, D, and H within the motif are cysteine, aspartate, and histidine respectively.

Underlined and bolded residues are those considered to be ligands in each cluster based

on X-ray structure, sequence homology, site directed mutagenesis, or spectroscopic

evidence (21, 42).

39

III. Ligation of iron-sulfur clusters

Cysteine is the major ligand in nature that coordinates iron atoms in iron-sulfur

clusters (62). Nonetheless, several different ligands, such as aspartate, histidine, or water,

have been identified. Direct information from crystal structures as well as through other

various spectroscopic studies has helped to identify the ligands involved in iron-sulfur

cluster coordination.

Aconitase is an enzyme containing a 4Fe-4S center that is not involved in redox

chemistry, but rather it is a substrate binding site (4, 49). It is an enzyme that catalyzes

the interconversion of citrate and isocitrate in the TCA cycle. The enzyme has been

characterized by different approaches. The crystallographic structures of the enzyme

alone, with substrates, and inhibitors have been determined. In the absence of substrate,

the active 4Fe-4S cluster is coordinated by 3 cysteines of the protein and an oxygen atom

from solvent is the fourth ligand (4, 74). In the presence of substrate, the crystal structure

shows a 4Fe-4S center in agreement with previous electron nuclear double resonance

(ENDOR) results. Both methods show that one Fe in the cluster has octahedral geometry

rather than tetrahedral. This octahedral geometry is due to additional ligands from the

carboxy and hydroxy groups of substrate and one water molecule (4, 49, 50).

The hydrogenase from Desulfovibrio gigas is another example of a non-cysteinyl

coordination that is shown by crystal structure. This protein contains a 4Fe-4S cluster

and Ni as prosthetic groups. The structure revealed the presence of three cysteines and

one histidine ligand to the 4Fe-4S center (83).

Albeit that the crystal structure is always a useful tool to determine ligand

identity, it is not easy to obtain the crystal structure for all proteins. Hence, other

methods often must be used. Iron-sulfur proteins are suspected of having non-cysteinyl

ligation if they have similar spectroscopic and catalytic characteristics to known non-

cysteinyl coordinated iron-sulfur proteins. Mostly, sequence comparisons with

homologues in combination with spectroscopic and site-directed mutagenesis studies

have been used to help identify the ligands for iron-sulfur clusters in proteins. For

40

example, electron spin echo envelope modulation (ESEEM) and ENDOR spectroscopy

indicate that Rieskie proteins contain 2Fe-2S centers that are coordinated by two

cysteines and two histidines (7, 18, 26, 27). Histidines and cysteines are conserved

among Rieskie type proteins. In addition, they exhibit higher reduction potentials than a

2Fe-2S cluster coordinating solely cysteines (55, 81).

Another mixed ligand coordination has been observed in [4Fe-4S] ferredoxins

from sulfate reducers and hyperthermophilic archaea. Ligands to the iron-sulfur clusters

in these proteins are cysteines and aspartate. The [4Fe-4S] binding motif in this group is

similar to the conventional cysteine motif (CX2CX2C and a distal C) except the second

cysteine is substituted by aspartate (8, 32, 64). These ferredoxins exhibit one similar

property which is the 3Fe-4S/4Fe-4S interconversion. Even though Pyrococcus furiosus

ferredoxin contains a [4Fe-4S] center, it exhibits anomalous spectroscopic properties

from clusters ligating with cysteine exclusively (9, 13).

A number of site-directed mutagenesis studies have contributed to an

understanding of iron-sulfur cluster ligation (62). Site directed mutagenesis provides not

only identification of the ligand residue, it also provides an understanding of the protein

environment which has a direct effect upon the iron-sulfur cluster structure and

properties. Replacement of one of the ligands can result in the variant protein being

either improperly folded or a variant with no iron-sulfur clusters. The conclusion from

this kind of result is almost always that the replacement amino acid is involved in cluster

coordination and that it also influences the protein folding (62, 79). Sometimes the

variant has similar properties as wild type and, as such, this result excludes the replaced

residue as a ligand to the iron-sulfur cluster. In several incidences, the residue replacing

cysteine serves as a ligand in which case the variants can contain iron-sulfur clusters with

altered electronic, redox potential and functional properties. The term "ligand exchange"

is applied where a new residue substitutes for the missing ligand. One form of ligand

exchange is the use of serine and aspartate in place of a cysteinyl ligand. Examples of

this are [4Fe-4S] center variants of PsaC and [2Fe-2S] center variants of ferredoxin from

C. pasteurianum (20, 43, 57). Another form of ligand exchange is the use of a free

41

cysteine from a different position to serve as a ligand rather than using the replacement

residue (23, 24, 79). This type of ligand exchange is referred to as "ligand swapping"

(23, 24). Ligand swapping often is associated with re-arrangement of the protein

conformation (23, 24, 79).

The phenomenon called "cluster conversion" occurs when the original iron-sulfur

cluster is converted into another structural type. Cluster conversions can result from

exposure to air, chemical oxidation-reduction, change of pH, and site-directed

replacement of ligands (42). A common form of cluster conversion occurs when a

cysteine ligand in a [4Fe-4S] center is replaced converting it to a [3Fe-4S] center.

Additionally, a [3Fe-4S] cluster may be converted into a [4Fe-4S] cluster by replacing a

noncysteinyl residue with cysteine that preserves the common cysteine motif (CX2CX2C

and a distal C) for coordination of a [4Fe-4S] center (1, 8, 44, 54). Another example of

cluster conversion is Clostridium pateurianum rubredoxin, in which the 1Fe cluster is

changed to a [2Fe-4S]-plant type cluster by replacing a cysteinyl ligand with alanine (58).

This result may be due to the structure around the metal site in rubredoxin which is

similar to the [2Fe-2S] Rieske protein. Another possibility is that the variant has a large

structural rearrangement allowing the additional incorporation of Fe and sulfides.

IV. Function of iron-sulfur centers.

A. Electron transfers.

Iron-sulfur clusters found in all domains of life are capable of serving several

functions. Most are electron carriers that accept, donate, and shift electrons (3).

Examples are ferredoxins and HIPIPs. Ferredoxins are well-studied proteins and their

structures from different organisms have been solved. The midpoint potentials of these

proteins are broadly varied from positive to negative values. This property allows them

to be either an electron donor or acceptor in various metabolic reactions. Examples of

enzymes and proteins that ferredoxins catalyze electron transfers are hydrogenase,

nitrogenase, cytochromes, nitrite reductase, nitrate reductase, sulfite reductase, pyruvate

oxidoreductase, and formate dehydrogenase. HIPIPs have a broad range of positive

42

midpoint potential values, which have been suggested to be correlated to the overall

charges of the polypeptide environment (30). HIPIPs are electron carriers between the

photoreaction center and the cytochrome bc1 complex in phototrophic microbes from

Bacteria domain (36, 37, 78). They also have been suggested to transfer electrons in

thiosulfate oxidation and iron oxidation (31).

B. Catalysis.

Many enzymes use iron-sulfur clusters as their substrate binding and activation

sites. For example, enzymes that perform dehydration and hydration activities in which

iron-sulfur clusters act as a Lewis acid in the reaction (4, 42). Aconitase is the best

characterized enzyme among this group. The catalytic Fe of the [4Fe-4S] center in

aconitase shows no redox changes during the reaction (2).

The catalytic role of iron-sulfur clusters which undergo redox changes is

exemplified in the radical based mechanism reactions. Examples are anaerobic

ribonucleotide reductase (RR) and biotin synthase (63). RR is involved in the first step in

DNA synthesis, ribonucleotide reduction to 2'-deoxyribonucleotide. Escherichia coli

uses specific RRs during aerobic and anaerobic growth conditions, both of which

generate radicals required for the reaction mechanism (73). The aerobic RR is a

heterodimer in which the smaller subunits contain µ-oxo diiron centers. The metal center

is used to oxidize a tyrosyl residue to generate a tyrosyl radical. The anaerobic RR

however, contains a [4Fe-4S] center at the interface of the two small subunits. This is the

third example in which an iron-sulfur cluster is located at the interface of two

polypeptides other than nitrogenase and Fx in photosystem I. The 4Fe-4S center of the

anaerobic RR reduces S-adenosyl methionine (SAM) to give a 5'-deoxyadenosyl radical

that generates a glycyl radical. Biotin synthase catalyses the last step of the biotin

synthesis pathway (77), in which a sulfur atom in the form of a thiol derivative is inserted

into dethiobiotin (16). The aerobically purified enzyme contains a [2Fe-2S] which is

proposed to be involved in the generation of the 5'-deoxy adenosyl radical. Recent

absorption, variable temperature magnetic circular dichroism (VTMCD), and EPR

43

spectroscopy indicated conversion of the [2Fe-2S] centers to a [4Fe-4S] center under

anaerobic conditions (16). The [4Fe-4S] center form of biotin synthase is postulated to

be involved in the radical generating reaction rather than the [2Fe-2S] cluster form. The

physiological significant of the cluster conversion may be a tool to regulate enzyme

activity due to oxidative stress (16). However, more experiments are needed to verify

this hypothesis.

C. Regulatory role

Iron-sulfur clusters also serve a regulatory role, in which they sense molecular

iron, oxygen, superoxide ion, and possibly nitric oxide concentrations (5, 22, 33, 35, 75).

For example, iron responsive binding protein (IRP) regulates the iron level in higher

organisms by controlling the gene expression of both iron storage ferritin and the

transferrin receptor (75). IRP recognizes a region called iron responsive element (IRE)

located at the 5' and 3' regions on the mRNA encoding ferritin and transferrin

respectively. When the cellular iron level is low, functional IRP binds IRE on both

ferritin and transferrin mRNA. This results in blocking ferritin translation and increasing

transferrin mRNA stability resulting in an increased production of transferrin (46, 56).

IRP is an isoform of aconitase, but contains no iron-sulfur clusters (28, 45). When iron is

limiting, IRP (apo-aconitase) increases. Conversely, when iron is abundant, holo-

aconitase with aconitase activity is increased while IRP activity is decreased. This

switching in activity of a bifunctional protein between enzyme catalysis (aconitase) and

RNA binding activity (IRP) occurs by assembly and disassembly of iron-sulfur clusters

which depends on the cellular iron level (3).

Another example of an iron-sulfur protein serving a regulatory role is fumarate

nitrate reductase regulatory protein (FNR), an oxygen sensor protein. FNR is a

transcription factor, which activates a set of genes under anaerobic conditions (80). The

functional enzyme form contains a [4Fe-4S] center. In the presence of oxygen, the

cluster is destroyed rapidly and FNR subsequently loses its DNA binding ability. Hence,

the regulatory function of FNR is controlled by the oxygen level in cells (76).

44

There is also a group of proteins with tandem repeats of leucine and aliphatic

residues in their sequence called leucine-rich repeats (LRRs) and leucine-rich variants

(LRVs). The X-ray crystal structure for an LRV was recently obtained. The structure

shows a 4Fe-4S cluster with a distinctive motif of CXXCX11CX5C located in a small

domain (67). This cysteine motif is different from the one described for the motif in

LRRs (CXCX20CX20C). The [4Fe-4S] centers of these LRRs and LRVs are highly

susceptible to oxygen (67), hence it is postulated that those proteins may function as an

oxygen sensor; however, more evidence is needed to support this hypothesis.

Sox R is another protein where the iron-sulfur cluster is proposed to serve a

regulatory role. The Sox R system is a mechanism developed to protect cells from

superoxide and nitric oxide. The mechanism is unknown at this time; however, the iron-

sulfur center seems to have a role in sensing these compounds (35, 84).

D. Iron storage role.

Polyferredoxin from Methanobacterium thermoautotrophicum contains 12[4Fe-

4S], and is thought to function as an iron storage protein or as an electron carrier (29, 72).

The postulated physiological role for polyferredoxin as an electron acceptor of the MV-

reducing hydrogenase is obscured, since the specific rate of reduction is very slow.

E. Structural role.

Another role proposed for iron-sulfur clusters involves maintaining protein

structure (42). The [4Fe-4S] centers in endonuclease III and Mut Y, members of the base

excision repair enzyme superfamily (47, 48, 59, 60), have no catalytic role and are

resistance to oxidation and reduction (14, 19). However, the enzyme activity is

dependent upon the presence of these iron-sulfur clusters. These observations suggest the

iron-sulfur cluster may be required for structural integrity. Although the [4Fe-4S] centers

of these enzymes are not directly involved in substrate binding or catalysis, they appear

45

to be required for efficient specific DNA binding. The iron-sulfur cluster may juxtapose

the catalytic or substrate binding sites to interact with DNA (69, 70). Another striking

feature for this superfamily of enzymes is that they contain a similar cysteine motif of

CX6CX2CX5C, which coordinates [4Fe-4S] centers (47, 48). This motif is one of the

most compact cysteine motifs known to date.

V. Iron-sulfur cluster assembly in proteins.

Understanding the in vivo formation of iron-sulfur clusters still falls behind, in

spite of the deep understanding of iron-sulfur cluster structure, reactivity, physiological

roles, and properties. Two scenarios for iron-sulfur cluster synthesis have been proposed.

The first is called spontaneous self-assembly. In the mechanism, iron-sulfur clusters are

thought to form spontaneously in the presence of ferric or ferrous salt in aqueous

mercaptoethanol with sodium sulfide (38). The logic for this process is that nature may

have synthesized iron-sulfur clusters by using the available chemistry of the geosphere

during evolution of the biosphere (52). This spontaneous process has been successful in

replicating known metalloprotein core clusters.

The other proposed scenario, assisted assembly, involves other protein

components for cluster formation and insertion into the proteins. The best characterized

are the Nif S, U, and M system from Azotobacter vinelandii. Nif S is a cysteine

desulfurase that converts L-cysteine to L-alanine and an activated sulfur (cysteinyl

persulfide). The enzyme contains pyridoxal phosphate, which is essential for catalysis

(87). Nif S is proposed to be a sulfur donor in the formation of the iron-sulfur cluster

core structure (87). The reconstitution of iron-sulfur clusters of apoproteins is facilitated

in the presence of Nif S (12, 86). The nif S gene is highly homologous and present in

different organisms, suggesting that it is a universal sulfur mobilizing agent (51, 87). Nif

U and Nif M are less well characterized, but they are hypothesized to be involved in

cluster formation.

46

References

1. Aono, S., D. Bentrop, I. Bertini, C. Luchinat, and R. Macinai. 1997. The

D13C variant of Bacillus schlegelii 7Fe ferredoxin is an 8Fe ferredoxin as

revealed by 1H-NMR spectroscopy. FEBS Lett. 412:501-5.

2. Beinert, H. 1990. Recent developments in the field of iron-sulfur proteins.

FASEB J. 4:2483-91.

3. Beinert, H., R. H. Holm, and E. Munck. 1997. Iron-sulfur clusters: nature's

modular, multipurpose structures. Science. 277:653-9.

4. Beinert, H., and M. C. Kennedy. 1989. 19th Sir Hans Krebs lecture.

Engineering of protein bound iron-sulfur clusters. A tool for the study of protein

and cluster chemistry and mechanism of iron-sulfur enzymes. Eur. J. Biochem.

186(1-2):5-15.

5. Beinert, H., and P. Kiley. 1996. Redox control of gene expression involving

iron-sulfur proteins. Change of oxidation-state or assembly/disassembly of Fe-S

clusters? [comment]. FEBS Lett. 382:218-9; discussion 220-1.

6. Beinert, H., and A. J. Thomson. 1983. Three-iron clusters in iron-sulfur

proteins. Arch. Biochem. Biophys. 222:333-61.

7. Britt, R. D., K. Sauer, M. P. Klein, D. B. Knaff, A. Kriauciunas, C. A. Yu, L.

Yu, and R. Malkin. 1991. Electron spin echo envelope modulation spectroscopy

supports the suggested coordination of two histidine ligands to the Rieske Fe-S

centers of the cytochrome b6f complex of spinach and the cytochrome bc1

complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine

heart mitochondria. Biochemistry. 30:1892-901.

8. Busch, J. L., J. L. Breton, B. M. Bartlett, F. A. Armstrong, R. James, and A.

J. Thomson. 1997. [3Fe-4S] <--> [4Fe-4S] cluster interconversion in

Desulfovibrio africanus ferredoxin III: properties of an Asp14 --> Cys mutant.

Biochem. J. 323:95-102.

9. Calzolai, L., C. M. Gorst, Z. H. Zhao, Q. Teng, M. W. Adams, and G. N. La

Mar. 1995. 1H NMR investigation of the electronic and molecular structure of

the four-iron cluster ferredoxin from the hyperthermophile Pyrococcus furiosus.

47

Identification of Asp 14 as a cluster ligand in each of the four redox states.

Biochemistry. 34:11373-84.

10. Carter, C. W., Jr., S. T. Freer, N. H. Xuong, R. A. Alden, and J. Kraut. 1972.

Structure of the iron-sulfur cluster in the Chromatius iron protein at 2.25

Angstrom resolution. Cold. Spring. Harb. Symp. Quant. Biol. 36:381-5.

11. Carter, C. W., Jr., J. Kraut, S. T. Freer, R. A. Alden, L. C. Sieker, E. Adman,

and L. H. Jensen. 1972. A comparison of Fe4S4 clusters in high-potential iron

protein and in ferredoxin. Proc. Natl. Acad. Sci. U S A. 69:3526-9.

12. Chen, S., L. Zheng, D. R. Dean, and H. Zalkin. 1997. Role of NifS in

maturation of glutamine phosphoribosylpyrophosphate amidotransferase. J.

Bacteriol. 179:7587-90.

13. Conover, R. C., A. T. Kowal, W. G. Fu, J. B. Park, S. Aono, M. W. Adams,

and M. K. Johnson. 1990. Spectroscopic characterization of the novel iron-sulfur

cluster in Pyrococcus furiosus ferredoxin. J. Biol .Chem. 265:8533-41.

14. Cunningham, R. P., H. Asahara, J. F. Bank, C. P. Scholes, J. C. Salerno, K.

Surerus, E. Munck, J. McCracken, J. Peisach, and M. H. Emptage. 1989.

Endonuclease III is an iron-sulfur protein. Biochemistry. 28:4450-5.

15. Davidson, E., T. Ohnishi, E. Atta-Asafo-Adjei, and F. Daldal. 1992. Potential

ligands to the [2Fe-2S] Rieske cluster of the cytochrome bc1 complex of

Rhodobacter capsulatus probed by site-directed mutagenesis. Biochemistry.

31:3342-51.

16. Duin, E. C., M. E. Lafferty, B. R. Crouse, R. M. Allen, I. Sanyal, D. H. Flint,

and M. K. Johnson. 1997. [2Fe-2S] to [4Fe-4S] cluster conversion in

Escherichia coli biotin synthase. Biochemistry. 36:11811-20.

17. Eaton, W. A., and W. Lovenberg. 1973. The iron-sulfur complex in rubredoxin.

Iron-sulfur proteins , Academic Press, Inc. (London) Ltd., ed. Lovenberg W. 2.

18. Fee, J. A., D. Kuila, M. W. Mather, and T. Yoshida. 1986. Respiratory proteins

from extremely thermophilic, aerobic bacteria. Biochim. Biophys. Acta. 853:153-

85.

48

19. Fu, W., S. O' Handley, R. P. Cunningham, and M. K. Johnson. 1992. The role

of the iron-sulfur cluster in Escherichia coli endonuclease III. A resonance raman

study. J. Biol. Chem. 267:16135-7.

20. Fujinaga, J., J. Gaillard, and J. Meyer. 1993. Mutated forms of a [2Fe-2S]

ferredoxin with serine ligands to the iron- sulfur cluster. Biochem. Biophys. Res.

Commun. 194:104-11.

21. Gao-Sheridan, H. S., H. R. Pershad, F. A. Armstrong, and B. K. Burgess.

1998. Discovery of a novel ferredoxin from Azotobacter vinelandii containing

two [4Fe-4S] clusters with widely differing and very negative reduction

potentials. J. Biol. Chem. 273:5514-9.

22. Gaudu, P., and B. Weiss. 1996. SoxR, a [2Fe-2S] transcription factor, is active

only in its oxidized form. Proc. Natl. Acad. Sci. U S A. 93:10094-8.

23. Golinelli, M. P., L. A. Akin, B. R. Crouse, M. K. Johnson, and J. Meyer.

1996. Cysteine ligand swapping on a deletable loop of the [2Fe-2S] ferredoxin

from Clostridium pasteurianum. Biochemistry. 35(27):8995-9002.

24. Golinelli, M. P., C. Chatelet, E. C. Duin, M. K. Johnson, and J. Meyer. 1998.

Extensive ligand rearrangements around the [2Fe-2S] cluster of Clostridium

pasteurianum ferredoxin. Biochemistry. 37:10429-37.

25. Gorst, C. M., Y. H. Yeh, Q. Teng, L. Calzolai, Z. H. Zhou, M. W. Adams, and

G. N. La Mar. 1995. 1H NMR investigation of the paramagnetic cluster

environment in Pyrococcus furiosus three-iron ferredoxin: sequence-specific

assignment of ligated cysteines independent of tertiary structure. Biochemistry.

34:600-10.

26. Gurbiel, R. J., C. J. Batie, M. Sivaraja, A. E. True, J. A. Fee, B. M. Hoffman,

and D. P. Ballou. 1989. Electron-nuclear double resonance spectroscopy of 15N-

enriched phthalate dioxygenase from Pseudomonas cepacia proves that two

histidines are coordinated to the [2Fe-2S] Rieske-type clusters. Biochemistry.

28:4861-71.

27. Gurbiel, R. J., T. Ohnishi, D. E. Robertson, F. Daldal, and B. M. Hoffman.

1991. Q-band ENDOR spectra of the Rieske protein from Rhodobactor

49

capsulatus ubiquinol-cytochrome c oxidoreductase show two histidines

coordinated to the [2Fe-2S] cluster. Biochemistry. 30:11579-84.

28. Haile, D. J., T. A. Rouault, J. B. Harford, M. C. Kennedy, G. A. Blondin, H.

Beinert, and R. D. Klausner. 1992. Cellular regulation of the iron-responsive

element binding protein: disassembly of the cubane iron-sulfur cluster results in

high-affinity RNA binding. Proc. Natl. Acad. Sci. U S A. 89:11735-9.

29. Hedderich, R., Albracht, S. P. J., Linder, D., Koch, J., and Thauer, R.K.

1992. Isolation and characterization of polyferredoxin from Methanobacterium-

thermoautotrophicum - The mvhB gene product of the methylviologen-reducing

hydrogenase operon. FEBS Letters. 298:65-68.

30. Heering, H. A., B. M. Bulsink, W. R. Hagen, and T. E. Meyer. 1995. Influence

of charge and polarity on the redox potentials of high- potential iron-sulfur

proteins: evidence for the existence of two groups. Biochemistry. 34:14675-86.

31. Heinrich, S., and P. Rosch. 1998. The structure of iron-sulfur proteins. Prog.

Biophys. & Mol. Biol. 70:95-136.

32. Heltzel, A., E. T. Smith, Z. H. Zhou, J. M. Blamey, and M. W. Adams. 1994.

Cloning, expression, and molecular characterization of the gene encoding an

extremely thermostable [4Fe-4S] ferredoxin from the hyperthermophilic archaeon

Pyrococcus furiosus. J. Bacteriol. 176:4790-3.

33. Hentze, M. W., and L. C. Kuhn. 1996. Molecular control of vertebrate iron

metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and

oxidative stress. Proc. Natl. Acad. Sci. U S A. 93:8175-82.

34. Herskovitz, T., B. A. Averill, R. H. Holm, J. A. Ibers, W. D. Phillips, and J. F.

Weiher. 1972. Structure and properties of a synthetic analogue of bacterial iron-

sulfur proteins. Proc. Natl. Acad. Sci. U S A. 69:2437-41.

35. Hidalgo, E., J. M. Bollinger, Jr., T. M. Bradley, C. T. Walsh, and B. Demple.

1995. Binuclear [2Fe-2S] clusters in the Escherichia coli SoxR protein and role of

the metal centers in transcription. J. Biol. Chem. 270:20908-14.

36. Hochkoeppler, A., P. Kofod, and D. Zannoni. 1995. HiPiP oxido-reductase

activity in membranes from aerobically grown cells of the facultative phototroph

Rhodoferax fermentans. FEBS Lett. 375:197-200.

50

37. Hochkoeppler, A., D. Zannoni, S. Ciurli, T. E. Meyer, M. A. Cusanovich, and

G. Tollin. 1996. Kinetics of photo-induced electron transfer from high-potential

iron- sulfur protein to the photosynthetic reaction center of the purple phototroph

Rhodoferax fermentans. Proc. Natl. Acad. Sci. U S A. 93:6998-7002.

38. Hong, J., and J. C. Rabinowitz. 1967. Preparation and properties of Clostridial

apoferredoxins. Biochem. Biophys. Res. Commun. 29:246-52.

39. Huber, C., and G. Wachtershauser. 1997. Activated acetic acid by carbon

fixation on (Fe,Ni)S under primordial conditions. Science. 276:245-247.

40. Huynh, B. H., J. J. Moura, I. Moura, T. A. Kent, J. LeGall, A. V. Xavier, and

E. Munck. 1980. Evidence for a three-iron center in a ferredoxin from

Desulfovibrio gigas. Mössbauer and EPR studies. J. Biol. Chem. 255(8):3242-4.

41. Huynh, V. 1998. Mössbauer spectroscopy. Inorganic biochemistry summer

workshop, University of Georgia.

42. Johnson, M. K. 1994. Iron-sulfur proteins. Encyclopedia of Inorganic Chemistry,

Johnson Wiley and Sons (King, R.B., ed). 4:1896-1915.

43. Jung, Y. S., I. R. Vassiliev, J. Yu, L. McIntosh, and J. H. Golbeck. 1997.

Strains of Synechocystis sp. PCC 6803 with altered PsaC. II. EPR and optical

spectroscopic properties of FA and FB in aspartate, serine, and alanine

replacements of cysteines 14 and 51. J. Biol. Chem. 272:8040-9.

44. Kemper, M. A., H. S. Gao-Sheridan, B. Shen, J. L. Duff, G. J. Tilley, F. A.

Armstrong, and B. K. Burgess. 1998. Delta T 14/Delta D 15 Azotobacter

vinelandii ferredoxin I: creation of a new CysXXCysXXCys motif that ligates a

[4Fe-4S] cluster. Biochemistry. 37:12829-37.

45. Kennedy, M. C., L. Mende-Mueller, G. A. Blondin, and H. Beinert. 1992.

Purification and characterization of cytosolic aconitase from beef liver and its

relationship to the iron-responsive element binding protein [published erratum

appears in Proc. Natl. Acad. Sci. U S A 1993; 90:2556]. Proc. Natl. Acad. Sci. U

S A. 89:11730-4.

46. Klausner, R. D., T. A. Rouault, and J. B. Harford. 1993. Regulating the fate of

mRNA: the control of cellular iron metabolism. Cell. 72:19-28.

51

47. Kuo, C. F., D. E. McRee, R. P. Cunningham, and J. A. Tainer. 1992.

Crystallization and crystallographic characterization of the iron- sulfur-containing

DNA-repair enzyme endonuclease III from Escherichia coli. J. Mol. Biol.

227(1):347-51.

48. Kuo, C. F., D. E. McRee, C. L. Fisher, S. F. O'Handley, R. P. Cunningham,

and J. A. Tainer. 1992. Atomic structure of the DNA repair [4Fe-4S] enzyme

endonuclease III. Science. 258:434-40.

49. Lauble, H., M. C. Kennedy, H. Beinert, and C. D. Stout. 1992. Crystal

structures of aconitase with isocitrate and nitroisocitrate bound. Biochemistry.

31:2735-48.

50. Lauble, H., M. C. Kennedy, H. Beinert, and C. D. Stout. 1994. Crystal

structures of aconitase with trans-aconitate and nitrocitrate bound. J. Mol. Biol.

237(4):437-51.

51. Leibrecht, I., and D. Kessler. 1997. A novel L-cysteine/cystine C-S-lyase

directing [2Fe-2S] cluster formation of Synechocystis ferredoxin. J. Biol. Chem.

272(16):10442-7.

52. Lippard, S. J., and J. M. Berg. 1994. Physical methods in bioinorganic

chemistry. Priciples of bioinorganic chemistry:75-100.

53. Lovenberg, W., and B. E. Sobel. 1965. Rubredoxin: a new electron transfer

protein from Clostridium pasteurianum. Proc. Natl. Acad. Sci. U S A. 54:193-9.

54. Manodori, A., G. Cecchini, I. Schroder, R. P. Gunsalus, M. T. Werth, and M.

K. Johnson. 1992. [3Fe-4S] to [4Fe-4S] cluster conversion in Escherichia coli

fumarate reductase by site-directed mutagenesis. Biochemistry. 31:2703-12.

55. Mason, J. R., and R. Cammack. 1992. The electron-transport proteins of

hydroxylating bacterial dioxygenases. Ann. Rev. Microbiol. 46:277-305.

56. Melefors, O., and M. W. Hentze. 1993. Iron regulatory factor--the conductor of

cellular iron regulation. Blood Rev. 7:251-8.

57. Meyer, J., J. Fujinaga, J. Gaillard, and M. Lutz. 1994. Mutated forms of the

[2Fe-2S] ferredoxin from Clostridium pasteurianum with noncysteinyl ligands to

the iron-sulfur cluster. Biochemistry. 33:13642-50.

52

58. Meyer, J., J. Gagnon, J. Gaillard, M. Lutz, C. Achim, E. Munck, Y. Petillot,

C. M. Colangelo, and R. A. Scott. 1997. Assembly of a [2Fe-2S]2+ cluster in a

molecular variant of Clostridium pasteurianum rubredoxin. Biochemistry.

36:13374-80.

59. Michaels, M. L., C. Cruz, A. P. Grollman, and J. H. Miller. 1992. Evidence

that MutY and MutM combine to prevent mutations by an oxidatively damaged

form of guanine in DNA. Proc. Natl. Acad. Sci. U S A. 89:7022-5.

60. Michaels, M. L., J. Tchou, A. P. Grollman, and J. H. Miller. 1992. A repair

system for 8-oxo-7,8-dihydrodeoxyguanine. Biochemistry. 31:10964-8.

61. Moore, G. D., G. W. Pettigrew, and N. K. Roger. 1986. Factors influencing

redox potentials of electron transfer proteins. Proc. Natl. Acad. Sci. USA.

83:4998-5000.

62. Moulis, J.-M., V. Davasse, M.-P. Golinelli, J. Meyer, and I. Quinkal. 1996.

The coodination sphere of iron-sulfur clusters: lessons from site-directed

mutagenesis experiments. J. Biol. Inorg.Chem. 1:2-14.

63. Mulliez, E., M. Fontecave, J. Gaillard, and P. Reichard. 1993. An iron-sulfur

center and a free radical in the active anaerobic ribonucleotide reductase of

Escherichia coli. J. Biol. Chem. 268:2296-9.

64. Okawara, N., M. Ogata, T. Yagi, S. Wakabayashi, and H. Matsubara. 1988.

Amino acid sequence of ferredoxin I from Desulfovibrio vulgaris Miyazaki. J.

Biochem (Tokyo). 104:196-9.

65. Orme-Johnson, W. H. 1973. Iron-sulfur proteins: structure and function. Annu.

Rev. Biochem. 42:159-204.

66. Orme-Johnson, W. H., and R. H. Sands. 1973. Probing Iron-sulfur proteins

with EPR and ENDOR spectroscopy. Iron-sulfur proteins , Academic Press, Inc.

(London) Ltd., ed. Lovenberg W. 2.

67. Peters, J. W., M. H. Stowell, and D. C. Rees. 1996. A leucine-rich repeat

variant with a novel repetitive protein structural motif [letter]. Nat. Struct. Biol.

3:991-4.

53

68. Phillips, W. D., M. Poe, J. F. Weiher, C. C. McDonald, and W. Lovenberg.

1970. Proton magnetic resonance, magnetic susceptibility and Mössbauer studies

of Clostridium pasteurianum rubredoxin. Nature. 227:574-7.

69. Porello, S. L., M. J. Cannon, and S. S. David. 1998. A substrate recognition

role for the [4Fe-4S]2+ cluster of the DNA repair glycosylase MutY.


70. Prince, R. C., and M. J. Grossman. 1993. Novel iron-sulfur clusters. Trends.

Biochem. Sci. 18:153-4.

71. Rao, K. K., R. Cammack, D. O. Hall, and C. E. Johnson. 1971. Mössbauer

effect in Scenedesmus and spinach ferredoxins. The mechanism of electron

transfer in plant-type iron-sulphur proteins. Biochem. J. 122:257-65.

72. Reeve, J. N., G. S. Beckler, D. S. Cram, P. T. Hamilton, J. W. Brown, J. A.

Krzycki, A. F. Kolodziej, L. Alex, W. H. Orme-Johnson, and C.T. Walsh.

1989. A hydrogenase-linked gene in Methanobacterium thermoautotrophicum

strain H encodes a polyferredoxin. Proc. Natl. Acad. Sci. USA. 86:3031-3035.

73. Reichard, P. 1993. From RNA to DNA, why so many ribonucleotide reductases?

Science. 260(5115):1773-7.

74. Robbins, A. H., and C. D. Stout. 1989. Structure of activated aconitase:

formation of the [4Fe-4S] cluster in the crystal. Proc. Natl. Acad. Sci. U S A.

86:3639-43.

75. Rouault, T. A., D. J. Haile, W. E. Downey, C. C. Philpott, C. Tang, F.

Samaniego, J. Chin, I. Paul, D. Orloff, and J. B. Harford. 1992. An iron-sulfur

cluster plays a novel regulatory role in the iron- responsive element binding

protein. Biometals. 5:131-40.

76. Rouault, T. A., and R. D. Klauser. 1996. Iron-sulfur clusters as biosensors of

oxidants and iron. Trends. Biochem. Sci. 21:174-177.

77. Sanyal, I., G. Cohen, and D. H. Flint. 1994. Biotin synthase: purification,

characterization as a [2Fe-2S]cluster protein, and in vitro activity of the

Escherichia coli bioB gene product. Biochemistry. 33:3625-31.

78. Schoepp, B., P. Parot, L. Menin, J. Gaillard, P. Richaud, and A. Vermeglio.

1995. In vivo participation of a high potential iron-sulfur protein as electron donor

54

to the photochemical reaction center of Rubrivivax gelatinosus. Biochemistry.

34:11736-42.

79. Shen, B., D. R. Jollie, T. C. Diller, C. D. Stout, P. J. Stephens, and B. K.

Burgess. 1995. Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I:

cysteine ligation of the [4Fe-4S] cluster with protein rearrangement is preferred

over serine ligation. Proc. Natl. Acad. Sci. U S A. 92:10064-8.

80. Spiro, S., and J. R. Guest. 1990. FNR and its role in oxygen-regulated gene

expression in Escherichia coli. FEMS Microbiol. Rev. 6:399-428.

81. Trumpower, B. L. 1990. Cytochrome bc1 complexes of microorganisms.

Microbiol. Rev. 54:101-29.

82. Tsang, H. T., C. J. Batie, D. P. Ballou, and J. E. Penner-Hahn. 1989. X-ray

absorption spectroscopy of the [2Fe-2S] Rieske cluster in Pseudomonas cepacia

phthalate dioxygenase. Determination of core dimensions and iron ligation.


83. Volbeda, A., M. H. Charon, C. Piras, E. C. Hatchikian, M. Frey, and J. C.

Fontecilla-Camps. 1995. Crystal structure of the nickel-iron hydrogenase from

Desulfovibrio gigas. Nature. 373:580-7.

84. Wu, J., W. R. Dunham, and B. Weiss. 1995. Overproduction and physical

characterization of SoxR, a [2Fe-2S] protein that governs an oxidative response

regulon in Escherichia coli. J. Biol. Chem. 270:10323-7.

85. Yoch, D. C., and R. P. Carithers. 1979. Bacterial iron-sulfur proteins.

Microbiol. Rev. 43:384-421.

86. Zheng, L., and D. R. Dean. 1994. Catalytic formation of a nitrogenase iron-

sulfur cluster. J. Biol. Chem. 269:18723-6.

87. Zheng, L., R. H. White, V. L. Cash, R. F. Jack, and D. R. Dean. 1993.

Cysteine desulfurase activity indicates a role for NIFS in metallocluster

biosynthesis. Proc. Natl. Acad. Sci. U S A. 90:2754-8.

55

CHAPTER 3

OBJECTIVES

Methanoarchaea obtain energy for growth by coupling electron transport

phosphorylation to methane formation. An undeveloped area of research has been the

identification of electron carriers. The iron-sulfur flavoprotein (Isf) from

Methanosarcina thermophila has been proposed to be an electron carrier in the acetate

fermentation pathway. The main objectives of my research were to characterize the

properties of the iron-sulfur center and FMN in Isf, identify the ligands required for

coordination of the iron-sulfur cluster, and to identify the electron acceptor of Isf.

Isf was previously shown to contain one FMN and iron-sulfur center per subunit;

however, the properties of these redox centers were not characterized. Various proteins

posses different iron-sulfur cluster types, each showing distinct properties. Therefore, the

identity of the iron-sulfur center in Isf is important for understanding how Isf functions as

a redox protein.

Most iron-sulfur clusters are ligated to the polypeptide backbone primarily via

cysteine residues. Several amino acid motifs for iron-sulfur cluster ligation have been

reported (refer to Chapter 2). These motifs can be used as a basis for the preliminary

prediction of ligands that bind to iron-sulfur cluster types in proteins. Most iron-sulfur

clusters are coordinated by four cysteine residues. Sequence comparisons among Isf

homologues reveal a novel motif with perfectly conserved cysteines which could possibly

coordinate the iron-sulfur center in Isf (refer to Chapter 4). This putative motif is the

most compact cysteine motif known for coordinating a redox active protein.

The discovery of Isf as an electron carrier in the acetate fermentation for M.

thermophila allows the possibility for a comprehensive study of the electron transport

components in this methanoarchaeon. Determination of the midpoint potential of the

iron-sulfur center and FMN will be useful in investigating the possibility of intra- and

inter-molecular electron transfer processes in the acetate fermentation pathway. The

56

presence of both a one-electron carrier and a two-electron carrier in the same protein

suggests a one/two electron switch function for Isf. The fact that Isf has a defined redox

potential and a possible one-two electron switch function provides a basis to identify the

electron acceptor for Isf.

57

CHAPTER 4

ELECTROCHEMICAL AND SPECTROSCOPIC PROPERTIES OF THE

IRON-SULFUR FLAVOPROTEIN FROM Methanosarcina thermophila

ABSTRACT

Based on spectroscopic analyses, a heterologously produced iron-sulfur

flavoprotein (Isf) from Methanosarcina thermophila contains two [4Fe-4S] centers and

two flavin mononucleotide (FMN) cofactors in the homodimeric protein. The midpoint

potentials (Em) for the [4Fe-4S]2+/1+ and FMN/FMNH2 are – 394 and –277 mV

respectively. Interestingly, the semiquinone form of FMN was not detected during the

potentiometric titration; however, a small amount of red semiquinone was found in

frozen reaction mixtures of CODH/ACS and Isf. The EPR spectrum of the reduced

protein showed g values characteristic of [4Fe-4S] center with additional g values (2.06,

1.93, 1.86 and 1.82) due to microheterogeneity among Isf molecules. A variety of

physiological 2-electron acceptors were examined for the ability to oxidize Isf, but none

are able to carry out this function. Extract from H2/CO2-grown Methanobacterium

thermoautotrophicum cells catalyzed either H2 or CO-dependent reduction of M.

thermophila Isf. Furthermore, the genomic sequences of M. thermoautotrophicum and

Methanocoocus jannaschii also contain Isf homologues. These results may suggest a

general role for Isf as an electron carrier in both acetate fermenting and CO2-reducing

methanoarchaea.

INTRODUCTION

Methane production resulting from the energy-yielding metabolism of the

methanoarchaea represents the final step in the anaerobic degradation of complex

materials (7). This process is unique and involves many enzymes and coenzymes found

only in methanoarchaea (26). Methanoarchaea produce methane by two major pathways

(7). The first is the CO2-reducing pathway in which CO2 is reduced to methane using

electrons derived from either H2 or formate (equations 1 and 2). Another pathway is

58

acetate fermentation, which contributes two-thirds of all biological methane produced in

nature (equation 3). In this pathway, acetate is cleaved in a CoA-dependent reaction and

the methyl group is reduced to methane using electrons derived from oxidation of the

carbonyl group to carbon dioxide (8).

CO2 + 4H2 à CH4 + 2H2O (1)

4 HCOO- + 4H+ à CH4 + 3 CO2 + 2 H2O (2)

CH3COO- + H+ à CH4 + CO2 (3)

Although the biochemistry and enzymology of carbon flow in these pathways are areas of

intense research, the electron transport carriers in methanogenesis are poorly understood.

Recently, a homodimeric iron-sulfur flavoprotein (Isf) was identified from an

acetate-utilizing methanoarchaeon, Methanosarcina thermophila. In the genome of M.

thermophila, the isf gene is located upstream and transcribed in the opposite direction

from the pta-ack operon encoding phosphotransacetylase and acetate kinase (14).

Comparison of the deduced Isf sequence with sequences in the available databases

suggested Isf was a novel iron-sulfur flavoprotein. Isf was hyperproduced in Escherichia

coli. The heterologously produced protein was partially characterized and in vitro

reconstitution experiments suggested Isf is a component in the electron transport chain

for methanogenesis from acetate (14). The absorption spectra and chemical analyses

suggested one iron-sulfur cluster and one FMN per subunit.

The present study reports the characterization of this iron-sulfur cluster and

unique FMN properties to offer insight into the role of Isf in electron transport.

Additionally, the context of Isf function in M. thermophila electron transport was

examined by testing several known electron carriers for interactions with Isf. Lastly,

information about the Isf homologs in other CO2-reducing methanoarchaea will be

discussed to help understand the physiological role of this electron carrier.

Portions of data contained in this section were obtained by Dr. S.W. Ragsdale and

Dr. D.F. Becker at the University of Nebraska, and Dr. K.K. Surerus at the University of

Wisconsin Milwaukee. Their contributions are noted in the appropriate sections.

59

MATERIALS AND METHODS

Isf sequence analysis. In the process of doing site-directed mutagenesis for

ligand identification, it was discovered that the sequence at the C-terminal was incorrect

from that previously reported (14) and therefore, the gene was re-sequenced. The coding

region of isf was amplified using the Polymerase Chain Reaction (PCR) from

Methansarcina thermophila genomic DNA. The sequence of the upstream primer was 5'-

GGTGCACATATGAAAATAACAGGAAT-3' and contained the recognition sequence

for NdeI. The sequence of the downstream primer was 5'-

CAACTGGATCCATGCGATCATAAAC-3' and contained the recognition sequence for

BamHI. The PCR product was restricted with NdeI and BamHI. The resulting DNA was

cloned into the BamHI and NdeI sites of the pT7-7 overexpression vector. The construct

was checked by sequencing using the automated dideoxy method at the Penn State

University nucleic acid facility. The λ-EMBL3 (M. thermophila genomic library

containing isf , (14)) constructs were also subjected to the same procedure as described

above. Lastly, plasmid pML701 (14) was sequenced to confirm this error.

Protein production and purification. All proteins were purified anaerobically.

M. thermophila Isf was overproduced in Escherichia coli BL21(DE3) and purified as

described (14). M. thermophila ferredoxin A was purified as described (25). The

following procedure was used to obtain 57Fe-enriched Isf for Mössbauer study. The57Fe-labeled Isf was produced by growing E. coli cells in defined medium supplemented

with 57Fe at the final concentration of 20 µM. A solution of 57Fe was obtained by

dissolved 42.5 mg 57Fe in 850 µl of 2 N H2SO4 in a tube capped with a rubber stopper

that has a small plastic tubing insert at the top to let the gas out during the reaction. The

reaction solution was heat at 60-65o C for a week. The iron concentration was

determined by ferrozine using standard Fe(II) solution as a standard curve. The defined

medium composition per liter was: 745 ml deionized water; 200 ml M-solution (in a 1 l

solution contains: 42 g MOPS, 4 g tricine, 14.6 g NaCl, 8 g KOH, 2.55 g NH4Cl); 2 ml

O-solution (in a 50 ml solution contains: 2.68 g MgCl2.6H2O, 1 ml T solution (in a 100

60

ml solution contains: 8 ml concentrate HCl, 18.4 mg CaCl2.2H20, 64 mg H3BO4, 40 mg

MnCl2.4H20, 340 mg ZnCl2, 605 mg Na2MoO4

.2H2O, 10mM 57Fe solution); 2 ml P-

solution (1 M KH2PO4); 1 ml S-solution (276 mM K2SO4); 0.5 ml of 0.2 % vitamin B

(thiamin); 20 ml of 20 % glucose; 40 ml of 3.75 % casamino acid; and 1 ml of 100

mg/ml ampicillin. The 57Fe solution was prepared by dissolving the iron metal in 2 N

H2SO4 for one week.

Cell extract of M. thermoautotrophicum used in the reduction of Isf was prepared

as follows. M. thermoautotrophicum strain Marburg was grown as described (21). Five

g (wet weight) cells resuspended in 50 mM Tris-Cl pH7.6 was lysed in a French pressure

cell at 20,000 psi. The lysate was centrifuged for 30 min at 33,000 x g, and the resulting

supernate (cell extract) was saved. All proteins were stored at –80o C until used.

UV-visible spectroscopy and potentiometric titrations. Potentiometric

measurements were performed as previously described (23, 24). All electrochemical

potentials are reported relative to the standard hydrogen electrode. Isf (3 - 4 µM) was

titrated at 20o C in 50 mM potassium phosphate buffer (pH 7.0 - 7.05) in a solution

containing methyl viologen (0.1 mM) as the mediator dye with phenosafarin (Em = -

0.252 V, pH 7.0) (5 µM) and benzyl viologen (Em= -0.362 V, pH 7.0) (5 µM) as the

indicator dyes. The pH measured after the experiment was recorded as the pH for the

titration. The visible spectra in each experiment were obtained and stored on an Olis-14

interfaced Cary spectrophotometer. The absorbance at 480 nm was used to monitor the

amount of oxidized and reduced FMN after correcting the spectra for turbidity. The

reduction potentials reported were determined by potentiometric measurements in the

reductive direction. After each potentiometric titration of the FMN chromophore, the

iron-sulfur flavoprotein was reoxidized completely using ferrocyanide (0.1 mM) as the

mediator dye. Equilibrium of the system in the UV-visible potentiometric measurements

was considered to be obtained when the measured potential drift was less than 1 mV in 5

min; this was typically around 1-2 h. The midpoint potentials (Em) and n values were

calculated using the Nernst Equation indicated below, where E is the measured

equilibrium potential at each point in the titration and n is the number of electrons. The

61

typical error in the reported reduction potential values was ± 2-3 mV.

E=Em+ (0.058/n) log ([ox]/[red])

All midpoint potential value determinations exhibited Nernstian behavior as indicated by

their n values.

EPR spectroscopy and potentiometric titrations. The EPR spectra were

recorded on a Bruker ESP 300E spectrometer equipped with an Oxford ITC4 temperature

controller and automatic frequency counter of a Hewlett Packard Model 5340 and Bruker

Gaussmeter. The spectroscopic parameters are given in the figure legends. Double

integration of the EPR signals was performed with copper perchlorate (1 mM) as the

standard. Isf was frozen in liquid nitrogen prior to EPR analyses.

For the power saturation studies, Isf (74 µM, pH 7.0) was reduced in the presence

of 50 mM methyl viologen with a 40-fold excess of sodium dithionite prepared freshly at

pH 9.0. The solution was immediately frozen in an EPR tube and stored in liquid

nitrogen. Spectra of the reduced [4Fe-4S] cluster were recorded at powers varying from

0.1- 200 mW at five different temperatures between 5 and 25 K. The power for half

saturation (P1/2) at each temperature was determined by a fit to a plot of log (S/P*e0.5)

versus log P using Equation 7, where S is the signal amplitude, P is the microwave power

incident on the cavity, and b is the inhomogeneity parameter. Best fits to the data were

obtained by using a b value of 1.2.

The zero field splitting constant (∆) was determined by a linear fit to a plot of ln

P1/2 versus 1/T according to Equation 8, where T is the temperature, P1/2 is the power

for half saturation, k is the Boltzmann constant, and A is a coefficient representative of

the phonon-spin coupling properties of the [4Fe-4S] cluster.

P1/2=Aexp(-∆/kT)

Potentiometric measurements of the [4Fe-4S] cluster were performed in an EPR-

0.5b*1/2 e)P/P/(1P S +=

62

spectroelectrochemical cell with a quartz EPR tube describes previously (9). Isf samples

(80 - 160 µM) in 50 mM potassium phosphate buffer (pH 7.0) were titrated at 20o C in

the presence of the mediator dyes, 150 µM benzyl viologen (Em = -0.362 V), 150 µM

methyl viologen (Em -0.440 V), 100 µM 1,1'-trimethylene-2,2’-bipyridyl (Em = -0.540

V), and 100 µM 4,4'-dimethyl-2,2'-dipyridyl (Em = -0.586 V). The intensity of the EPR

signal with a g-value of 1.93 was monitored to determine the redox state of the [4Fe-4S]

cluster during the titration. Potentiometric measurements were performed in the

reductive and oxidative directions. The system was considered to have reached

equilibrium when the measured potential drift was less than 1 mV in 2 min.

Mössbauer spectroscopy. Mössbauer spectra were recorded on a constant

acceleration spectrophotometer, MS-1200D, using a Janis Super Varitemp cryostat model

8DT with a Lakeshore temperature controller model 340 and a 57Co gamma source. The

experiments were done at 4.2 and 100 K. The reduced Isf was generated by adding 10-

fold excess of sodium dithionite.

Reduction of methanophenazine by Isf. The assay mixtures were

ananerobically equilibrated with 1.0 atm of CO in a stoppered 1.0 ml-cuvette maintained

at 35o C. The assay mixture (700 µl) contained: 50 mM Tris-Cl (pH 7.6), 2 mM

dithiothreitol, 1 mg/l resazurine, 25 µg carbon monoxide dehydrogenase /acetyl CoA

synthetase complex (CODH/ACS), and 9 µg M. thermophila ferredoxin A. After 10 min

incubation, 180 µg Isf were added to the assay mixtures and incubated for 10 min. The

reaction was initiated by the addition of 120 µM of 2-hydroxyphenazine (1). The

absorbance at 478 nm was measured in the Hewlett Packard 8452A diode array

spectrophotometer.

Reduction of Isf by M. thermoautotrophicum cell extract. The assay mixtures

were anaerobically equilibrated with 1.0 atm of CO, H2, or N2 in a stoppered 1.0 ml-

cuvette maintained at 35o C. The assay mixture (700 µl) contained: 50 mM Tris-Cl (pH

7.6), 2 mM dithiothreitol, 1 mg/l resazurine, 180 µg M. thermoautotrophicum cell extract,

63

and 13.5 µg M. thermophila ferredoxin A. After 10 min incubation, the reaction was

initiated by addition of 180 µg Isf. Ferredoxin A was omitted in some assays whereas

ferredoxin from Clostridium pasteurianum was substituted where indicated. The

absorbance at 476 nm was measured in the Hewlett Packard 8452A diode array

spectrophotometer.

RESULTS

Sequence analysis. Error in the previously reported isf sequence (14) was

detected during site-directed mutagenesis studies (refer to chapter 4). Figure 1 shows the

corrected sequence deduced from genomic DNA. The corrected Isf contains 191

residues, 81 fewer than previously reported. Additionally, the C-terminal residues177

KLCDVLELIQKNRDK191

in the corrected Isf sequence replace177

NSVMSWNLFRKIEIN191

in the previously reported Isf. Since the DNA library

containing isf gene was available, isf gene from this library was sequenced to see if there

was an error that may result in error in previous study. Identical sequences were found

both from genomic DNA and DNA library as reported in Figure 1. Re-sequencing

revealed the correct isf sequence in pML701 used for the heterologous production of Isf

reported here and previously (14).

64

ATG AAA ATA ACA GGA ATT TCA GGC AGT CCA CGA AAG GGC CAG AAC 45M K I T G I S G S P R K G Q N 15

TGT GAG AAA ATA ATT GGA GCT GCT CTT GAG GTT GCA AAA GAA AGA 90C E K I I G A A L E V A K E R 30

GGG TTT GAA ACT GAT ACC GTT TTT ATC TCA AAC GAG GAG GTT GCC 135G F E T D T V F I S N E E V A 45

CCC TGC AAA GCG TGC GGG GCT TGC AGA GAT CAA GAT TTC TGT GTG 180P C K A C G A C R D Q D F C V 60

ATT GAT GAT GAT ATG GAC GAG ATA TAT GAA AAA ATG AGG GCT GCA 225I D D D M D E I Y E K M R A A 75

GAC GGT ATA ATT GTT GCA GCT CCC GTA TAT ATG GGG AAT TAT CCT 270D G I I V A A P V Y M G N Y P 90

GCC CAG CTT AAA GCC CTT TTT GAC AGG AGT GTC CTG CTT CGC CGT 315A Q L K A L F D R S V L L R R 105

AAA AAC TTT GCA CTA AAA AAT AAA GTT GGG GCA GCT CTT TCA GTT 360K N F A L K N K V G A A L S V 120

GGG GGC TCA AGA AAC GGA GGA CAG GAA AAA ACA ATT CAG TCC ATA 405G G S R N G G Q E K T I Q S I 135

CAT GAC TGG ATG CAC ATT CAC GGA ATG ATT GTA GTC GGC GAT AAT 450H D W M H I H G M I V V G D N 150

TCC CAC TTC GGT GGA ATT ACG TGG AAC CCG GCA GAA GAG GAC ACT 495S H F G G I T W N P A E E D T 165

GTT GGA ATG CAG ACA GTT TCC GAA ACT GCA AAA AAA CTC TGT GAT 540V G M Q T V S E T A K K L C D 180

GTC CTG GAA CTT ATT CAG AAA AAT AGA GAT AAA TAA CAA AAT TCA 585V L E L I Q K N R D K * 191

TAA ATT ATA TAA GTC AGG GTA GAA TAA AAC AAA AAA TAT GAA TTT 630CCG AGA AGT AAA TTA GTT ATA TTA ATC TTA TTA TAT TGC ACA TTT 675CAA ACC CTG GCA ATC CTG TGC CAC TAT GCT ATA CCA AAA AGT CAT 720TTT GTT GAT ATA AAA TCA ATA AAA TGT CCA ATA ATC AAA TAT TAT 765TGT CCT ATA TTG GTA GGA GTT CAA AAA CCC CAG GAT AAT GGA GTA 810CAT CTC CCG GTT TGA 825

Figure 1. Corrected nucleic acid sequence and predicted amino acid sequence of isf from

M. thermophila. The DNA is presented in the 5' to 3' direction. The predicted amino

acid sequence of Isf is shown in single-letter code directly below the first base of each

codon. *, initial base of translation stop codon.

65

EPR spectroscopy of the iron-sulfur centers. Isf was purified and sent to Dr.

S.W. Ragsdale and Dr. D.F. Becker at the University of Nebraska for EPR analysis.

Their results, interpretations, and conclusions follow. The reduced heterologously

produced Isf displayed the rhombic EPR spectrum indicative of a [4Fe-4S]+1 type with g

values at 2.06, 1.93 and an unusual split signal at 1.86 and 1.82 (Fig. 2). Additional

evidence for the presence of the [4Fe-4S]1+ center is the disappearance of the g = 1.93 at

the temperatures above 25 K (20). Several lines of evidence suggest the

splitting signal observed at gmin region of EPR spectrum is derived from the

microheterogeneity within Isf molecules. For example, the presence of viologen dyes did

not contribute to this complex spectrum since the sodium dithionite-reduced sample

without dyes also give a similar spectrum. Since the flavin is diamagnetic in both the

oxidized and reduced state, this diamagnetic cofactor cannot give rise to this unusual

feature. The possibility that a strong hyperfine interaction between an unpaired electron

on the cluster and a strongly coupled proton produces a complex feature was examined.

Reduced protein after an extensive exchange with D2O also exhibited the same spectrum;

thus, this possibility was ruled out. In the power and temperature studies, the result

showed two separate and distinct species, one with g values at 2.06, 1.92, and 1.82, and

another one with g values at 2.03, 1.92, and 1.86 (refer to chapter 5). These results are

consistent with the conclusion of microheterogeneity that contributes to the unusual

spectrum.

Power saturation studies of the [4Fe-4S] cluster were examined at five difference

temperatures (5, 10, 15, 20, and 25 K). The half saturation powers (P1/2) are in the range

of 79 to 14.4 mV. The plot of P1/2 and 1/T showed a linear relationship at the

temperatures higher than 5 K (Fig. 3). The zero field splitting parameter (∆) was

observed at the values between 10 and 11.5 cm-1 (Fig. 3,inset).

66

Figure 2. EPR spectroscopy of the [4Fe-4S] cluster in Isf poised at various redox

potentials in 50 mM potassium phosphate buffer (pH 7.0). Experimental conditions were

as follows: temperature, 10 K: microwave power, 1.26 milliwatts; microwave frequency,

9.43 GHz; receiver gain, 2 x 104; modulation amplitude, 10 G; modulation frequency,

100 kHz. The derviative feature at g = 2.0 results from the mediator dyes.

67

Figure 3. Semilogarithmic plot of P1/2 versus 1/T, which shows a linear relationship

according to equation P1/2 = Aexp(-∆/kT). The slope (-∆/k = 1.5 +/- 0.73) yields an

estimate of 11.5 cm-1 for the zero field splitting value (∆). Inset, a nonlinear plot of P1/2

versus 1/T, which includes the data at 5 K. The slope (-∆/k = 14.1 +/- 1.8) yields an

estimate for ∆ of 9.8 cm-1.

68

Mössbauer spectroscopy. Prior to Mössbauer spectroscopy, Isf was

heterologously produced in E. coli cultured in media enriched with 57Fe. The 57Fe-

enriched protein was purified and sent to Dr. K.K. Surerus at the University of Wisconsin

Milwaukee for Mössbauer spectroscopy. Her results, interpretations, and conclusions

follow. The as-purified Isf exhibited a single broad quadrupole doublet Mössbauer

spectrum with an average isomer shift, δ = 0.45 mm/s, and an average quadrupole

splitting, ∆EQ = 1.22 mm/s, at 4.2 K (Fig. 4a). The quadrupole splitting decreased

slightly at 100 K (∆EQ = 1.12 mm/s). Dithionite-reduced Isf exhibited a single broad

nonsymmetrical quadrupole doublet with an average isomer shift, δ = 0.55 mm/s and an

average quadrupole splitting, ∆EQ = 1.30 mm/s at 100 K (Fig. 4b). These parameters are

indicative of values for the [4Fe-4S] center with 2+ and 1+ redox states. The

nonsymmetrical line shapes observed for both as-purified and reduced proteins,

especially for reduced protein, suggest the irons within the cluster are not identical.

Another reason may due to a microheterogeneous population of iron-sulfur clusters in the

Isf molecule. The Voigt (Gaussian distribution of a Lorenzian lineshape) lineshape of the

doublet, rather than Lorenzian, indicates a microheterogenous environment for the iron

sites. These finding are consistent with the conclusion drawn from EPR spectrum.

The property of electronic spin of the iron-sulfur cluster was studied by applying

the magnetic field either parallel or perpendicular to the γ beam. Mössbauer spectra of

the reduced Isf exhibited paramagnetic hyperfine structure indicative of reduced [4Fe-

4S]1+ cluster with S = ½ (Fig. 5). The shape of the spectra and the derived hyperfine

structure are typical of that observed in other [4Fe-4S] proteins (4, 15, 17).

69

Figure 4. Mössbauer spectra recorded at 100 K. A) oxidized Isf protein. B) reduced Isf

protein. The solid line is a least-square fit with a Voigt line shape.

70

Figure 5. Mössbauer spectra of reduced Isf protein recorded at 4.2 K and 450 G applied

parallel (A) or 450 G applied perpendicular (B) to the γ beam. The solid line is a

theoretical fit of an S = ½.

71

Potentiometric titrations of FMN and the [4Fe-4S] center. Potentiometric

measurements were performed by Dr. S.W. Ragsdale and Dr. D.F. Becker at the

University of Nebraska. The absorption spectrum of Isf was followed between 300 to

700 nm during potentiometric titration (Fig. 6). Reduction of FMN was monitored at 480

nm to avoid absorption interference from the [4Fe-4S] cluster. Absorption due to the

[4Fe-4S] cluster was measured at potentials below -305 mV to avoid interference from

the absorption of FMN. The FMN/FMNH2 couple showed a midpoint potential of –277

mV (Fig. 6, inset). There was no appearance of absorption around 500-600 nm, which is

generally present due to the semiquinone. This destabilization of the semiquinone is a

rare case for flavoprotein.

FMN has been shown to go through one electron reductions sequentially

generating a semiquinone and the hydroquinone. Although the semiquinone was not

detected during potentiometric titration, semiquinone may be stabilized in the

physiological system. Attempts to determine the semiquinone formation in the biological

system was performed in the mixture of CODH/ACS, Isf, and CO. The EPR spectra of

frozen reaction mixtures at different time points were observed. The maximum of only

2.5 % of semiquinone form was observed at 28 min (Fig. 7). Over the course of 50 min,

the cluster underwent reduction as the FMN was fully reduced to the hydroquinone form.

The semiquinone form occurred during this reaction is classified as the anionic or red

semiquinone, since the EPR signal yielded a line width with 16 gauss . Therefore, the

formation of semiquinone is possible under the physiological conditions.

The Em value for the +2/+1 couple state of the [4Fe-4S] center was determined

using spectroelectrochemical titration. When the data were analyzed using the Nernst

equation, the Em calculated for the [4Fe-4S]2+/1+ center was –394 mV and the slope was

53 mV (Fig. 2 and 8), results consistent with a one electron transfer carrier (the

theoretical value for a one-electron transfer is 58 mV). The redox reaction was fully

reversible since the reduction was titrated in both the oxidative and reductive directions.

Thus, the midpoint potential of the [4Fe-4S] cluster is more than 100 mV lower than that

of the FMN/FMNH2 couple (Fig. 6 and 8) and is in the range as the value reported for

72

Figure 6. Potentiometric titration of the FMN in Isf (3.2 µM) in 50 mM potassium

phosphate buffer (pH 7.0) at 20o C (curves 1-7, fully oxidized, -262, -272, -281, -290, -

305, and –342 mV respectively). Inset, Nernst plot of the potentiometric data.

73

Figure 7. EPR spectrum of Isf (170 µM dimer) was recorded at 10 K following

incubation for 17 min with CO and CODH (0.5 µM) at 25o C. The amount of FMN

hydroquinone and reduced iron-sulfur cluster at this time point were 46 and 3% (0/03

spin/mol), respectively. After the sample was frozen in liquid nitrogen, the spectrum was

recorded using the conditions described in figure 2)

74

Figure 8. A fit of the Isf midpoint potential data to a theoretical curve generated from

the Nernst equation for two redox centers with reduction potentials of –277 mV (n = 2)

and –394 mV (n = 1).

75

other low-potential [4Fe-4S] clusters (2). These results suggest electrons flow from the

4Fe-4S center to FMN.

Potential electron acceptors for Isf. A previous study indicated ferredoxin A is

a direct physiological electron donor for Isf (14); however, the physiological electron

acceptor is unknown. From the properties of redox centers in Isf described above, 2-

electron carriers are candidates to accept electrons from Isf. Isf could accept one electron

from ferredoxin A thorough the [4Fe-4S] center and two electrons are transferred to

FMN. FMN in the hydroquinone form donates the 2 electrons to the 2-electron carrier.

Several 2-electron carriers such as F420, NAD+, NADP, were included in reconstitution

electron transport assays composed of purified components of CO, CODH, ferredoxin A,

and Isf. The results showed none of these 2-electron carriers were reduced by Isf. Unless

an unknown 2-electron carrier accepts electrons from Isf, these results suggest Isf is not a

1- electron 2-electron switch.

The participation of methanophenazine as electron acceptor for Isf was examined.

2-hydroxyphenazine was a gift from Dr. U. Deppenmeier. Methanophenazine was

isolated from H2/CO2-grown Methanosarcina mazei Gö1 and shown to be involved in

reduction of CoB-S-S-CoM (1). The as-isolated methanophenazine was water insoluble,

thus 2-hydroxyphenazine (a water-soluble analogue) has been used in aqueous buffer

assays (1). The midpoint potential of 2-hydroxyphenazine is –255 mV (1). With the

assumption that the redox potential of methanophenazine is similar to this, this cofactor

should be able to act as an electron acceptor of Isf. However, the results revealed a

different data from the theoretical assumption. Reduction of 2-hydroxyphenazine in the

reconstitution electron transport system with CO, CODH/ACS, and ferredoxin A is

greater than the system containing those components and Isf (Fig. 9). In the

reconstitution system with CO, and CODH/ACS, no reduction of 2-hydroxyphenazine

was observed. These results indicate that ferredoxin A is an electron donor for both Isf

and 2-hydroxyphenazine. At this stage, we have been unable to identify the electron

acceptor for Isf.

76

Figure 9. Time course for reduction of methanophenazine with Isf from M. thermophila.

The standard assay mixtures were anaerobically equilibrated with 1.0 atm. of CO in a

stoppered 1.0 ml-cuvette maintained at 35 o C. The standard assay mixture (700 µl)

contained: 50 mM Tris-Cl (pH 7.6), 2 mM dithiothreitol, 1 mg/l resazurine, 25 µg

CODH/ACS and 120 µM methanophenazine (♦). The assay contained all components of

the standard assay plus 9 µg M. thermophila ferredoxin A ( ). The assay contained all

components of the standard assay plus 9 µg M. thermophila ferredoxin A and 180 µg Isf

( ).

0

0.1

0.2

0.3

0.4

0.5

0 2 4 6 8 10

Minutes

A47

8

77

Evidence for Isf homologs in phylogenetically and physiologically diverse

microbes. Two genomic sequences of the CO2-reducing methanoarchaea, M. jannaschii

and M. thermoautotrophicum, were recently completed (3, 22). M. jannaschii and M.

thermoautotrophicum are phylogenetically and physiologically distinct from M.

thermophila. Neither M. jannaschii or M. thermoautotrophicum can utilize acetate as

growth substrate, instead they evolve methane by reducing CO2 using H2 as electron

donor. Two open reading frames (ORF) from M. jannaschii, MJ0731 and MJ1083,

which encode 192 and 194 amino acid proteins have 40 and 49 % identity to Isf

from M. thermophila. The genome of M. thermoautotrophicum contains three ORFs,

MTH135, MTH1473, and MTH1595 with 41, 34, and 30 % identity to the M.

thermophila Isf. Comparisons of these Isf sequences also show a completely conserved

N-terminal cysteine motif (Fig. 10). These results suggest a general function for this

electron carrier in the methanoarchaea; thus, Isf-like proteins may be widespread electron

carriers for methanogenesis in diverse methanoarchaea. An electron carrier function of

Isf in methanoarchaea other than M. thermophila was examined to test this notion.

Extracts of H2/CO2 grown M. thermoautotrophicum were able to catalyze the reduction

of M. thermophila Isf with either H2 or CO as the electron donor (Fig. 11). These

results suggest Isf homologues are components of the electron transport chain in CO2-

reducing methanoarchaea. The reduction of Isf in the presence of CO is higher than that

of H2 suggesting Isf may be specific for electron transport coupled to CO oxidation. The

Isf reduction rate was stimulated by addition of ferredoxin A from M. thermophila. This

implies that ferredoxin A or a homologue is able to couple the oxidation of either H2 or

CO to the reduction of Isf. However, ferredoxin from C. pasteurianum cannot replace

ferredoxin A for Isf reduction in this system (Fig. 12); thus, the reaction appears to be

ferredoxin A specific.

78

Figure 10. Multiple amino acid sequence alignment of Isf from M. thermophila with

sequences deduced from open reading frames identified in the genomic sequences of M.

jannashii and M. thermoautotrophicum. Accession numbers for the M. jannashii and M.

thermoautotrophicum sequences and percentage identity (in parenthesis) with M.

thermophila Isf are as follows: MTH135 (41%), MTH1473 (34%0, MTH1595 (30%), MJ

1083 (49%), and MJ0731 (40%). Asterisks indicate conserved cysteine residues.

79

Figure 11. Time course for reduction of Isf with extract from M. thermoautotrophicum.

The assay mixture (700 µl) contained cell extract (180 µg protein), M. thermophila

ferredoxin (13.5 µg), 50 mM Tris (pH 7.6) and 2 mM dithiothreitol. Ferredoxin was

omitted in two of the assays (�, ♦). The assay mixtures were anaerobically equilibrated

with 1 atm. of CO (�, •), H2 ( ,,♦), or N2 (∆) in a stoppered 1.0-ml cuvette maintained at

35o C. After a 10-min incubation, the reaction was initiated be the addition of 180 µg of

Isf.

80

Figure 12. Time course for reduction of Isf with extract from M. thermoautotrophicum.

The assay mixture (700 µl) contained: cell extract (180 µg protein), either M.

thermophila ferredoxin A (•, ) or C. pasteurianum ferredoxin (o, ∆) (13.5 µg), 50 mM

Tris (pH 7.6) and 2 mM dithiothreitol. The assay mixtures were anaerobically

equilibrated with 1 atm. of CO (•, o), or H2 ( , ∆) in a stoppered 1.0-ml cuvette

maintained at 35o C. After a 10-min incubation, the reaction was initiated by the addition

of 180 µg of Isf.

0.00

0.05

0.10

0.15

0.20

0 5 10 15Minutes

∆∆A

476

81

DISCUSSION

The properties and physiological role of Isf was examined in this investigation.

Previous studies indicate an involvement of Isf in the electron transport chain of CO-

dependent CoM-S-S-CoB during methanogenesis of M. thermophila (14). However,

further characterization of the redox centers in Isf is necessary to provide insight into a

specific role for Isf in the electron transport chain. The iron-sulfur cluster type present in

the heterologously produced Isf was unequivocally demonstrated by EPR and Mössbauer

spectroscopy to be [4Fe-4S]. The Isf sequence reveals a completely conserved cysteine

motif which has the potential to serve as ligands for the [4Fe-4S] center; however, the

cysteine spacing in the motif is distinct from any known motifs accommodating known

[4Fe-4S] centers. The unusual cysteine motif in Isf may represent a novel class of

cysteine motif ligating the [4Fe-4S] center among the Isf-like sequences. The presence of

two negative absorption features in the EPR spectra of the reduced cluster is unusual.

The results presented here indicate that microheterogeneity within the population of Isf

molecules accounts for this atypical feature. This situation is similar to that of a class of

corinoid/iron-sulfur proteins from methanoarchaea and homoacetogenic anaerobes from

the Bacteria domain in which the reduced [4Fe-4S] cluster exhibits a broad absorption

feature in the same g value region (10, 12, 19). The Em results for the 4Fe-4S center and

FMN indicate that the intra-electron transfer from [4Fe-4S] to FMN is plausible. The Em

values from this investigation and other known Em values from other electron transfer

components are consistent with the electron flow as CODH (Em for center C and [4Fe-

4S] center = -540 and –444 mV, (16)) Õ ferredoxin (Em = -407 mV, (5)) Õ Isf [4Fe-4S] (Em

= -394 mV) Õ Isf [FMN] (Em = -277 mV) Õ an unknown electron carrier (Fig. 13). Most

flavin/Fe-S proteins stabilize four redox states: flavinox:FeSox, flavin semiquinone:FeSox,

flavin semiquinone:FeSred, and flavinred:FeSred (13, 18). Stabilization of the semiquinone

allows versatility of mediating both one-electron and two-electron transfer reactions. The

results reported here suggest only a transient stabilization of the FMN semiquinone;

however, stability of semiquinone may be increased when Isf interacts with its

physiological electron donors/acceptors.

82

Figure 13. Proposed electron transport pathway for oxidation of CO or the carbonyl

group of acetyl-CoA. Cdh A, subunit of the CODH/ACS; FdxA, ferredoxin A; ?,

postulated unknown electron carrier. Midpoint potentials are shown in mV.

CdhA

“C” -540 mV“B” -444 mV

CO

CO2

-518 mV FdxA

4Fe4S -407mV

Isf

4Fe4S -395 mVFMN -277 mV

?

83

Due to the high instability of the semiquinone of FMN, it is possible that the

physiological electron acceptor for Isf (which is unknown) could be a two-electron carrier

and Isf functions as a one-electron/two-electron switch. However, involvement of the

obligate two-electron carrier coenzyme F420, NAD, NADP in the electron transport chain

has been excluded. A role for the two-electron carrier methanophenazine (1) as electron

acceptor of Isf was ruled out. Indeed, methanophenazine competed with Isf for

ferredoxin A. Methanophenazine has never been isolated from acetate-grown M.

thermophila cells. Due to the reduction of methanophenazine by ferredoxin A of M.

thermophila, it is possible that this compound is present in cells and may function in

place of Isf under different growth conditions. Additional experiments are required to

confirm if the reduction of methanophenazine is physiologically significant.

The environment in the FMN binding site of Isf must be different from other

flavoproteins. To stabilize a negative charge of hydroquinone, an amino acid with

positive charge may be required. From the sequence comparisons, two positively

charged amino acids, K94 and R124, are highly conserved. These two residues may

flank the FMN binding site and would result in the thermodynamic stabilization of the

hydroquinone.

The presence of Isf-like sequences in the genomes of M. jannaschii and M.

thermoautotrophicum, and the use of either H2 or CO as electron donor for Isf reduction

by M. thermoautotrophicum cell extract, imply a physiological significance of Isf as a

primary electron carrier in different methanogenesis pathways. The greater reduction of

Isf using CO as electron donor and the ability of M. thermoautotrophicum to grow and

produce CH4 with CO as the sole energy source (6) indicate a physiological role for

CODH in the energy metabolism. Furthermore, this methanoarchaeon involves a CODH

in the synthesis of CO for incorporation into acetyl-CoA for cell carbon (11). The

CODHs from either M. thermoautotrophicum or M. jannaschii have not been purified

and, therefore, the electron acceptor is unknown. The results presented here are

consistent with ferredoxin as the electron acceptor; however, purification of the CODH is

necessary to prove this hypothesis. The implicated functions of Isf homologues in

84

diverse methanoarchaea are also supported by the gene organization in M. jannaschii.

The MJ0731 is located adjacent to MJ0722 and MJ0728, which encode an 8Fe

ferredoxin- and CODH/ACS-like proteins (3). However, there is no such gene

arrangement found in M. thermoautotrophicum (22).

ACKNOWLEDEGEMENT

I would like to thank Dr. D.F. Becker and Dr. S.W. Ragsdale at University of

Nebraska-Lincoln for performing the EPR spectroscopy, and Dr. K.K. Surerus at

University of Wisconsin Milwaukee for Mössbauer spectroscopy. I also thank Dr. U.

Deppenmeier for the 2-hydroxymethanophanize and Dr. J. M. Bollinger for the

suggestion on 57Fe solution preparation. Work described here was partially supported

National Institutes of Health Grant No. 1-R15-GM52666-01 (KKS), and by Department

of Energy Basic Energy Sciences Grants No. DE-FG02-ER20053 (SWR) and DE-FG02-

95ER20198 (JGF). UL was supported by a MOSTE grant from Thailand.

85

REFERENCES

1. Abken, H.-J., M. Tietze, J. Brodersen, S. Bäumer, U. Beifuss, and U.

Deppenmeier. 1998. Isolation and characterization of methanophenazine and the

function of phenazines in memebrane-bound electron transport of

Methanosarcina mazei Gö1. J.Bacteriol. 180:2027-2032

2. Bruschi, M., and F. Guerlesquin. 1988. Structure, function and evolution of

bacterial ferredoxins. FEMS Microbiol. Rev. 4:155-75.

3. Bult, C. J., O. White, G. J. Olsen, L. X. Zhou, R. D. Fleischmann, G.

G.Sutton, J. A. Blake, L. M. Fitzgerald, R. A.Clayton, J. D.Gocayne, A. R.

Kerlavage, B. A.Dougherty, J. F. Tomb, M. D. Adams, C. I. Reich, R.

Overbeek, E. F. Kirkness, K. G. Weinstock, J. M. Merric, A. Glodek, J. L.

Scott, N. S. M. Geoghagen, J. F. Weidman, J. L. Fuhrmann, D. Nguyen, T. R.

Utterback, J. M. Kelly, J. D. Peterson, P. W. Sadow, M. C. Hanna, M. D.

Cottoon, K. M. Roberts, M. A. Hurst, B. P. Kaine, M. Borodvsky, H.-P.

Klenk, C. M. Fraser, H. O. Smith, C. R. Woese, and J. C. Venter. 1996.

Complete genome sequence of the methanogenic archaeon, Methanococcus

jannaschii. Science. 273:1058-1073.

4. Christner, J. A., P. A. Janick, L. M. Siegel, and E. Munck. 1983. Mössbauer

studies of Escherichia coli sulfite reductase complexes with carbon monoxide and

cyanide. Exchange coupling and intrinsic properties of the [4Fe-4S] cluster. J.

Biol. Chem. 258:11157-64.

5. Clements, A. P., L. Kilpatrick, W.-P. Lu, S. W. Ragsdale, and J. G. Ferry.

1994. Characterization of the iron-sulfur clusters in ferredoxin from acetate-


6. Daniels, L., G. Fuchs, R. K. Thauer, and J. G. Zeikus. 1977. Carbon

monoxide oxidation by methanogenic bacteria. J. Bacteriol. 132:118-126.

7. Ferry, J. G. 1992. Biochemistry of methanogenesis. Crit. Rev. Biochem. Mol.

Biol. 27:473-503.

8. Ferry, J. G. 1992. Methane from acetate. J. Bacteriol. 174:5489-5495.

9. Harder, S. R., B. A. Feinberg, and S. W. Ragsdale. 1989. A

86

spectroelectrochemical cell designed for low temperature electron paramagnetic

resonance titration of oxygen-sensitive proteins. Anal. Biochem. 181:283-7.

10. Harder, S. R. L., W. P. Lu, B. A. Feinberg, and S.W. Ragsdale. 1989.

Spectroelectrochemical studies of the corrinoid iron-sulfur protein involved in

acetyl coenzyme-A synthesis by Clostridium thermoaceticum. Biochemistry.

28:9080-9087.

11. Hemming, A., and K. H. Blotevogel. 1985. A new pathway for CO2 fixation in

methanogenic bacteria. Trends Biochem. Sci. 10 :198-200.

12. Jablonski, P. E., W. P. Lu, S. W. Ragsdale, and J. G. Ferry. 1993.

Characterization of the metal centers of the corrinoid;iron- sulfur component of

the CO dehydrogenase enzyme complex from Methanosarcina thermophila by

EPR spectroscopy and spectroelectrochemistry. J. Biol. Chem. 268:325-329.

13. Johnson, M. K. 1994. Iron-sulfur proteins. Encyclopedia of Inorganic Chemistry,

Johnson Wiley and Sons (King, R.B., ed). 4:1896-1915.



271:24023-24028.

15. Lindahl, P. A., E. P. Day, T. A. Kent, W. H. Orme-Johnson, and E. Munck.

1985. Mössbauer, EPR, and magnetization studies of the Azotobacter vinelandii

Fe protein. Evidence for a [4Fe-4S]1+ cluster with spin S = 3/2. J. Biol. Chem.

260:11160-73.

16. Lu, W. P., P. E. Jablonski, M. Rasche, J. G. Ferry, and S. W. Ragsdale. 1994.

Characterization of the metal centers of the Ni-Fe-S component of the carbon-

monoxide dehydrogenase enzyme complex from Methanosarcina thermophila. J.

Biol. Chem. 269:9736-9742.

17. Middleton, P., D. P. Dickson, C. E. Johnson, and J. D. Rush. 1978.

Interpretation of the Mössbauer spectra of the four-iron ferredoxin from Bacillus

stearothermophilus. Eur. J. Biochem. 88:135-41.

18. Muh, U., I. Cinkaya, S. P. Albracht, and W. Buckel. 1996. 4-Hydroxybutyryl-

CoA dehydratase from Clostridium aminobutyricum: characterization of FAD and

iron-sulfur clusters involved in an overall non-redox reaction. Biochemistry.

87

35:11710-8.

19. Ragsdale, S. W., P. A. Lindahl, and E. Munck. 1987. Mössbauer, EPR, and

optical studies of the corrinoid;iron-sulfur protein involved in the synthesis of

acetyl-CoA by Clostridium thermoaceticum. J. Biol. Chem. 262:14289-14297.

20. Rupp, H., K. K. Rao, D. O. Hall, and R. Cammack. 1978. Electron spin

relaxation of iron-sulphur proteins studied by microwave power saturation.

Biochim. Biophys. Acta. 537(2):255-60.

21. Schonheit, P., J. Moll, and R. K. Thauer. 1980. Growth parameters (Ks, mu-

max, Ys) of Methanobacterium thermoautotrophicum. Arch. Microbiol. 127:59-

65.

22. Smith D. R. , L. A. Doucette-Stamm, C. Deloughery, H. Lee, J. Dubois, T.

Aldredge, R. Bashirzadeh, D. Blakely, R. Cook, K. Gilbert, D. Harrison, L.

Hoang, P. Keagle, W. Lumm, B. Pothier, D. Qiu, R. Spadafora, R. Vicaire,

Y. Wang, J. Wierzbowski, R. Gibson, N. Jiwani, A. Caruso, D. Bush, H.

Safer, D. Patwell, S. Prabhakar, S. McDougall, G. Shimer, A. Goyal, C. J.

Daniels, J. I. Mao, P. Rice, J. Nölling, and J. N. Reeve. 1997. The complete

genome sequence of Methanobacterium thermoautotrophicum strain ∆H:

functional analysis and comparative genomics. J. Bacteriol. 179:7135-7155.

23. Stankovich, M., and B. Fox. 1983. Redox potentials of the flavoprotein lactate

oxidase. Biochemistry. 22:4466-72.

24. Stankovich, M. T. 1980. An anaerobic spectroelectrochemical cell for studying

the spectral and redox properties of flavoproteins. Anal. Biochem. 109:295-308.



263:4080-4082.

26. White, R. H., and D. Zhou. 1993. Biosynthesis of the coenzymes in

methanogens. Methanogenesis, J.G. Ferry, ed. Chapman and Hall, New York.

409-444.

88

CHAPTER 5

A NOVEL 4Fe-4S CLUSTER BINDING MOTIF IN THE IRON-

SULFUR FLAVOPROTEIN OF Methanosarcina thermophila

ABSTRACT

Isf (Iron-sulfur flavoprotein) from Methanosarcina thermophila has been

produced in Escherichia coli as a dimer containing two [4Fe-4S] clusters and two FMN

(flavin mononucleotide). The deduced sequence of Isf contains six cysteines (C16, C47,

C50, C53, C59, and C180), four of which (C47, C50, C53, and C59) compromise a motif

perfectly conserved among several putative Isf sequences available in the databases. The

spacing of the four conserved cysteines is highly compact and atypical of motifs

coordinating known 4Fe-4S clusters; therefore, all 6 cysteines in Isf were altered to either

alanine or serine to obtain biochemical confirmation that the motif coordinates the 4Fe-

4S cluster and to determine the influence of the protein environment on the properties of

the cluster. All except the C16S variant were produced in inclusion bodies that required

solubilization and reconstitution of the iron-sulfur cluster and FMN. The UV-visible

spectra of all variants indicated the presence of iron-sulfur clusters and FMN. The

reduced C16X (X = A or S) variants showed the same EPR spectra as wild type Isf

whereas the reduced C180X variants showed EPR spectra similar to one of the 4Fe-4S

species present in the wild type Isf spectrum. EPR spectra of the oxidized C50A and

C59A variants showed g-values characteristic of a 3Fe-4S cluster. The spectra of the

C47A and C53A variants indicated a 4Fe-4S cluster but with g-values different from wild

type. The retention of 4Fe-4S cluster in the C47A and C53A variants suggests functional

replacement of the cysteines by 2-mercaptoethanol that was present in the reconstitution

buffer. The reduced C47S, C50S, and C53S exhibited EPR spectra of 4Fe-4S centers.

EPR spectrum of both C47S and C53S revealed two different 4Fe-4S ligand species,

which could be due to the replacement of the missing ligand by serine or 2-

mercaptoethanol. Taken together with strict sequence conservation, these results indicate

that C47, C50, C53 and C59 are ligands to the 4Fe-4S cluster, a result which identifies

the most compact cysteine motif know which ligates a redox-active 4Fe-4S cluster. The

89

results suggest C16 is important for maintaining a local conformation required for

transfer of electrons from ferredoxin A to FMN, and that C180 is essential for the overall

structural integrity of Isf. The reduction of FMN in the Isf variants by ferredoxin A was

either several-fold impaired or enhanced suggesting that the 4Fe-4S cluster serves to

transfer electrons from ferredoxin A to FMN.

INTRODUCTION

Two-thirds of the biologically produced methane in nature originates from the

methyl group of acetate in a pathway where acetate is cleaved and the methyl group is

reduced to methane with electrons derived from oxidation of the carbonyl group to

carbon dioxide (7). Much is known concerning the cleavage of acetate and one-carbon

transfer reaction (6); however, less is known regarding electron transport. Recently, a

novel iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila was characterized

which participates in electron transport during the methanogenic fermentation of acetate

(2, 13). The homodimeric Isf contains two FMN molecules and two 4Fe-4S clusters

unequivocally identified by EPR and Mössbauer spectroscopy. The midpoint-potential

values of the 4Fe-4S cluster and FMN are -394 and -277 mV, respectively. These results

are the basis for a postulated role for the cluster in electron flow from ferredoxin A, the

physiological electron donor for Isf, to the 4Fe-4S cluster and then to the FMN of Isf,

however, this proposal has not been tested. The physiological electron acceptor for Isf is

unknown. The deduced sequence of Isf contains six cysteines, four of which are in a

highly compact novel motif (CX2CX2CX4-7C) that is perfectly conserved among putative

Isf-like sequences identified in the databases suggesting the motif ligates the 4Fe-4S

cluster.

The cubane [4Fe-4S] cluster is ubiquitous in proteins from all domains of life (22)

where they mainly function in electron transfer. The sulfur atom of cysteine is the

prominent protein ligand coordinated to iron atoms in these clusters. Few examples of

variations from cysteine ligation include aconitase with oxygen ligation originating from

hydroxide, water, or substrate. The 4Fe-4S cluster in the ferredoxin from Pyrococcus

90

furiosus is ligated with oxygen from aspartate (27). An iron atom in the 4Fe-4S cluster of

hydrogenase from Desulfovibrio gigas is coordinated by a histidyl nitrogen (26). A single

motif (CX2CX2C plus a distal C in the polypeptide chain) coordinates all low potential,

redox active, 4Fe-4S clusters for which cysteine is the exclusive ligand. Possible

exceptions to this ubiquitous 4Fe-4S motif are found in the corrinoid/iron sulfur proteins

of M. thermophila and Clostridium thermoaceticum, and a putative iron-sulfur protein

from Rhodobacter capsulatus, where the sequence CX2CX4CX16C is perfectly conserved

(14); however, conclusive evidence for involvement of this motif in ligation of 4Fe-4S

clusters has not been reported. Thus, the highly conserved CX2CX2CX4-7C motif in Isf is

the most compact motif known with the potential to coordinate a 4Fe-4S motif. Although

the great majority of iron-sulfur proteins function in electron transfer reactions, the

clusters in a few function in non-redox catalysis or serve a structural role. Still other iron-

sulfur clusters bind nucleic acids or play a regulatory role (3, 10). Two of these,

endonuclease III and MutY, contain a redox inert 4Fe-4S cluster coordinated by a

compact cysteine motif (CX6CX2CX5C) (19, 20). Although the 4Fe-4S cluster of Isf has

reversible redox activity, conclusive evidence for a role in electron transfer has not been

reported.

Much has been learned regarding the coordination of iron-sulfur clusters utilizing

site-specific replacement of residues ligating the clusters. Changes in spectroscopic

properties and other characteristics have provided information regarding the polypeptide

environment of the cluster and the effects that the coordinating ligands have on the

biochemical properties of the cluster. Thus, a series of site-specific replacements in Isf

were performed to obtain biochemical evidence for the novel putative cysteine motif and

further characterize the biochemical and physiological properties of the 4Fe-4S cluster.

The results support involvement of the remarkably compact motif in coordination of the

4Fe-4S cluster that is surprisingly resistant to changes in ligation. The results also support

a role for the 4Fe-4S cluster in the transfer of electrons from the physiological electron

donor (ferredoxin A) to FMN.

91

Portion of the results contained in this section were obtained by Dr. J.H. Golbeck

and M. L. Antokine at Pennsylvania State University. Their contributions are noted in

the appropriate sections.

EXPERIMENTAL PROCEDURES

Sequence comparisons. Microbial genomic sequence databases were searched at

http://www.tigr.org. Sequences were aligned using the program Clustal X version 1.64b.

Plasmid construction and site directed mutagenesis. Plasmid pML701, which

contains the entire gene for Isf, was used as a template to construct mutants. Site directed

mutagenesis was performed using MORPH as described by the manufacturer (5 Prime →

3 Prime, Inc. 5603 Arapahoe, Boulder, CO 80303). Each construct was confirmed for

the intended mutation by sequencing using the automated dideoxy method at the Penn

State University nucleic acid facility.

Protein production and purification. Escherichia coli BL21 (DE3) cells

transformed with derivative expression plasmids carrying the designated isf mutations

were grown on LB broth supplemented with 100 µg/ml ampicillin. Once cells reached an

A600 of about 0.8, they were induced to produce high levels of the Isf variants by addition

of 1% (final concentration, w/v) Bacto-lactose for 2 h. The cells were harvested by

centrifugation at 11,800 x g for 10 min at 4oC. The cell pellets were frozen at -70oC.

The C16S variant and wild type was purified as described (13). All other variants

were purified as follows. Approximately 5 g (wet weight) of cells were suspended in 6

volumes (w/v) of buffer A (50 mM Tris-HCl pH 7.6, 200 µg/ml lysozyme, and 2 mM

DTT) and incubated for 20 min at 21oC. Cells were lysed by two passages through a

French pressure cell at 20,000 psi. The lysate was centrifuged at 10,000 x g for 30 min at

4oC. The pellet, containing inclusion bodies, was washed twice in 30 ml buffer B (50 mM

Tris-HCl, pH 7.6, 2 M urea, 1% Triton X-100, 2 mM DTT). The protein aggregates were

solubilized in 2 ml buffer C (50 mM Tris-HCl, pH 7.6, 6 M guanidine-HCl), and

incubated for 2 h at 21oC. Insoluble protein was removed by centrifugation at 10,000 x g

92

for 10 min at 4oC. The protein solution at this stage is termed "denatured". The soluble

fraction was then diluted 100-fold in buffer D (50 mM Tris-HCl, pH 7.6, 500 mM L-

arginine, 2 mM DTT), and incubated at 4oC for 12 h. In the following step, the sample

was concentrated using PEG 8000. The protein was dialyzed in buffer E (50 mM Tris-

HCl, pH 7.6, 250 mM L-arginine, 200 mM NaCl, 2 mM DTT) and then F (50 mM Tris-

HCl, pH 7.6, 200 mM NaCl, 2 mM DTT). The protein at this stage is defined as

"renatured". There was no apparent change in subunit size among wild type and the

variants as judged by migration in SDS-PAGE. The overall procedure resulted in

homogenous proteins as judged by SDS-PAGE.

Ethylenediaminetetraacetic acid (sodium salt) (EDTA)-treated wild type protein

was prepared by incubated 6 mg of as-purified wild type in buffer G (50 mM Tris-Cl

pH7.6, 400 mM NaCl, 2 mM EDTA and 2 mM DTT) for at 21o C. Then the EDTA-

treated protein was treated in buffer D, E, and F as stated above.

Reconstitution of iron-sulfur clusters and FMN into renatured apoprotein.

Reconstitution of iron-sulfur clusters and FMN was performed by adding 1 ml of 10 mM

FMN, 800 µl of 2-mercaptoethanol, 300 µl of 60 mM FeCl3, and 300 µl of 60 mM Na2S

to 100 ml renatured apoprotein solution (12 mg). All reagents were added drop-wise with

10 min intervals between steps, and the reconstitution reaction was incubated at 4oC for

12 h. The protein was concentrated with an ultrafiltration unit fitted with a YM 30

membrane (Amicon, Beverly, Mass.) and the unbound molecules were removed by a

PD10 gel filtration. The protein at this step is called "reconstituted". The recovery yield

after the overall processes (denatured, renatured, and reconstituted processes) was varied,

and in the range of 20-50 %.

Spectroscopy. UV-visible spectra were obtained with a Hewlett-Packard 8452A

diode array spectrophotometer. EPR signals of iron sulfur clusters were recorded with a

Bruker ECS 106 Electron Paramagnetic Resonance (EPR) X-band spectrometer operating

with an ER/4012 ST resonator and an Oxford liquid helium cryostat. The temperature

93

was controlled using an ITY4 Oxford temperature controller. The microwave frequency

was determined with a Hewlett-Packard 5340A frequency counter.

Reduction of Isf by ferredoxin A. Experiments were carried out in a stoppered

1.0 ml-cuvette equilibrated with an atmosphere of CO. Continuous reduction of

ferredoxin A was accomplished by including catalytic amounts of CODH/acetylCoA

synthetase. All protein components except the Isf variants were anaerobically purified as

previously described (13, 23, and 24). The assay mixture contained 27 µg

CODH/acetylCoA synthetase and 9 µg ferredoxin A in anaerobic 50 mM Tris-HCl (pH

7.6) containing 500 mM sucrose, 0.1 mg/l resazurine, and 2 mM DTT. After 10 min

incubation, 180 µg of the indicated Isf variant was added to the assay mixtures to initiate

the reaction. The absorbance at 476 nm was measured to follow the reduction of FMN

without interference from reduction of the iron-sulfur cluster. Ferredoxin A and

CODH/acetyl CoA synthetase were present in catalytic amounts such that any absorption

change in these proteins did not interfere with the assay.

RESULTS

Sequence comparisons of Isf from M. thermophila with putative Isf proteins.

Figure 1 shows that metabolically diverse species contain sequences with identity to M.

thermophila Isf suggesting the possibility that this electron carrier functions in carbon

dioxide-reducing (Methanococcus jannaschii and Methanobacterium

thermoautotrophicum) and sulfate-reducing (Archaeoglobus fulgidus) Archaea, and also

in metabolically diverse procaryotes from the Bacteria domain (Chlorobium vibrioforme,

Clorobium tepidum, and Clostridium difficile). Only Isf from M. thermophila has been

characterized; thus, the sequences shown in Figure 1 are putative Isf homologs.

Nonetheless, comparison of these sequences with Isf from M. thermophila shows an

unusually compact N-terminal cysteine motif with a strictly conserved spacing of

CX2CX2CX4-7C atypical of cysteine motifs required for 4Fe-4S coordination. The strict

conservation provides a strong indication for involvement in ligation of the 4Fe-4S

cluster; however, it is possible that any one of these conserved cysteines is essential for

94

16 47

MST ---------M KITGISGSPR KGQNCEKIIG AALEVAKERG FETDTVFISN EEVAP--CKA 49MCJ-2 ---------M KVIGISGSPR PEGNTTLLVR EALNAIAEEG IETEFISLAD KELNP--CIG 49MBT-1 MKQKEVDFMV KVIGICGSPR KNGNTEILLR EALDAAEEAG AETELVRLAG LDINP--CRA 58MBT-2 ---------- MILGICGSPR K-QATEHVLE RALSMLEDDG LETEFFTVRG KNISP--CRH 47AF-2 ---------- MIVGISGSPR R-KATEFVLG EALKMLEERG FETKFFTVRG KKISP--CQH 47MCJ-1 ---------M KVFGISGSPR L-QGTHFAVN YALNYLKEKG AEVRYFSVSR KKINF--CLH 48CV ---------M KVIGINGSPR PAGNTSIMLK TVFETLEQEG IETELIQVGG TDIKG--CRA 49CT ---------M KVIGINGSPR RAGNTSIMLK TIFEVLEDEG IETELIQVGG TNIKG--CRA 49AF-3 ---------M KLLAINGSPN K-RNTLFLLE VIAEEVKKLG HEAEIIHLKD YEIKE--CKG 48MBT-3 ---------- ---------- ------MVLE HCRDAIESHG VETDIISLRG MKIES--CRA 32AF-1 ---------M KAVGILGSPR KYGNASKMLD AALKELENSG FEVEKVHISS KKINY--CTG 49CD ---------M IITVMNGSPR KNGATSKVLT YLYKDIERLI PDVKINYFDL SEVNPSYCIG 51

50 53 59MST CGACRDQDF- -CVID-DDMD EIYEKMRAAD GIIVAAPVYM GNYPAQLKAL FDRSVLLRR- 105MCJ-2 CNMCKEEGK- -CPII-DDVD EILKKMKEAD GIILGSPVYF GGVSAQLKML MDRSRPLR-- 104MBT-1 CDSCKKTGE- -CAIE-DDLN RVVELAASAH GIIIGSPVYF GSVTAQTKMF MDRTRPLR-- 113MBT-2 CDYCLRNKE- -CVLK-DDMF PLYELLRRAA GIIIATPVYN GGVSAQIKAI MDRCRALGAE 104AF-2 CDYCLKHKE- -CRIK-DDMF ELYEMLKDAK GIVMATPVYN GGVSAQIKAV MDRCRALVAA 104MCJ-1 CDYCIKKKEG -CIHK-DDME EVYENLIWAD GVIIGTPVYQ GNVTGQLKTL MDRCRAILAK 106CV CYACIRNKNS KCSTK-DGFN EIFEKMVEAN GMILGSPVYF ADITPELKAL IDRSGFVSRT 108CT CYACIKNKNS ECSTKGDGFN EIFAKMVEAD GMILGSPTYF ADITPELKAL IDRAGFVSRT 109AF-3 CDACLKGD-- -CSQK-DDIY KVLEKMQEAD AIVIGTPTYF GNVTGIVKNL IDRSRMAR-M 103MBT-3 CLSCAKKHR- -CRID-DGLN DIIDRIRDSE GFIVATPVYF GTARGDLMAA LQRIGMVSRA 89AF-1 CGTCLAKGE- -CVQR-DDMD ELKRLVEESD AVILASPVYY LNVTAQMKTF IDRMLPYG-- 104CD CLNCYKMGK- -CINQNDKVE YIHDIITKSD GVIFGSPTYG SSVTGLFKVF TDRAHMML-- 107

MST KNFALKNKVG AALSVGGSRN GGQEKTIQSI HDWMHIHGMI VVGDNS---- HFGGI---TW 158MCJ-2 IGFQLRNKVG GAVAVGASRN GGQETTIQQI HNFFLIHSMI VVGDND-PTA HYGGT---GV 160MBT-1 SEFRLANRVG GAVTVGGSRN GGQETACRDI HSFFLIHEAA VVGNAS-PTA HYGGT---GV 169MBT-2 DYDSLRGKVG MGIAVGGDRC GGQEPALMQI HTFYILNGVI PVSGGS-FGA NLGAC---FW 160AF-2 DYDFFRGKVG MAIAVGGDRI GGQELAIQQI LTFYILNGVI PVSGGS-FGA NIGAT---FW 160MCJ-2 NPKVLRGRVG MAIAVGGDRN GGQEIALRTI HDFFIINEMI PVGGGS-FGA NLGAT---FW 162CV NGQLFRHKVG ASIVS--LRR GGGVHAYDSI NHLFQICQMF MVGSTY---W NLG-----FG 158CT NGQLFRHKVG ASVVS--LRR GGGIHAYDSI NHLFQICQMF MVGSTY---W NLG-----FG 159AF-3 GNYRLRNRVF APVVTSGLRN GGAEYAAMSL IVYALGQAML PVSIVE-NPI TTGTFPVGVI 162MBT-3 SDGFLSWKVG GPIAV--ARR GGHTATIQEL LMFYFINDMI VPGSTY-WNM VFG------- 139AF-1 HRPTLKGKYG GSIVVY-AGV GKPEEVAGYM NRVLKAWGIV PVGYAVGFGV IPGEVGDEDL 163CD ERLLYRKPCI AVTTY--ENA RGS-KAISFI KSMVLDSGGY VCGSLS---I KTG------F 155

180MST NPAE------ EDTVGMQTVS E-TAK--KLC D-----VLEL IQKNR----- -------DK- - 191MCJ-2 GKAP------ GDCKNDDIGL E-TAR--NLG K-----KVAE VVKLI----- -------KK- - 193MBT-1 GGAK------ GESADDMTGI E-TAR--NLG R-----RVAL LAARI----- -------HG- -- 202MBT-2 SRDT-L---- EVLKRTHMDS KPSKRPWACL KGSWTLKDPE ILFYS----- -------EFI -- 203AF-2 SRDT-L---- EGVKEDEEGF R-SLR--KTV K-----RFAE MLEKM----- -------EGV - 195MCJ-1 SKDRGK---- KGVEEDEEGL R-VLR--KTL N-----RFYE VLKEK----- -------RGL -- 198CV GRDG------ GEVVNDTEGM D-NMR--DLG K-----SMAF LLKKL----- -------NAS -- 192CT GRDG------ GEVVNDTEGM E-NMR--DLG H-----SMAF LLK------- ---------- -- 188AF-3 QGDAGW---- RSVKKDEIAI N-SAK--ALA KR--IVEVAE ATKNL----- -------RES -- 201MBT-3 -WAP------ GEVEDDSEGI E-TIR--RFG E-----NVAE LIKRI----- -------NGG S- 173AF-1 KKASQLGSKI AEAFESKYRM EPSDEDLELQ K-----QLLT LIKNYGHLMK ADYEFWKEKG FI 220CD NQNP------ ---------- ---------- ---------- ---------- ---------- -- 159

Figure 1. Multiple amino acid sequence alignment of Isf from M.thermophila (MST)

with sequences deduced from open reading frames identified in the genomic sequences of

M. jannaschii (MCJ), M. thermoautotrophicum (MBT), Archaeoglobus fulgidus (AF),

95

Chlorobium vibrioforme (CV), Chlorobium tepidum (CT), and Clostridium difficile (CD).

The numbers after the abbreviated name of organisms indicate the different protein

isoforms. Database codes for each protein are as follows, MST: Genbank U50189;

MCJ-1, -2 : Genbank C64391 (MJ0731), B64435 (MJ1083); MBT-1,-2,-3: Genbank

AE000802 (MTH135), AE000908 (MTH1473), AE000919 (MTH1595); AF1-,-2,-3:

AE0010041 (AF1438), AE0009971 (AF1519), AE0009721 (AF1896); CV: EMBL

Z83933.1; CT: C tepidum gct10; CD: CD shotgun.dbs cd2h6.q1t. Cysteines (C16, 47,

50, 53, 59, and 180) in Isf of M.thermophila are numbered at the top line. Residues

conserved in at least 7 out of 10 sequences are shaded in gray. Putative FMN binding

regions in Isf are underlined.

96

another function and other non-cysteinyl residues may ligate the 4Fe-4S cluster. Thus, we

undertook a biochemical approach to obtain experimental evidence for the proposed role

of the motif and investigate properties of the cluster dependent on the protein

environment.

Heterologous production, purification and reconstitution of wild type Isf and

variants. In the course of the investigation of cysteine ligation in the 4Fe-4S cluster, six

cysteines present in Isf (Fig. 1) were individually altered to either alanine or serine.

Except for C16S, the variants were contained in inclusion bodies. The soluble C16S was

purified the same as for wild type. The inclusion bodies were isolated by centrifugation

as a first step in purification of the remaining variants. The isolated inclusion bodies were

extracted in guanidine-hydrochloride to solubilize the proteins. At this juncture, no

discrete bands were detected by native PAGE (data not shown) suggesting the proteins

were denatured. The solubilized variants were diluted in buffer containing arginine to

prevent protein aggregation during renaturation (1, 5, and 25). After removal of the

arginine by dialysis, native PAGE indicated no discrete bands (data not shown)

suggesting the proteins had not achieved the native state. UV-visible spectra indicated the

proteins contained very low amounts of iron-sulfur clusters and FMN (data not shown);

thus, the apoproteins were incubated in the presence of ferric iron, sulfide, and FMN to

reconstitute the redox clusters. UV-visible spectroscopy (Fig. 4, 5) indicated

incorporation of flavin and iron-sulfur clusters. Native PAGE (Fig. 2) indicated a discrete

band for each variant migrating to approximately the same position as the purified wild

type, which suggested all are dimeric in accord with the initial characterization of the

wild type (13). These results suggested that either an iron-sulfur cluster or flavin, or both,

must be present to adopt a native conformation. There was no apparent change in subunit

size among wild type and the Isf variants as judged by migration in SDS-PAGE (data not

shown). A similar denaturation/renaturation/reconstitution process was performed for

wild type. As for the variants, only the reconstituted wild type exhibited a discrete band

after native PAGE (Fig. 2). There was intermittent success and low yields in the

reconstitution of both C59X and C180X (X = A or S) indicating they were unstable.

97

Figure 2. Coomassie blue stained native PAGE of wild type Isf and variants. Top

panel contains as-purified wild type Isf (25 µg), reconstituted Isf (25 µg), and cysteine

to alanine variants (25 µg; except 18 µg for 180A). Bottom panel contains as-purified

wild type Isf (18 µg), reconstituted cysteine to serine variants (25 µg for C16S and

C50S, 18 µg for C47S, C53S, and C180S).

98

Since the proteins showed less precipitation during refolding in the presence of 0.5 M

arginine, cofactors reconstitution in C59X and C180X in the presence of arginine were

performed. In some instances with no consistency, this method yields higher recovery of

reconstituted proteins, which is indicated in higher intensity of EPR signal (Fig. 8).

Spectroscopic characterization of reconstituted wild type Isf and variants.

The UV-visible spectra of denatured and renatured wild type Isf showed no absorbance

characteristic of either iron-sulfur clusters or FMN (Fig 3) suggesting the complete loss

of both redox components. The UV-visible spectrum of the reconstituted Isf was nearly

identical to the as-purified wild type suggesting the reconstituted protein might have

properties similar to as-purified Isf. The UV-visible absorption spectra for the

reconstituted variants were similar to the wild type (Fig. 4, 5); however, the ratio of FMN

absorbance at 378 nm relative to iron-sulfur cluster absorbance centered at 430 nm was

generally lower for all except C16A and C16S. These results suggest significantly lower

incorporation of FMN relative to iron-sulfur clusters for all variants except C16A and

C16S. The UV-visible spectrum of C59S contained no features characteristic of iron-

sulfur cluster incorporation, a result that was confirmed by EPR (data not shown).

EPR spectrum of wild type Isf, alanine and serine variants were recorded and

compared to determine cysteines that participate in ligating iron-sulfur cluster in Isf.

The reduced as-purified Isf exhibited an EPR spectrum with g-values of 2.06, 2.03, 1.92,

1.86, 1.81 (Fig. 6, Table 1), results that are nearly identical to a previous report (2) in

which the authors attributed the complexity of the spectrum to heterogeneity of the

sample (refer to chapter 3). We were able to distinguish two distinct species based on

power and temperature dependencies, one with g values of 2.06, 1.92, and 1.81 and

another with g values of 2.03, 1.92, and 1.86 (Fig. 6). The ratio of these species varied in

different Isf preparations suggesting the as-purified protein exists in two distinct

conformational states. Incubation of as purified Isf with ethylenediaminetetraacetic acid

sodium salt (EDTA) resulted in nearly complete destruction of 4Fe-4S center as followed

by EPR (Fig. 6). However, the overwhelming majority of the center reconstituted to the

g = 2.03, 1.92, and 1.81 species. This implies that under experimental conditions, this Isf

99

Figure 3. UV-visible absorption spectra of as-purified, denatured, and reconstituted wild

type Isf. The denatured protein was in 50 mM Tris-Cl (pH 7.6) containing 6 M

guanidine-HCl. The as purified, renatured, and reconstituted proteins were in 50 mM

Tris-Cl (pH 7.6) containing 200 mM NaCl. The spectra were recorded at 21o C. The

amount of protein used for as-purified Isf was 80 µg, all other were 150 µg.

280 330 380 430 480 530

Wavelength (nm)

as-purified

reconstituted

0.1 A

renatured

denatured

100

Figure 4. UV-visible absorption spectra of wild type Isf and alanine variants. The

samples (500 µl) were in 50 mM Tris-Cl (pH 7.6) containing 200 mM NaCl, and the

spectra were recorded at 21oC. The amount of protein used for wild type Isf was 80 µg;

all variants were 120 µg.

280 330 380 430 480 530

Wavelength (nm)

Isf

C16A

C47A

C50A

C53A

0.2 A

C59A

C180A

101

Figure 5. UV-visible absorption spectra of wild type Isf and serine variants. The

samples (500 µl) were in 50 mM Tris-Cl (pH 7.6) containing 200 mM NaCl, and the

spectra were recorded at 21oC. The amount of protein used for wild type Isf was 80 µg;

all variants were 120 µg.

280 330 380 430 480 530

Wavelength (nm)

0.2 A

Isf

C16S

C47S

C50S

C53S

102

Figure 6. EPR spectra of reduced wild type and reconstituted Isf. EPR conditions were

as follows: temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT.

Proteins were reduced by sodium dithionite.

103

conformation is energetically more favorable and it could be achieved more easily.

Reconstitution of wild type Isf that had been denatured and renatured also exhibited a

4Fe-4S EPR spectrum identical to wild type with evidence for both species. This result

indicated that the procedure for reconstituting the Isf apoprotein yielded a 4Fe-4S cluster

with an environment identical to the wild type.

The reduced C16A and C16S variants exhibited EPR spectra with g-values of

2.06, 2.04, 1.92, 1.86, 1.82 (Fig. 7, Table 1) characteristic of wild type Isf with two

distinct 4Fe-4S species. The reduced C180A and C180S variants showed EPR spectra

(Fig. 8, 9, Table 1) with line shapes and g-values (2.07, 2.04, 1.93, 1.86, 1.81) nearly

identical to one of the 4Fe-4S species present in the wild-type Isf spectrum. A minor

contribution of the other species was also observed as a shoulder at g 2.07. These results

strongly indicate that C16 and C180 do not participate in ligation of the 4Fe-4S cluster of

Isf. This conclusion is further supported by sequence comparisons showing that C16 and

C180 are not conserved with putative Isf proteins (Fig. 1). The predominance of one

4Fe-4S species in the EPR spectra of the C180 variants suggests one of the conformations

possible for wild type Isf is preferred in these variants. This proposal is consistent with

the instability of the C180A and C180S variants which suggests that C180 is important

for the overall structural integrity of the protein.

Cysteines 47, 50, 53 and 59 are strictly conserved among putative Isf sequences

(Fig. 1) as a compact motif CX2CX2CX4-7C suggesting the motif is essential and,

therefore, a candidate for ligation of the 4Fe-4S cluster. EPR spectroscopy of the reduced

C50A or C59A variants detected no [4Fe-4S]1+ cluster; however, spectra of the oxidized

variants had linewidths and g-values typical for [3Fe-4S]2+ clusters (Fig. 10, 11, Table 1).

These results strongly indicate that C50 and C59 are involved in ligation of the 4Fe-4S

cluster in Isf. The results also show that other ligands cannot substitute for C50 and C59

to preserve the 4Fe-4S cluster in these variants. A [4Fe-4S]1+ cluster was detected in

both of the reduced C47A and C53A variants by EPR spectroscopy; however, there were

significant differences in the line widths and g-values between the spectra of the two

104

2.3 2.2 2.1 2.0 1.9 1.8g -value

C16A/S

C16S C16A

g =

Figure 7. EPR spectra of reduced C16X (X = A or S). EPR conditions were as follows:

temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT. Proteins were

reduced by sodium dithionite.

105

2.3 2.2 2.1 2.0 1.9 1.8g -value

C180A

+Arg -Arg

g =

Figure 8. EPR spectra of reduced C180A. (+ Arg) The protein was denatured, refolded

as described in experimental procedures. However, the reconstitution process was

performed immediately after refolding with no dialysis to remove arginine. (–Arg) The

protein was denatured, refolded, dialyzed to remove arginine, and then reconstituted.

EPR conditions were as follows: temperature 15 K, microwave power 20 mW,

modulation amplitude 1 mT. Proteins were reduced by sodium dithionite.

106

2.3 2.2 2.1 2.0 1.9 1.8g -value

C180S

g =

Figure 9. EPR spectrum of reduced C180S. EPR conditions were as follows:

temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT. The protein

was reduced by sodium dithionite.

107

2.05 2.00 1.95 1.90 1.85

g -value

C50A

g =

Figure 10. EPR spectrum of as-purified C50A. EPR conditions were as follows:

temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT.

108

2.2 2.1 2.0 1.9

g -value

C59A

g =

Figure 11. EPR spectra of as-purified C59A. EPR conditions were as follows:

temperature 15 K, microwave power 1.26 mW, modulation amplitude 1 mT.

109

variants. The line widths and g-values for both variants were also significantly different

from the spectra of either of the two species present in the reduced form of as-isolated

wild-type Isf (Fig. 12, 13, Table 1). A low-intensity [3Fe-4S]2+ EPR signal was detected

in the oxidized C47A and C53A variants. These data strongly indicate that one or more

ligands to the 4Fe-4S cluster changed in these variants, a result suggesting that C47 and

C53 are ligands to the 4Fe-4S cluster in wild type Isf. The EPR results obtained for the

C47A, C50A, C53A, and C59A variants, combined with strict conservation of the

CX2CX2CX4-7C motif in putative Isf sequences from diverse species (Fig. 1), strongly

suggest involvement of the motif in ligation of the 4Fe-4S cluster in Isf.

Although other residues could replace cysteine as a ligand to the 4Fe-4S clusters

in C47A and C53A, we consider it most likely that 2-mercaptoethanol is an external

thiolate ligand in these variants for the following reasons. The buffer used for

reconstitution of the variants contained 2-mercaptoethanol which has been shown to

serve as an external ligand to the 4Fe-4S cluster in the C51D and C14G variants of PsaC

from Photosystem I in Synechocystis sp. PCC 6803 (11, 28). The reconstitution

conditions used in this work were nearly identical to the conditions used to reconstitute

iron-sulfur clusters in PsaC. Thiolate ligands in C53A and C47A are expected to occupy

different positions in the coordination sphere of the 4Fe-4S cluster consistent with

differences in the EPR spectra recorded for these variants. Thiolate ligation is also

consistent with differences in the EPR signals of the C53A and C47A variants compared

to wild-type Isf.

The EPR spectra of the reduced C47S, C50S, and C53S variants indicated the

presence of 4Fe-4S clusters (Fig. 14-16, Table 1). The EPR spectra of C47S, C50S and

C53S suggest the presence of two different ligand species. These features could

potentially derive from the use of substituted serine and 2-mercaptoethanol presence in

the reconstitution system. The role of introduced serine residues in ligation of iron-sulfur

clusters has been determined for several proteins by substitution with alanine in which

case the native cluster does not assemble if the replaced cysteine or serine residues are

110

2.3 2.2 2.1 2.0 1.9 1.8g -value

C47A

g =

Figure 12. EPR spectrum of reduced C47A. EPR conditions were as follows:



111

2.3 2.2 2.1 2.0 1.9 1.8g -value

C53A

g =

Figure 13. EPR spectrum of reduced C53A. EPR conditions were as follows:



112

2.3 2.2 2.1 2.0 1.9 1.8g -value

C47S

g =




113

2.3 2.2 2.1 2.0 1.9 1.8g -value

C50S

g =




114

2.3 2.2 2.1 2.0 1.9 1.8g -value

C53S

g =


temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT. Protein was

reduced by sodium dithionite.

115

Table 1. EPR properties of wild-type Isf and variants.

Protein g values Iron-sulfur center type

As-purified wild-type 2.06, 1.92, 1.81

2.03, 1.92, 1.86

[4Fe-4S]

Reconstituted wild-type 2.06, 1.92, 1.81

2.03, 1.92, 1.86

[4Fe-4S]

C16X

X = A or S

2.06, 1.92, 1.82

2.04, 1.92, 1.86

[4Fe-4S]

C47A

C47S

2.05, 1.93, 1.89

2.05, 1.94, 1.86, 1.80

[4Fe-4S]

[4Fe-4S]

C50A

C50S

2.01,1.99

2. 07, 2.04,1.92, 1.90,1.82

[3Fe-4S]

[4Fe-4S]

C53A

C53S

2.03,1.91,1.89

2.05, 1.99, 1.93, 1.91, 1.82

[4Fe-4S]

[4Fe-4S]

C59A 2.01,1.99 [3Fe-4S]

C180X

X = A or S

2.04, 1.93, 1.86

With minor contribution from

2.07, 1.93, 1.81

[4Fe-4S]

116

required ligands (16, 17). Thus, conversion of the 3Fe-4S cluster of C50A to a 4Fe-4S

cluster by replacement with serine suggests that serine can substitute for C50 in Isf.

Functional characterization of Isf variants. It has been proposed that electron

flow is from reduced ferredoxin A to the 4Fe-4S cluster of Isf and then to FMN based

only on midpoint potential values (2); thus, the ability of ferredoxin A to reduce FMN in

the stable variants was investigated to test this hypothesis (Table 2). The reduction of

FMN was followed at A476 to avoid interference due to reduction of the iron-sulfur

clusters. The as- purified and reconstituted Isf were reduced at similar rates (Table 2)

consistent with the spectroscopic characterizations indicating that the reincorporated 4Fe-

4S and FMN were functionally similar to as-purified wild-type Isf. The C47, C50, and

C53 variants showed either several-fold lower or higher rates compared to wild type

(table 2), a result which suggests that the 4Fe-4S cluster is required to transfer electrons

from ferredoxin A to FMN. This result is consistent with results suggesting that the

reconstitution of FMN into apo-protein is diminished by substitution of residues in the

motif (C47, C50, C53, and C59) coordinating the 4Fe-4S cluster. Local conformational

changes in the environment of the 4Fe-4S cluster could potentially influence the

reconstitution of FMN if it were adjacent to the 4Fe-4S cluster for electron transfer.

The FMN reduction in C47A and C50S was decreased relative to wild type

demonstrating that the 4Fe-4S clusters in these variants are able to function, albeit less

effectively, with the ligands that replaced C47 and C50. The several-fold higher rates

relative to wild type Isf for variants C53A and C47S is unexplained; however, the results

clearly indicate that the 4Fe-4S clusters in these variants are fully functional suggesting

that the ligands replacing C53 and C47 conserve essential properties of the wild type

4Fe-4S cluster. Midpoint potentials of 3Fe-4S clusters are generally less negative than

4Fe-4S clusters, the Em range for 3Fe-4S is +80 to – 420 mV while the 4Fe-4S center is

+80 to – 700 mV. Thus, it is hypothesized that the lower rate of FMN reduction

exhibited by C50A could possibly be influenced by the redox potential of the 3Fe-4S

cluster in this variant similar to that predicted for the 4Fe-4S cluster in the corrinoid/iron-

117

Table 2. Rates for reduction of FMN in wild type Isf and variants.

Protein cluster type Rate a

As-purified wild type 4Fe-4S 0.52 ± 0.03

Reconstituted wild type 4Fe-4S 0.59 ± 0.08

C16A 4Fe-4S 0.27 ± 0.01

C47A 4Fe-4S 0.14 ± < 0.01

C50A 3Fe-4S 0.14 ± < 0.01

C53A 4Fe-4S 1.53 ± 0.01

C16S 4Fe-4S 0.49 ± < 0.01

C47S 4Fe-4S 1.38 ± 0.26

C50S 4Fe-4S 0.21 ± < 0.01

C53S 4Fe-4S 0.50 ± 0.09

a:Change in absorbance at 476 nm/min/µmole FMN with ferredoxin A as the electron

donor (see Materials and Methods).

118

sulfur protein from C. thermoaceticum (15). Although the results suggest C16 is not

involved in ligation of the 4Fe-4S cluster, reduction of FMN in C16A was impaired

suggesting this residue indirectly influences the transfer of electrons from ferredoxin to

FMN.

DISCUSSION

The data presented here provides biochemical confirmation of a novel and

unusually compact motif (CX2CX2CX4-7C) for ligation of the 4Fe-4S cluster. Sequence

comparisons (Fig. 1) suggest that the spacing between the first and the second cysteines

in this motif is rigid while spacing between the third and the fourth cysteines is somewhat

variable; however, additional site-directed mutagenesis experiments are necessary to test

this hypothesis. This motif differs from those in ferredoxins (typically CX2CX2C and a

distal C) and high potential iron proteins (typically CX2CX16CX13C) where the ligands to

the 4Fe-4S clusters are more dispersed in the primary structure. Other examples of highly

compact motifs ligating a 4Fe-4S cluster are endonuclease III and MutY (CX6CX2CX5C);

however, these clusters are resistant to oxidation and reduction (19, 20) and function

instead to position basic residues for interaction with the phosphate backbone of DNA.

Another compact cysteine motif (CX2CX11CX5C) ligating a redox resistant 4Fe-4S

cluster is present in the LRR protein for which the physiological function is unknown

(18). Still other novel regulatory and enzymatic roles have been described for iron-sulfur

clusters (3, 10). The results reported here are consistent with a role for the 4Fe-4S cluster

in transfer of electrons from the physiological electron donor (ferredoxin A) to FMN;

however, alternate or additional roles cannot be ruled out.

This investigation has also provided insight into iron-sulfur cluster and FMN self-

assembly in Isf in vivo. The present work showed that Isf variants could be refolded in

the presence of arginine to a conformational state such that the variant could be

reconstituted by treating the protein with ferric ion, sulfide, and FMN. It is proposed that

arginine helps to reshuffle molecules trapped in non-productive reactions, which results

in increased refolding efficiency (4). Unfortunately, we were unable to identify

119

conditions to produce an appreciable yield of the reconstituted C59X and C180X (X = A

or S) variants. Refolding in the presence of ferric ion, sulfide, and FMN was performed

for these variants; however, the majority of the protein precipitated during concentration

and buffer exchange. Nevertheless, we were successful in characterizing these variant

spectroscopically (see Results). Replacement of cysteines in the proposed 4Fe-4S binding

motif (C47, C50, C53, and C59) suggests that integrity of the 4Fe-4S cluster is important

for reconstitution of FMN. Changes in these residues resulted in poor incorporation of

FMN relative to the iron-sulfur cluster.

The results presented here show that the biochemical and physiological properties

of the iron-sulfur cluster are remarkably stable to changes in ligation at the same time it is

very sensitive to the ligand environment. All of the serine variants contained a 4Fe-4S

cluster, except C59S and all of the alanine variants contained an iron-sulfur cluster, two

of which were of the 3Fe-4S type. Furthermore, the FMN of all the variants was reduced

by ferredoxin A at a significant rate compared to wild type, a result demonstrating that

the iron-sulfur clusters were competent in transferring electrons from ferredoxin A to

FMN. This resiliency of the 4Fe-4S cluster to changes in the ligation environment may be

a consequence of the unusually compact nature of the motif coordinating the cluster.

Examples of 4Fe-4S clusters bridging between protein subunits have previously

been shown for the nitrogenase Fe-protein and the Fx cluster in photosystem I. The

possibility that the cysteines of the motif which ligate the 4Fe-4S centers are shared

between subunits can not be completely ruled out based on available experimental data.

However, we see no evidence of spin coupling between two clusters, although that effect

is distance dependent.

The N-terminal half of the deduced sequence of Isf contains regions

(Fig. 1, underlined residues) with identity to the flavin-binding domain of flavodoxins

(13). The unusually compact nature of the cysteine motif coordinating the 4Fe-4S cluster

obviates the need for a remote cysteine suggesting the possibility that Isf could have

evolved by insertion of a small ancestral 4Fe-4S protein, containing the compact motif,

120

into the N-terminal half of an ancestral flavodoxin. A search of the databases revealed no

additional sequences with significant identity to residues 23-85 in the M. thermophila Isf

sequence (Fig. 1) that includes the cysteine motif.

In addition to the motif ligating the 4Fe-4S cluster, two other cysteines (C16 and

C180) are present in the Isf sequence (Fig. 1). Although the evidence suggests C16 and

C180 are not involved in coordination of the 4Fe-4S cluster, the C16A variant was

impaired in the ability to catalyze ferredoxin A-dependent reduction of FMN. A direct

role for C16 in electron transfer was ruled out by the observation that C16A is partially,

and C16S fully, competent in electron transfer from ferredoxin A to FMN compared with

wild type Isf. Sequence comparisons (Fig. 1) indicate that a threonine residue in Isf

homologs may replace C16 of the M. thermophila Isf. This finding suggests that a

hydroxyl or sulfhydryl group is important for maintaining a conformation required for

interaction of ferredoxin A with the 4Fe-4S cluster of Isf or intramolecular electron

transfer from the cluster to FMN. The results obtained for C180 suggest this residue is

not involved in iron-sulfur cluster ligation, however, instability of the C180X (X = A or

S) variants suggests that this cysteine is required only for the overall structural integrity

of the protein.

ACKNOWLEDGEMENTS

We would like to thank M.L. Antokine and Dr. J.H. Golbeck at Pennsylvania

State University for performing the EPR spectroscopy. We thank Dr. R.C. Thauer for

suggestion on the arginine refolding method and a special thank you to R.D. Miles for her

critical reading of the manuscript. Work described here was partially supported by the

Department of Energy Basic Energy Sciences Grant No. DE-FG02-95ER20198 (JGF)

and MCB-9723661 (JHG). UL was supported by MOSTE grant from Thailand.

121

REFERENCES

1. Ahn, J. H., Y. P. Lee, and J. S. Rhee. 1997. Investigation of refolding condition

for Pseudomonas fluorescens lipase by response surface methodology. J.

Biotechnol. 54:151-60.

2. Becker, D. F., U. Leartsakulpanich, K. K. Surerus, J. G. Ferry, and S. W.

Ragsdale. 1998. Electrochemical and spectroscopic properties of the iron-sulfur

flavoprotein from Methanosarcina thermophila. J. Biol. Chem. 273:26462-9.

3. Beinert, H., R. H. Holm, and E. Munck. 1997. Iron-sulfur clusters: nature's

modular, multipurpose structures. Science. 277(5326):653-9.

4. Buchner, J., I. Pastan, and U. Brinkmann. 1992. A method for increasing the

yield of properly folded recombinant fusion proteins: single-chain immunotoxins

from renaturation of bacterial inclusion bodies. Anal. Biochem. 205:263-70.

5. Buchner, J., and R. Rudolph. 1991. Renaturation, purification and

characterization of recombinant Fab- fragments produced in Escherichia coli.

Biotechnology (N Y). 9:157-62.

6. Ferry, J. G. 1997. Enzymology of the fermentation of acetate to methane by

Methanosarcina thermophila. Biofactors. 6:25-35.

7. Ferry, J. G. 1992. Methane from acetate. J Bacteriol. 174:5489-95.

8. Golinelli, M. P., L. A. Akin, B. R. Crouse, M. K. Johnson, and J. Meyer.

1996. Cysteine ligand swapping on a deletable loop of the [2Fe-2S] ferredoxin

from Clostridium pasteurianum. Biochemistry. 35:8995-9002.

9. Golinelli, M. P., C. Chatelet, E. C. Duin, M. K. Johnson, and J. Meyer. 1998.

Extensive ligand rearrangements around the [2Fe-2S] cluster of Clostridium

pasteurianum ferredoxin. Biochemistry. 37:10429-37.

10. Johnson, M. K. 1998. Iron-sulfur proteins: new roles for old clusters. Curr. Opin.

Chem. Biol. 2:173-81.

11. Jung, Y. S., I. R. Vassiliev, F. Qiao, F. Yang, D. A. Bryant, and J. H. Golbeck.

1996. Modified ligands to FA and FB in photosystem I. Proposed chemical rescue

of a [4Fe-4S] cluster with an external thiolate in alanine, glycine, and serine

mutants of PsaC. J. Biol. Chem. 271:31135-44.

122

12. Kemper, M. A., H. S. Gao-Sheridan, B. Shen, J. L. Duff, G. J. Tilley, F. A.

Armstrong, and B. K. Burgess. 1998. Delta T 14/Delta D 15 Azotobacter

vinelandii ferredoxin I: creation of a new CysXXCysXXCys motif that ligates a

[4Fe-4S] cluster. Biochemistry. 37:12829-37.



271:24023-8.

14. Maupin-Furlow, J. A., and J. G. Ferry. 1996. Analysis of the CO

dehydrogenase/acetyl-coenzyme A synthase operon of Methanosarcina


15. Menon, S., and S. W. Ragsdale. 1998. Role of the [4Fe-4S] cluster in reductive

activation of the cobalt center of the corrinoid iron-sulfur protein from

Clostridium thermoaceticum during acetate biosynthesis. Biochemistry. 37:5689-

98.

16. Meyer, J., J. Fujinaga, J. Gaillard, and M. Lutz. 1994. Mutated forms of the

[2Fe-2S] ferredoxin from Clostridium pasteurianum with noncysteinyl ligands to

the iron-sulfur cluster. Biochemistry. 33:13642-50.

17. Moulis, J.-M., V. Davasse, M.-P. Golinelli, J. Meyer, and I. Quinkal. 1996.

The coodination sphere of iron-sulfur clusters: lessons from site-directed

mutagenesis experiments. J. Biol. Inorg.Chem. 1:2-14.

18. Peters, J. W., M. H. Stowell, and D. C. Rees. 1996. A leucine-rich repeat

variant with a novel repetitive protein structural motif [letter]. Nat. Struct. Biol.

3:991-4.

19. Porello, S. L., M. J. Cannon, and S. S. David. 1998. A substrate recognition

role for the [4Fe-4S]2+ cluster of the DNA repair glycosylase MutY.


20. Prince, R. C., and M. J. Grossman. 1993. Novel iron-sulfur clusters. Trends.

Biochem. Sci. 18:153-4.

21. Shen, B., D. R. Jollie, T. C. Diller, C. D. Stout, P. J. Stephens, and B. K.

Burgess. 1995. Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I:

123

cysteine ligation of the [4Fe-4S] cluster with protein rearrangement is preferred

over serine ligation. Proc. Natl. Acad. Sci U S A. 92:10064-8.

22. Sticht H, R. P. 1998. The structure of iron-sulfur proteins. Prog. Biophys. Mol.

Biol. 70:95-136.



263:4080-2.

24. Terlesky, K. C., M. J. Nelson, and J. G. Ferry. 1986. Isolation of an enzyme

complex with carbon monoxide dehydrogenase activity containing corrinoid and

nickel from acetate-grown Methanosarcina thermophila. J. Bacteriol. 168:1053-8.

25. Tsumoto, K., K. Shinoki, H. Kondo, M. Uchikawa, T. Juji, and I. Kumagai.

1998. Highly efficient recovery of functional single-chain Fv fragments from

inclusion bodies overexpressed in Escherichia coli by controlled introduction of

oxidizing reagent--application to a human single-chain Fv fragment. J. Immunol.

Methods. 219:119-29.

26. Volbeda, A., M. H. Charon, C. Piras, E. C. Hatchikian, M. Frey, and J. C.

Fontecilla-Camps. 1995. Crystal structure of the nickel-iron hydrogenase from

Desulfovibrio gigas. Nature. 373:580-7.

27. Zhou, Z. H., and M. W. Adams. 1997. Site-directed mutations of the 4Fe-

ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus: role of the

cluster- coordinating aspartate in physiological electron transfer reactions.


28. Antonkine, M. L., C. Falzone, A. Hansen, F. Yang, and J. H.Golbeck.

Chemical rescue of site-modified ligands to the iron-sulfur clusters of PsaC in

Photosystem I. Proceedings of XIth international congress in photosynthesis,

Budapest, 1998, Kluwer academic publishers, (in press).

124

CHAPTER 6

SUMMARY AND FURTURE DIRECTIONS

Advances in understanding the biochemical reactions involved in carbon

transformations during methanogenesis have inspired investigations of how electrons are

transported to generate energy for the cell. The characterization of a novel redox protein

(Isf) in this study provides a better understanding of electron transport in the acetate

fermentation pathway.

Information derived from the sequence of the heterologously produced iron-sulfur

flavoprotein (Isf) from the archaeon Methanosarcina thermophila shows many striking

features distinct from any known iron-sulfur proteins or flavoproteins. The sequence

reveals a novel cysteine motif that has been shown by site-directed mutagenesis and

spectroscopic analyses to accommodate a 4Fe-4S center. An unusual higher stability of

hydroquinone relative to semiquinone prompts an investigation of the environment in the

FMN binding site that results in hydroquinone formation. The three-dimensional

structure of this archaeal Isf is being solved using X-ray crystallography in collaboration

with Dr. C. Bremnane and Prof. D. Rees at Caltech. Since Isf of M. thermophila is the

prototype for this protein family and there is no significant sequence identity with known

proteins, the structure may help to elucidate the environment of redox centers. The

structure may support the proposed functions of these two cofactors by showing if both of

them are able to participate in electron transfer as expected. This will lead to a better

understanding of the mechanism for electron transfer.

The heterologously produced Isf has been studied, but the native Isf from M.

thermophila has not yet been purified. Thus, it is not possible to propose with confidence

that they share the same properties. Attempts to identify Isf from M. thermophila using

Western blots were not successful. This protein may be expressed only at very low

levels and only under certain growth conditions. Northern blot analysis is an alternative

approach to examine the expression and regulation of the isf gene.

125

Although the spectroscopic techniques and site-directed mutagenesis reported

here provide structural information for Isf, the function of Isf remains an issue. The

involvement of Isf in electron transport CO-dependent CoM-S-S-CoB reduction has been

shown, and there is evidence that ferredoxin A is a direct electron donor for Isf. In

contrast, the physiological oxidative partner is not known. Passing M. thermophila cell

extract through an affinity column to which Isf is bound may be used to identify proteins

that interact with Isf and possibly function as its electron acceptor.

Biochemical characterization of Isf-like sequences from other metabolically

diverse microbes may reveal their roles in these microbes. These sequences may provide

an opportunity to study evolutionary convergence of Isf.

126

Curriculum Vista UBOLSREE LEARTSAKULPANICH

Department of Biochemistry and Anaerobic Microbiology Virginia Polytechnic Institute and State University Blacksburg, VA 24061

204 S.Frear Bld. 447 W. Clinton Ave., # 408BMB, Penn State University State CollegeUniversity Park PA 16801PA 16802-4500 [email protected]

PERSONAL

Date of birth: 2/10/71 (Bangkok, Thailand)Place of birth: Bangkok, Thailand

EDUCATION:

Aug 1993 - present:Doctoral candidate, Department of Biochemistry and Anaerobic MicrobiologyVirginia Polytechnic Institute and State University (VPI&SU), Blacksburg, VA.

Aug 1995 - present:Department of Biochemistry and Molecular BiologyPennsylvania State University, University Park, PA.In absentia from Virginia Polytechnic Institute and State University

Apr 1992 – 1993MS. candidate, Department of Biochemistry, Mahidol University,Bangkok, Thailand

Jun 1988 - 1992B.S. Biochemistry, Chulalongkorn University, Bangkok, Thailand

PROFESSIONAL EXPERIENCE

Jun 1994 - Aug 1994:Graduate Teaching Assistant, Department of Biochemistry and AnaerobicMicrobiology. VPI&SU.Laboratory instructor for BAM5104 Advanced Methods of Biochemical Analysis

Apr 1991 – May 1991:Student training at Chareonpokapun group (CP), Samut Prakarn, Thailand

HONORS AND AWARDS:

Royal Thai Scholarship for Ministry of Science, Technology and Energy(MOSTE)

127

National Science and Development Agent scholarship (NSDA)The first rang student of the 1992 MS. candidate class, Mahidol UniversityThe first range student for 1992 MS. entrance examination, Mahidol UnversityBS. with Second class honor degree, Chulalongkorn University

PUBLICATIONS:

Becker D.F., Leartsakulpanich U., Surerus K.K., Ferry J.G., and S.W. Ragsdale.1998. Electrochemical and spectroscopic properties of the iron-sulfurflavoprotein from Methanosarcina thermophila. J. Biol Chem. 273:26462-26469

Leartsakulpanich U, Antokine M.L., Golbeck J.H., and J.G Ferry. A novel [4Fe-4S] iron-sulfur cluster binding motif in the iron-sulfur flavoprotein ofMethanosarcina thermophila (in preparation)

ABSTRACTS AND PRESENTATIONS:

Inorganic Biology Summer Workshop (IBSW 98), Athens, GAJul 25 – Aug 5, 1998Leartsakulpanich U, Antokine M.L., Golbeck J.H., and J.G. Ferry. 1998. Ligandsto the 4Fe-4S center in the iron-sulfur flavoprotein from Methanosarcinathermophila and proposed physiological function

Penn State Sixteenth Summer Symposium in Molecular Biology MicrobialStructural Biology: Novel Enzymes from Diverse MicrobesAug 7-9, 1997Leartsakulpanich U., Becker D.F., Ragsdale S.W., Borup B., Aldrich H.C., andJ.G.Ferry. Characterization of the iron-sulfur flavoprotein (Isf) fromMethanosarcina thermophila.

Date post:	11-Aug-2019
Category:	Documents
Upload:	duongphuc
View:	214 times
Download:	0 times

Ubolsree Leartsakulpanich - vtechworks.lib.vt.edu · Chapters 1 and 2 are intended to serve as an...

Documents