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1

Thesis Submitted for the Degree of

Candidatus Scientiarum

Electron Paramagnetic Resonance Studies of the Mixed Valence Diiron-Oxygen Cluster in the Mouse Ribonucleotide Reductase R2 Protein

by

Åsmund Kjendseth Røhr

Department of Biochemistry

The Faculty of Mathematics and Natural Sciences

University of OsloOslo 2001

Electron Paramagnetic Resonance Studies of the Mixed

Valence Diiron-Oxygen Cluster in the Mouse Ribonucleotide

Reductase R2 Protein

III

AcknowledgmentsThe work presented in this thesis has been carried out at the Department of

Biochemistry, University of Oslo under supervision of Professor K. Kristoffer

Andersson.

Professor K. Kristoffer Andersson has provided valuable supervision and given me

the opportunity to learn about the interesting field of metalloprotein research. He has

also provided financial support to me when visiting conferences. I am very grateful

for all his care during my last years at the University of Oslo.

I will also thank my co-supervisor Dr. Peter Paul Schmidt for all help and

encouragement. He trained me in EPR spectroscopy and the basic lab work.

Dr. Morten Sørlie and Cand. Scient. Kari Røren Strand have read through this thesis. I

want to thank them for correcting my language and for helpful discussions regarding

the scientific presentation of my work.

Dr. Joshua Telser has kindly provided computer software for analysis of EPR spectra,

and valuable advise on how to make it work. Professor Einar Sagstuen helped me to

compile and modify the source code from Dr. Telser. I want to thank them for their

valuable help.

Thanks to far, mor, Torolf, and Kristin for all support and encouragement!

Oslo, 20.05.2001

Åsmund Kjendseth Røhr

V

Table of Contents

1 Abstract____________________________________________________________1

2 Introduction_________________________________________________________32.1 Preface________________________________________________________________3

2.2 Ribonucleotide Reductase________________________________________________42.2.1 General Introduction_________________________________________________________42.2.2 The Diiron-Oxygen Cluster and Tyrosyl Radical in RNR R2__________________________6

2.3 Methane Monooxygenase________________________________________________92.3.1 General Introduction_________________________________________________________92.3.2 The Diiron-Oxygen Cluster in MMOH__________________________________________10

2.4 Uteroferrin___________________________________________________________122.4.1 General Introduction________________________________________________________122.4.2 The Diiron-Oxygen Cluster in Uteroferrin_______________________________________12

2.5 Spectroscopical Properties of Diiron-Oxygen Clusters_______________________142.5.1 General Spectroscopic Properties of Different Oxidation States_______________________142.5.2 Application of EPR to Diiron-Oxygen Clusters___________________________________15

2.6 Aims for the Thesis____________________________________________________17

3 Methods___________________________________________________________193.1 Expression of the Mouse R2 Gene in E.coli_________________________________19

3.2 Purification of Mouse R2 Protein_________________________________________203.2.1 Lysis of Bacteria___________________________________________________________203.2.2 Precipitation of DNA________________________________________________________203.2.2 Precipitation of R2 Protein with Ammonium Sulfate_______________________________213.2.3 Gel Filtration Chromatography________________________________________________213.2.4 Anion Exchange Chromatography______________________________________________223.2.5 Sodium Dodecyl Sulfate Polyacrylamide Electrophoresis___________________________223.2.6 Ultra Filtration_____________________________________________________________23

3.3 Protein Quantification__________________________________________________243.3.1 Quantification Using UV/vis Spectrophotometry__________________________________243.3.2 Bio-Rad Protein Assay_______________________________________________________24

3.4 Buffer Exchange and Ultra Filtration_____________________________________25

3.5 Reconstitution of the Diiron-Oxygen Cluster and the Tyrosyl Radical__________25

3.6 Quantification of Dithionite_____________________________________________26

3.7 Anaerobic Reduction of Phenazine Methosulfate____________________________26

3.8 Reduction of the Diiron-Oxygen Cluster and the Tyrosyl Radical______________27

3.9 Electron Paramagnetic Resonance Spectroscopy____________________________293.9.1 Introduction to EPR Theory___________________________________________________293.9.2 The Electronic Zeeman Effect_________________________________________________303.9.3 Zero Field Splitting_________________________________________________________333.9.4 Exchange Coupling_________________________________________________________343.9.5 The Spin Hamiltonian for a Dinuclear Coupled Metal Cluster________________________353.9.6 Relaxation of a Paramagnetic System___________________________________________353.9.7 EPR Instrument Parameters Used in the Experiments_______________________________373.9.8 EPR Sample Preparation_____________________________________________________373.9.9 Quantification of Spin in an EPR Sample________________________________________37

3.10 Circular Dichroism Spectroscopy________________________________________38

VI

4 Results and Analysis_________________________________________________394.1 Protein Purification____________________________________________________39

4.2 Reconstitution of Mouse RNR-R2________________________________________39

4.3 Redox Studies of Phenazine Methosulfate__________________________________404.3.1 Purpose of the Experiments___________________________________________________404.3.2 The Equilibrium between Different Redox Forms of PMS___________________________41

4.4 Reduction of the Tyrosyl Radical and the Diiron-Oxygen Cluster______________424.4.1 Purpose of the Experiments___________________________________________________424.4.3 Estimation of the Midpoint Potential Em’ of the Diiron-Oxygen Cluster________________44

4.5 Interactions between Alcohols and the Diiron – Oxygen Cluster_______________484.5.1 Purpose of the Experiments___________________________________________________484.5.2 Affinity of Various Primary Alcohols to the Mixed Valence Cluster___________________484.5.3 Estimation of Binding Constants for Methanol and Ethanol with Mouse R2_____________504.5.4 Effect of Isotope Labeled Alcohols_____________________________________________524.5.5 Microwave Powersaturation Behavior of the Novel EPR Signals_____________________52

4.6 Theoretical Studies of the Mouse R2 Mixed Valence Cluster__________________554.6.1 Purpose of the Theoretical Studies_____________________________________________554.6.2 Simulation of Experimental EPR Spectra________________________________________564.6.3 Ligand Field Calculations____________________________________________________604.6.3 Summary of Results From Theoretical Calculations________________________________63

4.7 CD and Light Absorption Studies of the Mouse R2 Diferric Cluster____________644.7.1 Purpose of the Experiments___________________________________________________644.7.2 Reconstituted Mouse R2 in the Presence of Methanol______________________________64

5 Discussion_________________________________________________________675.1 Introduction__________________________________________________________67

5.2 Tyrosyl Radical Content in Reconstituted Mouse R2_________________________67

5.3 Redox Chemistry of PMS and Mouse R2__________________________________67

5.4 Small Alcohols might Bind to the Mouse R2 Mixed Valence Cluster____________69

5.4.1 Relevance of Results and Further Experiments____________________________75

6 Appendix__________________________________________________________776.1 Materials_____________________________________________________________77

6.2 The Culture Medium and Buffers________________________________________79

6.3 Input Parameters for the Program ddpowjea_______________________________82

Terms and Abbreviations_______________________________________________83

References___________________________________________________________85

VII

VIII

1 Abstract

The enzyme ribonucleotide reductase (RNR), important for all life, catalyses the

reduction of ribonucleotides to their corresponding deoxyribonucleotides. The R2

homodimer of the enzyme complex contains an -oxo bridged diiron-oxygen cluster

and a tyrosyl radical that are essential for the enzymatic reaction. Similar diiron-

oxygen clusters exist in the enzymes methane monooxygenase hydroxylase (MMOH)

and uteroferrin. We have studied the mixed valence Fe(II)-Fe(III) oxidation state of

the diiron-oxygen cluster in mouse R2 by electron paramagnetic spectroscopy (EPR).

From the results of introductory redox studies we estimate a midpoint potential (Em’)

to be between 52 mV and 62 mV (versus the SHE) for the reduction of the diiron-

oxygen cluster from the Fe(III)-Fe(III) oxidation state to Fe(II)-Fe(II). These

experiments resulted in a maximum yield of 0.56 mixed valence clusters per R2

dimer.

A novel interaction between alcohols and the mouse R2 mixed valence cluster was

characterized. Results from titration experiments indicate binding constants of Kb,

methanol = 0.24 0.02 M and Kb, ethanol = 0.60 0.03 M. These constants were estimated

from the observed perturbations of experimental EPR spectra induced by methanol

and ethanol, respectively. Our hypothesis that alcohol interact with the mixed valence

cluster is supported by theoretical calculations. These calculations indicate that the

ligand field of the ferrous iron in the mixed valence cluster is modulated by the

interaction of methanol and ethanol.

Altogether, our results suggest that the mixed valence diiron-oxygen cluster in mouse

R2 and MMOH have similar properties. The Em’ value determined for the reduction of

the diiron-oxygen cluster in MMOH (OB3b)1 is close to our estimate for the mouse

R2. Results form theoretical calculations performed at the methanol-mixed valence

cluster in MMOH2 and mouse R2 are comparable. Thus we suggest that mouse R2 is

an adequate starting point when engineering a MMOH like enzyme from R2.

1

2 Introduction

2.1 Preface

In this thesis, all experimental work has been performed using the R2 subunit of

mouse ribonucleotide reductase (RNR). The research has been focused around the

properties of the diiron-oxygen cluster in this protein and not the overall function of

the enzymatically active R1-R2 complex. Thus, it is natural to give a general

overview of several proteins that contain diiron-oxygen clusters similar to the one

found in mouse R2. The proteins to be introduced are listed in Table 2.1 with their

catalytic properties. A detailed presentation of the mixed valence Fe(II)-Fe(III)

oxidation states of the metal clusters in these proteins are given at the end of this

chapter. The discussion of the results presented in this thesis will be related to the

properties of the different diiron-oxygen clusters presented here.

Table 2.1 Diiron-oxygen proteins have different functions a

Enzyme Reaction type Catalytic reaction

Ribonucleotide reductase R2 1e- oxidation

Methane monooxygenase b hydroxylation

Uteroferrin hydrolysis of phosphate ester

a Information obtained from Solomon et al.3 b Methane hydroxylation is catalyzed by the hydroxylase component of the methane monooxygenase complex.

Other proteins such as stearoyl-acyl carrier protein 9 desaturase and the invertebrate

oxygen transporting protein hemerythrin also contain diiron-oxygen clusters

comparable to the one in R2. Solomon et al.3 have recently published an excellent

review regarding the geometric and electronic structure of the diiron-oxygen clusters

in these proteins.

3

Chapter 2 Introduction

2.2 Ribonucleotide Reductase

2.2.1 General Introduction

Class I ribonucleotide reductases are found in eukaryotic and prokaryotic organisms,

and some viruses that have nucleic acids coding for viral RNR. Other classes of RNR

also exist. These are described in the literature4,5 and will not be discussed here. The

holoenzyme of RNR catalyses the reduction of ribonucleotides to

deoxyribonucleotides (Scheme 2.1).

The catalytic active form of class I RNR is composed of two homodimers named R1

(2 x ~85 kD) and R2 (2 x ~43 kD). From X-ray crystallography studies, it has been

determined that the R1 monomer contains / barrel, -helical and + domains. The

active and regulatory sites are also located in this subunit.6,7 The R2 monomer has

mainly an -helical bundle tertiary structure. It can bind two ferrous iron atoms that

can react with dioxygen to form a diferric -oxo bridged diiron-oxygen cluster and a

tyrosyl radical. This radical is essential for enzymatic activity.8-10 A proposed structure

of the E.coli R1-R2 holoenzyme is visualized in Figure 2.1.5

A radical based reaction mechanism has been proposed for the ribonucleotide

reduction.12 According to this suggestion, a radical should be either on the 3’ or

2’carbon in the substrate, or on the catalytic active cysteines in the R1 subunit during

catalysis. This hypothesis has been supported by studies where cytidine analogs were

used to trap a radical at the R1 active site.13-15

An hydrogen atom or electron/H+ transfer16 pathway from the tyrosyl radical located

in the R2 subunit to the active site at R1 has been proposed in order to explain how

the radical can be translocated the distance of ~35 Å separating the tyrosyl radical and

the active site during catalysis.5

BASEO

O

OHOH

P

O

OH

OH

OH

O

O

P BASEO

O

OH

P

O

OH

OH

OH

O

O

P

Scheme 2.1

4


The hydrogen bonded amino acid sidechains suggested to be involved in the radical

transfer have been altered by site directed mutagenesis in order to verify that

hypothesis. Results from experiments performed with both E.coli and mouse R2

confirmed that the proposed amino acids were essential for catalysis.5,17 The

importance of an intact hydrogen bonded pathway in R2 has also been demonstrated

for the reaction between the ferrous iron atoms and a dioxygen molecule in the R2

subunit.17,18 It should also be mentioned that the proposed radical transfer pathway

includes one of the iron atoms in the diiron-oxygen cluster. Thus, a mixed valence

Fe(II)-Fe(III) oxidation state of the diiron-oxygen cluster might has a physiological

relevance.

Detailed information regarding the radical and the diiron-oxygen clusters in R2

proteins will be of great value for medical research when inhibitors specific for the

Figure 2.1 A proposed model of the E.coli R1-R2 holoenzyme. The model is based on an illustration published by B.-M. Sjöberg.{4} -helixes are colored yellow and blue in the R1 and R2 homodimers, respectively. -sheets are colored white in both homodimers. Red spacefill representations indicate important sites. The model was created from the PDB files 1RIB and 2R1R with the program MolMol.11

Active site

Diiron-oxygen cluster and Tyr

Regulatory site

R1

R2

5


radical and the metal cluster are to be constructed. Inhibition of RNR results in

depletion of the deoxyribonucleotide pools in the cells, thus arresting cell division.

Three common approaches for RNR inhibition are; inhibitors that binds to the active

sites at the R1 homodimer,19 peptides that resembles the R2 interface to the R1

homodimer and prohibit the indispensable R1 – R2 interaction,20 and third; inhibitors

that quench the radical and the metal center in the R2 homodimer that is essential for

catalysis.21,22 An example of such an inhibitor is hydroxyurea that eliminates both the

redox active tyrosyl radical and the diiron-oxygen cluster in mammalian R2.21,22

When the E.coli RNR enzyme is used as a model for the mouse RNR in this thesis,

the amino acid numbering is given as e.g. Y122(177), which point towards tyrosine

122 in E.coli and tyrosine 177 in mouse.

2.2.2 The Diiron-Oxygen Cluster and Tyrosyl Radical in RNR R2

In the active holo-RNR enzyme complex, the function of the R2 subunit is to supply a

radical to the active site at R1 during catalysis. R2 also provide a protected

environment for the radical when no substrate is bound at R1.

The most extensively studied class I RNR R2 protein is the one from E.coli. An

approach for the in vitro ferrous iron (Fe2+) dioxygen reaction in E.coli R2 was

published by Brown et al.23 in 1969. Results from early Mössbauer and light

absorption experiments showed that a diferric (Fe3+-Fe3+) antiferromagnetically

coupled cluster was formed in addition to the tyrosyl radical.24 Since then, numerous

studies have been focused at the iron-oxygen reaction, and its intermediates, that ends

with the generation of a tyrosyl radical. A detailed illustration of the proposed steps in

the in vitro reconstitution reaction is illustrated in Scheme 2.2 mainly adopted from

Andersson et al.25

6


It is necessary to explain the individual steps in

Scheme 2.2, and E.coli R2 amino acid

numbering is used throughout. At the starting

point D84, E238, E204, H241, E115, and H118

bind two ferrous iron atoms. The low

coordination numbers of the irons allow

binding of dioxygen to one of the irons.26

Crystallographic studies have shown that azide

(N3-) binds in a specific fashion to the reduced

diiron-oxygen cluster, and the O2 bound

complex have been modeled from these

results.25 During dioxygen cleavage, a peroxo

species have been observed in the mutant D84E

R2 protein.27 This intermediate, called

compound P, is proposed in the native R2 iron-

oxygen reaction as well. In the next step, the

peroxo bridge is reduced to a water molecule

and a -oxo bridge by two electrons. One

electron is provided from one of the ferric irons

of the cluster and the second electron is donated

from an external source that usually is Fe2+ in

the in vitro reconstitution reaction. This results

in a -oxo bridged Fe(IV)-Fe(III) mixed

valence cluster with a S = ½ ground state that

has been observed by electron paramagnetic

resonance (EPR) and Mössbauer

spectroscopy.28,29 This high-valence diiron-

oxygen cluster, termed compound X, is not

stable, and is assumed to abstract an electron

from Y122(177) to form the unprotonated

tyrosyl radical.3

Scheme 2.2

Diferrous

Peroxo

O2 bound

Compound X

Diferric + Tyr

7


When the three dimensional structure

of E.coli R2 was published in 1990 by

Nordlund et al.,8 it became clear that

the radical containing Y122(177) was

located ~5 Å away from the diiron-

oxygen cluster. The crystal structure of

the E.coli R2 monomer with a diferric

cluster is shown in Figure 2.2. As in

several other diiron-oxygen proteins,

carboxyls and histidines inside a four-

helix bundle ligate the metal cluster. In

its diferric form, both irons are six-

coordinated (Figure 2.3). It is not

agreed upon whether the reduced form

have two four-coordinated irons26 or

one four and one five-coordinated

iron.30 From crystallographic studies it

have been shown that only the

positions of the amino acid sidechains

involved in coordination of the diiron-

oxygen cluster are changed for the apo,

the diferrous, and the diferric forms of

E.coli R2.31 In 1996, the crystal

structure of mouse R2 was solved by

Kauppi et al.32 Unfortunately only one

iron was observed to be bound inside

the protein, and the crystal was grown

at the unphysiological pH 4.7. Hence,

there is no atom coordinates for the mouse R2 diiron-oxygen cluster available.

However, it was observed that the pocket where the diiron-oxygen cluster is located is

solvent exposed. This is not the case for E.coli R2 where the diiron-oxygen cluster is

inaccessible. The observations are supported by the fact that the mouse R2 tyrosyl

radical is more sensitive to scavengers than the E.coli R2 radical.33,34

Figure 2.2 Structure of E.coli R2 (monomer) containing a diferric cluster. Y122(177) is purple while iron ligating sidechains are red. The illustration was created from the PDB file 1RIB with the program MolMol.11

Figure 2.3 The diferric -oxo bridged diiron-oxygen cluster from E.coli R2. Each iron is coordinated by one His and one water molecule. All other ligands are carboxyls. The illustration was created from the PDB file 1RIB with the program MolMol.11

8


Other experiments have also confirmed differences between mouse and E.coli R2.

First, results from high field EPR experiments have suggested the presence of a

hydrogen bond to the unprotonated Y177 sidechain in mouse R2 that is not present in

E.coli R2.35 Second, the two irons in mouse R2 obviously have different redox

potentials because a mixed valence Fe(II)-Fe(III) state can be obtained by chemical

reduction of the Fe(III)-Fe(III) state.36,37 In native E.coli R2, the two irons are reduced

simultaneously at a lower redox potential than suggested for the diiron-oxygen cluster

in mouse R2.38 These differences suggest that even though the iron coordinating

amino acids are highly conserved, slight variations of the structures give the diiron-

oxygen clusters in mouse and E.coli R2 individual chemical and physical properties.

2.3 Methane Monooxygenase


Anaerobic methanogenic bacteria living in oceans, lakes, and wet soils produce large

amounts of methane. In the aerobic environment above the methanogenic bacteria

habitat, methanotropic bacteria metabolize methane into methanol. Methanol is the

only carbon source for these methanotropic bacteria, and it is converted into biomass

and CO2.39

In this chapter, only the soluble forms of the enzyme methane monooxygenase

(MMO) from Methylococcus capsulatus (Bath) and Methylosinus trichosporium

(OB3b) will be discussed. The reaction catalyzed by methane monooxygenase is

shown in Scheme 2.3.

Scheme 2.3

In brief, three protein components are required for catalytic activity:39

1. Hydroxylase (MMOH), a hetrotrimer (~245 kD) with a quaternary structure

()2 and a diiron-oxygen cluster active site located in subunit .

9


2. Reductase (MMOR), a monomeric protein (~38 kD) with a Fe2S2 cluster that

transfer electrons from NAD(P)H to the MMOH diiron-oxygen cluster.

3. Component B, a regulatory monomeric protein (~15 kD) able of inducing

conformational changes in the hydroxylase.

2.3.2 The Diiron-Oxygen Cluster in MMOH

In 1986 it was suggested that MMOH

(Bath) probably contained a diiron-

oxygen cluster.40 Mössbauer studies of

MMOH (OB3b) suggested that the

resting state of the enzyme had a

hydroxide-bridged diferric cluster.2

Pulsed EPR studies also revealed that

the one electron reduced mixed

valence Fe(II)-Fe(III) form also had a

hydroxide bridge.41 The ligation of the

mixed valence clusters in both OB3b42

and Bath43 MMOH was further

investigated by spectroscopy, and

histidine coordination of the diiron-

oxygen was suggested for both types

of MMOH. The first crystal structures

of Bath44 and OB3b45 MMOH were

determined in 1993 and 1997,

respectively. Both proteins had high

structural similarity and a diiron-

oxygen cluster in the -subunit. It was

also concluded that either two

hydroxides, or one hydroxide and one

water molecule, bridged the two

irons.45

Figure 2.4 Structure of the -subunit of MMOH. The diferric cluster and its protein ligands are orange and red, respectively. The illustration was created from the PDB file 1MTY with the program MolMol.11

Figure 2.5 The diferric cluster in MMOH is very similar to the one in E.coli R2. The illustration was created from the PDB file 1MTY with the program MolMol.11

10


The structure of the MMOH (Bath) subunit determined at 1.7 Å resolution

published in 1997 by Rosenzweig et al.46 is shown in Figure 2.4. The diferric clusters

of MMOH (illustrated in Figure 2.5) and E.coli R2 (Figure 2.3) show a high degree of

structural similarity. Instead of a -oxo bridge as observed in E.coli R2, a water and a

hydroxide have bridging positions in the diferric MMOH. In E.coli R2, one of the

carboxyl ligands is an aspartate (D84). This amino acid is a glutamate in MMOH.

Recently, new structures of MMOH (Bath) have been determined with the diiron-

oxygen cluster in oxidized Fe(III)-Fe(III), mixed valence Fe(II)-Fe(III) and reduced

Fe(II)-Fe(II) forms, revealing that the coordination numbers of the irons depend on

the oxidation state.47

The catalytic cycle of MMO results in

hydroxylation of the substrate. As in class I

RNR-R2, a high valent diiron-oxygen cluster

carry out the substrate oxidation.

Characterized key intermediates in the

MMOH iron-oxygen reaction are illustrated

in Scheme 2.3, which is taken from Shu et

al.48 Several similarities are found for the

diferrous R2 and MMOH dioxygen

activation process. Intermediate P, a

peroxide species characterized in MMOH,49

resembles compound P in R2. While

compound X in R2 has a Fe(III)-Fe(IV) oxidation state, the relative in MMOH,

termed intermediate Q, has been determined to be a Fe(IV)-Fe(IV) species.48

Cryoreduction of intermediate Q by -radiation results in a Fe(III)-Fe(IV) iron-

oxygen cluster that resembles compound X.50 By considering these data, it is apparent

that in addition to very similar iron coordination environments in R2 and MMOH, and

that the two proteins activate dioxygen in an analogous fashion.

It has been observed that the product release from diferric MMOH is the rate limiting

step during catalysis.51 The alcoholdiferric cluster complex termed compound T (T

for terminal adduct) has been studied by X-ray crystallography, and it was shown that

methanol was bound to both irons whereas ethanol was coordinated to a single iron.52

Scheme 2.3

11


Radiolytic reduction of the diferric cluster by -irradiation at 77 K in presence and

absence of methanol followed by EPR studies also indicated that methanol interacted

with the diferric cluster.53 Electron nuclear double resonance (ENDOR) analysis of the

mixed valence Fe(II)-Fe(III) cluster in the presence of methanol, demonstrated that

the alcohol was coordinated to the ferrous iron.54 A simple ligand field model was

used to explain the methanol-ferrous iron interaction on the basis of a combined

Mössbauer/ EPR study of the MMOH (OB3b) mixed valence cluster.2 This ligand

field model has also been used to explain a novel interaction between alcohols and the

mouse R2 mixed valence cluster in this thesis.

2.4 Uteroferrin


Uteroferrin (~35 kD) is a purple acid phosphatase that can be isolated from pig

allantoic fluid. Purple acid phosphatases are a highly homologous group of enzymes

that catalyze nonspecific cleavage of phosphoester bonds as illustrated in Scheme 2.4,

and are found in plants, fungi, and animals.55

Scheme 2.4

The mammalian type has been suggested to be involved in osteoporosis56 and

immunologic response to pathogens.57

2.4.2 The Diiron-Oxygen Cluster in Uteroferrin

12


In 1983, the first evidence for a

dinuclear iron-cluster in uteroferrin

was obtained from EPR

spectroscopy.58 Subsequent

Mössbauer spectroscopy studies

verified the presence of an

antiferromagnetically coupled diiron

cluster.59 In contrast to R2 and

MMOH, the active form of uteroferrin

contain a mixed valence Fe(II)-Fe(III)

diiron-oxygen cluster, and dioxygen

activation is not a part of the catalytic mechanism.55 It was suggested that the

antiferromagnetic coupling was weak from results obtained from magnetic circular

dichroism (MCD)60 and Mössbauer61 experiments. These indicated a bridging

hydroxide in the active Fe(II)-Fe(III) cluster.

A plausible reaction mechanism for the hydrolysis of phosphate suggested by Yang et

al.60 is depicted in Scheme 2.5. The crystal structure of uteroferrin was solved in 1999

and is illustrated in Figure 2.6.62 It shows a different tertiary organization of the

polypeptide chain compared to R2 and MMOH.

While R2 and MMOH have mainly helical structure, uteroferrin has a -sandwich

structure that is common to purple acid phosphatases. The diiron-oxygen clusters in

R2 and MMOH are buried in four helix bundles while the active site in uteroferrin is

located at the surface of the protein. The diiron-oxygen cluster in uteroferrin is

illustrated in Figure 2.7, and the ligand arrangement is different from the one in R2.

Scheme 2.5

Figure 2.6 The crystal structure of uteroferrin determined at 1.55 Å resolution. The -sandwich structure distinguishes this protein from the mainly helical R2 and MMOH proteins. The illustration was created from the PDB file 1UTE with the program MolMol.11

Figure 2.7 The diferric oxygen-bridged cluster in uteroferrin have quit different ligation than the R2 and MMOH cluster. The illustration was created from the PDB file 1UTE with the program MolMol.1113


An additional histidine coordinates to the uteroferrin diiron-oxygen cluster, and a

tyrosine and an asparagine have replaced two carboxyl ligands. The purple color of

concentrated enzyme solutions originates from charge transfer transitions between the

ferric iron and its tyrosine ligand.55

2.5 Spectroscopical Properties of Diiron-Oxygen Clusters

2.5.1 General Spectroscopic Properties of Different Oxidation States

Characterized binuclear iron clusters in proteins mainly have either oxygen or sulfur

based bridges. The proteins introduced in the previous sections contain binuclear iron

clusters that have oxygen, hydroxide, water, and carboxyl bridges. The properties of

these clusters are generally not well understood compared to Fe2S2 clusters.

The irons in the diiron-oxygen clusters are generally high spin. This means that the

electrons are distributed in the three non-bonding and the two antibonding d-orbitals.

This results in S = 5/2 for high spin Fe(III) and S = 2 for high spin Fe(II). When two

such irons have one or more bridging ligand(s), the spins are either ferromagnetically

or antiferromagnetically coupled. Such couplings implicate that the two spin vectors

of each iron are added or subtracted, and that the total spin is the resultant vector (see

Chapter 3.9.4 for a detailed explanation). Possible spin combinations in a diiron

cluster are listed in Table 2.2.

Table 2.2 Spin states in coupled high spin diiron clusters

Oxidation state Individual spins Stotal (antiferromagnetic)a Stotal (ferromagnetic)a

Fe(III)-Fe(III) 5/2……5/2 0 5

Fe(II)-Fe(III) 2…...…5/2 ½ 9/2

Fe(II)-Fe(II) 2…….…2 0 4a Only ground states are given here.

For mouse R2,36,37,63 MMOH,40,64 and uteroferrin,58,65 the diiron-oxygen clusters in

Fe(III)-Fe(III) and Fe(II)-Fe(III) oxidation states are antiferromagnetically coupled.

The Fe(III)-Fe(III) cluster of E.coli R2 is also antiferromagnetically coupled.63

14


However, the Fe(II)-Fe(III) form of the E.coli R2 cluster obtained by radiolytic

reduction by -irradiation at 77 K can be ferromagnetically coupled.66

The magnitude of the magnetic coupling between the irons is given by the exchange

constant J (see Chapter 3.9.4). Antiferromagnetic and ferromagnetic coupling

constants have values J < 0 and J > 0, respectively. Coupling constants for diiron-

oxygen clusters in different oxidation states are given in Table 2.3. A value of J larger

than approximately -35 cm-1 and lower than about -5 cm-1 is considered as a signature

of a protonated oxo-bridge.67

Table 2.3 Exchange coupling constants for diiron-oxygen clusters in proteins

Protein J (cm-1)

Fe(III)-Fe(III) Fe(II)-Fe(III) Fe(II)-Fe(II)

Mouse R237,63 -77 -30 < J < -10a J > 0b

E.coli R230,63,66 -92 6 ~ -0.5

MMOH2,68 -7.5 -30 ~ 0.5

Uteroferrin58,60,61,65 -300 < J < -80 -16 < J < -5 -a J has not been properly determined. This interval is assumed on the basis of the work presented in this

thesis. b Kari Røren Strand, personal communication.

The values presented in Table 2.3, and other parameters describing the electronic

structures of these diiron-oxygen clusters, have been estimated by spectroscopical

analysis. Mössbauer (57Fe -radiation absorption) and magnetic circular dichroism

(MCD) are excellent techniques for studying diiron-oxygen clusters in the redox-

states shown in Table 2.2. Electron paramagnetic resonance (EPR is a suitable

technique when studying half-integer spin systems. Complimentary information

regarding the electronic structure of a spin system can be obtained by using the three

techniques mentioned above.

2.5.2 Application of EPR to Diiron-Oxygen Clusters

The diiron-oxygen clusters in R2, MMOH and uteroferrin all have one or more

paramagnetic EPR active oxidation states. Information is especially obtained from the

15


mixed valence Fe(II)-Fe(III) oxidation

states, and EPR spectra recorded for

these species are briefly discussed

below.

Low temperature EPR spectra

recorded for the mouse R2 are

illustrated in Figure 2.8. Only the

narrow S = ½ tyrosyl radical signal,

which is expanded in Figure 2.8, is

detected for the Fe(III)-Fe(III) active

form (spectrum A). When reduced, the

tyrosyl radical signal at geff = 2

disappears, and two new signals arise;

1) the mixed valence Fe(II)-Fe(III)

form that has a S = ½ ground state, is

identified by the rhombic EPR signal

with effective g-values 1.92, 1.73 and

1.60. 2) an integer S = 4 signal

originating from ferrous iron is

observed at geff = 12.5.

In the proceeding chapters, the mixed

valence EPR spectra obtained with the

mouse R2 will be compared to spectra

that previously have been obtained

with MMOH and uteroferrin. Thus,

these spectra have been depicted in a

shared illustration to make

comparisons easier. In Figure 2.9, the

effective g-values for the various

mixed valence species are; MMOH

(Bath)42 (1.94, 1.86, 1.76), mouse R2

(1.92, 1.73, 1.60), and uteroferrin58

1000 2000 3000 4000

2.004

1.60

1.73

1.92

gobs= 12.5

EPR

FIR

ST D

ER

IVA

TIV

E

Field (G)

A

B

Figure 2.8 EPR spectra of mouse R2 in different oxidation states. Spectrum A; active R2 where only the tyrosyl radical is detected. Spectrum B; reduced forms of mouse R2. The mixed valence form and the diferrous form have g- values 12.5 and (1.92, 1.73, 1.60), respectively. A narrow signal at geff = 2 in spectrum B originates from the electron mediator that was used to reduce the active form. Conditions: (A); 15 K, 100W. (B); 4 K, 20 mW. Microwave frequency 9.6 GHz used for both spectra.

Figure 2.9 Mixed valence Fe(II)-Fe(III) X-band EPR spectra of A; MMOH, B; mouse R2 and C; uteroferrin. All spectra have been recorded at low temperature. See references in text.

A

C

B

16


(1.93, 1,74, 1.59). The large variations in the g-values are related to the structure of

the clusters. In brief, a combination of the exchange coupling occurring via the Fe(II)-

Fe(III) bridge and the ligand field induced by coordinating atoms strongly affects the

broadness of the signal and the positions of the peaks. The theory concerning EPR and

ligand field are described and discussed in Chapter 3.9 and 4.6, respectively.

2.6 Aims for the Thesis

Conduct introductory studies of the redox properties of the diiron-oxygen cluster

in mouse R2. The main purposes of these studies was to:

1. Achieve a high yield of the mixed valence oxidation state of the diiron-oxygen

cluster.

2. Estimate the midpoint potential for the reduction of the Fe(III)-Fe(III) cluster

to the Fe(II)-Fe(II) oxidation state.

Characterize the nature of a novel interaction between alcohol and the mouse R2

mixed valence cluster.

17

3 Methods

All materials utilized are listed in Chapter 6.1. The culture medium and buffer recipes

are described in Chapter 6.2. Abbreviations for these solutions are used throughout

the text.

3.1 Expression of the Mouse R2 Gene in E.coli

The E.coli bacteria strain BL21 (DE3) expressing the recombinant mouse RNR-R2

gene69 was kindly provided by Lars Thelander, University of Umeå.

Principle:

The mouse R2 gene is expressed in a T7 RNA polymerase expression system being

activated by Isopropyl--D-Thiogalactopyranoside (IPTG).70,71

Procedure:

The following procedure have been described by Mann et al..69 As a rule the bacteria

were grown in LB culture medium containing 50 g/mL carbenicillin (CARB) and 30

g/mL chloramphenicol (CAP).1. Incubate E.coli bacteria that have been stored in LB culture medium containing 20 % glycerol at -

70C, at a petri dish containing LB culture medium, agar, CARB and CAP at 37C for about 24

hours.

2. Select one colony for overnight incubation in 150 mL LB culture medium added CARB and CAP,

at 37C in a shaker at 230 rpm.

3. Distribute the overnight culture equally in five 5 L erlenmeyer flasks containing 1.5 L LB culture

medium added CARB and CAP.

4. Mount the five flasks in a shaker and incubate at 37C and 200 rpm until the optical density reach a

value of 0.5-0.6.

5. Add IPTG from a freshly prepared stock solution that has been filtered with a Millex-GP 0.22 m

unit to a final concentration of 0.5 mM in each flask.

6. Harvest the bacteria after 4 hours of further incubation by centrifugation at 5000 rpm (JA10), 4C

for 10 minutes.

7. Suspend the bacteria paste in buffer A (2.5 mL buffer A/ gram bacteria paste), distribute the

suspension in 50 mL centrifuge tubes and freeze in liquid nitrogen.

8. Store frozen bacteria at -74C.

19

3.2 Purification of Mouse R2 Protein

All manipulations, mainly carried out as described by Mann et al.,69 were performed

in a cold room (4C) or in a refrigerated centrifuge to avoid protein denaturation.

3.2.1 Lysis of Bacteria

Principle:

Lysozyme, constitutivelly produced in the bacteria, was able to leak out when thawing

the bacteria. Thus the peptidoglycan layer protecting the bacteria were broken down.

Procedure:

1. Thaw bacteria in a water bath at 5-10C.

2. Centrifuge at 20 000 rpm (JA25.50), 4C for 1.5 hours.

3. Collect the supernatant (and measure the volume) for further purification of R2 protein.

3.2.2 Precipitation of DNA

Principle:

Streptomycin sulfate precipitate DNA.

Procedure:

1. Prepare a 10% (w/v) streptomycin sulfate solution (volume = ¼ of supernatant).

2. Adjust the pH to 7.0 by adding ammonia (NH3 (aq)).

3. Add the streptomycin solution drop by drop over 10 minutes to the supernatant from 3.2.1 while

stirring carefully.

4. Stir carefully for 15 minutes.

5. Centrifuge at 15 000 rpm (JA25.50), 4C for 20 minutes.

6. Collect the supernatant (and measure the volume) for further purification.

20

Chapter 3 Methods

3.2.2 Precipitation of R2 Protein with Ammonium Sulfate

Principle:

Proteins have polar surfaces and interact with water mainly through hydrogen bonds

and other dipolar interactions. Water molecules are arranged in a specific manner

around the solvated proteins, and a chaotropic salt as (NH4)2SO4 (ammonium sulfate)

disrupts the matrix formed by the water molecules. This is because chaotropic salts

attract water for their own hydrating shells, and above a certain salt concentration

there is not sufficient amounts of water left to hydrate the proteins. The most

energetically favorable interactions are then found between the proteins themselves,

thus large aggregates of native proteins precipitate. This “salting out” effect is

described by A.A. Green.72

Procedure:

1. Add 0.243 g (NH4)2SO4/ mL supernatant (about 40% saturation) during a period of 15 minutes

while stirring carefully.

2. Stir carefully for 30 minutes.

3. Centrifuge at 10 000 rpm (JA14), 4C for 50 minutes.

4. Dissolve precipitate in 2-3 mL buffer A.

5. Add 20 L 10 mM phenylmethylsulfonyl fluoride (PMSF) dissolved in ethanol and mix carefully.

3.2.3 Gel Filtration Chromatography

Principle:

Ion exchange chromatography was used as one of the purification steps. When the ion

strength in the protein solution is to high, the protein will not bind to the column

material. Therefore the salt has to be removed by applying the protein solution at a

G25 column.

Gel filtration chromatography separates molecules by their size.73 The column

material (G25) is composed of small spherical particles with pores, and large

molecules such as proteins are not able to penetrate the particles while small

molecules like salts enter the pores. This results in a longer traveling distance for

small molecules than for large molecules. Large molecules such as proteins will be

eluted in the solvent used to equilibrate the column.

21

Chapter 3 Methods

Procedure:

1. Equilibrate a 30 mL G25 column with 150 mL buffer A.

2. Apply the dissolved (NH4)2SO4 precipitate (~3.5 mL).

3. Elute with buffer A and collect fractions with A280 nm > 0.5.

4. Wash the G25 column with 300 mL buffer A.

3.2.4 Anion Exchange Chromatography

Principle:

Solvent exposed protein surfaces are highly populated by polar and charged amino

acid sidechains. Negatively charged sidechains will bind to a positively charged

column material at low ionic strength.73 Variations in the populations of negatively

charged sidechains at protein surfaces among proteins result in selective affinity

between the proteins in the solution and the column material.

At high ionic strengths, counterions of the buffered solution will replace the proteins

that are subsequently eluted.

Procedure:

1. Degas 7.0 g of the weak anion exchanger DE52 (dietylaminoetyl) dissolved in stock solution A for

30 minutes.

2. Transfer the degassed column material to a 10 mL column and equilibrate with 200 mL degassed

buffer B.

3. Apply fractions containing desalted protein collected from the G25 column.

4. Wash with degassed buffer B (10-15 mL) until A280 nm< 0.1.

5. Elute with buffer C and collect 5 mL fractions until A280 nm< 0.4.

3.2.5 Sodium Dodecyl Sulfate Polyacrylamide Electrophoresis

All fractions collected from the anion-exchange column with A280> 0.4 was analyzed

by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) with respect

to the purity of R2. The Pharmacia PhastSystem and was used for this purpose.

Principle:

SDS-PAGE separates denatured macromolecules by their molecular mass.74,75

22

Chapter 3 Methods

Procedure:

1. Mix 14 L protein solution with 8 L Phast loadmix in 1.5 mL eppendorf tubes.

2. Seal the eppendorf tubes and boil for 10 minutes. Spin at 12000 rpm in micro centrifuge.

3. Prepare the PhastSystem instrument with Phast gel 8-25% and PhastGel buffer strips and set the

temperature to 16C.

4. Use the application comb to position 1 L of each prepared sample above the gel in the instrument.

5. Start the PhastSystem instrument and prepare the development unit of the instrument for staining

(Phast staining solution), destaining (Phast destaining solution), and preservation (Phast

preservation solution) of the gel.

6. Develop the gel and collect those fractions containing pure mouse R2.

3.2.6 Ultra Filtration

Fractions collected in step 3.2.5 had a concentration of about 1 mg/mL. Further

experiments required protein concentrations of about 50 mg/mL, thus a concentration

of the protein solution was necessary.

Principle:

The protein is concentrated using a collodion bag with molecular weight cutoff

(MWCO) 12000 D attached to a vacuum pump extracting the solvent, salt, and buffer

molecules through the semi-permeable membrane leaving the protein inside the bag.

Procedure:

1. Equilibrate the collodion bag in Milli Q filtered and ion-exchanged H2O (mqH2O) for 1 hour.

2. Mount the vacuum dialysis system in the cold room and start the system with only buffer A inside

the collodion bag.

3. Remove excess of buffer A after 5 minutes of dialysis and apply the fractions that passed the SDS-

PAGE control.

4. Let the system run overnight, dissolve concentrated protein in 200 L of buffer D, and freeze in

liquid nitrogen. Store at -74C.

23

Chapter 3 Methods

3.3 Protein Quantification

3.3.1 Quantification Using UV/vis Spectrophotometry

The extinction coefficient (280 -310 nm = 124000 M-1cm-1) for the apo mouse R2 dimer at

280 nm was determined in a spectrophotometric experiment from the A280 nm – A310 nm

value and the amino acid composition of mouse R2 in 1991.69 This obtained value was

used to quantify the R2 concentration.

Principle:

Aromatic amino acid sidechains, especially tyrosine and tryptophan, absorb UV-

radiation at 280 nm. This is due to excitation of (bonding) orbital electrons to *

(antibonding) orbitals.

Procedure:

1. Record a baseline of buffer A at the spectrophotometer.

2. Dilute 5 L concentrated apo mouse R2 to 500 L with buffer A.

3. Measure A280 nm and A310 nm and calculate the protein concentration in molar.

3.3.2 Bio-Rad Protein Assay

The Bio-Rad protein assay (Bradford assay76) was occasionally used as a second

method for determining protein concentrations.

Principle:

Coomassie Brilliant Blue G-250 is stabilized in its anionic form when interacting with

basic and aromatic amino acid sidechains in proteins. The anionic form has an Amax at

595 nm in a spectrophotometric experiment while other forms of the dye that do not

bind to proteins absorb light at other wavelengths.

Procedure:

The procedure for the Bio-Rad protein assay that was obtained from Bio-Rad is well

described in the accompanying manual.

24

Chapter 3 Methods

3.4 Buffer Exchange and Ultra Filtration

Buffer exchange for small sample volumes were carried out by using 1 mL spin

columns.

For larger sample volumes, NAP-5 or PD-10 (G25) columns (from Pharmacia) were

used for buffer exchange. If necessary, the protein concentration was increased by

ultra filtration (Centricon, MWCO 50.000 D). The procedures were described in detail

in the datasheets accompanying the different kits.

3.5 Reconstitution of the Diiron-Oxygen Cluster and the Tyrosyl

Radical

Several procedures for the reconstitution of the diiron-oxygen cluster and the tyrosyl

radical in E.coli and mouse R2 have been described in the literature. A novel

approach, based on a method described for E.coli R229,77,78 was applied to the mouse

R2 protein.

Principle:

The apo mouse R2 protein reacts with two ferrous atoms and dioxygen as described in

the introduction (see Chapter 2.2.2). Oxidation of Fe2+ at low pH is kinetically

forbidden. This allows addition of an acidic aerobic Fe2+ solution to the protein

solution without unnecessary loss of Fe2+.

Procedure:

1. Exchange protein solvent to buffer D as described in (Chapter 3.4) and quantify the concentration

of R2 protein in the solution (Chapter 3.3.1and 3.3.2).

2. Prepare a Fe2+ solution with [Fe2+] = 150 [R2] by dissolving ammonium iron(II) sulfate

hexahydrate in mqH2O with pH adjusted to ~2.3 by concentrated nitric acid (HNO3).

3. Add the Fe2+ solution to the protein solution until a final [Fe2+] seven times higher than [R2] is

obtained and mix carefully. After a few seconds, the protein solution will turn green indicating a

successful reconstitution of the tyrosyl radical and the iron-oxygen cluster.

4. Blow O2(g) over the surface of the protein solution for 5 minutes to ensure that there is a sufficient

amount of O2 present in the solution for the reconstitution reaction. This step is especially

important while working with high protein concentrations.

25

Chapter 3 Methods

3.6 Quantification of Dithionite

Disodium dithionite (DT) is a strong reductant with a midpoint potential at pH 7 Em’ =

- 420 mV.79 DT was used to reduce an electron transfer mediator, which in turn

reduced the tyrosyl radical and the iron-oxygen cluster. It was very important to

control the amount of primary reductant in the redox system, thus stock solutions of

DT were freshly prepared and quantified before each experiment.

Principle:

Fe(CN)63- has an extinction coefficient of 420 nm,ox = 1040 M-1cm-1 while the value for

the reduced form, Fe(CN)64-, is lower (420 nm,red = 2 M-1cm-1).80 Monitoring the change

in absorbance at 420 nm when adding DT to the Fe(CN)63- solution makes us able to

determine the concentration of DT in the stock solution.

Procedure:

1. This procedure works for DT stock solutions in the range of 2-100 mM.

2. Dissolve the DT salt in 200 mM HEPES, pH = 7.5 in sealed vials.

3. Record a baseline of a cuvette containing 200 mM HEPES, pH = 7.5.

4. Add small amounts of the DT solution to the sealed cuvette containing 1.0 mL 2.00 mM Fe(CN)63-,

200 mM HEPES, pH =7.5 until A420 nm ~1.3. There is no oxygen left inside the sealed cuvette at

this point.

5. Then add 3-8 L, depending of [DT], of the DT solution with a 10 L gas tight syringe to the

cuvette, mix well and read of the A420 nm value.

6. Calculate the concentration of DT in the solution by the formula

, where A is the change in absorbance and the volumes VFe(III) (volume of

the Fe(CN)63- solution) and VDT (volume of the DT solution) are given in L.

7. Repeat the procedure three times to determine the DT concentration properly.

3.7 Anaerobic Reduction of Phenazine Methosulfate

The electron transfer mediator phenazine methosulfate (PMS) was used as the primary

reductant and redox buffer when reducing the tyrosyl radical and the diiron-oxygen

cluster. It was important to understand the properties of the PMS redox buffer when

26

Chapter 3 Methods

studying the equilibrium of the mouse R2 diiron-oxygen cluster oxidation states in a

PMS/ DT solution.

Principle:

When the PMS salt is dissolved in water, a peak in the light absorbance spectrum at

388 nm originating from oxidized PMS can be observed (Figure 3.1). By monitoring

the decrease of the 388 nm peak when known amounts of DT is added, the redox

buffering properties of PMS can be elucidated.

Procedure:

PMS is light sensitive and decomposes when

exposed to light with wavelengths below 500

nm. Thus, all manipulations were carried out in

a dark room, and all equipment used for

handling the PMS solution was wrapped in

aluminum foil. All spectra were recorded using

a diode array spectrophotometer. 1. Prepare a 38 M PMS solution and a 3.8 mM DT

solution, both buffered with 50 mM Tris-HCl pH =

7.5, anaerobically.

2. Quantify the DT soulution as described in Chapter 3.6.

3. Evacuate a 2 mL quarts cuvette sealed with a rubber septum and flush with Ar (g).

4. Transfer 1 mL of the anaerobic PMS solution to the cuvette using a 1 mL gas tight syringe flushed

with Ar (g).

5. Record the spectrum of the oxidized form of PMS before adding 1-10 L DT solution with a gas

tight 10 L Hamilton syringe. Shake the cuvette and record spectrums at 10 seconds intervals until

equilibrium between DT and PMS is reached.

3.8 Reduction of the Diiron-Oxygen Cluster and the Tyrosyl Radical

The ferric irons in reconstituted mouse R2 are antiferromagnetically coupled. This

results in a S = 0 state for the diiron-oxygen cluster. For EPR spectroscopy, it is

necessary to have a paramagnetic half integer or integer spin system. However, it is

possible to reduce the diiron-oxygen cluster to a mixed valence state that has a ground

state S = ½ spin system that is EPR active.

350 400 4500.0

0.5

1.0

1.5

388 nm

Abs

orba

nce

Wavelength (nm)

Figure 3.1 Absorption spectrum of oxidized phenazine methosulfate.

27

Chapter 3 Methods

Principle:

Electron transfer mediators are often employed to transfer electrons from either a

working electrode or a chemical reductant the metal cluster(s) in the protein

examined.

Procedure:

All manipulations are carried out at 0-4C.1. Bubble 100 mL of 50 mM HEPES, 100 mM KCl, 20% glycerol, pH = 7.5 with O2 free Ar (g) for

one hour to remove O2(aq).

2. Reconstitute the protein in a glass vial as described in 3.5 before sealing with a butyl septum.

3. Evacuate and flush the sealed vial with O2 free Ar (g) at least 10 times, and leave under 2-3 Bar

Ar(g) when finished.

4. Prepare two 1 mL glass vials with proper amounts of DT and PMS, respectively, seal with butyl

septa and carry out 5 cycles of evacuation and Ar (g) flushing.

5. Inject 0.5 mL anaerobic HEPES buffer prepared in step 1 applying a 1 mL gas tight syringe. Be

aware that solvated PMS is photosensitive and must be protected from light.

6. Mix a proper amount of PMS solution into the reconstituted protein solution anaerobically with a

gas tight Hamilton syringe.

Sample Preparation for the Redox Equilibrium Studies

Add DT solution directly into the glass vial containing the anaerobic protein-PMS

solution, or use gas tight Hamilton syringes equipped with long needles and mix the

solutions simultaneously in an EPR tube. Final concentrations of R2, DT, and PMS in

the EPR tubes: [R2]= 50 – 100 M, [PMS]= 2 – 5 mM, [DT]= 0.12 – 1.65 mM.

In the introductory studies of the interactions between the mixed valence cluster and

alcohols, a 50%-50% alcohol – water mixture was added to the samples from the

kinetic studies and incubated at 4 C for 1 minute.

Sample Preparation for the Alcohol Titrations

Add 20 L of 50%-50% alcohol (methanol, ethanol, propanol, and butanol) – water

mixture to each of the EPR tubes and set up the homebuilt EPR sample mixer with

N2(g), a 1 mL gas tight syringe with protein-PMS and a 1 mL syringe with DT

solution. The N2 (g) flow in the homebuilt EPR sample mixer keep the syringes in an

anaerobic atmosphere and removes the air from the EPR tube that is to be filled.

28

Chapter 3 Methods

Final concentrations of R2, DT and PMS in the EPR tubes: [R2]= 100 M, [PMS]= 5

mM, [DT]= 1.7 mM.

3.9 Electron Paramagnetic Resonance Spectroscopy

3.9.1 Introduction to EPR Theory

Magnetic resonance is the phenomenon characterized by the change of the sign of a

spin experiencing a static magnetic field when absorbing electromagnetic radiation.

Electron paramagnetic resonance (EPR) spectroscopy is a magnetic resonance

technique not very different from nuclear magnetic resonance (NMR). With NMR the

nuclear magnetic moments of atoms with nuclear spin I 0 are detected, while the

magnetic moments of unpaired electrons in radicals and metal ions are detected by

EPR.

An electron is an elementary particle that is characterized by its mass, charge, and

angular momentum quantum numbers (Table 3.1).

Table 3.1 Angular momentum quantum numbers81

Name Representation Value

Orbital angular momentum a L 0, 1, 2…

Orbital magnetic a Ml 0, 1, 2,…, L

Spin angular momentum a S

Spin magnetic a Ms

a Upper case letters are used for orbital and spin angular momentums larger than 1 and ½, respectively, else lower case letters are used.

In general, spin and orbital angular momentum quantum numbers are eigenvalues of

the eigenvalue equation , where is the general spin or orbital angular

momentum operator (operators always have ^ “hats” in this thesis), jz is the

eigenvalue (quantum number), and the eigenfunction of .82 Later in this section,

the spin Hamiltonian operator will be described, hence the operator representation

of spin and orbital angular momentum will be used instead of quantum numbers

throughout the text. Information obtained from an EPR spectrum is usually analyzed

both qualitatively and quantitatively. By considering the positions, shapes, and

29

Chapter 3 Methods

linewidths of the peaks in the EPR spectrum, preliminary information about the spin

system is obtained. Further analysis usually involves quantum mechanical

calculations using a phenomenological spin Hamiltonian operator at a set of

wavefunctions in order to obtain possible energies for the transitions observed in the

experimental EPR spectrum. Thus, phenomenological spin Hamiltonians are used to

parameterize experimental EPR spectra. By simulating an experimental EPR

spectrum, physical properties can be assigned to the paramagnetic EPR active spin

center. Parameters for the spin Hamiltonian used in this thesis are described below,

and their values can be obtained by simulating EPR spectra.

3.9.2 The Electronic Zeeman Effect

Classically, the circulation of a charge results in a magnetic field, and the z-

component of the magnetic moment of the electron represented by the operator can

be explained by considering the rotation of a point charge. Though the classical

explanation is not entirely correct, the magnetic moment from the classical model is a

factor 2 lower than expected from quantum mechanics, it is still a reasonable

illustration.

In a static magnetic field, the z-component of the magnetic moment vector of the

electron can be oriented either along or against the field, and this phenomenon is

termed the electronic Zeeman effect. The energy level difference between these two

states is very small, and both states will be populated almost equally. Due to the small

population difference between the two energy levels, an EPR detectable net magnetic

moment appears. The population difference is described by the Boltzmann

distribution and depends on the temperature T and the separation of the energy levels

E as shown in Equation 3.1.82

Eq. 3.1

Ni and Nj are the populations of spin aligned along or against of the field direction,

respectively, and kb is the Boltzmann constant.

30

Chapter 3 Methods

The spin Hamiltonian for a free electron in a static magnetic field is given by:

Eq. 3.2

where B is the magnitude of the static magnetic field, e the electron gyromagnetic

ratio, the Bohr magneton, and the z-component of the electron spin operator.82

The energy levels for a free electron in a magnetic field are obtained by letting

operate at the electron spin functions |+1/2 and |-1/2 as illustrated in Equation 3.3

and 3.4 .82 This function annotation is termed Dirac notation, where < f | is the

complex and | f > the real function.

Eq. 3.3

Eq. 3.4

Equations 3.3 and 3.4 can be written as:

E = + ½ Bge Eq. 3.5

E = - ½ Bge Eq. 3.6

E and E given in Equation 3.5 and 3.6 are the energy levels of the electrons where

the z-components of the spins are oriented against and along the field, respectively. In

order to achieve electron paramagnetic resonance, the electrons have to absorb

microwave photons with the discrete energy E = E - E. This results in the

formulation of the resonance condition;

E = E - E = h Eq. 3.7

Useful relations

, where e is the elementary charge, me the electron mass, and ge the free electron g-factor.

, where h is the Planck constant.

31

Chapter 3 Methods

where is the microwave frequency.82

In EPR, the magnetic field (B) is

varied while the frequency is kept

constant. The electronic Zeeman

effect is illustrated in Figure 3.2,

where the resonance condition is

fulfilled when the magnitude of B

reaches a certain level.

The g-value of the free electron ge is 2.002319. Observed, effective g-values usually

deviate from ge, and especially in the case of transition metals. This can be explained

by:

1. The g-values are related to the splitting of the d-orbitals of a coordinated metal

atom.83

2. Modulations of the ligand field due to molecular vibrations that disturb the

electronic environment (this is the Jahn-Teller effect, which will not be further

discussed in this thesis).83

The symmetry of a spin center is reflected in its EPR signal g anisotropy, and some

examples of symmetries giving characteristic EPR signals is illustrated in Figure 3.3,

adapted from Hanson and Solomon.67

The Hamiltonian for a free electron in Equation 3.2 is not adequate for describing

complicated spin systems where the orientation of the spin system to the magnetic

B = 0 Increasing B

- ½ Bge

+ ½ Bge

E E = h

Figure 3.2 The energy levels E and E increase and decrease with slopes of + ½ Bge and - ½ Bge, respectively.

Figure 3.3 Different geometries of coordinated transition metal ions found in metallo proteins; A; pure octahedral (gx = gy = gz), B; rhombic (gx gy gz), C; square planar (gx = gy gz), D and E; distorted tetrahedral (gx gy gz).

32

Chapter 3 Methods

field and the orbital distribution of electrons have to be considered. In order to include

anisotropy, the electronic term of the spin Hamiltonian is written as

Eq. 3.8

where the static magnetic field is given by the vector , the g values is given in the

3x3 tensor with the diagonal as the principal g-values (gx, gy, gz), and the spin

operator .

3.9.3 Zero Field Splitting

For transition metals with S > ½, interactions between the electrons themselves within

the metal atom contributes to additional splittings of the Zeeman energy levels. The

origin of this effect is the electron-electron interaction that is induced by the metal

ligands and mediated by the spin orbit coupling.83 By decreasing the temperature, the

ground state get more heavily populated, and the influence of the exited states

mediated through the spin orbit coupling will change. Thus a temperature dependence

of this type of splitting can be observed. The spin Hamiltonian term for this

phenomenon is given as

Eq. 3.9

where is a traceless, symmetric 3x3 tensor. Since the magnetic field is not included

in this term, the splitting of the energy levels is independent of the static magnetic

field, hence it is called the zero field splitting (ZFS) term.

Equation 3.9 can be rewritten to

Eq. 3.10

33

Chapter 3 Methods

where S is the total spin and . The D and E-values are named the axial and

rhombic zero field splitting parameters, respectively.84 By considering Equation 3.10,

it can be noticed that the D-value describes ligand-induced symmetry distortions

along the z-axis of the spin system, while the E-value describes the deformations

along the x and y-axis. High spin non heme ferric irons usually have |D|-values below

1 cm-1 while high spin non heme ferrous irons have much higher values (|D| 5 – 15

cm-1).

3.9.4 Exchange Coupling

Dinuclear metalloproteins usually have bridging ligands linking the metal atoms

together. Such a bridging ligand couples the two spins (Sa and Sb) either

ferromagnetically or antiferromagnetically depending on the nature of the bridge.

When coupled antiferromagnetically, the spins are added together producing

Stotal = Sa + Sb, Sa + Sb –1, Sa + Sb – 2, . . . , |Sa - Sb| Eq. 3.11

spin states where |Sa - Sb| has the lowest energy. The Heisenberg, Dirac, Van Vleck

(HDVV) exchange Hamiltonian for a coupled spin system is given by

Eq. 3.12

where J is the Heisenberg coupling constant.84 By solving Equation 3.12 the energy

levels for the spin states calculated in Equation 3.11 can be obtained (Equation 3.13).

ES-total = -J[Stotal(Stotal + 1) – Sa(Sa + 1) – Sb(Sb + 1)] Eq. 3.13

3.9.5 The Spin Hamiltonian for a Dinuclear Coupled Metal Cluster

To this, point several spin Hamiltonian terms have been presented. In order to

describe the paramagnetic properties of a dinuclear coupled metal cluster these terms

34

Chapter 3 Methods

must be summed up. The electronic spin Hamiltonian (from the Zeeman effect) and

the zero field splitting spin Hamiltonian are defined for each of the atoms while the

exchange Hamiltonian link the two spin systems together as in Equation 3.14.83

Eq. 3.14

3.9.6 Relaxation of a Paramagnetic System

An important aspect of EPR is the way a spin system is excited by absorption of

microwaves and how it relaxes again to reach thermal equilibrium. Equation 3.1

describes the distribution of ms = + ½ and ms = - ½ in a static magnetic field, and N

= Ni – Nj is the number of electrons contributing to the net magnetic moment. When

the resonance condition (Equation 3.7) is satisfied, electrons in the ms = + ½ or ms = -

½ state will change the sign of their spins. Because the probability for changing spin

sign is equal for both states, N will approach 0 when the absorption of microwave

photons occur at a higher rate than the relaxation back to the equilibrium given in

Equation 3.1. When N decrease, the magnetic moment also decrease in magnitude,

and the EPR signal disappears.

The spin system is characterized as unsaturated when the spin relaxation is faster than

the microwave absorption and saturated when the microwave power is high enough to

overcome the spin relaxation.83 The double integral of the EPR first derivative

spectrum is proportional to the square root of the microwave power when the spin

system examined is unsaturated, and under such conditions the amount of spins of

different unsaturated paramagnetic species can be compared.

The relaxation rate is usually described by the spin-lattice (T1) and spin-spin (T2)

relaxation times. Spin-lattice relaxation is mainly due to vibrational interactions

between the paramagnetic cluster and the lattice while spin-spin relaxation involves

interactions between two or more paramagnetic centers. Pure spin-lattice relaxation

results in homogeneously broadened Lorentzian shaped EPR absorption lines. When

relaxation is mediated through both spin-spin and spin lattice interactions, the EPR

35

Chapter 3 Methods

absorption lines are inhomogeneously broadened and a Gaussian lineshape is

expected.83

For a complex paramagnetic system such as a bridged dinuclear cluster, the relaxation

theory is complicated. Because both atoms in the dinuclear cluster are paramagnetic,

distance and orientation dependent spin-spin interactions are expected to contribute to

the relaxation of the paramagnetic system in addition to spin-lattice interactions.83

3.9.7 EPR Instrument Parameters Used in the Experiments

Different parameter sets were used, depending on what kind of sample investigated.

The parameters and their applied values are described briefly in Table 3.2.

Table 3.2 EPR instrumental parametersParameter Description

Frequency Microwave frequency (9.6 GHz)

Power Microwave power (200 nW – 200 mW)

36

Chapter 3 Methods

Sweep width The magnetic field sweep range (0-5000 G).

Center field The magnetic field is swept about this value (0-5000 G).

Resolution Number of data points collected (1024 ).

Receiver gain Amplification of detector signal (2103 - 1106).

Modulation frequency The frequency which the magnetic field is modulated (100 kHz)

Modulation amplitude Amplitude of the field modulation (3 – 9 G).

Modulation phase Detection phase of the detector (90 degrees)

Conversion time Time to convert analog voltage to digital value (81.92 ms).

Time constant Time to filter the analog signal (40.96 ms).

Sweep time Time to record spectrum (83.89 s).

Harmonic Selection between first or second derivative detection mode (1 or 2)

3.9.8 EPR Sample Preparation

An anaerobic EPR sample is either prepared inside a sealed anaerobic EPR tube or

transferred into a tube with a long-needle gas tight Hamilton syringe. The EPR tubes

used have the dimensions 25 cm x 3.8 mm (inner diameter), and the sample volume

were in the range between 180 and 200 L.

Prepared samples are sealed with butyl septa and stored in N2 (l) at 77 K.

3.9.9 Quantification of Spin in an EPR Sample

A standard procedure was used to determine the quantities of unpaired electrons.

Typically, the first derivative of the EPR absorption curve is recorded. Thus enhanced

resolution of the spectra is achieved. The double integration of the EPR first

derivative absorption spectra acquired for an unknown sample can be compared to

that of a standard, and the amount of unpaired electrons in the unknown sample can

be determined. A 1 mM Cu(II) standard, from which a S = ½ EPR signal can be

recorded, was used for all spin quantifications.

3.10 Circular Dichroism Spectroscopy

37

Chapter 3 Methods

In Circular Dichroism (CD) spectroscopy the difference in absorbance between left

circularly polarized and right circularly polarized light is detected. Thus, this

technique can be used to study chiral molecules.85 The origin of the CD effect is that

in symmetrical molecules the electronic and magnetic transition dipoles are

perpendicular to each other, while that is not the case in asymmetric molecules. The

rotational strength (the change in the extinction coefficients Left and Right as functions

of the wavelength), which is measured in CD spectroscopy, depends on the angle

between the electronic and magnetic transition dipoles.

CD is expressed as either the difference in absorbance between the left and the right

circularly polarized ligth A = ALeft – ARight or the ellipticity that represents the

rotational strength. The relationship between A and is given by A = / 32.98.

3.10.2 CD instrumental parameters

Table 3.10.1 Parameters used for the CD experiments

Parameter Value

Band with 1 nm

Response 1 s

Sensitivity Standard

Measurement range 460 – 290 nm

Data pitch 0.5 nm

Scanning speed 50 nm/ min

Accumulation 1

Temperature 4 C

38

4 Results and Analysis

4.1 Protein Purification

The mouse R2 protein purification procedure, which is described in Chapter 3.1 and 3.2,

was published by Mann et al.69 in 1991. This procedure has routinely been carried out in

our laboratory since 1994, and the protein yield and purity are mainly the same for all the

purifications. For all EPR and CD sample preparations, the protein purity was considered

to be higher than 95 % by visual inspection of protein samples separated by SDS-PAGE.

The results from the experiments involving protein purification (Chapter 3.1 and 3.2),

protein quantification (Chapter 3.3) and solvent manipulations (Chapter 3.4) will not be

further discussed.

4.2 Reconstitution of Mouse RNR-R2

A novel approach was chosen for the reconstitution of the diiron-oxygen cluster and the

tyrosyl radical in mouse R2. The motivation for exploring new reconstitution techniques

was to save time and reduce the risk of adding oxidized iron (Fe3+) to the apo-protein

solution. The method previously reported for mouse R2 was based on initial binding of

ferrous (Fe2+) in the protein in the presence of ascorbate before molecular oxygen was

added.69 In the case of E.coli R2 protein reconstitution, O2 was already present in both

the protein solution and the acidic ferrous iron solution,29,77,78 or Fe2+ and ascorbate were

added anaerobically.10 We based our novel routine on an aerobic approach utilizing an

acidic Fe2+ solution. The tyrosyl radical yields that were reproducibly obtained are

presented in Table 4.1 and compared to published results for both mouse and E.coli R2.

39

Table 4.1 Results from the reconstitution of mouse R2

Method Tyrosyl radical per R2 dimer

Fe2+ and ascorbate added anaerobically (mouse R2) 69 1.2 – 1.5

Fe2+ and ascorbate added anaerobically (E. coli R2) 10 1.2

Acidic Fe2+ solution added aerobically (E. coli) 78 1.2 0.1

Acidic Fe2+ solution added aerobically (mouse R2) a 1.7 0.1a This work.

4.3 Redox Studies of Phenazine Methosulfate

4.3.1 Purpose of the Experiments

The electron transfer mediator phenazine methosulfate (PMS) was used when reducing

the diferric cluster and tyrosyl radical in reconstituted mouse R2.

It was important to understand the redox buffering properties of PMS before starting

upcoming experiments.

The redox chemistry of PMS is

complex. Different forms of PMS are

illustrated in Figure 4.1, where PMS+

(the oxidized cationic form) and

PMSH (the two electron reduced,

protonated form) dominate at pH

7.5.86 The one electron reduced

semiquinoid forms PMS and

PMSH+ are only stable at pH < 3.5,

and only small amounts are detected

at pH 7.5.86,87 When both PMS+ and

PMSH are present in the same

solution, formation of a complex (PMSHPMS)+ that is not reduced by NADH

(nicotinamide-adenine dinucleotide) has been suggested.86,87 The equilibrium of the

40

Figure 4.1 Phenazine methosulfate (PMS) can have

different protonation and redox levels.

Chapter 4 Results and Analys is

proposed complex formation where Keq = (1.3 0.2) 104 M-1,86 is illustrated in Scheme

4.1.

Scheme 4.1

4.3.2 The Equilibrium between Different Redox Forms of PMS

The spectral changes observed when two reducing equivalents per PMS+ molecule were

added to an anaerobic PMS+ solution are shown in Figure 4.2. Small amounts of DT were

added after virtually full

reduction of PMS+ was achieved,

and no further changes in the

spectrum appeared. This indicates

that spectrum B in Figure 4.2

represents PMSH. When 0.5, 0.8,

1.0, 1.6 reducing equivalents per

PMS+ were added, a linear

relationship between the decrease

of A388 nm (A388 nm) and the

amount of reductant was observed

(Figure 4.2, inset). The initial

concentration of PMS+ was

quantified using the extinction

coefficient PMS+ = 26.3 mM-1 cm-

1.86 DT solutions were quantified using the method described in Chapter 3.6 before and

after the experiment.

Semiquinoid forms of PMS have absorption maximums different than PMS+ and PMSH,

and were not observed at pH = 7.5 by light absorption spectroscopy. An intense EPR

signal originating from the PMS semiquinoid forms was observed in the samples

Figure 4.2 Anaerobic reduction of 38 M PMS by 38 M dithionite in 50 mM Tris at pH 7.5. Spectra A and B are fully oxidized and fully reduced PMS, respectively. Inset: the decrease of A388 nm as a function of reducing equivalents added.

350 400 450 500

0.25

0.50

0.75

0.5 1.0 1.5 2.0

0.2

0.4

0.6

0.8

Reducing equivalents

A38

8 nmA

B

Abs

orba

nce

Wavelength (nm)

41


containing mouse R2 mixed valence cluster. However, the amount of semiquinoid PMS

forms represented only (0.28 0.06) % of the initial PMS concentration in these

samples. Thus, no significant effect from the semiquinoid forms at the PMS+/ PMSH

redox couple equilibrium should be expected at pH 7.5.

Halaka et al.86 did not observe the equilibrium described in Scheme 4.1 directly. They

assumed that the absorption spectrum of (PMSHPMS)+ was the sum of the PMS+ and the

PMSH absorption spectra. Another assumption made by Halaka et al.86 was that the

(PMSHPMS)+ complex did not react with NADH in the observed time window. Hence,

they could explain that the complete reduction of PMS+ was not achieved when more

than equimolar amounts of NADH were added.86

Our results, however, show a linear dependence between the reduction of PMS+ to PMSH

and the amount of reductant added. Thus, it is reasonable to assume that no

(PMSHPMS)+ complex unable to react with DT is formed and that the redox couple

PMS+/ PMSH dominates the redox equilibrium in a PMS solution when DT is added

under anaerobic conditions.

4.4 Reduction of the Tyrosyl Radical and the Diiron-Oxygen Cluster


When studying metal clusters in proteins by spectroscopy, it is important that there is a

certain concentration of the redox state of interest in the sample. The spectroscopic

methods Mössbauer and MCD that are common in bioinorganic chemistry require 1-2

mM protein. EPR is usually more sensitive than Mössbauer and MCD, depending on the

broadness of the EPR signal. Usually, protein concentrations of 50 – 300 M are

sufficient for EPR spectroscopy.

42


The mixed valence oxidation state of the diiron-oxygen cluster in mouse R2 was the main

research object of this project. This redox form had to be obtained by reducing the

reconstituted protein. The previously reported yields of the mixed valence form obtained

by chemical reduction were not high.88,89 This was the initial motivation for the

introductory studies of the redox properties of the diiron-oxygen cluster in mouse R2.

As illustrated in Figure 4.3, the various reduction steps have dissimilar redox potentials.

The diferric (Figure 4.3 A) and the diferrous (Figure 4.3 C) form of E.coli R2 have been

characterized by crystallography.8,26 As indicated in Figure 4.3 the ligands of the irons are

partly exchanged when the diiron-oxygen cluster is reduced. Is should be emphasized that

the formal redox potentials E1’ and E2’ describes the redox properties of the irons and not

the thermodynamics of the rearrangement of the iron ligands.

In Figure 4.3, the reduction of the tyrosyl radical and the diiron-oxygen is expressed as

one reaction that is described by the redox potential E1’. This is not an accurate

description of the reaction because the reduction of the tyrosyl radical and the diiron-

oxygen cluster are two independent reactions. The tyrosyl radical have been suggested to

have a redox potential between 700 and 1000 mV.38 Thus, it was assumed that the tyrosyl

radical was reduced prior to the diiron-oxygen cluster and that an equilibrium between

the various oxidation states of the diiron-oxygen cluster and the redox buffer was formed.

Knowing estimates of E1’ and E2’ would able us to increase our yields of the mixed

valence cluster in mouse R2.

Figure 4.3 Different redox states of the diiron-oxygen cluster and the redox active tyrosine in RNR R2; reconstituted R2 (A), mixed valence form (B) and the fully reduced form (C).

OO

OO

Fe2+

Fe2+

Glu

Glu

O O

O

Fe3+

Fe3+

Glu

H

O O

O

Fe2+

Fe3+

Glu

Tyr-OHTyr-O' Tyr-OH

A B C

2 e-, 2 H+

e-, H +

E1’ E2’

43


4.4.3 Estimation of the Midpoint Potential Em’ of the Diiron-Oxygen Cluster

The observation of the mixed valence form of the mouse R2 by EPR made it clear that

the two irons in the diiron-oxygen cluster had individual redox potentials.36

Consequently, the presence of the three redox-states of the diiron-oxygen cluster; the

diferric, the mixed valence, and the diferrous, will depend on the redox potential of the

solution (Esol’).

A simple system for estimating the redox potentials E1’ and E2’ was established. A large

excess of an electron transfer mediator (M) compared to reconstituted R2 was added

anaerobically to the protein solution. The redox potential of the solution (Esol’) was then

adjusted by changing the ratio of the redox couple Mox/ Mred (Mox and Mred are the

oxidized and the reduced forms of the electron transfer mediator, respectively) with the

potent reductant DT. Since [M] was 20-100 times higher than [R2] in all our experiments,

it was assumed that the Mox/ Mred redox couple determined the redox potential of the

solution and that contributions from the diiron-oxygen cluster could be neglected.

The reduced forms of the two electron transfer mediators used in these experiments; PMS

and Toluidine Blue O (TB), are both 2 electron reductants at pH 7.5.90 At pH 7 PMS and

TB have the midpoint potentials (Em’) 85 and 34 mV, respectively.90-92 All the redox

potentials mentioned in this thesis are reported versus the standard hydrogen electrode

(SHE). The midpoint potential of an electron transfer mediator is defined as the redox

potential of the solution when [Mred] = [Mox].

When using the electron transfer mediator PMS, redox equilibrium between the various

redox states of the diiron-oxygen cluster and the electron transfer mediator was reached

within one minute (representative example in Figure 4.4 A). TB was only used in one

experiment, and 15 minutes incubation times for the samples were necessary before the

mouse R2 mixed valence form was stabilized (Figure 4.4 B). The yields of mixed valence

cluster were quantified by double integration of the EPR absorption first derivative

spectra.

44


Values of Esol’ were calculated using the Nernst equation:

Eq. 4.1

where R is the gas constant, T the temperature, n the number of electrons involved in the

reaction, F the Faraday constant, [Mox] the concentration of oxidized mediator, [Mred] the

concentration of reduced mediator, and pHEm the pH at which the midpoint potential of

the electron transfer mediator (Em’) had been determined. The last term of the Equation

4.1 equals -15 mV when pH – pHEm = 0.5, n = 2, and one proton is involved in the

reaction.

The Esol’ values and the yields of the mixed valence form obtained using various

concentrations of reactants are listed in Table 4.2.

10 20 30

0.2

0.4

A

Fe(I

I)Fe

(III

)/ R

2

Time (min.)10 20 30

0.2

0.4

B

Fe(I

I)Fe

(III

)/ R

2

Time (min.)

Figure 4.4 Formation of the mouse R2 mixed valence form. The black line indicates the progression of the reaction. The diiron-oxygen cluster and tyrosyl radical were reduced by: A; 5 mM PMS, 1.25 mM DT, B; 5 mM TB, 1.25 mM DT. Conditions; 100 M R2 (dimer), 20 % glycerol, 50 mM HEPES pH = 7.5, 100 mM KCl.

45


Table 4.2 Calculated redox potentials and mixed valence yields

[R2]a [Mediator] [DT]b [Mox] /[Mred] E’sol (mV)c [FeII-FeIII] / [R2]d

100 M 2 mM (PMS) 120 M 15.6 102 0.031 0.001

110 M 2 mM (PMS) 240 M 7.3 93 0.081 0.02

100 M 5 mM (PMS) 1 mM 4.0 86 0.18

100 M 5 mM (PMS) 1.25 mM 3.0 83 0.21 0.2

100 M 5 mM (PMS) 1.5 mM 2.3 80 0.2

50 M 5 mM (PMS) 1.65 mM 2.0 78 0.28 0.1

100 M 5 mM (TB) 1.25 mM 3.0 32 0.22 0.2a The concentrations of R2 were determined as described in Chapter 3.3.b The DT solutions were quantified (Chapter 3.6) before they were added.c The values were calculated form Equation 4.1 and are reported versus the SHE.d The amount of mixed valence cluster per R2 monomer (1 is the theoretical maximum value). Each value is calculated from 3 – 5 data points after redox equilibrium was reached (see representative examples in Figure 4.4).

In order to estimate the redox potentials E1’ and E2’, it was assumed that only the three

redox states depicted in Figure 4.3; the diferric, the mixed valence, and the diferrous form

were present in the samples. This assumption is supported by the fact that all three

depicted redox states of the diiron-oxygen cluster have been observed by either X-ray

crystallography8,26 or EPR spectroscopy.36,37 The Nernst equations for the different species

were then rearranged such that the fraction of the mixed valence form [FeII-FeIII] /

[R2monomer] was expressed by the redox potentials E1’, E2’, Esol’, and the number of

electrons involved in the first (n1) and the second (n2) electron transfer (Equation 4.2).93

Eq. 4.2

The constants in Equation 4.2 have been introduced previously. This equation was used

to estimate E’1 and E’2 from the data listed in Table 4.2. The results from the fitting

processes are shown in Figure 4.5. Because the oxidation of PMSH to PMS+ involves a

two-electron transfer, the best fits of the data in Table 4.2 was obtained with n1 = 2 and n2

= 1 or 2.

46


We will not speculate on the

mechanism of the electron

transfer from PMSH to the

diferric cluster. However, it

has been demonstrated that

the reaction between the

electron transfer mediator

phenazine ethosulfate (Em’ =

55 mV, 2 electron transfer at

pH 790), which is similar to

PMS, and the diiron-oxygen

cluster is reversible.88

Table 4.3 Estimated redox potentials for the reduction of the mouse R2 diiron-oxygen

cluster (versus the SHE) a

n –values E1’ (mV) E2’ (mV) Em’ b

n1 = 2, n2 = 2 70 43 57

n1 = 2, n2 = 1 71 53 62

n1 = 1, n2 = 1 55 48 52a The values are calculated from Equation 4.2 and the plots are shown in Figure 4.5. b Em’ is the midpoint potential given by E1’ + E2’/ 2, and is the redox potential where the highest yield of the mixed valence form is obtained by the different models.

It should be emphasized that these experiments were conducted only to estimate the

redox potential Em’ of the reduction of the Fe(III)-Fe(III) cluster to the Fe(II)-Fe(II)

oxidation state, and that the values presented in Table 4.3 may not be accurate.

The estimates of the Em’ value of the reduction of the diiron-oxygen cluster made us able

to adjust the redox potential of the solution in our samples so that Esol’ ~ Em’. A maximum

yield of the mixed valence oxidation state is obtained when Esol’ = Em’.

0 40 80 1200.0

0.1

0.2

0.3

0.4

[FeII

FeII

I ]/ [R

2 mon

omer

]

E'sol (mV)Figure 4.5 The data listed in Table 4.3 was fitted with Equation 4.3, and different values of n1 and n2 were tested. Dotted (…) line; n1 = n2 =2, solid line; n1 = 2 and n2 = 1, dashed line (---); n1 = n2 = 1. The arrows indicate the different midpoint potentials (Em’).

47


4.5 Interactions between Alcohols and the Diiron – Oxygen Cluster


A novel EPR spectrum was observed when a mouse R2 mixed valence sample containing

5 % (v/v) ethanol was investigated. The novel signal looked like a perturbed native mixed

valence signal and the shifts from the effective (or observed) geff values were large,

indicating an interaction between the mixed valence diiron-oxygen cluster and ethanol.

In order to characterize the possible interaction, various primary alcohols were added to

samples containing mouse R2 mixed valence diiron-oxygen cluster prior to EPR analysis.

4.5.2 Affinity of Various Primary Alcohols to the Mixed Valence Cluster

It was of interest to investigate the effect that selected alcohols had with respect to the

electronic environment surrounding the diiron-oxygen cluster. Primary alcohols with

varying lengths of alkyl chains were chosen for this experiment. Samples containing

mixed valence cluster were incubated for one minute in presence of 1 M (about 5 %

(v/v)) methanol, ethanol, 1-propanol, and 1-butanol, respectively. EPR spectra of the

samples were recorded at 4 K and compared to the native mixed valence signal (Figure

4.6 A-E).

48


EPR is a very powerful technique for probing the electronic structure of a spin center, and

a perturbed EPR signal indicates a change in the electronic structure of a compound.

Consequently, binding of small molecules like alcohols and anions to diiron-oxygen

clusters have been detected by

EPR.2,42,53,54,94,95

As shown in Figure 4.6, all four

alcohols perturbed the electronic

structure around the diiron-oxygen

cluster in the mixed valence state.

The effective g-values are listed in

Table 4.4. Trends of the perturbations

of the EPR spectra caused by the

alcohols were observed. First, large

shifts in the effective gxeff and gy

eff

values, at ~3600 and ~4000 gauss,

respectively, were observed for

methanol and ethanol. These gxeff and

gyeff shifts were not that pronounced

when 1-propanol and 1-butanol were

added. Second, when the length of

the alkyl chain increased, a shoulder

at ~4000-4150 gauss appeared. This

shoulder is the gzeff peak that overlaps

with the gyeff peak when methanol is

added and is resolved in the presence

of 1-butanol. From these observed

trends, a preliminary conclusion can

be formulated: the affinity of the

alcohol to the mixed valence cluster decrease with increasing numbers of carbons in the

linear alkyl chain. It was of interest to investigate the nature of the interaction between

3600 4000 4400

E

D

C

B

A

EPR

FIR

ST D

ERIV

ATI

VE

gzeff

gyeff

gxeff

Field (G)Figure 4.6 Addition of 1 M (about 5 % (v/v) alcohol to the mixed valence samples. A; native mixed valence signal, B; 1 M methanol, C; 1 M ethanol, D; 1M 1-propanol, E; 1 M 1-butanol. The samples were incubated with the alcohol for 1 minute at 4 C. EPR parameters; 5 mW, 4 K, 9.6 GHz.

49


the alcohols and the mixed valence cluster. Hence it was decided to carry out simulations

of the experimental EPR spectra and relate the results to possible alterations in the

structure of the diiron-oxygen cluster by ligand field calculations (see Chapter 4.6).

Table 4.4 Shifts in effective g-values upon alcohol addition

Mixed valence species gxeff gy

eff gzeff

Nativ 1.917 1.726 1.600

1 M methanol 1.945 1.743 1.652

1 M ethanol 1.945 1.753 1.660

1 M 1-propanol a 1.938 1.716 1.610

1 M 1-butanol a 1.933 1.735 1.610a Data obtained from a single experiment.

4.5.3 Estimation of Binding Constants for Methanol and Ethanol with Mouse R2

The interesting novel interactions between the selected alcohols and the mouse R2 mixed

valence cluster were further investigated. From the introductory experiments described in

Chapter 4.5.2, it appeared that methanol and ethanol had a higher affinity to the mixed

valence cluster than 1-propanol and 1-butanol did. Therefore, titrations of methanol and

ethanol into solutions of mouse R2 were carried out to observe the alcohol induced EPR

spectrum perturbation. Interestingly, shifts of the g-values were already observed when

50 mM methanol and 100 mM ethanol, respectively, were added to the protein solution.

In the range between 0 and 2 M methanol, the effective gxeff value shifted from 1.92 to

1.95. When the relative change of the gxeff value, grel (Equation 4.3), was plotted against

the concentration of alcohol, the data points formed a hyperbola.

Eq. 4.3

gxeff is the observed gx

eff value during the titration, gxeff

,min the gxeff value observed for the

native mixed valence signal, and gxeff

,max the apparent gxeff value when the diiron-oxygen

cluster is saturated with alcohol.

50


The data points were fitted with an equation describing the binding of a single ligand to a

complex (Equation 4.4, Figure 4.7).96 From this, the binding constant Kb was estimated.

Eq. 4.4

The titration experiment was repeated using ethanol instead of methanol. Equation 4.3

and 4.4 were also applied to the data from this experiment (Figure 4.7).

The results from these experiments are summarized in Table 4.5. Our hypothesis, that

alcohols are able to bind in a specific manner to the mouse R2 mixed valence cluster is

supported by our observations.

The observation that Kb, methanol < Kb, ethanol supports the preliminary conclusion presented in

Chapter 4.5.2 that the affinities of the alcohols to the mixed valence cluster decrease with

the length of the alkyl chain. However, these results should be considered preliminary

because the titration experiments have not been repeated.

Table 4.5 Estimated binding constants for alcohols to the mixed valence cluster

0.0 0.5 1.0 1.5 2.0

0.0

0.2

0.4

0.6

0.8

1.0

g rel

[MetOH] (M)0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

g rel

[EtOH] (M)

Figure 4.7 Titration with methanol and ethanol. The curves are the one site binding equation plots that was used to estimate the binding constants of methanol and ethanol to the mouse R2 mixed valence cluster. Fitting parameters: methanol; Chi2 = 0.00096, R2 = 0.992, Bmax = 1.13 and Kb = 0.24 0.02 M. ethanol; Chi2 = 0.0028, R2 = 0.981, Bmax = 1.6 and Kb = 0.60 0.03 M.

51


Alcohol Concentration range (M) R2 Kb (M)a

Methanol 0 – 2 0.992 0.24 0.02

Ethanol 0 – 1 0.981 0.60 0.03a Standard deviation obtained by fitting Equation 4.4 to the data plotted in Figure 4.7.

4.5.4 Effect of Isotope Labeled Alcohols

Additional evidence for an interaction between the alcohols and the mixed valence cluster

could possible be acquired by using spin labeled alcohols. Regular alcohols consists

mainly of 1H, 12C, and 16O isotopes, where carbon and oxygen both have nuclear spin I =

0 and hydrogen I = ½. By using deuterium (D, I = 1) and 13C (I = ½) labeled alcohols,

additional perturbations of the EPR spectra due to coupling between the spin active

isotope atoms and the diiron-oxygen cluster would further indicate an interaction between

the alcohols and the mouse R2 mixed valence cluster. Unfortunately no changes were

observed when CD3CD2OD, CD3OD, and 13CH3OH were added to the samples. The

distance between the spin active nuclei and the mouse R2 mixed valence cluster may

have been to long for a signal distortion to be resolved.

4.5.5 Microwave Powersaturation Behavior of the Novel EPR Signals

Structural changes around the spin center of interest can be probed by progressive

microwave saturation studies. As mentioned in Chapter 3.9.6, the spin relaxation times T1

and T2 represent spin-lattice and spin-spin relaxation pathways, respectively. Distortions

of the ligand structure around a paramagnetic cluster due to complexation might open

new lattice relaxation pathways, resulting in altered power saturation behavior compared

to the native, undisturbed structure.

The double integral of the EPR absorption first derivative spectrum is proportional to the

square root of the applied microwave power when unsaturated. When the applied

microwave power is high enough and the EPR signal becomes saturated and loose

52


intensity, the microwave absorption rate has become faster than the relaxation rate.

Progressive microwave saturation at a fixed temperature is used to determine the half-

saturation point (P1/2) of a paramagnetic species. Half-saturation values of several species

obtained at the same temperature can be directly compared, and differences in relaxation

properties can be discovered. An empirical equation that are readily used to determine P1/2

values is

Eq. 4.5

where K is a scaling factor, P the variable microwave power, and b the inhomogeneity

parameter.97 The b factor is fixed to 1 or 3 when the dominating lineshape is Gaussian

(spin-spin and spin lattice relaxation) or Lorentzian (spin-lattice relaxation), respectively.

An intermediate value of b (3 b 1) is allowed when a mixture of lineshapes is

observed.

Values of b below 1 have no physical relevance in the model presented above. However,

it is considered a signature of paramagnetic clusters that relaxes through a dipolar

exchange coupling with a nearby paramagnetic species.98

0.1 1 10 100

1 A

(Dou

ble

Inte

gral

/P1/

2 )/ (D

oubl

e In

tegr

al /P

1/2 ) 0

Power (mW)0.1 1 10 100

1

B

(Dou

ble

Inte

gral

/P1/

2 )/ (D

oubl

e In

tegr

al /P

1/2 ) 0

Power (mW)

Figure 4.8 Progressive power saturation of the native (A) and the ethanol-perturbed (B) mixed valence signals. A; solid line; P1/2 = 11.1 2.2 mW, b= 0.77 0.06, dotted line; P1/2 = 19.6 1.2 mW, b=1.0. B; P1/2 = 16.4 2.4, b= 0.99 0.07. Both data sets were recorded at 4.4 K, and double integrals of EPR first derivatives were compared with a 1 mM Cu(ClO4)2 standard.

53


The native and the ethanol-perturbed mixed valence clusters were characterized by

progressive power saturation. Equation 4.5 was used to analyze the data (Figure 4.8).

As illustrated in Figure 4.8 A, the best fit for the native mixed valence data was obtained

using Equation 4.5 with b = 0.77 0.06 and P1/2 = 11.1 2.2 mW (solid line). This

indicates the presence of a weak dipolar exchange coupling between the mixed valence

cluster and a nearby paramagnetic center not being a radical.99 . In Figure 4.8 A, it can be

noticed that the difference between the solid line (b = 0.77, P1/2 = 11.2) and the dotted line

(b = 1, P1/2 = 19.6) is not very large. Thus, it is not appropriate to speculate whether there

is an dipolar exchange coupling present between the mixed valence cluster and a nearby

paramagnetic cluster or not without additional experimental data. Analysis of the

progressive microwave saturation of the ethanol-perturbed EPR yielded P1/2 = 16.4 2.4

mW and b = 0.99 0.07 (Figure 4.8 B).

The methanol-perturbed EPR signal was also studied by progressive power saturation

(Figure 4.9). When the data was fitted

using Equation 4.5, the b value was fixed

to 1 and P1/2 was allowed to float in order

to obtain comparable P1/2 values.

The two P1/2 values were comparable in

magnitude; 22.8 1.0 mW and 28.6

2.3 mW for the native and methanol-

perturbed EPR signals, respectively. This

indicates that the relaxation properties of

the mixed valence cluster did not change

significantly in the presence of methanol.

The results from the progressive power

saturation studies of the mixed valence

cluster are summarized in Table 4.6.

1 10 100

1

(Dou

ble

Inte

gral

/P1/

2 )/ (D

oubl

e In

tegr

al /P

1/2 ) 0

Power (mW)

Figure 4.9 Progressive powersaturation of native (solid line) and methanol-perturbed (dotted line) mixed valence signal. Data was fitted with Equation 4.6, without methanol; P1/2 = 22.8 1.0 mW, with methanol; P1/2 = 28.6 2.3 mW. The b-value was fixed to 1.Temperature 4.2 K.

54


From these results it can be suggested that neither methanol nor ethanol change the

relaxation properties of the mixed valence cluster significantly. Hence, the progressive

powersaturation studies neither verified nor rejected the hypothesis that alcohol can

interact directly with the mouse R2 mixed valence cluster.

Table 4.6 Results from progressive microwave power saturation studies

Species b- value a P1/2 (mW) a

Mouse R2 mixed valence 0.77 0.06 11.1 2.2 (T = 4.2 K)c

Mouse R2 mixed valence in 1 M ethanol 0.99 0.07 16.4 2.4 (T = 4.2 K)c

Mouse R2 mixed valence 1 b 22.8 1.0 (T = 4.4 K)c

Mouse R2 mixed valence in 1 M methanol 1 b 28.6 2.3 (T = 4.4 K)c

a Standard error given by fitting program. b Few data points were not compatible with a floating b-value during the fitting procedure, hence it was fixed to 1. c The temperature measuring device have a constant positive unknown offset. Thus the temperatures tabulated here are those displayed by the measuring device and not the real ones.

4.6 Theoretical Studies of the Mouse R2 Mixed Valence Cluster

4.6.1 Purpose of the Theoretical Studies

In the preceding chapter, it was shown that the addition of primary alcohols induced

perturbations in the mouse R2 mixed valence EPR spectra. Titration experiments with

methanol and ethanol indicated that alcohol can bind to the mixed valence cluster. It was

of interest to connect the native and alcohol-perturbed EPR signals to the structure of the

diiron-oxygen cluster. To do that, advanced calculations to estimate the spin Hamiltonian

parameters that are related to ligand field theory were performed.

The study described here consists of two elements; simulations of experimental EPR

spectra using a phenomenological spin Hamiltonian and ligand field calculations based

on second order perturbation theory.

55


4.6.2 Simulation of Experimental EPR Spectra

Information regarding the properties of a paramagnetic spin system can be obtained by

simulation of experimental EPR spectra using a phenomenological spin Hamiltonian. The

elements of the spin Hamiltonian for a dinuclear coupled transition metal cluster are

briefly explained in Chapter 3.9. The operator is given as

(Equation 3.14).

The source code for the program ddpowjea100 written in Fortran 77 was kindly provided

by Dr. Joshua Telser, Roosevelt University, Illinois. The program creates an energy

matrix from the input parameters briefly described in Chapter 6.3. A dinuclear Sa = 2, Sb

= 5/2 antiferromagnetic coupled cluster yield a 30x30 energy matrix when all 30 possible

spin transitions are included (calculated from (2Sa+1)*(2Sb+1) = 30). Matrix

diagonalization yields transition energies (eigenvalues of the matrix) and transition

probabilities (eigenvectors of the matrix) that are combined with linewidth functions to

generate a theoretical EPR spectrum. Initial assumptions applied when calculating spin

Hamiltonian values for diiron-oxygen mixed valence clusters with low isotropic

exchange coupling (-30 cm-1 < J) are:

1. The ferric g-tensor (gx, gy, gz) is set to (2.0, 2.0, 2.0) due to the 6S ground state of the

ferric iron.2,101

2. Zero field splitting values D and E of the non heme ferric iron is very small compared

to those of the non heme ferrous iron, and a value of |D| < 1 cm-1 can be

assumed.2,60,102

When calculating spin Hamiltonian parameters of mixed valence or diferrous diiron-

oxygen clusters from EPR, Mössbauer, and MCD data, two relationships between the

ferrous zero field splitting values (D and E), the ferrous g-tensor(s) and the spin orbit

coupling ( = -100 cm-1) are usually employed:2,3,60,101,103,104

Eq. 4.6

56


Eq. 4.7

Equation 4.6 and 4.7 are only valid when the 1/3 E/D 0.

When applying the assumptions regarding the ferric iron and the relationships between

the ferrous g-tensor and zero field splitting values (Equation 4.6 and 4.7), the number of

spin Hamiltonian parameters that could be varied were reduced considerably. The

parameters varied during the simulations were: the z-component of the ferrous iron g-

tensor gz, the ferrous iron axial zero field parameter D, the ratio between the ferrous iron

rhombic and axial zero field splitting parameters E/D, and the isotropic exchange

coupling constant J.

General trends were noticed:

1. The gz value controlled the position of the observed effective g-value, gxeff.

2. The D and E values mainly determined the positions of gyeff and gz

eff.

3. The ratio of D/J determined the broadness of the spectra. The range of possible J

values were estimated to -30 cm-1 < J < -10 cm-1 by considering the spectra in Figure

4.10 and 4.11 of the limit 5 cm-1 < D < 8 cm-1. Larger values of D are in principle

possible. However, they are excluded by the fact that J for the mouse R2 mixed

valence cluster are not lower than the value found for the mixed valence methane

monooxygenase hydroxylase (J = -30 cm-1)2 that has a narrower EPR signal (see

Figure 2.9). Estimated D-values of ferrous irons are usually larger than 5 cm-1.

The isotropic exchange constant J for the mouse R2 mixed valence cluster was assumed

to be -16.4 cm-1 ( ). This assumption was necessary, and is built on the

experimental values estimated for uteroferrin. The observed geff values (1.93, 1,74, 1.59)

for uteroferrin are similar to those observed for the mouse R2 mixed valence cluster (see

Figure 2.9). Uteroferrin, a purple acid phosphatase isolated from pig allantoic fluid, has a

mixed valence Fe(II)-Fe(III) active form. The diiron-oxygen cluster in uteroferrin has

been characterized by EPR,58 Mössbauer,59,61,65 and MCD60 spectroscopy.

Different values of J, D and E/D for this cluster have been estimated (Table 4.7), and

significant deviations between the determined values are apparent. EPR spectra simulated

using plausible parameters are compared to geff values observed for the native mixed

57


valence mouse R2 EPR signals in Figure 4.10 and 4.11. From those illustrations it can be

suggested that the J value for the mouse mixed valence cluster is in the range –30 cm-1 < J

< -10 cm-1. Given the range of published J-values for uteroferrin, an analysis of the mouse

mixed valence EPR spectra with J = -16.5 cm-1 is not unreasonable.

Table 4.7 Spin Hamiltonian parameters for

uteroferrin

Reference Technique J (cm-1) ( ) D (cm-1) E/D

Yang et al.60 MCD < -5 5.17 0.17

Antanaitis et al. 58 EPR -7 - -

Sage et al. 65 Mössbauer -10 -8 0.18

Rodriguez et al. 61 Mössbauer -17.3 +10.8 0.29

The simulated (SIM) spectrum of the

experimental native mixed valence EPR

signal (EXP) is shown in Figure 4.12 A.

Using a Gaussian lineshape, a good

simulation was obtained with the parameters

3600 4000 4400

EXP

EXP

SIM

SIM

EXP

SIM

C

B

A

EPR

FIR

ST D

ERIV

ATI

VE

Field (G)Figure 4.12 Simulated (SIM) and experimental (EXP) EPR spectra of mouse R2 mixed valence cluster with A; nothing added, B; 1 M methanol, C; 1 M ethanol. Experimental spectra were recorded at 4 K, 5 mW microwave power, and 9.65 GHz.

Figure 4.10 Simulated EPR spectra where the ferrous ZFS parameters D and E/D were fixed at 5 cm-1 and 0.2, respectively. The isotropic exchange parameter was varied from –5 to -50 cm-1. By considering the position of the gx

eff =1.92 and gyeff=

1.73 of mouse R2 mixed valence in respect to the simulated spectra, only –30 <J<-10 makes sense.

3600 4000 4400

-50-40-30-20

-10

-5

J (cm-1)

EPR

FIR

ST D

ERIV

ATI

VE

geff=1.73geff=1.92

Field (G)3600 4000 4400

6

7

8

910

11

D (cm-1)

EPR

FIR

ST D

ERIV

ATI

VE

geff=1.73geff=1.92

Field (G)Figure 4.11 Simulated EPR spectra where E/D and J were fixed to 0.2 and –30 cm-1, respectively. The ferrous D value was varied from 6-11 cm-1. In the limit –30 cm-1 < J, the possible D values are found between approximately 6 and 8 cm-1.

58


listed in Table 4.8. For the alcohol perturbed EPR signals, the lineshape seemed to consist

of both Gaussian and Lorentzian elements, and the intensities of the first derivative peaks

were not possible to reproduce. However, the spin Hamiltonian parameters are obtained

when the effective g-values of the experimental and simulated spectra are the same,

regardless of the intensity of the spectral peaks. The same J value was kept during all

simulations so that the g-tensors and zero field splitting parameters could be compared

for all mixed valence species. Simulated spectra of methanol and ethanol perturbed EPR

spectra are shown in Figure 4.12 B and C, respectively. The spin Hamiltonian parameters

for the mixed valence cluster interacting with methanol or ethanol are listed in Table 4.8.

Table 4.8 Spin Hamiltonian parameters for the mouse R2 mixed valence cluster

Mixed valence species Fe(II) g-tensor (x,y,z) J (cm-1) Fe(II) D (cm-1) E/D

Nativ (2.167, 2.222, 2.075) -16.5 5.97 0.23

+ Methanol (2.154, 2.198, 2.052) -16.5 6.20 0.175

+ Ethanol (2.150, 2.193, 2.052) -16.5 5.97 0.18

4.6.3 Ligand Field Calculations

The zero field splitting D and E spin Hamiltonian parameters obtained for the mouse R2

mixed valence species in Chapter 4.6.2 were related to ligand field theory through second

order perturbation theory (Equation 4.6 and 4.7).

Thus, the coordinating environment of the ferrous iron in the Fe(II)-Fe(III) cluster can be

estimated provided that second order theory is valid for these systems.

Since the magnitude of the isotropic exchange coupling between the ferric and the ferrous

irons initially was guessed, the axial zero field splitting parameter D for the ferrous iron

determined by spin Hamiltonian simulations contain errors.

59


The main purpose of the spin Hamiltonian calculations and ligand field calculations was

to estimate the changes of the ligand field of the ferrous iron that could occur when an

alcohol interacted with the mixed valence cluster.

In a free ferrous iron the d-orbitals are

degenerate, meaning they posses the same

energy (see Figure 4.13). When a ferrous iron

is octahedrally coordinated (Oh) by ligands

donating electrons to the 4s, 4p, 3d(z2) and

3d(x2-y2) bonding orbitals, the energy levels of

the antibonding 3d(z2)* and 3d(x2-y2)*

orbitals are separated from the non-bonding

3d(xy), 3d(xz), and 3d(yz) orbitals by 10Dq

(about 12000 cm-1). The non-bonding d(xy),

d(xz) and d(yz) orbitals, termed the 5T2g

orbital set, are lower in energy compared to

the antibonding 5Eg orbital set consisting of

3d(z2)* and 3d(x2-y2)*. Since Fe(II) is a d6

atom, 6 electrons are distributed among the 5 d-orbitals of the 5Eg and 5T2g sets. The 5Eg

and 5T2g sets contain 2 and 4 electrons, respectively, when the energy splitting between 5Eg and 5T2g is smaller than the electron pairing energy. Such an electron distribution

results in a total spin S =2 and is referred to as the high spin state of Fe(II). When the

ferrous iron experience interactions along the z-axis, the energy of the d(z2)*, d(xz), d(x2-

y2)*, and d(yz) orbitals increase compared to the d(xy) orbital that is less affected by this

axial distortion. The energy difference between the doubly degenerated d(xz), d(yz)

orbital set and d(xy) are given by . If the direction of the axial distortion is tilted with

respect to the z-axis, the d(xz) and d(yz) orbitals will be separated by an energy V. Thus

the ferrous iron experience a rhombic ligand field.

Figure 4.13 Splitting of the ferrous iron d-orbitals. Pure octahedral coordination (Oh) split the orbitals into the 5Eg and 5T2g set while axial and rhombic distortion results in further splitting.

60


Approximate energies of d-orbitals split by octahedral and distorted octahedral ligand

fields are given in Figure 4.14. Figure 4.13 and 4.14 were taken (and modified) from

Solomon et al.105

The values of the calculated spin Hamiltonian parameters presented in Chapter 4.6.2 are

directly connected to ligand field theory for strong axial distortion of a octahedral

coordinated complex by the following equations:2,67,106

Eq. 4.8

Eq. 4.9

Figure 4.14 Ligand field splittings of the d-orbitals according to A; pure octahedral, B; strong axial and C; weak axial coordination.

61


The energies E(xz) and E(yz) are given with respect to the ground state orbital d(xy),

which has the energy E(xy) = 0. In all three equations above, the many electron spin orbit

coupling constant is 100 cm-1 (only for those equations), ge is the free electron g-value

and gx, gy, and gz are the elements of the ferrous iron g-tensor.

The expressions for the 5T2g zero field parameters in Figure 4.13 are then given by

Eq. 4.10

Eq. 4.11

All energies obtained from the ligand field calculations using Equations 4.8-11 are

summarized in Table 4.9, and the splitting of the 5T2g set is illustrated in Figure 4.15.

Table 4.9 Ligand field energies of the ferrous iron in the mixed valence clustera

Mixed valence species E(yz) (cm-1) E(xz) (cm-1) (cm-1) V (cm-1)

Nativ 912 1216 1064 304

+ Methanol 1024 1317 1170 293

+ Ethanol 1051 1357 1204 306a All energies are given in respect to the d(xy) ground state.

62


4.6.3 Summary of Results From Theoretical Calculations

Due to uncertainty in the exchange coupling constant J used throughout the spin

Hamiltonian calculations, the zero field parameters and thus ferrous g-tensors obtained

contain errors. These errors consequently affect the results from the ligand field

calculations. However, the theoretical calculations were performed to evaluate possible

scenarios for the alcohol-mixed valence cluster interaction that could be compared to

very similar results from studies of other mixed valence diiron-oxygen proteins.

With this perspective the theoretical calculations were successful. A further analysis is

presented in the discussion.

Figure 4.15 Ligand field splittings of the T2g set of the ferrous iron in the mouse R2 mixed valence cluster. An increase in are observed when alcohol was added while the values of V did not change much.

0

500

1000

1500

Vxzxz

xzyzyz

yz

xyxyxy

+Ethanol+MethanolNativ mixed valence

Ener

gy (c

m-1)

63


4.7 CD and Light Absorption Studies of the Mouse R2 Diferric Cluster


The exciting observation that alcohols interacted with the mixed valence cluster in mouse

R2 encouraged us to investigate whether the alcohols interacted with the active diferric

form of mouse R2. Since EPR mainly detects half integer spins, a different method had to

be used for this purpose. For reconstituted mouse R2, charge transfer transitions between

the ferric irons and the oxygen bridge can be observed in the 300-450 nm region (based

on observations for E.coli R2).69,107 In addition, a band originating from a vibronic

progression of a ~1500 cm-1 C-O vibration of the unprotonated tyrosyl radical appear at

416 nm.107 An interaction between methanol and the active R2 diferric cluster could

possible be probed by CD and light absorption spectroscopy, thus spectra were recorded

in the presence and the absence of methanol.

4.7.2 Reconstituted Mouse R2 in the Presence of Methanol

A titration of 160 M reconstituted

mouse R2 with methanol in the range

from 50 mM to 1 M gave no changes

in the CD spectrum (Figure 4.16).

However, the CD spectra were

recorded after only 1 minute

incubation with methanol, and the CD

bands between 290 and 320 nm were

not possible to detected due to

interference from the protein in that

region.

Figure 4.16 CD spectra of 160 M reconstituted mouse R2; solid line; nothing added, dotted line; 1 M methanol added. No difference in peak positions or signal intensity was observed. Spectra were recorded at 4 C.

300 350 400 450

Rel

ativ

e el

liptic

ity

Wavelength (nm)

64


When studying the diferric cluster by light absorption spectroscopy, the difference

spectra were recorded in order to eliminate all spectral contributions from the protein.

The sample was incubated in the presence of 0.5 M methanol in a sealed cuvette for about

12 hours at room temperature. In Figure 4.17, a difference can be noticed in the spectra of

reconstituted mouse R2 in the presence and the absence of methanol. Remarkably, the

signal seems to be unchanged in the low wavelength region while a decrease in intensity

is observed in the higher wavelengths.

Two explanations for these observations can be suggested:

1. Some of the iron-oxo charge transitions and tyrosyl radical bands are influenced by

addition of alcohol.

2. The long incubation time at room temperature resulted in disassembly of the diiron-

oxygen cluster.

The former explanation is supported by the

observation that the iron-oxo charge

transfer band at ~315 nm seems unchanged

after the long incubation with methanol,

indicating that the diiron-oxygen cluster is

intact. However, the experiment has not

been repeated.

Thus, the conclusion from the CD and light

absorption experiments must be that there

is a possibility that methanol interacts with

the diferric cluster. Further studies should

be undertaken.

Figure 4.17 UV-absorbance difference spectra of ~100 M active mouse R2 (solid line) and the same sample after 12 hours incubation in presence of 0.5 M methanol at ~20 C (dotted line).

300 350 400 450 500

0.5

1.0

1.5

2.0

Abs

orba

nce

(nm)

65

5 Discussion

5.1 Introduction

The work presented in this thesis has mainly been focused on the redox and spectroscopic

properties of the mixed valence redox state of the diiron-oxygen cluster in mouse R2.

Introductory studies such as R2 reconstitution and redox studies are considered in the first

two sections of this chapter, while the novel interaction between alcohols and the mixed

valence cluster and possible implications of this finding are discussed in the last sections.

5.2 Tyrosyl Radical Content in Reconstituted Mouse R2

Theoretically, both monomers of the R2 homodimer bind two ferrous irons that

subsequently react with dioxygen to form a diiron-oxygen cluster and a tyrosyl radical.

Consequently, quantification of the tyrosyl radical content should yield 2 radicals per R2

dimer. Yields of 1 – 1.5 radicals have been reported earlier.69,88,89 Our novel approach for

mouse R2 reconstitution yielded a radical content of 1.7 0.1 radicals per R2 dimer. The

obtained high radical yield confirmed that the reconstitution of the diiron-oxygen cluster

and tyrosyl radical in mouse R2 was successful.

5.3 Redox Chemistry of PMS and Mouse R2

The main purpose of the redox studies of the electron transfer mediator PMS and the

diiron-oxygen cluster in mouse R2 was to establish a system where reproducible large

amounts of the mouse R2 mixed valence cluster could be obtained. High yields of the

mixed valence cluster are important when employing techniques such as EPR,

Mössbauer, ENDOR, and MCD spectroscopy. It was also of interest to estimate the

67

midpoint potential for the reduction of the diiron-oxygen cluster in mouse R2 and relate

the obtained values to midpoint potentials determined for other diiron-oxygen proteins.

Earlier attempts to determine the redox potentials of the diiron-oxygen cluster in mouse

R2 by using low concentrations of electron transfer mediators (~0.1 mM) combined with

a voltmeter, electrodes, and EPR have failed (K.K. Andersson, personal communication).

Such an experiment usually requires about 10-20 hours, depending on how many samples

to be made. It has been suggested that the mouse R2 diiron-oxygen cluster disassembles

within that time window (K.K. Andersson, personal communication). Thus it has been

difficult to determine the redox potentials of the diiron-oxygen cluster.

Based on the rapid redox equilibration observed when 5 mM of the electron transfer

mediator PMS was used (see Figure 4.4), we suggest that the experiment mentioned

above should be repeated using such a concentration of PMS. Then disintegration of the

diiron-oxygen cluster during the experiment might be avoided.

The various redox states of the mouse R2 diiron-oxygen cluster can be obtained by

varying the redox potential of the protein solution. A high yield of mixed valence cluster

was obtained by utilizing a redox buffer to adjust the redox potential of the solution (Esol’)

to a value close to the Em’ estimated for the reduction of the mouse R2 diiron-oxygen

cluster.

When studying the redox equilibrium, it was important to understand the properties of the

redox buffer determining the redox potential of the solution. Oxidized and reduced PMS

constituted the redox buffer, and Esol’ was adjusted by adding the potent reductant DT.

The Esol’ values were calculated from the Nernst equation (Equation 4.1) on the basis of

the midpoint potentials previously determined for the PMS+/PMSH redox couple. Thus, it

was important to find out whether the PMS+/PMSH redox couple was formed in a

predictable fashion when DT was added or not.

In a previous study of PMS, nicotine amide dinucleotide (NADH) was used to reduce

PMS+.86 Addition of equimolar amounts of NADH to a PMS solution did not result in

complete reduction of PMS+ and a complex formation between PMS+ and PMSH was

68

Chapter 5 Discussion

suggested.86 When investigating the reduction of PMS by DT, we found a linear

dependence of the reduction of PMS+ to PMSH in respect to the concentration of DT.

Non of our observations confirmed a presence of a (PMSHPMS)+ complex. Thus, we

suggest that that the [PMS+]/ [PMSH] ratio is formed in a predictable fashion in the

presence of DT, and that the redox potential of a PMS/ DT solution can be estimated by

using the Nernst equation.

Samples containing reconstituted mouse R2, PMS, and DT were prepared and the yield of

mixed valence cluster was quantified by double integration of the EPR absorption first

derivative spectra. Esol’ values were calculated for each sample using the Nernst equation.

Three models, differing in the number of electrons involved in the electron transfers,

were used to explain the data. This gave Em’ values between 52 and 62 mV (versus the

SHE), which are within previous estimates where –70 mV < Em’ < 80 mV.108 With E.coli

R2, both ferric irons are reduced simultaneously in the presence of DT and electron

transfer mediators. The redox potential for this reduction has been determined to be –115

mV versus the SHE.38 Thus, the estimated midpoint potential for mouse R2 is more

similar to the one determined for MMOH (OB3b), which is 48 mV (versus the SHE).1

The regulatory protein, termed component B, of the MMO complex has been suggested

to alter the structure of the diiron-oxygen cluster containing MMOH protein.109 The

midpoint potential for the diiron-oxygen cluster in MMOH (OB3b) is shifted to –84 mV

(versus the SHE) in the presence of component B.1 It would be of interest to estimate the

midpoint potential for the diiron-oxygen cluster in mouse R2 in the presence and absence

of alcohol to pursue a possible structural effect of alcohol on the metal cluster.

5.4 Small Alcohols might Bind to the Mouse R2 Mixed Valence Cluster

Both MMOH (Bath and OB3b) and mouse R2 contain analogous diiron-oxygen clusters

that can have diferric Fe(III)-Fe(III), mixed valence Fe(II)-Fe(III) and diferrous Fe(II)-

Fe(II) redox states. A common approach when investigating structural properties of these

69


clusters is to probe the ability of exogenous ligands to interact with the diiron-oxygen

cluster in several oxidation states.

The scientific relevance of these studies can be justified by:

1. The dissociation of hydroxylated substrate from the diiron-oxygen cluster is the rate-

limiting step in the MMO catalytic cycle.51 Thus, the productdiiron-oxygen cluster

complex (compound T) is an important intermediate in this cycle. Compound T and

similar alcoholdiiron-oxygen cluster complexes have been characterized by

spectroscopy2,53,54 and crystallography.52 Interestingly, our results indicate that the

nature of the interaction between alcohols and the mouse R2 mixed valence cluster

might resemble compound T. This is fascinating in respect to the unrelated functions

of the diiron-oxygen clusters in mouse R2 and MMOH.

2. The ability of small molecules to interact with the diiron-oxygen cluster in R2 is also

of medical interest. When constructing R2 diiron-oxygen cluster specific inhibitors,

important elements to consider are the nature of the interaction and the affinity of the

inhibitors towards the diiron-oxygen cluster. Data on how small molecules interact

with the diiron-oxygen cluster form a basis for understanding these complex

interactions.

Our hypothesis, based on the titration experiments and the theoretical calculations, is that

primary alcohols can bind to the mixed valence cluster in mouse R2. By comparing our

results to information achieved from studies of MMOH, we can suggest plausible models

to explain our observations.

Previous studies of the diiron-oxygen clusters in MMOH and R2 have revealed that the

affinity of various exogenous ligands to the diiron-oxygen cluster depends on the redox

states of the two irons. The diferrous cluster in MMOH (OB3b), in which both irons are

5-coordinated,47,110 does not bind any anionic ligands (e.g. azide) or enzymatic products

tested.110 In E.coli and mouse R2, however, azide bind to the diferrous cluster.25,37,111

The mixed valence cluster of MMOH (Bath) binds methanol to the ferrous iron,54 but no

binding of anionic ligands to this cluster have been reported. The presence of 500 mM

70


azide does not alter the mouse R2 mixed valence cluster (K.K. Andersson, personal

communication). Results presented in this thesis indicate that primary alcohols can.

Recent crystallographic studies of MMOH (Bath) have shown that methanol and ethanol

bind in a specific manner between the two ferric irons in the oxidized diiron-oxygen

cluster.52 One might speculate that methanol interacts with the diferric cluster in mouse

R2 on the basis of the light absorption study presented in Chapter 4.7. Further

experiments are required to verify or reject this hypothesis.

The binding curves for methanol and ethanol illustrated in Figure 4.9 suggest a specific

interaction between the two alcohols and the mouse R2 mixed valence cluster. Since the

Kb values are 0.24 M and 0.6 M for methanol and ethanol, respectively, it can be argued

that the shifts of the effective g-values originates from a general solvent effect that

disturbs the protein tertiary structure. However, our hypothesis is supported by several

observations:

1. Research focused at protein stability in various solvents indicates that the alcohol

concentrations used in our studies do not denaturate the mouse R2 protein (see Table

5.1).

2. When the binding of methanol and ethanol to the diferric cluster in MMOH was

studied by crystallography, the protein crystals were soaked in 1 M methanol and 0.9

M ethanol, respectively prior to data collection.52 In these two crystal structures, only

slight changes in the iron coordinating environment due to alcohol binding were

observed.

3. The effect of 1 M methanol and 1 M ethanol on the mouse R2 mixed valence EPR

spectrum is more pronounced than for 1 M concentrations of the more denaturing

alcohols 1-propanol and 1-butanol.

Thus, we suggest that the perturbations of the mouse R2 mixed valence EPR signals

recorded in the presence of the selected alcohols originate from an interaction of alcohol

with the diiron-oxygen cluster.

Table 5.1 Denaturation midpoints for proteins in organic solvents112

Protein [Methanol] (M) [Ethanol] (M) [1-propanol] (M) [1-butanol] (M)

71


Myoglobina 12.4 5.3 2.0 0.8

Cytochrome c a 12.5 7.4 4.0 -

-Chymotrypsinogenb 7.2 3.8 1.6 0.7a pH = 5.7. b pH = 2.8. All values are determined at 25 C.

The weaker affinity of 1-propanol and 1-butanol to the mixed valence cluster can

possibly be explained by their larger size compared to methanol and ethanol. Since the

diiron-oxygen cluster is positioned inside a pocket, insufficient space in the pocket may

lead to exclusion of 1-propanol and 1-butanol.

The theoretical calculations also support our hypothesis that alcohol can bind to the

mixed valence cluster. Analogous calculations have been performed at the alcohol

perturbed mixed valence EPR signals recorded for MMOH (OB3b).2 Simulations of the

native and alcohol perturbed mouse R2 mixed valence EPR spectra resulted in sets of

spin Hamiltonian parameters. Those parameters could be related to the ligand field

energies for the ferrous iron of the cluster when assuming a strong axial interaction.

Ligand field energies calculated from spin Hamiltonian parameters for mouse R2 (this

work) and MMOH (OB3b)2 are compared in Figure 5.1. Only the orbitals belonging to

the T2g set that is described by the zero field parameters and V are included in that

illustration. As expected, the energies of the native, unperturbed mixed valence clusters in

mouse R2 and MMOH (OB3b) are not similar due to structural differences. Upon

methanol addition to the native MMOH (OB3b) mixed valence cluster, the zero field

splitting parameter was lowered by 190 cm-1 and the change in V was 5.7 %.2 For

mouse R2, the changes in were 106 and 140 cm-1 in the presence of methanol and

ethanol, respectively. The relative variations of V were also small for the mouse R2

cluster (3.7 and 0.6 % in the presence of either methanol or ethanol).

The comparable magnitudes of the changes in the ligand field energies upon alcohol

addition observed for the MMOH (OB3b) and mouse R2 mixed valence clusters suggest

that methanol and ethanol can bind to the ferrous iron in the mouse R2 cluster. However,

since the theoretical calculations were based on the set of assumptions given in Chapter

72


4.6.2, the calculated parameter set should only be considered as one of several possible

solutions.

Thus, no certain conclusions can be made based on the ligand field treatment.

Possible binding of methanol and ethanol to the mouse R2 mixed valence cluster can be

modeled by considering data that have been published for E.coli R2 and MMOH because:

1. All amino acids involved in iron binding and the radical transfer pathway are highly

conserved in E.coli and mouse R2.32

2. X-ray structures of mouse32 and E.coli8 R2, show that the positions of the iron

coordinating amino acid sidechains are comparable in both proteins.

3. The coordination and structure of the diiron-oxygen clusters in MMOH and R2 are

very similar. Structures of MMOH (Bath) with methanol or ethanol bound to the

diferric cluster52 might resemble alcohol bound to the mouse R2 mixed valence

cluster.

4. Binding of methanol to the ferrous iron of the mixed valence cluster in MMOH

(Bath) have been observed by ENDOR.54 Results from EPR and Mössbauer studies

0

1000

2000

V

V

xz

xz yz

yz

xyxy+MetOH+EtOH+MetOH NativNativ

Ener

gy (c

m-1)

MMOHMouse R2

Figure 5.1 Calculated ligand field energies for the T2g orbital set for the ferrous iron in the mouse R2 and MMOH (OB3b) mixed valence cluster.

73


also support the binding of methanol to the mixed valence cluster in MMOH

(OB3b).42,54

Three plausible modes of alcohol binding to the mouse R2 mixed valence cluster are

illustrated in Figure 5.2. The crystal structures of E.coli R2 and MMOH (Bath and OB3b)

all show that one water is terminally bound to each of the irons in the diferric oxidation

state. In our models it is assumed that the geometry of the iron coordinating environment

in mouse R2 is conserved upon reduction to the mixed valence state, and that an alcohol

replaces the terminally bound water when binding to the cluster.

However, geometric flexibility of the iron coordinating amino acid sidechains have been

shown for both E.coli R2113 and MMOH.47,113 Such a flexibility of the endogenous ligands

of the irons is not included in our models. It is also not known whether it is the alcohol or

alcoxide that might binds to the iron cluster. However, it is not possible to suggest more

precise models due to lack of experimental evidence.

Glu233

O

O

Glu267O

O

Glu170

O O

O

Fe1 Fe2

N

NHis173

N

N His270

OH2OH2

Asp139

O

OH

Glu233

O

O

Glu267O

O

Glu170

O O

O

Fe1 Fe2

N

NHis173

N

N His270

OOH2Asp139

O

O HCH3

Glu233

O

O

Glu267O

O

Glu170

O O

O

Fe1 Fe2

N

NHis173

N

N His270

O

Asp139

O

O H

CH3Glu233

O

O

Glu267O

O

Glu170

O O

O

Fe1Fe2

N

NHis173

N

N His270

OH2OAsp139

O

OCH3 H

A B

C D

Figure 5.2 Proposed mode of binding of methanol to the mixed valence cluster in mouse R2. Amino acid sidechains are labeled using mouse R2 amino acid numbering. A; native mixed valence cluster, B; water replaced by methanol at Fe2, C; water replaced by methanol at Fe1, C; methanol replaces both waters and forms a bridge between Fe1 and Fe2.

74


Considering the results from the spin Hamiltonian calculations in Chapter 4.6.2, it is to be

expected that the alcohol interact with the ferrous iron in the mixed valence cluster. Even

though, it is not known which of the irons (Fe1 or Fe2, Figure 5.2) that are reduced. Thus,

Figure 5.2 B is most correct if Fe2 is reduced and Figure 5.2 C when Fe1 is reduced. If the

alcohol binds in a bridging position, Figure 5.2 D is a plausible model. The hydroxide

bridge of the mouse R2 mixed valence diiron-oxygen cluster has been proposed by Atta

et al.37

Similar models to those presented in Figure 5.2 can be used to illustrate possible ethanol

binding to the mixed valence cluster.

5.4.1 Relevance of Results and Further Experiments

The redox experiments show that it is possible to efficiently reduce the diferric form of

mouse R2 cluster to a mixed valence oxidation state by using a high concentration of a

redox buffer. By reproducing the high yield of mixed valence it is now possible to make

samples suitable for Mössbauer spectroscopy.

Interestingly, the midpoint potential Em’ for mouse R2 seems to be close to the one

determined for the diiron-oxygen cluster in MMOH (OB3b). Further experiments,

involving accurately determination of the midpoint potential of the mouse R2 diiron-

oxygen cluster, might verify similarities of the redox behavior of the mouse R2 and the

MMOH diiron-oxygen clusters.

Considering the results obtained from both titration experiments and theoretical

calculations, we suggest that both methanol and ethanol can bind to the mouse R2 mixed

valence cluster. ENDOR, Mössbauer, and MCD spectroscopy are suitable techniques that

can be used to verify this hypothesis. No such experiments have been conducted yet.

Those techniques can also be used to confirm whether the diferrous and diferric oxidation

states can bind alcohol or not.

75


Since the proposed electron transport pathway is suggested to include one of the irons in

the mouse R2 diiron cluster, the mixed valence form might be of physiological relevance.

The presence of alcohol might influence the activity of the R1-R2 holoenzyme. Thus,

studying the RNR enzyme kinetics in the presence of alcohol would be of interest.

Considering the properties of mouse R2 and MMOH that are not shared with E.coli R2, it

might be that the mouse protein is a more appropriate fundament for constructing a

MMOH similar enzyme from R2 than the bacterial protein. Since the major goal of the

Iron-Oxygen Protein Network is to convert R2 to an enzyme capable of substrate

hydroxylation, knowledge of the analogous properties of R2 and MMOH form an

important basis for this work.

76

6 Appendix

6.1 MaterialsChemicals Source

1-Butanol (> 99.5 %) Merck1-Propanol (> 99.5 %) Merck2-Mercaptoethanol SigmaAcetic Acid (100 %, p.a.) MerckAlbumin, Bovine (> 96 %) SigmaAmmonia (25 %, p.a.) MerckAmmonium Iron(II) sulfate Hexahydrate (p.a.) MerckAmmonium Sulfate (p.a.) MerckArgon ((g), 99.9997 %) AGABacto Agar DifcoBacto Tryptone DifcoBacto Yeast Extract DifcoBromphenol Blue SigmaCarbenicillin SigmaChloramphenicol SigmaDi-Potassium Hydrogen Phosphate MerckEDTA (> 99 %) SigmaEthanol (Absolutt Prima) ArcusEthyl-d5 Alcohol-d (>99 atom % D) AldrichGlycerol (87 %, p.a.) MerckHelium (lq) AGAHEPES (> 99.5 %, Free Acid) SigmaHydrochloric Acid (36 %, p.a.) ProlaboIsopropyl -D-Thiogalactopyranoside SigmaLow Molecular Weight Standard PharmaciaMethanol (>99.5 %, p.a.) MerckMethyl-13C Alcohol (99 atom % 13C) AldrichMethyl-d3 Alcohol-d (99.8 atom % D) AldrichNitric Acid (69 %, p.a.) AppliChemNitrogen (lq) AGAOxygen (g) AGAPhastGel Blue R (tablets) PharmaciaPhenazine Methosulfate (92 %) SigmaPhenylmethylsulfonyl Fluoride (> 99 %) SigmaPotassium Chloride (p.a.) MerckPotassium Di-Hydrogen Phosphate (p.a.) MerckPotassium Ferricyanide(III) (>99 %) AldrichPotassium Hydroxide (p.a.) MerckSodium Chloride (p.a.) Merck

77

Sodium Chloride (p.a.) MerckSodium Dithionite (~80 %) SigmaSodium Dodecyl Sulfate (> 99 %) SigmaSodium Hydroxide (p.a.) MerckStreptomycin Sulfate SigmaToluidine Blue O (80 %) Sigma Tris(hydroxymethyl)aminomethane (> 99.9 %) Sigma

Column materials Source

Diethylaminoethyl Cellulose (DE52) WhatmanSephadex G-25 (medium) Pharmacia

Equipment Source

Bio-Rad Protein Assay Bio-RadButyl Septa (various sizes) Norton VerneretCentricon (YM-50) MilliporeCollodion Bags (12000 MWCO) SartoriusCuvettes (quarts) TeknolabEPR tubes (707-SQ, 25 cm) WilmadFolding Filter (Ø185mm) S&SHamilton Syringes TeknolabMillex-GP 0.22 m filter MilliporeNAP-5 Columns PharmaciaNitrocellulose Filters (0.45 m) MilliporePD-10 Columns PharmaciaPhastGel (8-25 Gradient) PharmaciaPhastGel SDS Buffer Strips PharmaciaPolyethylene tubing (various diameters) Becton DickinsonSpin Columns (1 mL) Bio-Rad

Instruments Manufacturer

ER 4113 HV Liquid Helium Control System Oxford InstrumentsHP8452A Spectrophotometer Hewlett PackardpH meter (420A) OrionSpectropolarimeter (J-810) JascoPhastSystem Pharmacia ESP 300E 10/12 X-band spectrometer Bruker

78

Chapter 6 Appendix

Scientific Computer Software Source

MolMol 2k.1 Koradi, Retoddpowjea 4.0 Telser, JoshuaRasmol 2.6 Sayle, RogerWinEPR 2.0 Bruker

6.2 The Culture Medium and Buffers

In all mediums and other solutions, Milli Q filtered and ion-exchanged H2O (mqH2O) was used. Buffer solutions were filtered with a nitrocellulose filter (0.45 m) and degassed for 20 minutes before they were used.

8 L of LB culture medium:

80 g Bacto Tryptone80 g Sodium Chloride20 g Bacto Yeast Extract

Add mqH2O until 8 L. Adjust pH to 7.5 by adding 12 M NaOH. Autocleave at 120 C for 25 minutes. When preparing petri dishes add 15 g Bacto Agar per liter LB culture medium prior to autoclavation.

Buffers

Stock solution A 1.0 M Phosphate BufferpH 7.0

Prepare by mixing a 1.0 M K2HPO4 solution with a 1.0 M KH2PO4 solution until pH = 7.0.

Buffer A

50 mM Tris1 mM EDTA pH = 7.5

Adjust pH by adding concentrated HCl to the basic Tris solution.

79

Chapter 6 Appendix

Buffer B

10 mM Phosphate Buffer1 mM EDTA30 mM KClpH = 7.0

Use stock solution A as a basis for the phosphate buffer.

Buffer C

10 mM Phosphate Buffer1 mM EDTA70 mM KClpH = 7.0

Use stock solution A as a basis for the phosphate buffer.

Buffer D

50 mM HEPES100 mM KCl20 % glycerolpH=7.5

Adjust pH by adding12 M KOH to the acidic HEPES solution.

PhastSystem Solutions Phast Loadmix

500 L 10 % SDS solution100 L 2-Mercaptoethanol200 L 0.1 % Bromphenol Blue solution

8 L loadmix is added to a 14 L buffered protein sample before boiling.

Phast Staining Solution

mL 0.2 % PhastGel Blue R solution40 mL 20 % Acetic Acid, 80 % mqH2O

80

Chapter 6 Appendix

Phast Destaining Solution 90 mL Methanol30 mL Acetic Acid180 mL mqH2O

Phast preservation solution

8 mL Glycerol8 mL Acetic Acid64 mL mqH2O

81

Chapter 6 Appendix

6.3 Input Parameters for the Program ddpowjeaParameter value Description2 Spin one (Fe2+)2.5 Spin two (Fe3+)2.167, 2.222, 2.075 g- tensor (gx, gy, gz) for spin one (Fe2+)5.97 2nd order axial ZFS parameter (D) for Fe2+ (cm-1)1.373 2nd order rhombic ZFS parameter (E) for Fe2+ (cm-1)0 3rd order Zeeman parameter for Fe2+ 0 4th order axial ZFS parameter for Fe2+ (cm-1)0 4th order cubic ZFS parameter for Fe2+ (cm-1)0 4th order rhombic ZFS parameter for Fe2+ (cm-1)2.0, 2.0, 2.0 g- tensor (gx, gy, gz) for spin two (Fe3+)N No rotation of spin one in respect to spin two0.1 2nd order axial ZFS parameter (D) for Fe2+ (cm-1)0 2nd order rhombic ZFS parameter (E) for Fe2+ (cm-1)0 3rd order Zeeman parameter for Fe2+ 0 4th order axial ZFS parameter for Fe2+ (cm-1)0 4th order cubic ZFS parameter for Fe2+ (cm-1)0 4th order rhombic ZFS parameter for Fe2+ (cm-1)35 b Isotropic exchange coupling J (cm-1) 0 2nd order isotropic exchange coupling J2 (cm-1)0 Axial part of dipolar exchange coupling (cm-1)0 Rhombic part of dipolar exchange coupling (cm-1)N No rotation of dipolar coupling tensors0,0,0 Anisotropic exchange coupling Ja (cm-1) 9.6516 Microwave frequency (GHz) 0 Perpendicular mode (direction of microwave magnetic component)Y Include population weighting of transition intensities4 Temperature in K300 Number of spectral points80 Igloo grid interval (accuracy of integration) 30 Rho grid interval (accuracy of integration)N No specific intervals for integration (full powder spectra) 1 Minimum state from which to calculate transitions29 Maximum state from which to calculate transitions2 Minimum state to which to calculate transitions30 Maximum state to which to calculate transitionsG Gauss is the unit of the magnetic field3300 Minimum field (G)4600 Maximum field (G)1 EPR absorption outputG Gaussian lineshape 195,235,145 Linewidths in MHz 1000 Linewidth cutoff 1 Electronic linewidth strain factor for Ms = 2 1 Electronic linewidth strain factor for Ms = 11 Electronic linewidth strain factor for Ms = 01 Electronic linewidth strain factor for Ms = 5/21 Electronic linewidth strain factor for Ms = 3/21 Electronic linewidth strain factor for Ms = 1/2 xxx.txt Parameter output filexxx.dat Data output file .

82

Terms and Abbreviations

A Absorption The Bohr magneton

Magnetic field vectorB Magnetic fieldBath Indicates Methylococcus capsulatusCD Circular dichroismCompound P Peroxo Fe(III)-Fe(III) form of the metal cluster in mouse R2Compound T Product Fe(III)-Fe(III) cluster complex in MMOHCompound X Fe(IV)-Fe(III) oxidation state of the metal cluster in mouse R2 Energy splitting of the 5Eg and 5T2g orbital setsD Axial zero field splitting parameter

Zero field splitting tensorDT Sodium dithionite E Rhombic zero field splitting parameterE1’ First formal redox potential of a reactionE2’ Second formal redox potential of a reactionEm’ Midpoint potential for a redox reactionENDOR Electron nuclear double resonanceEPR Electron paramagnetic resonanceEsol’ Redox potential of the solutionF Faraday constantFe2S2 Disulfur bridged diiron clusterFerric Fe(III) oxidation state of ironFerrous Fe(II) oxidation state of irone Electron gyromagnetic ratio

Electron g-tensorge Free electron g-valuegeff Effective (observed) g-values

Spin Hamiltonian operatorHDVV Heisenberg, Dirac, and Van Vleck

HDVV exchange operatorI Nuclear spinIntermediate P Peroxo Fe(III)-Fe(III) form of the metal cluster in MMOHIntermediate Q Fe(IV)-Fe(IV) oxidation state of the metal cluster in MMOHJ Exchange coupling constantKb Binding constant Many electron spin orbit coupling constantLMW Low molecular weight standardMCD Magnetic circular dichroismMMO Methane monooxygenaseMMOH Methane monooxygenase hydroxylaseMMOR Methane monooxygenase reductaseMox Oxidized electron transfer mediator

83

mqH2O Milli-Q filtered and ion-exchanged waterMred Reduced electron transfer mediator

Magnetic moment operator of an electronMWCO Molecular weight cutoff Frequencyn Number of electrons involved in a redox reactionNADH Nicotinamide-adenine dinucleotideNADPH Nicotinamide-adenine dinucleotide phosphateOB3b Indicates Methylosinus trichosporiumP1/2 Half saturation pointPDB Protein data bankPMS Phenazine methosulfatePMS+ Oxidized PMSPMSF Phenylmethylsulfonyl fluoridePMSH Reduced PMSR Gas constantR1 Ribonucleotide reductase R1 homodimerR2 Ribonucleotide reductase R2 homodimerRNR Ribonucleotide reductaserpm Revolutions per minute

Electron spin operatorS Spin angular momentum quantum numberSDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresisSHE Standard hydrogen electrodeT1 Spin lattice relaxation timeT2 Spin-spin relaxation timeT TemperatureTB Toluidine Blue OUV/ vis Ultraviolet/ visible light absorption spectrophotometry V Energy of the rhombic splitting of the 5T2g orbital setZFS Zero field splitting

Standard Amino acids

Alanine Ala A Leucine Leu LArginine Arg R Lysine Lys KAsparagine Asn N Methionine Met MAspartic Acid Asp D Phenylalanine Phe FCysteine Cys C Proline Pro PGlutamine Gln Q Serine Ser SGlutamic Acid Glu E Threonine Thr TGlycine Gly G Tryptophan Trp WHistidine His H Tyrosine Tyr YIsoleucine Ile I Valine Val V

84

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