Flavin Redox Switching and Proline

• Flavins and redox switching

• Electrochemical Methods

June 16, 2011

• Proline Utilization A (PutA)

Flavin Enzyme Diversity

1. Flavins support one-electron and two-electron transfer processes

2. Reactions catalyzed by flavoenzymes:

Dehydrogenation

Electron transfer

Hydroxylation

Dehalogenation

DNA repairDNA repair

Disulfide reduction

Luminescence

Histone demethylation

3. Biological Processesenergy metabolism, amino acid metabolism, DNA synthesis, fatty acid

metabolism, cholesterol, neuroactive compounds, chromatin remodeling, and

bioremediation of polychlorinated aromatics

4. Regulate protein function- protein interactions, DNA binding, membranes

Discovery of flavin mononucleotide

(FMN) by a Swedish biochemist

Showed the biochemical basis for riboflavin as a vitamin

O O

N

N

N

N

NH2FAD

Nobel Prize in Physiology or Medicine 1955

Axel Hugo Theodor TheorellN

N

NH

NH3C

H3C

O

O

CH2

CHHO

CHHO

CH

H2C

HO

O P O P O

O-

O O

O-

CH2O

H

OH OH

H

Nobelprize.org

Redox Chemistry of Flavin

Flavin semiquinone is EPR active (S=1/2).

Flavin Redox Switch

ExamplesNifL

Azotobacter vinelandii

PutAEscherichia coli

Vivid (VVD)Neurospora

Circadian clock

ox ox

Nitrogen fixation Proline metabolism

red

red

+ WC-1

WC-1Inhibitory complex

dimer

[carotenoid biosynthesis]

Per-Arnt-Sim (PAS) Domain Family1. In all kingdoms of life

2. Mediate protein-protein interactions and act as a primary sensors

3. Bind different redox cofactors flavin, heme, 4Fe-4S clusters

4. Sense oxygen levels, light, metals, and energy status

5. Regulate a diverse set of functions, including nitrogen fixation, gene expression,

phototropism, and chemotaxis

Crane, Biochemistry 47: 7012-9, 2008

LOV (light, oxygen, voltage)

Vivid (VVD) and NifL

oxygen

VVD

N

N

NH

N

O

O

R

Cys-SH

N

N

NH

N

O

O

R

Cys-S-

N

N

NH

N

O

O

R

H

Moffat, Biochemistry 46: 3614-23, 2007

NifL

N

N

NH

N

O

O

R

N

N

NH

N

O

O

R

H

H

O2

(

e-

source ?

Crane, Nat Chem Biol 5: 827-34, 2009

ExamplesNifL

Azotobacter vinelandii

PutAEscherichia coli

Vivid (VVD)Neurospora

Circadian clock

ox ox

Nitrogen fixation Proline metabolism

red

red

+ WC-1

WC-1Inhibitory complex

dimer

NADP+ + Pi

- H2O

ProlineProline

P5C P5C

P5CR P5CR FAD

NADPH

NADP+

PRODH PRODH

GSA GSA

FADH2

PutAPutA

+ H2O

Figure 1

NADP + Pi

γγγγ-GP γγγγ-GP

ADP

ATP

NADPH

Glutamate Glutamate

P5CDH P5CDH P5CS P5CS

GPR GPR

GK GK

NAD+ + H2O

NADH + H+

Proline Metabolism

PRODH

Ornithine

CitrullineArginineMitochondrion

Cytosol

Urea cycle

P5CDH

OAT

Glutamate

P5C ProlineP5CR

PRODH

NH2

COO-+

NH

COO-+

ProlineP5C

NADPH NADP+

P5CS

P5CDH

Human enzymes in proline metabolism

Mitsubuchi et al., 2008. J. Nutr.

PYCR1-related autosomal recessive cutis laxa a.k.a wrinkly skin syndrome

Mutations in the PYCR1 gene (P5CR) found in wrinkly skin syndrome

Nat Genet. 2009 Sep;41(9):1016-21. Mutations in PYCR1 cause cutis laxa with progeroid features

PYCR1-related autosomal recessive cutis laxa a.k.a wrinkly skin syndrome

• P5CR localizes to mitochondria.

• Fibroblasts from affected individuals have altered mitochondrial morphology

• Increased membrane potential and apoptosis upon oxidative stress

Organization of Proline Catabolic Enzymes

PutA

PRODH P5CDHD

PRODH P5CDH

E. coli 1-

B. jap. 1-

-1320

-999

PRODH P5CDHD

PRODH P5CDH

E. coli 1-

B. jap. 1-

-1320

-999

putP putA

PutR

putA+

Tanner, JJ. Amino Acids. 2008 Nov;35(4):719-30.

+

Electrochemistry Methods for Studying Redox Proteins

Potentiometry

An important thermodynamic parameter is the establishment of equilibrium between all

redox active species. The redox potential (Eo’) or midpoint potential (Em) is determined

along with the number of electrons (n) transferred in the reduction step. Measurement is

taken at zero current flow.

Emeas = Eo’ +(RT/nF) ln (ox/red)

Spectroelectrochemistry

Combines electrolysis, potentiometry, and spectroscopy

(UV-visible, electron spin resonance, IR, etc.)

Coulometrynumber of electrons (n) transferred to a protein

molar absorptivity (εεεε)

Q = nFVC

Cyclic voltammetry

“Electronic scanning” Potential is scanned while current

is measured

Prosthetic Groups are buried in ProteinsFAD

Space-Filling

Model

Electrochemical Methods

Emeas=

m(free)+ ln [ox]

[red]RTnF

E

Flavin Binding Domain of PutA

12

14

0.25

0.30

po

ten

tia

l (V

)

-0.10

-0.08

-0.06

-0.04

-0.02

1

2e-

N

N

NH

N

O

OR

H

H

N

N

NH

N

O

OR

2e-

N

N

NH

N

O

OR

H

H

N

N

NH

N

O

OR

Em(free) = -0.076 V (pH 7.5)

Wavelength (nm)

300 400 500 600

0

2

4

6

8

10

12

εε εεm

M-1

cm-1

Becker and Thomas (2001) Biochemistry 40, 4714-4721.; Vinod et al. (2002) Biochemistry 41, 6525-6532.; Lee et al., (2003) Nat. Struct. Biol. 10, 109-114; Zhang et al., (2004) Biochemistry 43, 12539-12548.

Wavelength (nm)

300 400 500 600 700

Ab

sorb

an

ce

0.00

0.05

0.10

0.15

0.20

percent reduced

0 20 40 60 80 100

po

ten

tia

l (V

)

-0.14

-0.12

-0.10

7

PRODH Active Site

(S)-Tetrahydro-2-furoic acid (THFA)

Kd =0.24 mM

O CO O H

R556

Becker and Thomas (2001) Biochemistry 40, 4714-4721.; Vinod et al. (2002) Biochemistry 41, 6525-6532.; Lee et al., (2003) Nat. Struct. Biol. 10, 109-114; Zhang et al., (2004) Biochemistry 43, 12539-12548.

R555

R431

Structures of PutA

E. coli

PRODH P5CDHD1- -1320PRODH P5CDHD1- -1320

P5CDH

PRODH PRODH

P5CDH

PRODH PRODH

PRODH P5CDH1- -999PRODH P5CDH

B. jap.B. jap.

1- -999

Structure of full-length bifunctional PutA

P5CDH

PRODH PRODH

P5CDH

PRODH PRODH

Tanner, JJ. PNAS 2010.

Strategy for testing channeling

P5CDH

PRODH

P5C

GSAGlu

NADHNAD+

P5CDH P5CDH

wt BjPutA

Non-channeling BjPutA variants

Pro

PRODH

P5CDH P5CDH P5CDH P5CDH

Non-channeling BjPutA variants

C792A R456M

Evidence for channeling in BjPutA

PutA-DNA Binding

PutA

0 0.3 0.2 0.3 0.5 0.8 1.0 1.5 4.0PutAK9M

E. coli (1-47)

RHH consensus (1-43)

helix A helix Bstrand

MGTTTMGVKLDDATRERIKSAATRIDRTPHWLIKQAIFSYLEQLENS

--MKRITVRLPDELYEALEELAAERGRSRSELIREAIREYLEEEE--

(µM)

PutA-DNA complex

put control DNA (5 nM)

complex

Gu et al., (2004) J. Biol. Chem. 279:31171-31176.

Larson et al., (2006) Protein Sci.15: 1-12.

[PutA, µµµµM] 0 0.25 0 0.25 0.5 0.9

wt DNA ∆∆∆∆12345 DNA

PutA-

Identification of DNA Binding Sites

GTTGCA GTCATA

- + - + - + - + - + - +∆5 ∆4 ∆3 ∆2 ∆1 WTEcPutA52

(500 nM)

DNA-Binding

Sites

putP putA1 2 3 4 5

put

control

DNA,

2 nM

PutA-

DNA

complex

put DNA

(2 nM)

PutA52-DNA

Complexes

Zhou et al., J Mol Biol. 2008 Aug 1;381(1):174-88.

Structural PutA-DNA Footprint

9 bp

Kd = 210 nM

Kd = 3100 nM

O2

Zhou et al., J Mol Biol. 2008 Aug 1;381(1):174-88.

PutA Functional SwitchingL-proline

PutP

PutA

red red

H+

red red

red redputP

putA

redred

red red

ox

ox

ox

ox

putP

putA

- proline + proline

red red

red

UbiquinoneUbiquinol

O

O

CH 3

R

O

O

H3C

H3C

OH

OH

CH 3

R

O

O

H3C

H3C

NH

N

NH

HN

O

OR

N

N

NH

N

O

OR

FADox FADred

P5CProline

Inner

Membrane

Becker, DF and Thomas, E. (2001) Biochemistry 40, 4714-4721.

Protein-DNA Interactions

The binding of molecule (L) to the oxidized and reduced forms of a protein

can be linked to differences in the potential of the protein in the presence

(Em(bound)) and absence (Em(free)) of the ligand by the following

thermodynamic box.

Em(free)

+ 2e-Proteinox Proteinred

Kd(ox)L L

Em(bound)

Kd(red)

*L + 2e-Proteinox * L Proteinred

There are two binding equilibria:Pox + L ↔↔↔↔ Pox*L Pred + L ↔↔↔↔ Pred*L

Kdox = [Pox][L]

[Pox*L]

Kdred = [Pred][L]

[Pred*L]

PutA Redox Properties in the Presence

of put Intergenic DNA

0.10

0.15

0.20

percent reduced0 20 40 60 80 100

pote

nti

al

(V)

-0.14

-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

1

Ab

sorb

an

ce

2 e-PutAox PutAred

Em = -76 mV

Em(bound) = -0.086 V (pH 7.5)

Wavelength (nm)

300 400 500 600 700

0.00

0.05

0.10

8

Wavelength (nm)

Ab

sorb

an

ce

PutAox-DNA

Kd = 45 nM DNA DNA

2 e-

2 e-

PutAred-DNA

Em = -86 mV

Kd = 98 nM

Becker and Thomas. (2001) Biochemistry 40, 4714-4721.

Only ~ 2-fold increase in Kd

Proline Dependent Binding to Lipid Bilayers (Surface Plasmon Resonance Study)

Res

pon

se (

RU

)

Time (s)

40

60

80

100

120

PutA + proline (5 mM)

Res

pon

se (

RU

)

-100 0 100 200 300 400 500

-40

-20

0

20

Time (s)

PutA (ox)

Zhang and Becker (2004) Biochemistry 43, 13165-13174.; Zhang et al., (2007) Biochemistry 46:483-91.

Sensorgrams of oxidized PutA (20 nM) and PutA (20 nM) with 5 mM

proline binding on E. coli polar extract lipids. The arrows indicate the

starting and ending of injection of protein sample. Buffer: 10 mM

HEPES, 150 mM NaCl, pH 7.4

Controlled Potentiometric Titration

Emeas (mV) OX -31 -36 -48 -60 –66 -74 -85 RED Re-OX

135 kDa

119 kDa111 kDap

ote

nti

al

(V)

log ([ox]/[red])

-1.0 -0.5 0.0 0.5 1.0-0.09

-0.08

-0.07

-0.06

-0.05

-0.04

-0.03

Em(Conf) = - 58 mV

(pH 7.5)

Em (FAD) = - 76 mV

Zhu, W. and Becker, D. F. (2003) Biochemistry 42, 5469-5477.

PRODH P5CDHD1- -1320

PutA

- -

Conformational change

Trp 211Ser 216Arg234

Flu

ore

scen

ce in

ten

sity

(au

)

150

200

250

oxB 1

2

Fl (

au)

0.08

0.12

0.16

Kinetics of Conformational Change

PRODH P5CDHD1- -1320

E. coli

- -

Wavelength (nm)

300 350 400 450 500F

luo

resc

ence

inte

nsi

ty (

au)

0

50

100

150

red 0 2 4 6 8 100.04

Time (s)

0.6 s-1

Zhu and Becker, (2005) Biochemistry 44, 12297.

E-FADox + S E-FADoxS E-FADredPslow

DCPIPoxDCPIPred

fast

fast

E*-FADredP membrane

binding

55 s-1

FAD Conformational Changes

THFA-Bound Dithionite reduced crystal

Zhang et al., (2007) Biochemistry 46:483-91.

Kinetic Properties of Reconstituted and Mutant PutA Enzymes

Enzyme Km (mM)

kcat (s-1)

kcat/Km (s-1M-1)

Wild-type 100 8 80

5-deaza-FAD NA (< 1 %) NA

R431M 174 1.2 7

2-deoxy-FAD 103 4 39

R556M > 1 M 1.4 < 1.4

a Parameters were estimated by best-fit analysis to the Michaelis-Menten equation.

N(5) Position is Critical

putC:lacZ

PutA + pro

- PutA

PutA R431M

Zhang et al., (2007) Biochemistry 46:483-91.

.25

PutA mechanism based inhibitor

4-methylene-L-proline N-propargylglycine

(N-PPG)Tetrohydro-2-furoic acid

(THFA)

EcPutA + 0.5 mM N-PPG Inhibition of full-length E. coli PutA with N-PPG100

0.25 mM PPG

Wavelength (nm)

300 350 400 450 500 550 600

Ab

s

0.00

.05

.10

.15

.20

Zhu, W et al., 2002; Zhang, M et al., 2004; Tritsch, D. et al.,1993; Szewczuk, L et al., 2007; White, T et. al., 2008

Time (min)

0 20 40 60 80 100 120

Re

lative A

ctivity (

%)

0

20

40

60

80

0.25 mM PPG

0.50 mM PPG

1.00 mM PPG

2.50 mM PPG

ki = 0.13 min-1, Ki = 1.46 mM

Structure of PPG inactivated PutA86-630

• Covalent linkage formed between K329, N-PPG and N5 of FAD,

• Flavin isoalloxazine butterfly bending

• 2-OH’ group of ribityl moiety rotates 90°C and forms new H-bond to N1-FAD

Lys3

29

Proposed mechanism of inactivation

SDS treated

PPG mimics proline reduced PutA

M

200 kD

150 kD

100 kD

PutA OX PRO THFA PPG

119 kD

Limited proteolysis Liposome binding

75 kD

50 kD

90 kD

Oxidized and PPG Structures

Glutamate cluster

E372E373

C-terminal membrane binding domain ?

E.coli AR----GESNILLERLYIERSLSVNTAAAGGNASLMTIG- 1320 E.coli AR----GESNILLERLYIERSLSVNTAAAGGNASLMTIG- 1320

S.typhimurium AR----GESNILLERLYIERSLSVNTAAAGGNASLMTIG- 1319

K.pneumoniae AR----GETNLLLERLYIERSLSVNTAAAGA--------- 1312

Y.enterocolitica AR----GETNILLERLLIEHSLSVNTAAAGGNASLMTIG- 1323

P.luminescens AR----GETNLLLERLLHERSLSINTAAAGGNASLMTIG- 1326

P.syringae SH----GETNVPLERLVIERALSVNTAAAGGNASLMTIG- 1300

P.putida SS----GDHQIALERLVIERAVSVNTAAAGGNASLMTIG- 1317

R.solanacearum PH----GGQGLALERLLIERSLSVNTAAAGGNASLMTIG- 1325

B.pertussis SADALAAGASYAPDRLLAERSISVNTAAAGGNASLMTIG- 1273

B.japonicum -----------------TEQTVTINTAAAGGNAALLAGEE 999

~ 30 Å

~ 15 ÅYL L RL V AS

hydrocarbon core

polar solvent

inte

rfac

e

Zhou et al., Amino Acids. 2008 Nov;35(4):711-8.

PutA(1-1308)DNA binding (yes)

PRODH activity (yes)

P5CDH Activity (yes)

Substrate Channeling (yes)

Membrane binding (NO)

Summary

• Changes in hydrogen bonding at the flavin N(5) is a key feature flavin switches

• Hydrogen bond networks that link the flavin N(5) to the surface of the protein is a common theme.the protein is a common theme.

• Internal hydrogen bond rearrangements and electrostatic networks are critical for transmitting redox signals out of the flavin active site

• Electrochemistry is a versatile tool for studying redox regulation and conformational changes