Flavin Redox Switching and Proline

June 11, 2012

• Flavins and electrochemical methods

• Proline Metabolism

• Proline Utilization A (PutA)

• Flavin redox switching of 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 repair

Disulfide reduction

Luminescence

Histone demethylation

3. Biological Processes

energy 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

Nobel Prize in Physiology or Medicine 1955

Axel Hugo Theodor Theorell N

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

N

N

N

N

NH2

FAD

Nobelprize.org

Redox Chemistry of Flavin

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

Flavins support one-electron and two-electron transfer processes involving the N(1), C(4a) and N(5)

positions of the isoalloxazine ring system.

Because flavins are involved in catalyzing oxidative and reductive mechanisms, characterizing the redox potentials of the

substrates, products, and flavins are important. The redox potential of free flavin is -219 mV (pH 7). However, when bound to an

enzyme, the redox potential can vary anywhere from + 100 mV to – 400 mV. Thus, the active site environment has a tremendous

influence on the redox and electronic properties of the flavin coenzyme in order to optimize catalysis.

Electrochemistry Methods for Studying Redox Proteins

Coulometry

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)

number of electrons (n) transferred to a protein

molar absorptivity ()

Q = nFVC

Spectroelectrochemistry

Combines electrolysis, potentiometry, and spectroscopy

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

Cyclic voltammetry

“Electronic scanning” Potential is scanned while current

is measured

Prosthetic Groups are buried in Proteins FAD

Space-Filling

Model

Electrochemical Methods

E meas =

m(free) + ln [ox]

[red] RT nF

E

Redox potential of bound FAD in PutA

Wavelength (nm)

300 400 500 600

0

2

4

6

8

10

12

14

m

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

0.25

0.30

percent reduced

0 20 40 60 80 100

po

ten

tia

l (V

)

-0.14

-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

7

1

2e-

N

N

NH

N

O

O

R

H

H

N

N

NH

N

O

O

R

2e-

N

N

NH

N

O

O

R

H

H

N

N

NH

N

O

O

R

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

Redox linked protein-ligand 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.

Kd(ox) L L

Em(free)

Em(bound)

Kd(red)

*L + 2e- Proteinox

+ 2e- Proteinox

* L Proteinred

Proteinred

There are two binding equilibria:

Pox + L Pox*L Pred + L Pred*L

Kdox = [Pox][L]

[Pox*L]

Kdred = [Pred][L]

[Pred*L]

Flavin Redox Switch

NADP+ + Pi

- H2O

g-GP

ADP

ATP

NADPH

Proline

P5C

Glutamate

P5CR FAD

NADPH

NADP+

PRODH

GSA

FADH2

PutA

P5CDH P5CS

GPR

GK

+ H2O

NAD+ + H2O

NADH + H+

Proline Metabolism

Human enzymes in proline metabolism

Mitsubuchi et al., 2008. J. Nutr.

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

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

Organization of PRODH and P5CDH

Bradyrhizobium japonicum (BjPutA) short bifunctional

Escherichia coli (EcPutA) trifunctional (Functional switching)

Tanner and Becker, Proline Metabolism and PutA. Handbook of Flavins, (2012) in press.

15

PutA (PRODH/P5CDH) and bioenergetics

COO-COO- COO-COO-

PRODH FAD FADH2

CoQ

COO-COO-

± H2O

-OOC COO--OOC COO-

NAD+, H2O NADH + H+

PutA

Pro P5C

GSA Glu

reductive

half-reaction

CoQH2

P5CDH

oxidative

half-reaction kcat = 5 s-1

Km (pro) = 34 mM

Km (CoQ1) = 124 mM

kcat/Km = 1400 M-1s-1

Proline is an important energy

source in various species

Bacteria (e.g., H. pylori)

Cuttlefish

Insects

Beetle:www.reptileforums.co.uk

Reductive Half-Reaction

E + Pro E-Pro k (506 M-1s-1) 1

f + P5C k (255 s-1) -1

k (27.5 s-1) 2

k (1.6 s-1) -2

k (2.2 s-1) 3

k (0.19 s-1) -3

F-P5C f-P5C k (95.5 s-1) 4

k (4600 M-1s-1) -4

Oxidative Half-Reaction

f + CoQ f-CoQ k (2100 M-1s-1) 5

k (2.9 s-1) -5

k (5.4 s-1) 6 k (4.7 s-1) 7 e-CoQH2 E-CoQH2

Moxley and Becker (2012) Biochemistry 51:511-520.

Stopped-flow kinetics of PutA

Slow step in the overall reaction is k6.

PROLINE

FADH2

FAD

P5C PRODH

PROLINE ETCox

ETCred

ATP/NADPH

Apoptosis

Cell Survival

ROS

cyt c release Nrf2

AKT/Foxo3a

Proline/PRODH and Cell Survival

Prostate cancer cells (PC3)

Knockdown of

PRODH decreases

P-AKT and stress

survival of PC3 cells In worm, PRODH

activates Nrf2 leading to

increased catalase/SOD

and lifespan

Cell Metab. 15:451-65; 2012. FRBM. In revision, 2012.

PROLINE

FADH2

FAD

P5C PRODH

PROLINE ETCox

ETCred

ATP/NADPH

Apoptosis

Cell Survival

ROS

cyt c release Nrf2

AKT/Foxo3a

Proline/PRODH and Cell Death

The gene encoding PRODH is

inducible by p53

Upregulation of PRODH by p53

results in formation of proline-

dependent ROS and induction of

intrinsic and extrinsic apoptotic

pathways

Co-expression of Mn-SOD blocks

apoptosis induced by PRODH

Overexpression of PRODH

suppresses tumorigenesis in

xenografted nude mice

Nature. 389:300-5. 1997; Cancer Res. 61:1810-5. 2001; Oncogene. 25:5640-7. 2006;

Cancer Res. 69:6414-22. 2009; PNAS USA. Epub ahead of print May, 2012.

PutA Functional Switching L-proline

PutP

PutA

red

red

red

red

H+

red red

red red putP

putA

red

red

red red

ox

ox

ox

ox

putP

putA

Ubiquinone Ubiquinol

O

O

CH3

R

O

O

H3C

H3C

OH

OH

CH3

R

O

O

H3C

H3C

NH

N

NH

HN

O

O

R

N

N

NH

N

O

O

R

FADox FADred

P5C Proline

Inner

Membrane

- proline + proline

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

Flavin Redox Switch

Structures of PutA

Mem CoQ4 CoQ2 CoQ1 Mena Duro DCPIP

Wt PutA

Km

(pro, mM)

1.5 ± 0.29

2.25 ± 0.53

9.38 ± 1.6

35 ±4

67 ± 6

27 ± 2

100

kcat

(s-1)

0.40 ± 0.02

0.18 ± 0.01

0.4 ± 0.01

1.3 ± 0.03

2.4 ± 0.06

0.99 ± 0.02

5-8

kcat/Km

(mM-1 s-1)

0.293 0.08 0.043 0.04 0.036 0.0372 0.03-0.08

Mem CoQ4 CoQ2 CoQ1 Mena Duro DCPIP

Wt PutA

Km

(pro, mM)

1.5 ± 0.29

2.25 ± 0.53

9.38 ± 1.6

35 ±4

67 ± 6

27 ± 2

100

kcat

(s-1)

0.40 ± 0.02

0.18 ± 0.01

0.4 ± 0.01

1.3 ± 0.03

2.4 ± 0.06

0.99 ± 0.02

5-8

kcat/Km

(mM-1 s-1)

0.293 0.08 0.043 0.04 0.036 0.0372 0.03-0.08

E. coli

PRODH P5CDH D 1 - - 1320 PRODH P5CDH D 1 - - 1320

P5CDH

PRODH PRODH

P5CDH

PRODH PRODH

PRODH P5CDH 1 - - 999 PRODH P5CDH

B. jap . B. jap .

1 - - 999

PutA-DNA Binding

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

PRODH Active Site

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.

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

Kd =0.24 mM

O C O O H

R555

R556

R431

Structure of full-length bifunctional PutA

P5CDH

PRODH PRODH

P5CDH

PRODH PRODH

Tanner, JJ. PNAS 2010.

24

Model of EcPutA and EcPutA-DNA complex

Singh et al. J. Biol. Chem. 2011. 286: 43144-43153.

A

Non-Channeling Control

PRODH

S I P

Channeling

C792A

Channeling Test in PutA

R456M

P5CDH

PRODH

S I

P5CDH

PRODH P5CDH

I P

B

Channeling in PutA Enzymes

PutA Functional Switching L-proline

PutP

PutA

red

red

red

red

H+

red red

red red putP

putA

red

red

red red

ox

ox

ox

ox

putP

putA

Ubiquinone Ubiquinol

O

O

CH3

R

O

O

H3C

H3C

OH

OH

CH3

R

O

O

H3C

H3C

NH

N

NH

HN

O

O

R

N

N

NH

N

O

O

R

FADox FADred

P5C Proline

Inner

Membrane

- proline + proline

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

Flavin Redox Switch

PutA Redox Properties in the Presence

of put Intergenic DNA

Wavelength (nm)

300 400 500 600 700

0.00

0.05

0.10

0.15

0.20

percent reduced 0 20 40 60 80 100

pote

nti

al

(V)

-0.14

-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

8

1

Wavelength (nm)

Ab

sorb

an

ce

PutAox-DNA

Kd = 45 nM DNA DNA

2 e-

2 e- PutAox PutAred

PutAred-DNA

Em = -76 mV

Em = -86 mV

Kd = 98 nM

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

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

Only ~ 2-fold increase in Kd

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

Res

po

nse

(R

U)

Time (s)

-100 0 100 200 300 400 500

-40

-20

0

20

40

60

80

100

120

PutA + proline (5 mM)

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

Where is the membrane binding domain?

Conserved C-terminal Motif (CCM)

Zhou et al., Amino Acids. 2008 35:711.

PutA 1-1308 BjPutA

EcPutA

Singh et al. J. Biol. Chem. 2011. 286: 43144-43153.

PutA1-1308

55-fold lower activity

PutA1-1308 Deficient in Membrane Associations

M S L S L S L S L

OX RED OX RED

WT 1-1308

Zhu, unpublished, 2012

Conformational change in PutA with FAD

reduction

po

ten

tia

l (V

)

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

135 kDa

119 kDa 111 kDa

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 P5CDH D 1 - - 1320

PutA

- -

Conformational

change

Trp 211

Ser 216

Arg234

Wavelength (nm)

300 350 400 450 500 F

luo

rescen

ce in

ten

sit

y (

au

)

0

50

100

150

200

250

ox

red 0 2 4 6 8 10

B 1

2

Fl (a

u)

0.04

0.08

0.12

0.16

Time (s)

kobs = 0.6 s-1

Kinetics of Conformational Change

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

PRODH P5CDH D 1 - - 1320

E. coli

- -

PutA switching from a transcriptional repressor to a membrane-bound

enzyme most likely occurs on the timescale of seconds, which is

consistent with movements of large domains and the folding and

unfolding of sections of the polypeptide chain.

33

How does FAD control PutA conformation

and function?

Yellow

Oxidized EcPutA PRODH domain

(PDB code 1TIW)

Gray

Reduced EcPutA PRODH domain

(PDB 3ITG)

Structural changes induced by reduction of the FAD in PutA

Structural changes: 2’-OH group rotates 90o

FAD(N)5-R431 and

R431-D370 interactions are

disrupted

Srivastava et al. 2010 Biochemistry 49:560. Tanner and Becker, Proline Metabolism and PutA. Handbook of Flavins, (2012) in press.

putC:lacZ

PutA R431M

PutA + pro

- PutA

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

FAD N(5)-R431 is important for activation of membrane binding

Removal of 2’-OH group (2’-deoxyFAD) and D370 (D370A) also disrupt membrane

binding

N(5) Position is Critical

35

Working model of redox switch in PutA

FAD reduction leads to release of C-terminal peptide

Summary • Flavins support one-electron and two-electron transfer processes

involving the N(1), C(4a) and N(5) positions of the isoalloxazine ring system.

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

• Proline/PRODH generates mitochondrial ROS and influences signaling pathways that determine cell survival and cell death outcomes

• PutAs have fused PRODH and P5CDH domains that are connected by a substrate channeling cavity

• FAD reduction induces conformational changes in EcPutA that lead to activation of membrane binding

• Hydrogen bonding interactions at the flavin N(5) are disrupted in reduced structures of the EcPutA PRODH domain

• Redox signals originating at the flavin N(5) are likely transmitted to the surface of EcPutA via internal hydrogen bond rearrangements

• C-terminal domain of EcPutA may be involved in membrane binding