Protein Film Electrochemistry

Fraser Armstrong Department of Chemistry, Oxford University

Introduction to Protein Film Electrochemistry

The concept: investigating electron transfer proteins attached to an electrode

Measure catalytic current = turnover rate* and lock rates to potential (E)

Control chemistry by modulating electrode potential and spinning the electrode

e.g. hydrogenase

electrons

catalytic voltammetry

individual centres

gas binding

E-step kinetics of changes in states

H2, H+

CO, X, Y, Z

Under hydrodyamic control

Water in

Water out

E lectrode rotator

P t counter electrode

R eference electrode

G as out

A B

G as flow meters

G as in

O ptional light source enzyme

PGE electrode

reactant product

e-

Water in

Water out

E lectrode rotator

P t counter electrode

R eference electrode

G as out

A B

G as flow meters

G as in

O ptional light source enzyme

PGE electrode

reactant product

Water in

Water out

E lectrode rotator

P t counter electrode

R eference electrode

G as out

A B

G as flow meters

G as in

O ptional light source enzyme

PGE electrode

reactant product

e-

The sealed electrochemical cell with rotating disc electrode, controlled gas flow, and injection port

In most experiments protein is adsorbed on rotating disc electrode of area 0.03 cm3. Less than 10 femtomole of enzyme is addressed, and numerous consecutive experiments can be conducted on same sample.

Enzyme being applied to electrode surface

Simple and coupled electron transfers

Normalised current

Simple Electron Transfer

O + n e- R

n x potential/volts

Signals from redox centres are sharp and finite. For reversible case, Nernst equation predicts

δ α 1/n, ipeak α n2

O + n e- RSimple Electron Transfer

reduction

oxidation

normalised current

-1

-0.5

0

0.5

1

-0.3 0 0.3

δ potential /V

-0.15

0

0.15

-1 -0.5

[3Fe-4S]+/0

[4Fe-4S]2+/+

[3Fe-4S]0/2-

Potential/ V vs SHE

Current/µA

subtract baseline

Duff et al. JACS 118, 8593 (1996).

Early application was to look at FeS clusters

The simplest transformation between Fe-S clusters is addition of a metal ion (such as Fe) to [3Fe-4S].

[3Fe-4S] [M3Fe-4S]

Butt et al. JACS, 113, 6663 (1991).

Rapid transformations of Fe-S clusters in a small protein – an early study

0.0 0.2 0.4 0.6-6

-3

0

3

6

Curre

nt /

(µA)

Potential / (V vs NHE)

Azurin: layer on Au electrode modified with 1-decanethiol SAM Scan rate 10 volts/sec

Plot of peak potentials vs scan rate

‘Trumpet plot’ fit gives k0 = 320 s−1.

The Basics: Electron transfer kinetics reductive pre-polarisation oxidative pre-polarisation

Hirst & Armstrong, Anal Chem.70, 5062 (1998) Jeuken & Armstrong. J. Phys. Chem. 105, 5271 (2001)

0.01 0.1 1 10 100 10000.30

0.35

0.40

0.45

Peak

Pot

entia

l / (V

vs S

HE)

Scan Rate / (V s-1)

-1.5

-1

-0.5

0

0.5

1

-0.3 0 0.3

Coupled reactions perturb the signal: detection of coupled gated electron transfer

[3Fe-4S]1+ [3Fe-4S]0

[3Fe-4S]1+

e-

H+

H

Fast scan rate red voltammogram recorded at pH<pK

Interpreting ‘Trumpet Plot’ for coupled/gated ET reaction: example is a coupled proton transfer

-0.1

-0.05

0

0.05

0.1

0.15

0.01 0.1 1 10 100

Peak potential /Volts

Scan rate / V s-1

‘potentiometric’ region

gating region

uncoupled ET region

pH << pKR

pH >> pKR

Nature 405, 814 (2000)

Electrocatalysis by enzymes

position of membrane

Possible structure of Hyd-1 in membrane

cytochrome b in labe γ subunit(s)

Volbeda et al. PNAS 2012 Structure 2013

Structure of Hydrogenase-1 (Hyd1) from E. coli. Typical of enzymes now studied by PFE.

k E

k cat

e-

e-

e-

electrode

substratesox red

cur r ent

Enzymes as modular electrocatalysts

genetic engin. spectroscopy crystallography PFE... ....binding acid-base PCET long-range ET redox potentials surface attachment orientation ?

Use rough electrode surface such as pyrolytic graphite edge (PGE)

Define electrocatalysts as those catalysing half-cell reactions. A ideal electrocatalyst will show ‘reversible’ behaviour (blue trace).

electrons

electrode

electrocatalyst

The ‘red’ voltammogram is for an electrocatalyst with a large overpotential requirement.

Ox Red

Living organisms Redox reactions are basis of energy conversions. Chains of reactions occur rapidly within a narrow free energy range. Reactions are usually catalysed by electron-transport enzymes that catalyse half-cell reactions

0 V

O2 /H2O

CO

NADH H2 QH2

cyt c

-1 V 1 V fats, CHO

FAD

turnover rate(current)

driving potential

Electrode potential

Protein film electrochemistry measures efficiency of redox enzymes

F. A. Armstrong & J. Hirst, PNAS 108, 14049 (2011)

Ni

CO

CN-

CN- Fe

Glu

Arg

His

Otherwise only reversible at Pt Here we see H2 cycling at pH 6, 1 atm H2, at a carbon electrode modified with a [NiFe]-hydrogenase.

Electrode potential / V vs SHE

Cur

rent

/ µA

Estimated range for H2ase active site based on data for several different enzymes and assumptions on electroactive coverage

How active are hydrogenases as electrocatalysts? Where would [FeFe]- and [NiFe]-active sites lie on an volcano plot for electrochemical activation, noting the very large footprint of their host environments?

hydrogenases

Vincent, Parkin & Armstrong Chemical Reviews 107, 4366-4413 (2007).

A unified model for surface electrocatalysis based on observations with enzymes Suzannah V. Hexter, Thomas F. Esterle and Fraser A. Armstrong PhysChemChemPhys. (2014) DOI: 10.1039/c3cp55230f

potentialdependent

potentialindependent

potentialindependent

Primary event:electrons enter or exit enzyme

Secondary event:low potential-substrate isreduced

Secondary event:high potential-substrate isoxidized

Note: absolute k2 values may depend on potential

k1 values set potential at which electrons enter or leave enzyme

k2 values determine how fast electrons are used within enzyme

The simplest model

Cur

rent

/ µA

Electrode potential / V vs SHE

[NiFe]-hydrogenases show three regions of characteristic activity.

H2 oxidation

H2 production

Lukey et al. JBC 2010 Armstrong & Hirst PNAS 2011

Cyclic voltammogram of a hydrogenase measured under 1 atm H2

Each region can be probed to establish kinetics of reactions that affect catalysis

Fits 2H+/H2 electrocatalysis by different hydrogenases over small temp. range. Hexter et al. PNAS 109, 11516 (2012)

[FeFe]

[FeFe]

[NiFe]

[NiFe] (O2-tolerant)

T

Multi-state model for an (enzyme) electrocatalyst – catalytic activity depends on potential controlling crucial step of catalysis and prevailing oxidation states of sites

Analogous off/on switch processes modeled for enzyme and surface catalysts

-0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

i norm

E (V vs. SHE) -0.2 0.0 0.2 0.4 0.6 0.8-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

i norm

E (V vs. RHE)

Ru (0001) [NiFe]-hydrogenase (Hyd-2)

NiIII-OH

Run+-OH

Hexter, Esterle & Armstrong PCCP in press

Test case 1 [NiFe]-hydrogenases

position of membrane

Possible structure of Hyd-1 in membrane

cytochrome b in γ subunit(s)

Volbeda et al. PNAS 2012 Structure 2013

Structure of Hydrogenase-1 (Hyd1) from E. coli. The enzyme is easily prepared and engineered.

Volbeda et al. PNAS 2012 Roessler et al. JACS 2012

The Fe-S electron relay of Hyd-1

Wt Ec Hyd-1, pH6.0 at 30 °C, 1000 scc/min total flow rate, Ar carrier gas, electrode rotation rate 3500 rpm: see Evans et al. JACS 2013

For an O2-tolerant [NiFe]-hydrogenase, O2 acts as a competitive inhibitor, H2 oxidation activity is attenuated not destroyed

100% 10% 100% H2 level:

When Hyd-1 variant with mutated defect proximal and medial Fe-S clusters is exposed to just 1% O2, the catalytic H2 oxidation current drops to zero and no activity is regained spontaneously when O2 removed.

NiII FeS

S

SSH CO

CN

CN

Cys

CysCys

Cys

NiIII FeS

S

SSH CO

CN

CNHO

Cys

CysCys

CysO2

3 electrons

1 electron

H2

H2O

2 H+

H2O

NiIII FeS

S

SSH CO

CN

CNHO

Cys

CysCys

Cys

O2

< 3 electrons

[O]

catalyst is damaged

fast

cycling

O2-tolerant [NiFe]-hydrogenases are H2/O2 oxidases – catalysing wet combustion

O2-tolerant respiratory [NiFe]-hydrogenases catalyse 2 H2 + O2 2 H2O

P. Wulff, C. C. Day, F. Sargent, and F. A. Armstrong PNAS, 111, 6606-6611 (2014).

H218O by MS

H2O2 /O2- by

chemical analysis

How the proximal [4Fe-3S] cluster defends active site against O2

normal electron transfer during H2 oxidation

O2 attack ! two electrons fired back rapidly from proximal cluster

This assumes O2 attacks active site in reduced state

[4Fe-3S] 4+/3+ [4Fe-3S] 5+

C17

C120

C149

E76

C20 C115

C120

C149

C115 E76

C19

C20

C17 C19

Proximal cluster can transfer two electrons

Higuchi et al. Nature 2011, Volbeda et al. PNAS 2012

[Fe4S3(SR)6]3-

EPR EPR 30 mV 230 mV [Fe4S3(SR)6]2- [Fe4S3(SR)6N]2-

Vincent et al PNAS 102, 16951 (2005).

gadget

ANODE – O2-tolerant hydrogenase

CATHODE – Bilirubin oxidase or laccase

Air with low-level H2 H2 O2

electrolyte/gel overlays two electrodes

electrons electrons

Concept - Miniature H2 fuel cells – no membranes

The Hydrogen House - Christmas 2013

Powered by membrane-free enzyme fuel cell operating on non-explosive H2/O2 mixture

See Xu and Armstrong, Energ.Env. Science 2013

-30mV/decade ρ(H2) -60 mV/pH

e

ELECTRODE

e-

e-

H2 2H+

0 mV/ ρ(H2) ~0 mV/pH

How E.coli makes H2 ? The effect of ρ(H2) and pH on Hyd-1

600s-1

Murphy et al. EES in press

500 s-1 500 s-1

Excellents fits are obtained using basic model of Hexter et al PNAS 2012, in which electron entry-exit site determines direction and onset potential

Cyanide inhibits [NiFe]-hydrogenases, How is this observation interpreted and followed up?

Cycle number. KCN injected at start of 3

-0.6 -0.4 -0.2 0.0 0.2 0.4

0

1

2

3

4

5

HCN

86543

1,2

curre

nt (µA

)

E (V vs. SHE)

12

EcHyd1

KCN solution

Test case 2 [FeFe]-hydrogenases

Cys503

[4Fe-4S]

CO

CN

CN

CO

H2O

A

CN

CN

CO

COCys382

[4Fe-4S]

FePFed

B

CO

COFed

FeP

Cys503

[4Fe-4S]

CO

CN

CN

CO

H2O

A

CN

CN

CO

COCys382

[4Fe-4S]

FePFed

B

CO

COFed

FeP

Hox in the hydrogenase from Clostridium pasteurianum (CpI) shown in oxidised active state (formally FeI, FeII)

Hred in the hydrogenase from Desulfovibrio desulfuricans DdHydAB shown in reduced state (formally FeI, FeI)

[FeFe] hydrogenases react irreversibly with O2

I II

I

I Postulate: Hox binds H2, CO (and O2) Hred binds H+

Stripp et al. PNAS 106, 17331-17336 (2009)

0

0.1

0.2

0 50 100 150[Inhibitor] / µM

k i/ s

-1CO

O2

B

0

0.1

0.2

0 50 100 150[Inhibitor] / µM

k i/ s

-1CO

O2

B

0.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Cur

rent

10%

O2

in (i

ii)

Flus

h ou

t O2

(i)

Flus

h ou

t CO

(ii,

iii)

A 0 2000 3000 40001000

0.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Cur

rent

10%

O2

in (i

ii)

Flus

h ou

t O2

(i)

Flus

h ou

t CO

(ii,

iii)

A 0 2000 3000 40001000

20% N2

20% N2

10% CO

0 2000 3000 4000

Time / s

10% CO(ii) Inhibition

(iii) Protection

1000

20% N2

10% O2

(i) Inactivation

10% O2

20% N2

20% N2

10% CO

0 2000 3000 4000

Time / s

10% CO(ii) Inhibition

(iii) Protection

1000

20% N2

10% O2

(i) Inactivation

10% O2

Stripp et al. PNAS 106, 17331-17336 (2009)

These experiments, (in which catalytic current is recorded as the gas composition is changed) -measure the ability of CO (a reversible, competitive inhibitor of [FeFe]-hydrogenases) to protect against irreversible damage by O2

Formaldehyde is a reversible inhibitor of H+ reduction

•Final concentration 44.7 mM formaldehyde (20 µM anhydride) •Cell rinsed with 50 mL of buffer at 900 s (exchange takes approx. 1 min) •Conditions: 10 °C, 80% H2 / 20 % N2, pH 6, 2500 rpm,

After 300 s in the presence of 44.7 mM formaldehyde, H+ reduction activity is fully recoverable

Wait et al JACS 2011

Inject formaldehyde

Rinse cell with 50 mL buffer Ca HydA

H+ reduction at -556 mV

curr

ent

time/s

The potential dependence of inhibition by HCHO is complementary to that of CO inhibition. Data fit to two n=1 processes, linking prevailing states shown

Increase HCHO level

Hox Hox-1 Hox-2

Foster et al. JACS 2012

Ru

H N

NH

X(R3P)2

H H

O

H-cluster ?

Noyori

Formaldehyde blocks H2 evolution by H-cluster

Foster et al. JACS 134, 7553 (2012).

Test case 3 Carbon monoxide dehydrogenase

Catalysed at active site of carbon monoxide dehydrogenase

[Ni4Fe-4S] cluster with CO2 bound

The simplest CO2 reduction reaction CO2 + 2 e- + 2 H+ CO + H2O

E 0 = -0.53 V at pH 7

CO2/CO interconversion by CODH attached to an electrode

Inhibition of CODH I by cyanide CO oxidation CO2 reduction

-0.8 -0.6 -0.4 -0.2 0.0 0.2-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

i norm

E (V vs. SHE)

Cyanate (NCO−), an analogue of CO2 , binds to most reduced state, called Cred2

NCO− blocks CO2 reduction and introduces onset overpotential for CO oxidation

Hexter et al.PCCP in press

Potential-selective binding of substrates and inhibitors to CODH

How inhibitors intercept CODH catalysis

Hydrogenases since 2010 Gabrielle Goldet Carina Foster Suzannah Hexter Michael Lukey Bonnie Murphy Maxie Roessler (now lecturer at QMULondon) Sara Wehlin Annemarie Wait Philip Wulff Lang Xu Dr Kylie Vincent (now Ast Prof. at Oxford) Dr Erwin Reisner (now Ast Prof. at Cambridge) Dr Rhiannon Evans Dr Alison Parkin (now lecturer at York) Dr Gopan Krishnan* (now Ast. Prof at Okla. State)

Collaborations Jeffrey Harmer, Oxford Juan Fontecilla-Camps, Grenoble Bärbel Friedrich, Oliver Lenz, HU Berlin Thomas Happe, RU Bochum Steve Ragsdale (Michigan State) Frank Sargent, Dundee John McGrady, Oxford

Funding BBSRC, EPSRC Supergen ESF

Acknowledgements