barton et al, chem rev, 104 4867 (2004) (special …bip.cnrs-mrs.fr/bip06/pdf/leger_ise2010.pdf ·...

Marseille

Nice

Southampton

FR

UK

Laboratory of Bioenergetics

and Engineering of Proteins

CNRS, Marseille

Slides can be downloaded at

http://bip.cnrs-mrs.fr/bip06/publications




Barton et al, Chem Rev, 104 4867 (2004) (special issue on Batteries and Fuel cells)

I. Background: the structures of redox enzymes and their cofactors

II. Electrodes for direct electron transfer (ET) to proteins and enzymes

III. « Non catalytic » voltammetry

• at slow scan rates: measuring reduction potentials

• at fast scan rates: measuring rates of interfacial ET and coupled chemical events

IV. Catalytic voltammetry for measuring the enzyme’s activity & studying the mechanism

• principle

• learning about the mechanism from steady-state measurements

• deviation from steady-state

V. Using redox enzymes

• Limitations

• Fuel cells and photoelectrochemical cells

• Heterogenesous catalysts based on conducting particles

• Biosensors

I) Background: structural biology

Proteins (= polypeptides) are linear polymers made of

hundreds of α-aminoacids joined by peptides bonds

There are 21 natural α-aminoacids

which differ by their side chain « R »average Mw of an amino acid = 130 Da

Mw of a protein in the range 10-1000 kDa

Amino Acid Short name Abbrev. Side chain R Hydrophobic pKa Polar

Alanine A Ala -CH3 X - -Cysteine C Cys -CH2SH X 8.18 -Aspartic acid D Asp -CH2COOH - 3.90 X

Glutamic acid E Glu -CH2CH2COOH - 4.07 X

Phenylalanine F Phe -CH2C6H5 X - -

Glycine G Gly -H X - -

Histidine H His -CH2-C3H3N2 - 6.04 X

Isoleucine I Ile -CH(CH3)CH2CH3 X - -

Lysine K Lys -(CH2)4NH2 - 10.54 X

Leucine L Leu -CH2CH(CH3)2 X - -

Methionine M Met -CH2CH2SCH3 X - -

Asparagine N Asn -CH2CONH2 - - X

Proline P Pro -CH2CH2CH2- X - -

Glutamine Q Gln -CH2CH2CONH2 - - X

Arginine R Arg -(CH2)3NH-C(NH)NH2 - 12.48 X

Serine S Ser -CH2OH - - X

Threonine T Thr -CH(OH)CH3 - - X

Selenocysteine U Sec -CH2SeH X 5.73 -

Valine V Val -CH(CH3)2 X - -

Tryptophan W Trp -CH2C8H6N X - -

Tyrosine Y Tyr -CH2-C6H4OH - 10.46 X

http://en.wikipedia.org/wiki/Proteinogenic_amino_acid

http://en.wikipedia.org/wiki/Alanine

http://en.wikipedia.org/wiki/Cysteine

http://en.wikipedia.org/wiki/Aspartic_acid



http://en.wikipedia.org/wiki/Glutamic_acid



http://en.wikipedia.org/wiki/Phenylalanine

http://en.wikipedia.org/wiki/Glycine

http://en.wikipedia.org/wiki/Histidine

http://en.wikipedia.org/wiki/Isoleucine

http://en.wikipedia.org/wiki/Lysine

http://en.wikipedia.org/wiki/Leucine

http://en.wikipedia.org/wiki/Methionine

http://en.wikipedia.org/wiki/Asparagine

http://en.wikipedia.org/wiki/Proline

http://en.wikipedia.org/wiki/Glutamine

http://en.wikipedia.org/wiki/Arginine

http://en.wikipedia.org/wiki/Serine

http://en.wikipedia.org/wiki/Threonine

http://en.wikipedia.org/wiki/Selenocysteine

http://en.wikipedia.org/wiki/Valine

http://en.wikipedia.org/wiki/Tryptophan

http://en.wikipedia.org/wiki/Tyrosine

The sequence of aminoacids in each protein

(also called primary structure)

is defined by the DNA (by the sequence of the gene)

Example: the sequence of the small protein of the enzyme « hydrogenase »ltakhrpsvvwlhnaectgcteaairtikpyidalildtisldyqetimaaageaae

aalhqalegkdgyylvvegglptidggqwgmvaghpmiettkkaaakakgiicigtc

sayggvqkakpnpsqakgvsealgvktinipgcppnpinfvgavvhvltkgipdlde

ngrpklfygelvhdncprlphfeasefapsfdseeakkgfclyelgckgpvtynncp

kvlfnqvnwpvqaghpclgcsepdfwdtmtpfyeqg

Site-directed mutants are proteins in which one or several

amino acids have been changed, using genetic

engineering

4 different representations of the 3D structure of an enzyme:

NiFe hydrogenase

Space-filling

model

Sticks Backbones Secondary structures

• Complex of two proteins (one large & one small)

• 90 kDa

• 8nm diameter

• 800 Amino acids, 10000 atoms

including 1 Ni and 12 Fe

α-helices

The protein may bind redox cofactors (= prosthetic groups)

required for the protein’s biological activity

FAD Heme Copper site FeS cluster

The protein may bind redox cofactors (= prosthetic groups)

required for the protein’s biological activity

Organic cofactors (Mw < 1 kDa), e.g NAD(P), quinones,

FAD (flavin)

e.g. in the enzyme « glucose oxidase »

quinone (oxidized) /

semiquinone (half reduced) /

hydroquinone (2-electron reduced)

ISE 2010: electrochemistry of cytochromes, see the talks of

Sean J. Elliott, Tuesday, Symp 3, 11:20, Yoon-Bo Shim, Tuesday, symp3, 17:20

Franziska Wegerich, Thursday, symp 11, 15:20, G. Almeida, Monday, symp 3, 11:20

Organic cofactors with bound metal ionsHemes = Fe bound to porphyrin,

in proteins (e.g. cytochrome c, hemoglobin) or enzymes (e.g.

peroxydases)

here the heme of a c-type cytochrome

FeIII/II

His

Met

Inorganic cofactors, e.g. copper sites

in proteins (e.g. azurin) or enzymes (e.g. laccase, billirubin oxidase)

here the type 1 (« blue ») copper site of azurin

His

His

Cys

Met

CuII/I

ISE 2010: electrochemistry of multicopper oxidases, see the talks of

Y. Beyl, Wednesday, Symp 14, 09:40, P. Atanassov, Wednesday, Symp 14, 10:40,

S. Shleev, Monday, Symp 3, 11:00

Inorganic cofactors, e.g. FeS clusters

in proteins « ferredoxins » and enzymes (e.g. hydrogenases)

here a typical [4Fe4S] cluster, the most common FeS cluster

Also [2Fe2S], [3Fe4S] and also much more complex clusters…

[4Fe4S]2+ /+

(2Fe3+,2Fe2+) : [4Fe4S]2+

(Fe3+,3Fe2+): [4Fe4S]+

Cys

Cys

Cys

Cys

4 Fe ions

The biological redox scale at pH 7

(in OEC)

Hydrogenases oxidize thousands of H2 per second

H2 (the substrate of the reaction) → 2H++2e- (the products)

Active site chemistryProton transferElectron transfersIntramolecular diffusion (H2, CO, O2)

Ni

Fe

The chemical reaction occurs at an active site

(may consist of a few amino acids, or a cofactor)

Other cofactors are used for electron transfers

amino acids and water molecules for proton

transfers

Take home message 1

Redox enzymes are large, complex

and very efficient molecular catalysts

Efficient interfacial electron transfer requires that the active

site or at least one redox cofactor is exposed at the surface

of the protein

II) Electrodes for adsorbing redox proteins and enzymes.Case study: adsorbing hydrogenases

Connection with electrodes at the distal cluster

ISE 2010: see the talk of Elisabeth Lojou: Monday, symp 3, 14:40

Examples of electrodes for wiring hydrogenases

1) Au covered with carbon black

INTRODUCTION: « The investigation of enzymes as catalysts for

electrochemical processes is a novel area of chemical enzymology. (…)

Besides, it is also of interest to study electrochemically the mechanism of the

redox enzyme action ».

ISE 2010: see the talk of Arkady Karyakin, Wednesday, Symp 3, 10:00

Yaropolov et al, Bioelectrochem Bioenerg. 12 267 (1984)

Amstrong et al., Chem. Soc. Rev. 26 169 (1997)

Blanford et al., J. Solid. State Electrochem. 10 830 (2006)

SEM image after abrasion Formation of a proteic filmGraphene layers

2H+

electrons

H2

1μm


2) Pyrolytic graphite “edge”

2H+

electrons

H2

H2ase de A. vinosum adsorbed on stationary PGE electrode,

under 1 atm. Ar

Pershad et al, Biochemistry 38 8992 (1999)



1 atm H2, RDE, fast scan rate: 200mV/s (one cycle in 8s)

Léger et al., Biochemistry 41 15736 (2002), J. Phys. Chem. B 106 13058 (2002)



1atm H2, RDE, slow scan rate: 0.3mV/s (this cycle in 1h30)

Jones et al., J. Am. Chem. Soc. 125 8505 (2003)

Fourmond et al., J. Am. Chem. Soc. 132 4242 (2010)



Inactive

Oxidation

Reduction

Active

Glutamate residues near the surface

exposed 4Fe4S cluster (carboxylates)

Favors favorable orientation

Rüdiger et al., J. Am. Chem. Soc. 127 16008 (2005)

NO2

NO2

NO2

Allongue et al.,

J. Am. Chem. Soc. 119 201

(1997)

Protein SurfaceCarbodiimide

Amide bond


3) Covalent linkage on functionalized graphite

Carbodiimide coupling for covalent attachment


3) Covalent linkage on functionalized carbon nanotubes

Alonso-Lomillo et al., Nano Letters 7 1603 (2007)

Adsorption

Covalent grafting

H2ase de D. gigas, pH 7, 1 atm H2, E=-280mV

50μm

5μm

ISE 2010: see the poster of Cristina Gutiérrez-Sánchez, s14-P-001

Lojou et al., J. Biol. Inorg. Chem, 14 127500 (2009)

ISE 2010: see the talk of Elisabeth Lojou: Monday, symp 3, 14:40

Rüdiger et al, Electroanalysis 22 776 (2010)

Hydrogenase covalently attached to a SAM of 4-amino-thiophenol

ISE 2010: see the poster of Cristina Gutiérrez-Sánchez, s14-P-001


4) Covalent linkage on functionalized gold

Examples of electrodes for wiring

hydrogenases

5) SAM on Au

→ ready for in situ spectroscopy

D. vulg

aris

Miy

azaki H

2ase, pH

5.5

, 1m

V/s

Millo et al, J. Phys. Chem. B 113 15344 (2009)

Self assembled

monolayer

(SAM) of thiols

HS-(CH2)6-NH2

Ar

H2

Hoeben et al., Langmuir, 24 5925 (2008)

Au + PM + H2ase A. vinosum,

pH 9, 1mV/s

0.13 - 1.3 pmol/cm2

(from kcat en solution)

Mica + Au(111) + PM + H2ase A. vinosum

Image AFM (tapping mode, in air)

0.23 pmol/cm2

PM


6) Gold nanoelectrode covered with polymyxin

ISE 2010: see the poster of H.A. Heering s14-P-026

Hoeben et al, ACS NANO 12 2497 (2008)A vinosum H2ase, pH 6, 1.5mV/s

Si + 500nm SiO2 + 30nm Au

+ 300nm PMMA

100x100 nm2


6) Gold nanoelectrode covered with polymyxin

→ studying hydrogenase at the single molecule level

(nearly there)

ISE 2010: see the poster of H.A. Heering s14-P-026

(1 atm Ar)

hydrophobic physiological substrate, interacts with T1 copper site

Blanford et al, Chem Comm 1710 (2007)

Mono- vs multilayers: see M. Sosna et al, Phys. Chem. Chem. Phys 12 10018 (2010)

An electrode for wiring laccasesReviews: M. Sosna et al, Phys. Chem. Chem. Phys 12 10018 (2010), and also Shleev et al, Biosens Bioelectron 20 2517 (2005)

7) anthracene mono(?)layer on PGE or GC

ISE 2010: talks of T. Ruzgas, Mon, Symp 3, 14:00, & S. Shleev, Mon, Symp 3, 11:00

8) Electrodes for plugging into

heme-enzymes, e.g. horseradish peroxydase (HRP)

L. Gorton et al, Anal. Chim. Acta 400 91 (2000)

H. Zimmermann et al, Chem. Eur. J. 6 592 (2000)

FAD- and NADH-dependent enzymes: Willner et al, J. Am. Chem. Soc. 118 10321 (1996)

Kayats et al, J. Am. Chem. Soc. 124 14724 (2002)

9) Using natural or engineered surface exposed

cysteins or histidines for attachment on goldhere horseradish peroxidase

Kartashov et al, PCCP 12 10098 (2010)

Hasan et al, J. Biol. Inorg. Chem. 11 651 (2006)

ISE 2010: see the poster of E. Ferapontova s03-P-047

Take home message 2

Adsorbing the protein or enzyme may be easy,

You must make sure that it is not denatured

by the proximity of the electrode surface

- redox proteins:is the reduction potential consistent with independent determinations (if

available, e.g. potentiometric titrations followed by spectroscopy)

- redox enzymes: simply add the substrate!

is the enzyme still able to catalyse the physiological reaction at a

significant rate, and in a reasonable range of electrode potential?

Part III.

Non catalytic voltammetry (redox proteins and enzymes)

Slow scan voltammetry to measure reduction potentials

Fast scan voltammetry to measure rates of interfacial ET,

Fast scan to measure rates of reactions coupled to ET

Voltammetry of a diffusive redox species

Bard and Faulkner, Electrochemical methods, Wiley, 2004

Example: measuring the reduction potential of a cytochrome

Rhodopseudomonas

palustris

cytochrome c2

~100 residues,

1 surface exposed

heme,

Mw=14kDa

A single redox transition

FeIII/FeII

Geremia et al, Protein Sc. 11 6 (2002)

Example: measuring the reduction potential of a cytochrome

Rhodopseudomonas

palustris

cytochrome c2

at a 4-mercaptopyridine surface-

modified Au electrode

Peak separation

59±2 mV

C=0.2mM,

V=0.5mL,

that is 100nmol of

protein (1.5mg)

Battistuzzi et al, Biochemistry 36 16247 (1997)

Voltammetry of an adsorbed redox species

Bard and Faulkner, Electrochemical methods, Wiley, 2004

Example : a type 1 « blue » copper site

Pseudomonas

aeruginosa azurin

~120 residues,

1 surface exposed

copper site

Mw=14kDa

a single redox transition

CuII/CuI

Example : a type 1 « blue » copper site

Pseudomonas

aeruginosa azurin

adsorbed on PGE

CV recorded at

ν=50mV/s

Jeuken et al. J. Phys. Chem. B. 106 2304 (2002)

ISE 2010: see the talk of Lars Jeuken, Monday, Symp 3, 17:20

The case of a single two-electron redox couple (e.g. FAD)3 redox states O (ox), I (intermediate) and R (reduced)

E0O/I - E0

I/R = 0.4 to -.2 V

Plichon and Laviron, J. Electroanal. Chem. 71 143 (1976)

Example: the 2-electron signal of the flavin in

glucose oxidase

Aspergillus niger

glucose oxidase

~600 residues,

70kDa

1 buried FAD

4 glycosylation sites

16-25 wt %

pdb 1CF3 , Wohlfahrt et al, Acta Crystallogr. D. 55 969 (1999)

ISE 2010: see the talk of Nicolas Mano: Monday, Symp. 3, 10:00

ISE 2010: see the talk of Nicolas Mano: Monday, Symp. 3, 10:00

Example: the 2-electron signal of the flavin in

glucose oxidase

Courjean et al, Angew. Chem. Int. Ed. 48 5897 (2009)

(Adsorbed enzyme catalyses glucose oxidation)

Aspergillus niger

glucose oxidase

~600 residues,

70kDa

1 buried FAD

at a glassy carbon electrode

after enzymatic

deglycosylation

CV recorded at ν=20mV/s

WHH=52mV

hence 2-electron

2-electron signals from disulfide bonds

Ex. E. coli thioredoxin, ~100 residues, 2e-/2H+ transferring protein

pdb 2TRX, 3TRX, Katti et al, J. Mol. Biol. 212 167 (1990)

OxidizedReduced

ISE 2010: see the talk of Sean J. Elliott, Tuesday, Symp 3, 11:20

2-electron signals from disulfide bonds

Comparison of various thioredoxins adsorbed on PGE

ISE 2010: see the talk of Sean J. Elliott, Tuesday, Symp 3, 11:20

Chobot et al, Angew. Chem. Int. Ed. 46 4145 (2007)

-60mV/pH

Azodobacter vinelandii

ferredoxin I

~ 100 residues

2 redox centers

[3Fe4S] +/0 and 0/2-

and [4Fe4S] 2+/+

(3 redox couples)

Schipke et al, Biochemistry 38 8228 (1999)

Example: a ferredoxin containing [3Fe4S] and [4Fe4S] clusters

Example: a ferredoxin containing [3Fe4S] and [4Fe4S] clusters

Azodobacter vinelandii

ferredoxin I

~ 100 residues

2 redox centers

[3Fe4S] +/0 and 0/2-

and [4Fe4S] 2+/+

(3 redox couples)

Here at a PGE electrode

Armstrong et al, Biochem. J. 264 265 (1989)

Iverson et al, Science 284 1961 (1999)

Example

E. coli fumarate

reductase,

2 proteins600+200 residues

Mw=100kDa

1FAD,

[2Fe2S]

[4Fe4S]

[3Fe4S] surface exposed

In the case of large enzymes, non catalytic signals are small


Léger et al, Biochemistry 2001

Example

E. coli fumarate

reductase,

2 proteins600+200 residues

Mw=100kDa

1FAD,

[2Fe2S]

[4Fe4S]

[3Fe4S] surface exposed

Here at a PGE electrode

pdb 3ABG, Mizutani et al, Acta Crystallogr. F, 66 765 (2010)

Example

Billirubin

oxidoreductase

500 residues

1 trinuclear copper site

(active site for O2

reduction)

1 surface exposed

type-I copper site

(for electron transfer)


Ramirez et al, Biochim Biophys Acta, 1777 1364 (2008)

ISE 2010: see the talk of Sergey Shleev, Monday, Symp 3, 11:00

Example

Billirubin

oxidoreductase

500 residues

1 surface exposed

type-I copper site

(for electron transfer)

1 trinuclear copper site

(active site for O2

reduction)


here on bare spectrographic graphite


The relation with protonation and ligand binding


Measuring rates of chemical reactions coupled to ET

Part III.


Reduction potentials are affected by ligand binding

ligand = proton, substrate, inhibitor, or even the apo-protein

one-electron one-proton coupled electron transfer:

« square scheme » and the corresponding pourbaix diagram

n-electron reduction coupled to the uptake of m protons:

Example: the effect of pH on the reduction potential of

the type-1 copper site in azurin

Jeuken et al., J. Biol. Inorg. Chem. 4 257 (1999)

ISE 2010: see the talk of Lars Jeuken, Monday, Symp 3, 17:20

Example: complete pourbaix diagram of a

« Rieske-type » 2Fe2S ferredoxin

Zu et al, JACS 123 9906 (2001)



The relation with electron transfer rates



Part III.


Intramolecular electron transfer chain in redox enzymes

are sometimes « roaller coasters »

E.g. NiFe hydrogenase:

E0+50mV

E0-350mV

E0-350mV[4Fe4S]2+/+

[4Fe4S]2+/+

[3Fe4S]+/0

Page et al, Nature 402 47 (1999)

Electron transfer betwen two distant redox centers

Marcus’ « inverted region » is very important in biology

Dutton et al, J. Bioenerg. Biomemb. 27 263 (1995)

Interfacial electron transfer, betwen a metallic electrode and a redox center

Electron transfer betwen a metallic electrode and a redox center

Chidsey, Science 251 919 (1991), plotted by Jeuken in Biochim Biophys Acta 1604 67 (2003)

η=E-E0 (V)

(ferrocene terminated thiols self-assembled on Au)






Part III.


ΔEp=0 at small scan rate

(ideally, but residual

small peak separation is

common)

ΔEp is all the greater than

ν is large and/or k0 small

The peaks separate

when

NB: since ΔEp depends on

log(ν/k0), only the order of

magnitude of k0 can be

determined

Measuring k0 from peak splitting against scan rate

Laviron, JEAC 101 19 (1979)

Hirst et al, Anal. Chem. 70 5062 (1998)

Reported values of k0 range from…

very small (~ 0.1s-1) or slower

to very large (up to ~ 10 000 s-1)

Exemple

c555m=

type-1cytochrome

from A. aeolicus.

has a flexible tail

(62 residues)

with terminal cysteine

c555s = soluble version,

no tail

Baymann et al, FEBS Letters, 539 91 (2003)

SAM of hexanethiol on gold

Reported values of k0 range from…

very small (~ 0.1s-1) or slower

to very large (up to ~ 10 000 s-1)

Baymann et al, FEBS Letters, 539 91 (2003)

Two different attachements of the same cytochrome c555

c555m = with the tail attached to Au

c555s = no tail (cleaved before adsorption)

0.01 0.1 1 10 100 1000

0.1

0.2

0.3 c555s

c555m

peak p

ositio

n (

mV

)

scan rate (V/s)

@ 8V/s





Electronic coupling with the electrode

Reorganization energies and gated electron transfers


Part III.


How k0 depends on distance from the

electrode

k0 measured by electrochemistry or spectroelectrochemistry in: Song et al, J. Phys. Chem. 97 6564 (1993)

Murgida et al, JACS 123 4062 (2001), Chi et al, J. Phys. Chem. B 105 3088 (2001)

Reviewed in: Jeuken, Biochim Biophys Acta 1604 67 (2003)

Murgida et al, ChemPhysChem 11 1225 (2010)

How k0 depends on distance from the electrode

Conformational rearrangement or thermal fluctuations

may limit the rate of (i.e. « gate ») interfacial ET

Reviewed in: Jeuken, Biochim Biophys Acta 1604 67 (2003)

Chi et al, PNAS 102 16203 (2005)

Murgida et al, ChemPhysChem 11 1225 (2010)

ISE 2010: see the talk of Q. Chi, Thursday, Symp 3, 17:00

Upper limit to ET rate at short distance or high overpotential

decreases the apparent value of λ, when the data are

analyzed with the Marcus model

Jeuken, Biochim Biophys Acta 1604 67 (2003), and refs therein

Jeuken et al, J. Phys. Chem. B, 106 2304 (2002)







An evidence for kinetic dispersion


Part III.


Davis et al, J. Phys. Chem. B, 110 20651 (2006)

Salverda et al, Angew. Chem. Int. Ed. 49 1 (2010)

Azurin adsorbed on Au + SAM of hexanethiol

The protein is labeled with a dye

whose fluorescence is quenched

by nonradiative Energy Transfer (FRET)

when the Cu site is oxidized

Fluorescence detected using a TIRF microscope

ISE 2010: see the poster of H A Heering, s14-P-026


Salverda et al, Angew. Chem. Int. Ed. 49 1 (2010)ISE 2010: see the poster of H A Heering, s14-P-026










e.g. protonations

Part III.


Proton transfer is a fundamental process in bioenergetics

Respiratory

enzyme

ATP

Synthase

Reduced substrate O2

H+

H+

ATP + H2O ADP + Pi

Peter Mitchell (1920-1992, Nobel 1978): ATP synthesis uses a gradient of proton concentration

« Chemiosmotic theory » of oxidative phosphorylation

electronsprotons

Inner mitochondrial membrane

driving force for

ATP synthesis:

[H+] gradient

Nicholls and Ferguson « Bioenergetics 3 » Academic Press (2002)

Hydrolysis ΔrG<<0

Proton transfer to the buried [3Fe4S] cluster of a ferredoxin

The pH-dependence of the reduction potential of the [3Fe4S]+/0

demonstrates proton coupled ET, pKred ~ 7.5

The [3Fe4S] cluster is not exposed to solvent

Surface exposed glutamateChen et al, Nature 405 814 (2000)

Camba et al, Biochemistry 42 10589 (2003)

Hirst et al, J. Am. Chem. Soc. 120 7085 (1998)


Molecular dynamics simulations show that the surface exposed glutamate

can approach the clusterChen et al, Nature 405 814 (2000)




Chen et al, Nature 405 814 (2000)


Properties of the D15E mutant


koff (s-1) kon (s

-1M-1)

WT 300 8 109

D15E 2.5 2 107

D →E

Part IV.

Catalytic voltammetry (enzymes)

Using direct electrochemistry to measure the enzyme’s activity

(turnover rate)

and learning about all aspects of the catalytic mechanism:

• active site chemistry

• interfacial electron transfer

• intramolecular electron transfer

• slow (in)activation processes

• substrate transport along substrate channels

• …

H+ H2

Reduced MV

(blue)

Oxidized methyl

viologen

(color-free)

electrons

Solution assay of the activity (=turnover rate) of

hydrogenase

Easy experiment but:

- Characteristic time of the

measurement: 10-20 minutes (steady

state conditions)

- The driving force is not usually varied

H+ H2

Current = [enzyme] x turnover

Reference electrode

Working electrode

Counter electrode

H2

Rotating disc electrode

Measuring the activity using direct electrochemistry

electrons

RDE

Léger et al., Chem. Rev. 108 2379 (2008)

Vincent et al., Chem. Rev. 107 4366 (2007)

H+ H2

Current = [enzyme] x turnover

Rotating disc electrode

Léger et al., Chem. Rev. 108 2379 (2008)

Vincent et al., Chem. Rev. 107 4366 (2007)

electrons

Major drawback:

The current is proportional to

[enzyme]×turnover rate, but the

electroactive coverage cannot

be accurately determined

Advantages:

The activity (the current) can be

sampled at high frequency

The electrode potential is a

useful control parameter

Measuring the activity using direct electrochemistry

The dependance of activity on electrode potential

may be complex, we must make it informative

Taken from: Léger et al, Biochemistry 40 11234 (2001), Heffron et al, Biochemistry 40 3117 (2001), Fourmond et al, JACS. 132 4848 (2010)

Hudson et al, JACS 127 6977 (2005), Fourmond et al, J. Phys. Chem. B 114 3341 (2010), Léger et al, J. Phys Chem. B 106 13058 (2002)

Fumarate reductase

oxidizing succinate (top)

reducing fumarate

(bottom)

DMSO (top) and

nitrate (bottom)

reductasesHydrogenases

100mV

All at RDE,

rotating at a high

rate ( > 3 krpm)

Succinate oxidation by E. coli fumarate reductase (at RDE)

electrode

Fumarate

Current = turnover rate

Succinate

Léger et al., Biochemistry 40 11234 (2001)

Michaelis-Menten kinetics, Michaelis constant Km

Henri, C.R. Acad. Sc.135 916 (1902)

Michaelis et al., Biochemisches Zeitschrift 49 333 (1913)

Michaelis-Menten equation

[Succinate]

[Succinate] / Km

Michaelis-Menten kinetics for i

lim



The dependence of current on E gives the original information: the reduction potential of the AS under turnover conditions



Varying the concentration of succinate

at constant pH

Pourbaix and pourbaix-like diagrams for the change in

E0(FAD) against pH and [succinate] give

acidity and dissociation constants of the active site


(@ 1mM [succinate]) (@ pH 7)

In many cases, the current does not reach a plateau at

high overpotential but rather varies in proportion to E

Léger et al, J. Phys Chem. B 106 13058 (2002)

Reveals distribution of orientations

(and thus interfacial ET rates)

Hydrogen oxidation by A. vinosum NiFe

hydrogenase, 3krpm, pH7, 1 bar H2

fitdata

Léger et al, section 2.2.5.4 in Chem Rev 108 2379 (2008)

and J. Phys Chem. B 106 13058 (2002)

Modelisation:

1) choose model for interfacial ET

2) current as a function of k0

3) distribution of orientations

4) distribution of k0

5) current integrated across all values of k0

The case of slow intramolecular ET

(between electron relay and active site)

Exemple of enzyme

with a single relay:

sulfite oxidase

Heme

+ Mo active site

oxidizes sulfite to sulfate

Kisker et al, Cell 91 973 (1997)

The position of the wave when the enzyme has one redox relay

kf

kb ele

ctr

odeAS Rk-1

k1

Léger et al, J. Am. Chem. Soc. 128 180 (2006)

The model predicts a normal current equations but with

reduction potential that takes apparent values

(if k0

very fast)

kithe slowest intramolecular ET rate cst


Fast ET Slow ET

Application to sulfite oxidase (one heme mediates electrons from Mo active

site, where sulfite SO32- is oxidized to sulfate SO4

2-)

Using kcat

=95 s-1 , one finds ki=350 s-1

Flash photolysis experiments: 800 s-1

(Enemark 1993)


Voltammetry: catalytic wave

whose position does not depend much

on pH

Elliott et al, J. Am. Chem. Soc. 124 11612 (2003)E1

Simple diagnosis of slow interprotein ET in cytc/cyt

cc oxidase

complex adsorbed on SAM/Au

Haas et al, J. Phys. Chem. B 105 11351 (2001)

Cytochrome c oxidase

cytochrome c

Fast scan rate: 500mV/s

Slow scan rate: 5mV/s

The current (activity) drops at high

potential (formation of an oxidized

inactive state called NiB) and it is

recovered on the return scan

(reductive reactivation)

at pH 7, 1bar H2, 40oC, RDE

Oxidative inactivation of NiFe hydrogenase,

and the meaning of the « switch potential »

Inactive (“NiB”)

Oxidation

Reduction

Active

E0 values: M. Pandelia et al., Unpublished .

Voltammetry of A. aeolicus NiFe hydrogenase

E0 values: M. Pandelia et al., Unpublished . Fourmond et al, JACS 132 4848 (2010)

Voltammetry of A. aeolicus NiFe hydrogenase

Fourmond et al, JACS 132 4848 (2010)

Chronoamperometry for studying the inactivation mechanism

Fourmond et al, JACS 132 4848 (2010)

Oxidative inactivation of NiFe hydrogenase,

and the meaning of the « switch potential »

Example of complex catalytic wave shape: nitrate reduction by nitrate reductase,

has 2 hemes and 1 [4Fe4S] cluster

and one Mo active site where nitrate is reduced to nitrite

Fourmond et al, Biochemistry 49 2424 (2010)

The electrochemical behavior is mirrored in solution assays where the driving force

provided by reduced MV decreases as a function of time

Frangioni et al, JACS 126 1328 (2004)

Bertrand et al, JPCB 111 10300 (2007)

The electrochemical waveshape may result from the

competition between multiple reaction pathways

The steady-state catalytic signal for nitrate reduction is complex

In addition, strong hysteresis at high nitrate concentration

Fourmond et al, J. Phys. Chem. B 112 15478 (2008)

Fourmond et al, J. Phys. Chem. B 114 3341(2010)

Fourmond et al, J. Phys. Chem. B 114 3341(2010)

Evidence for substrate inhibition at moderately low potential

both in chronoamperometry and solution assays

Diffusion along the substrate channel of NiFe hydrogenase

Active site

Tunnel

L122

V74

Ni

Fe

There are « gas »

tunnels

in many other enzymes

- cytochrome c oxidase

(O2 reduction)

- nitrogenase (N2

reduction)

- CO dehydrogenase

(CO2 reduction)

- photosystem II (H2O

oxidation)

“channel mutants” of hydrogenase

Review: Lautier et al., Faraday Discussions 148 (2011)

H2

CO

Kinetics of reaction with CO:

Chronoamperometry at constant electrode potential

Cell flushed with H2


H2

CO

instant inhibition



Cell flushed with H2, CO injected

Result obtained

with the WT enzyme


H2



Cell flushed with H2, CO injected, and then flushed away by H2

instant inhibition, reversible


Result obtained

with the WT enzyme

CO

H2

Kinetics of reaction with CO, with a mutant whose

inhibition by CO is slow


Slow inhibition

→ slow binding of CO

Result obtained

with a mutant

whose channel is

partly blocked

Leroux et al. PNAS 105 11188 (2008), Liebgott et al, Nat. Chem. Biol. 6 63(2010)

H2




Slow

release of CO

Slow

binding

of CO

Result obtained

with a mutant

whose channel is

partly blocked





Result obtained

with a mutant

whose channel is

partly blocked

Fit returns

Almeida et al, FEBS Letts 581 284 (2007)


and

The rates of inhibition are strongly affected by mutations in the

channel

Liebgott et al., Nature Chemical Biology 6 63 (2010)

CONH2 ← COOH

+1CH2

Take home message 3

Electrochemistry has ben used to study all aspects

related to protein function

Active site redox chemistry, substrate binding,

Electron transfer (interfacial, intramolecular),

Proton transfer,

Substrate channels,

(in)activation,

Inhibitition, …

Léger and Bertrand, Chem. Rev. 108 2379 (2008)

Part V.

Using redox enzymes in biotech devices

Intrinsic limitations of DET-based electrodes

Examples of applications:

Biofuel cells (using hydrogenases, GOX, laccases, billirubin oxidases)

Photoelectrochemical fuel cells (hydrogenases)

Particles coupling two redox enzymes

Biosensors (nitrite reductase)

Barton et al, Chem Rev, 104 4867 (2004) (special issue on Batteries and Fuel cells)

Fuel cells and photoelectrochemical cells:

primary challenge is increased biocatalytic power

An ideal monolayer of a typical enzyme

100nm2 cross section, 500 electrons /s → 80μA/cm2

Practical fuel cells operating at > 10mA/cm2

would require thousands of layers

(that is loading in the mg/cm2 range, assuming MW=100kDa)→ mediated electron transfer is more promissing in this respect

Some devices that take advantage of the high selectivity

of the enzymes for their substrates.

1842 1969 2007

E.g. H2/O2 fuel cell

October 2007

special issue on H2

and hydrogenases

Comparison between Pt and hydrogenase adsorbed on

graphite as H2 fuel cell anode catalysts

1/ω1/2 (rpm ½)

Jones et al. Chem Comm. 866 (2002)

A. vinosum H2ase

H2 oxidation at RDE, E=.24V/SHE

C. acetobutylicum H2ase

Oxidation/production of H2 on RD graphite E

Hambourger et al. JACS 130 2015 (2008)

1 bar H2, pH 7, 50mV/s, 3000rpm1 bar H2, pH 7, 45oC, E=0.24V vs SHE

ISE 2010: see the talk of K. Vincent, Thursday, Symp 14, 09:40

Hydrogenase

Pt

H2/O2 fuel cell: avoiding short-circuit requires expensive

membranes

Using selective enzyme electrodes:

Hydrogenase at the anode (H2 oxidation)

laccase or billirubin oxidase at the cathode (O2 reduction)

to design membraneless fuel cells

Vincent et al. Chem. comm. 5033 (2006) & PNAS 102 16951 (2005)

A membraneless H2/O2 biofuel cell using

a hydrogenase that is able to oxidize traces of H2 in air

and a fungal laccase, both connected to PGE

ISE 2010: see the talk of K. Vincent, Thursday, Symp 14, 09:40

Hambourger et al. JACS 130 2015 (2008), Photochem. Photobiol. 81 1015 (2005)

Renewable hydrogen production, in a photoelectrochemical cell

that uses the FeFe hydrogenase from C. acetobutylicum

(instead of Pt)

Kamitaka et al, Phys.Chem.Chem.Phys 9 1793 (2007)

A one-compartment fructose/dioxygen non-mediated biofuel cell

based on fructose dehydrogenase and laccase, up to 0.8 mW/cm2

Enzymatic catalysis on conducting particles

Vincent et al, Nat Chem Biol 3 761 (2007)

Reisner et al, JACS 131 18457 (2009)

H2 as electron source

Photoproduction of H2

~5μm2

Bullen et al, Biosensors and bioelectronics, 21 2015 (2006)

No need to reach high current densities to design biosensors

Silveira et al, Biosensors and bioelectronics 25 2026 (2010)

ISE 2010: see the talk of M. Gabriela Almeida: Monday, symp 3, 11:20

A non-mediated nitrite biosensorbased on penta-heme nitrite reductase NO2

- + 6e- + 8H+ → NH4+ + 2H2O

+ 1 buried

active site heme

4 electron-transfer hemes

2 of which surface exposed

Silveira et al, Biosensors and bioelectronics 25 2026 (2010)

ISE 2010: see the talk of M. Gabriela Almeida: Monday, symp 3, 11:20

A non-mediated nitrite biosensorbased on penta-heme nitrite reductase

encapsulated in a porous silica matrix on graphite

@E= -.5 -.7 and -.9V/AgCl

At -.9V, linear response

in the range 0.12μM to 50 μM nitrite, stable for 2 weeks

Further readings

Willner and Katz, « Integration of layered redox proteins and conductive supports

for bioelectronic applications » Angew Chem Int. Ed. 39 1180 (2000)

Cracknell, Vincent and Armstrong « Enzymes as working or inspirational

electrocatalysts for fuel cells and electrolysis » Chem. Rev. 108 2439 (2008)

Léger and Bertrand « Direct electrochemistry of redox enzymes as a tool for

mechanistic studies » Chem. Rev. 108 2379 (2008)

A free software to analyse electrochemical data

Available for download at : http://bip.cnrs-mrs.fr/bip06/software.html

Works on Mac OS X (one-click installer available) or Linux. Not Windows.

Good for : noise filtering, baseline subtractions, fitting

Fourmond et al, Bioelectrochem. 76 141 (2009)

http://bip.cnrs-mrs.fr/bip06/software.html





D. Fructosovorans: Sébastien Dementin, Pierre Pol Liebgott, Marc Rousset

A. Aeolicus: Pascale Infossi, E. Lojou, M.Thérèse Giudici-Orticoni

EPR: Bénédicte Burlat, Emilien Etienne, Bruno Guigliarelli,

Electrochemistry: Pierre-Pol Liebgott, Fanny Leroux, Vincent Fourmond,

Carole Baffert, Pierre Ezzano, Patrick Bertrand

C. acetobutylicum: Thomas Lautier, I. Meynial, Ph. Soucaille (INSA Toulouse)

R. sphaeroides: David Pignol, Monique Sabaty, Pascal Arnoux

Crystallography: Anne Volbeda, Juan Fontecilla Camps (CEA, UJF)

H/D kinetics: Laurent Cournac (CEA, Cadarache)

FTIR: Antonio De Lacey (D. fructosovorans, CSIC, Madrid),

FTIR, EPR: Maria Pandelia, Wolfgang Lubitz (A. Aeolicus, Max Planck, G)

Collaborators

Slides can be downloaded at http://bip.cnrs-mrs.fr/bip06/publications






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