“Two-Step” Chronoamperometric Method for Studying the Anaerobic Inactivation of an Oxygen Tolerant NiFe Hydrogenase

Fourmond, Vincent; Infossi, Pascale; Giudici‐Orticoni, Marie‐Thérèse; Bertrand, Patrick; Léger, Christophe

doi:10.1021/ja910685j

Cited by 66 publications

(157 citation statements)

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“…For example, if the ratio k i ðEÞ=k a ðEÞ increases as the electrode potential increases, the enzyme inactivates after a step up. We have shown that, to independently determine the two rate constants from CA experiments, one must simultaneously interpret the time constant and the magnitude of the transients (15). The result of such detailed investigation of the oxygen-tolerant hydrogenase from A. aeolicus in ref.…”

Section: Resultsmentioning

confidence: 99%

“…After each step down, the catalytic current immediately drops and then slowly increases as activity is recovered. A straightforward analysis of these experiments consists of fitting each current transient to an exponential function to measure the time constant τðEÞ = 1=ðk i ðEÞ + k a ðEÞÞ at any potential (4,15); the results are shown as squares in Fig. 1F.…”

Section: Resultsmentioning

confidence: 99%

“…Changing the electrode potential also triggers the conversion between active and inactive states. In chronoamperometry (CA) experiments, the (in)activation is seen as current transients following potential steps (14,15). In cyclic voltammetry (CV) experiments, the oxidative inactivation gives the electrochemical response the complex shape that is shown in Fig.…”

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Relation between anaerobic inactivation and oxygen tolerance in a large series of NiFe hydrogenase mutants

Abou‐Hamdan

Liebgott

Fourmond

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Nickel-containing hydrogenases, the biological catalysts of H 2 oxidation and production, reversibly inactivate under anaerobic, oxidizing conditions. We aim at understanding the mechanism of (in)activation and what determines its kinetics, because there is a correlation between fast reductive reactivation and oxygen tolerance, a property of some hydrogenases that is very desirable from the point of view of biotechnology. Direct electrochemistry is potentially very useful for learning about the redox-dependent conversions between active and inactive forms of hydrogenase, but the voltammetric signals are complex and often misread. Here we describe simple analytical models that we used to characterize and compare 16 mutants, obtained by substituting the position-74 valine of the O 2 -sensitive NiFe hydrogenase from Desulfovibrio fructosovorans. We observed that this substitution can accelerate reactivation up to 1,000-fold, depending on the polarity of the position 74 amino acid side chain. In terms of kinetics of anaerobic (in)activation and oxygen tolerance, the valine-tohistidine mutation has the most spectacular effect: The V74H mutant compares favorably with the O 2 -tolerant hydrogenase from Aquifex aeolicus, which we use here as a benchmark.electrocatalysis | direct electron transfer | protein film voltammetry | hydrogen T he nickel-iron hydrogenases that have been crystallized and/ or thoroughly studied so far are very similar from a structural point of view: They all are either soluble heterodimers or heterodimers isolated from a membrane-associated complex. The amino acids that surround the NiFe active site are conserved (1) and yet the kinetic properties of these enzymes are diverse. For example, some NiFe hydrogenases can oxidize and produce H 2 , whereas others preferentially catalyze one direction of the reaction (2-4). Another property of some hydrogenases that has attracted considerable interest is their sensitivity (and sometimes their resistance) to O 2 . This interest stems from the fact that hydrogenases could be used for H 2 oxidation in fuel cells or H 2 production in photo-electrochemical cells if they were functional under aerobic conditions (5).The NiFe hydrogenases that have been studied first, referred to as "standard," were purified from Allochromatium vinosum or Desulfovibrio species. Upon exposure to O 2 , they convert into two inactive forms called NiA and NiB, where an oxygenated ligand bridges the Ni and the Fe. The NiB and NiA states can be reactivated by reduction, the former more quickly than the latter (6). The membrane-bound NiFe hydrogenases from, e.g., Ralstonia eutropha or Aquifex aeolicus are reversibly inhibited by O 2 and termed "O 2 resistant." Apparently, this resistance results from (i) the enzyme reacting with O 2 to form only the NiB state and (ii) this NiB state reactivating much more quickly than in standard hydrogenases (4, 7). The most patent differences between O 2 -resistant and O 2 -sensitive hydrogenases are the structures and redox properties of the FeS clu...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Relation between anaerobic inactivation and oxygen tolerance in a large series of NiFe hydrogenase mutants

Abou‐Hamdan

Liebgott

Fourmond

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…These characters are coincident with those described in a previous study using tolerant A. aeolicus [NiFe]-hydrogenase. 37 The inactivation process involves the one-electron oxidation of the Ni(II) in the [NiFe] catalytic center and the hydroxide ligand coordination to the Ni(III). 2,22,26 The fact that k I is independent of the electrode potential means that the rate of the hydroxide ligand coordination to the Ni(III) (the coordination of water on the [NiFe]-active site and the succeeding deprotonation) is the ratedetermining step of the inactivation.…”

Section: Kinetic Analysis Of Reversible Anaerobic Inactivationmentioning

confidence: 99%

Kinetic Analysis of Inactivation and Enzyme Reaction of Oxygen-Tolerant [NiFe]-Hydrogenase at Direct Electron-Transfer Bioanode

Kitazumi

Shirai

et al. 2014

Bulletin of the Chemical Society of Japan

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Membrane-bound [NiFe]-hydrogenase (MBH) from Hydrogenovibrio marinus is an O 2 -tolerant enzyme and allows direct-electron-transfer (DET) bioelectrocatalysis for H 2 -oxidation. MBH is a promising bioelectrocatalyst for bioanode of enzymatic H 2 O 2 biofuel cells. From the practical viewpoint of electricity production, the H 2 -depletion near the electrode surface and the oxidative and reversible inactivation called "anaerobic inactivation" of [NiFe]-hydrogenases limit the H 2 -oxidation at high potentials. We have already proposed a gas-diffusion system to avoid inactivation in our previous study. In this research, we have analyzed the kinetics of the electrochemically induced anaerobic inactivation and the DET bioelectrocatalytic reaction of MBH on electrodes. When the inactivation is considered as a competitive inhibition-like reaction, the maximum value of the apparent Michaelis constant reaches 6.5 mM (at Ketjen Black-modified electrode) as analyzed in our kinetic model. Since the value is larger than the saturated H 2 -concentration in solution (0.74 mM), we conclude that high-speed H 2 -supply realized by a gas-diffusion electrode is essential to compete with the inactivation. Furthermore, a gas-diffusion bioanode with MBH can eliminate the H 2 -depletion near the electrode surface and has reached about 10 mA cm ¹2 at 0 V (vs. Ag«AgCl«sat. KCl electrode) under quiescent (passive) and H 2 -atmospheric conditions.Hydrogenases are enzymes which catalyze the interconversion between hydrogen and proton.1 The two major classes of hydrogenases are known as [FeFe] or [NiFe] according to the metals in their buried active sites.1,2 Since hydrogenases are highly active, with turnover frequencies for H 2 -oxidation in excess of thousands of molecules of H 2 per second at 30°C, 3 they can be alternative catalysts instead of platinum as an H 2 O 2 fuel cell.4 From the aspect of bioelectrochemistry, hydrogenases have received a lot of attention, and the bioelectrocatalytic properties have extensively been characterized all over the world. 1,2,58 Enzymatic biofuel cells, which are electric generators that utilize redox enzymes as catalysts, can generally operate under mild conditions (neutral pH, atmospheric pressure, and room temperature). They are classified into two types: mediated electron transfer (MET)-type and direct electron transfer (DET)-type.912 MET-type cell utilizes artificial mediators to shuttle the electron between the enzyme and the electrode to reduce the kinetic hindrance in the interfacial electron transfer. On the other hand, DET-type cell does not require, in principle, any separator or membrane between the electrodes due to the mediator-less configuration. In addition, the DET-type one can reduce the possible health hazard problems caused by artificial mediators and the thermodynamic loss ascribed to the difference in the redox potentials between the mediator and the active site of the enzyme. Furthermore, the DET-type system enables us to construct a very simple and compact enzymatic biofuel ...

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“…47 It has been shown that its value depends on the scan rate of the CV measurement, and on thermodynamic (formal potential of the equilibrium between the active and inactive redox states of the hydrogenase bi-metallic active site) and kinetic parameters (the rate constant of the inactive state reduction) of the reactivation process. 48 The …”

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Oriented Immobilization of a Membrane-Bound Hydrogenase onto an Electrode for Direct Electron Transfer

et al. 2011

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The ACS journal of surfaces and colloids 27: 6449-6457 (2011) 2 ABSTRACTThe interaction of redox enzymes with electrodes is of great interest for studying the catalytic mechanisms of redox enzymes and for bioelectronic applications. Efficient electron transport between the biocatalysts and the electrodes has achieved more success with soluble than with membrane enzymes due to the higher structural complexity and instability of the latter proteins. In this work we report a strategy for immobilizing a membrane-bound enzyme onto gold electrodes with a controlled orientation in its fully active conformation. The immobilized redox enzyme is the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough, which catalyzes H 2 -oxidation reversibly and is associated to the cytoplasmic membrane by a lipidic tail. Gold surfaces modified with this enzyme and phospholipids have been studied by atomic force microscopy (AFM) and electrochemical methods. The combined study indicates that by a two-step immobilization procedure the hydrogenase can be inserted via its lipidic tail onto a phospholipidic bilayer formed over the gold surface, only allowing mediated electron transfer between enzyme and electrode. On the other hand, a one-step immobilization procedure favours formation of a hydrogenase monolayer over the gold surface with its lipidic tail inserted in a phospholipid bilayer formed on top of the hydrogenase molecules. This latter method has allowed for the first time efficient electron transfer between a membrane-bound enzyme in its native conformation and an electrode.

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“Two-Step” Chronoamperometric Method for Studying the Anaerobic Inactivation of an Oxygen Tolerant NiFe Hydrogenase

Cited by 66 publications

References 43 publications

Relation between anaerobic inactivation and oxygen tolerance in a large series of NiFe hydrogenase mutants

Relation between anaerobic inactivation and oxygen tolerance in a large series of NiFe hydrogenase mutants

Kinetic Analysis of Inactivation and Enzyme Reaction of Oxygen-Tolerant [NiFe]-Hydrogenase at Direct Electron-Transfer Bioanode

Oriented Immobilization of a Membrane-Bound Hydrogenase onto an Electrode for Direct Electron Transfer

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