Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

Hexter, Suzannah V.; Grey, Felix; Happe, Thomas; Climent, Vı́ctor; Armstrong, Fräser A.

doi:10.1073/pnas.1204770109

Cited by 160 publications

(240 citation statements)

References 35 publications

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“…All experiments were carried out at 0°C, with 0.10 M sodium phosphate buffer at pH 7.0, under strictly anaerobic conditions, using a rotating disk pyrolytic graphite edge (PGE) electrode at which CpI and CrHydA1 adsorb tightly on the rough surface in a highly electroactive state (2). Under 100% H 2 , catalytic activity is close to optimal for each enzyme and electrode rotation at 1,000 rpm ensured that H 2 mass transport is not limiting (24).…”

Section: Resultsmentioning

confidence: 99%

“…Attached to an electrode, these enzymes catalyze rapid oxidation and reduction, with only a minimum overpotential being required to swap the current direction either side of the equilibrium potential (2)(3)(4)(5). Of particular interest are hydrogenases, which catalyze the oxidation and production of H 2 with activities that may rival platinum (6)(7)(8)(9)(10)(11) and are inspirational in the quest for future electrocatalysts as well as in artificial photosynthesis (12,13).…”

mentioning

confidence: 99%

“…For enzymes, PFE has focused entirely on net catalytic electron flow that is observed as direct current (DC) in voltammograms (2,3,10). Use of DC cyclic voltammetry as opposed to single potential sweeps alone helps to distinguish rapid, steady-state catalytic activity at different potentials from relatively slow, potential-dependent changes in activity that are revealed as hysteresis (10).…”

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confidence: 99%

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Frequency and potential dependence of reversible electrocatalytic hydrogen interconversion by [FeFe]-hydrogenases

Pandey

Islam

Happe

et al. 2017

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

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The kinetics of hydrogen oxidation and evolution by [FeFe]hydrogenases have been investigated by electrochemical impedance spectroscopy-resolving factors that determine the exceptional activity of these enzymes, and introducing an unusual and powerful way of analyzing their catalytic electron transport properties. Attached to an electrode, hydrogenases display reversible electrocatalytic behavior close to the 2H + /H 2 potential, making them paradigms for efficiency: the electrocatalytic "exchange" rate (measured around zero driving force) is therefore an unusual parameter with theoretical and practical significance. Experiments were carried out on two [FeFe]-hydrogenases, CrHydA1 from the green alga Chlamydomonas reinhardtii, which contains only the active-site "H cluster," and CpI from the fermentative anaerobe Clostridium pasteurianum, which contains four low-potential FeS clusters that serve as an electron relay in addition to the H cluster. Data analysis yields catalytic exchange rates (at the formal 2H + /H 2 potential, at 0°C) of 157 electrons (78 molecules H 2 ) per second for CpI and 25 electrons (12 molecules H 2 ) per second for CrHydA1. The experiments show how the potential dependence of catalytic electron flow comprises frequency-dependent and frequency-independent terms that reflect the proficiencies of the catalytic site and the electron transfer pathway in each enzyme. The results highlight the "wire-like" behavior of the Fe-S electron relay in CpI and a low reorganization energy for electron transfer on/off the H cluster.hydrogen | electrocatalysis | impedance spectroscopy | hydrogenase | electron transfer

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Frequency and potential dependence of reversible electrocatalytic hydrogen interconversion by [FeFe]-hydrogenases

Pandey

Islam

Happe

et al. 2017

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

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“…signals resulting from the catalytic oxidation or reduction of the enzyme's substrate at the electrode. The resulting catalytic current, which is proportional to the enzymatic activity, lends itself well to quantitative interpretation: one can monitor its evolution over time to learn about inactivation/activation processes [4][5][6][7], or model its dependence on electrode potential to learn about the steps in the catalytic cycle [8][9][10]. In catalytic PFV, the substrate is consumed at the electrode by the catalytic reaction.…”

Section: Introductionmentioning

confidence: 99%

Reliable estimation of the kinetic parameters of redox enzymes by taking into account mass transport towards rotating electrodes in protein film voltammetry experiments

Merrouch

Hadj-Saïd

Léger

et al. 2017

Electrochimica Acta

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Reliable estimation of the kinetic parameters of redox enzymes by taking into account mass transport towards rotating electrodes in protein film voltammetry experiments. Electrochimica Acta, Elsevier, 2017Elsevier, , 245, pp.1059Elsevier, -1064Elsevier, . 10.1016Elsevier, /j.electacta.2017 AbstractIn Protein Film Voltammetry, a redox enzyme is immobilized on a rotating electrode in a configuration allowing fast, direct electron transfer. This technique is used to probe the mechanism of enzymes by quantitatively interpreting the response in current as a function of the experimental conditions. Limitation by mass transport of the substrate towards the electrode may obscure important features and complicate the analysis of the enzymatic response, so much so that the enzyme has high activity. In this work, we derive equations taking into account mass transport of substrate, for the steady-state current generated by an enzyme following Michaelis-Menten kinetics and immobilized onto a hydrodynamic (e.g. rotating) electrode. We use these equations to model the current response of films of CO-dehydrogenase, a metalloenzyme that catalyzes the oxidation of CO, to transient exposures to its gaseous substrate. We show that neglecting transport yields poor fits and overestimated, unreliable, values of K m (even when using the Koutecky-Levich approximation), whereas taking into account transport yields much better fits and more reliable parameters. We reinterpret previously published data by taking into account transport limitations.

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“…Despite a detailed biochemical and structural understanding of these enzymes (11,15), the importance of individual isoforms of hydrogenases to cellular physiology is often not known. In particular, hydrogenase-1 (Hyd-1), encoded by hyaAB of the hyaABCDEF operon, is one of two hydrogenases that function in H 2 oxidation under anaerobic conditions in Escherichia coli (30).…”

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ArcA and AppY Antagonize IscR Repression of Hydrogenase-1 Expression under Anaerobic Conditions, Revealing a Novel Mode of O ₂ Regulation of Gene Expression in Escherichia coli

et al. 2012

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Transcription of the Escherichia coli hydrogenase-1 operon (hyaABCDEF) is increased by the transcription factors ArcA and AppY under anaerobic growth conditions. However, IscR, which represses transcription of the hyaA promoter (P hyaA ) under aerobic conditions, was not known to repress transcription of this promoter under anaerobic conditions. Here, we report that ArcA and AppY increase P hyaA expression under anaerobic conditions by antagonizing IscR binding at P hyaA , since IscR repression is observed when either ArcA or AppY is eliminated. The ability of ArcA and AppY to act as antirepressors of IscR repression of P hyaA depended on IscR levels, suggesting that IscR competes with ArcA and/or AppY for binding. In support of this competition model, electrophoretic mobility shift assays and DNase I footprinting showed that the ArcA and IscR binding sites overlap and that binding of ArcA and IscR is mutually exclusive. Unexpectedly, IscR with a C92A mutation (IscR-C92A), which mimics the clusterless form of the protein that is present predominantly under aerobic conditions, was a better repressor under anaerobic conditions of both P hyaA and a constitutive promoter containing the IscR binding site from P hyaA than wild-type IscR, which is predominantly in the [2Fe-2S] form under anaerobic conditions. This observation could not be explained by differences in DNA binding affinities or IscR levels, so we conclude that [2Fe-2S]-IscR is a weaker repressor of P hyaA than clusterless IscR. In sum, a combination of ArcA and AppY antirepression of IscR function, lower levels of IscR, and weak repression by [2Fe-2S]-IscR leads to increased P hyaA expression under anaerobic conditions. H ydrogenases, which catalyze the reversible oxidation of H 2 , are widespread throughout the prokaryotic world (36). Despite a detailed biochemical and structural understanding of these enzymes (11, 15), the importance of individual isoforms of hydrogenases to cellular physiology is often not known. In particular, hydrogenase-1 (Hyd-1), encoded by hyaAB of the hyaABCDEF operon, is one of two hydrogenases that function in H 2 oxidation under anaerobic conditions in Escherichia coli (30). Recent studies have demonstrated that, unlike most hydrogenases, Hyd-1 is O 2 tolerant (20), raising the question of whether it might also have a role under O 2 -limiting conditions. Expression of E. coli hya is also subject to various physiological inputs (i.e., anaerobiosis, stationary phase, nitrate, and phosphate starvation) (2, 5, 31), suggesting that it could have specialized roles.It is well established that expression of the hya operon is increased under anaerobic fermentative conditions and decreased under aerobic or anaerobic respiratory conditions with nitrate, consistent with the proposal that Hyd-1 functions in fermentation, not respiration (30). NarL and NarP appear to be responsible for repression of hya expression in response to nitrate (5, 31), whereas the anaerobic transcription factors ArcA and AppY are required for the increased express...

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Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

Cited by 160 publications

References 35 publications

Frequency and potential dependence of reversible electrocatalytic hydrogen interconversion by [FeFe]-hydrogenases

Frequency and potential dependence of reversible electrocatalytic hydrogen interconversion by [FeFe]-hydrogenases

Reliable estimation of the kinetic parameters of redox enzymes by taking into account mass transport towards rotating electrodes in protein film voltammetry experiments

ArcA and AppY Antagonize IscR Repression of Hydrogenase-1 Expression under Anaerobic Conditions, Revealing a Novel Mode of O ₂ Regulation of Gene Expression in Escherichia coli

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Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

Cited by 160 publications

References 35 publications

Frequency and potential dependence of reversible electrocatalytic hydrogen interconversion by [FeFe]-hydrogenases

Frequency and potential dependence of reversible electrocatalytic hydrogen interconversion by [FeFe]-hydrogenases

Reliable estimation of the kinetic parameters of redox enzymes by taking into account mass transport towards rotating electrodes in protein film voltammetry experiments

ArcA and AppY Antagonize IscR Repression of Hydrogenase-1 Expression under Anaerobic Conditions, Revealing a Novel Mode of O 2 Regulation of Gene Expression in Escherichia coli

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ArcA and AppY Antagonize IscR Repression of Hydrogenase-1 Expression under Anaerobic Conditions, Revealing a Novel Mode of O ₂ Regulation of Gene Expression in Escherichia coli