Understanding and Tuning the Catalytic Bias of Hydrogenase

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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confidence: 99%

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

Pandey

Islam

Happe

et al. 2017

“…They are also very active under conditions optimized for a particular direction, operating at up to 20,000 s −1 for H 2 production (5) and 10,000 s −1 for H 2 oxidation (6). Under conditions where reversibility is optimized (3,(7)(8)(9)(10), net turnover frequencies (TOFs) are typically slower. For hydrogenases attached to electrode surfaces, the TOFs are not always quantitated due to uncertainty in surface coverage (9), but in one example, TOFs for a series of mutants of Desulfovibrio fructosovorans [NiFe]-hydrogenase were reported, ranging from 3 to 500 s −1 for H 2 production and 600 to 1,000 s −1 for H 2 oxidation under one set of conditions (8).…”

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confidence: 99%

“…Although the mechanistic details controlling fast, reversible catalysis in hydrogenases are likely different, the Léger group demonstrated that catalytic bias in [NiFe]-hydrogenase is controlled not by the redox properties of the active site, but by residues remote from the active site that control either H 2 release (H 2 production) or electron transfer (H 2 oxidation) (8). In this model system, we demonstrate that we are controlling H 2 addition and proton transfer with groups remote from the active site, providing a conceptual parallel between the role of the outer coordination sphere in the molecular model and the role of the protein scaffold in the enzymatic system.…”

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confidence: 99%

Amino acid modified Ni catalyst exhibits reversible H ₂ oxidation/production over a broad pH range at elevated temperatures

Dutta

DuBois

Roberts

et al. 2014

116

137

Hydrogenases interconvert H 2 and protons at high rates and with high energy efficiencies, providing inspiration for the development of molecular catalysts. Studies designed to determine how the protein scaffold can influence a catalytically active site have led to the synthesis of amino acid derivatives of [Ni(P 2+ (CyAA). It is shown that theseCyAA derivatives can catalyze fully reversible H 2 production/ oxidation at rates approaching those of hydrogenase enzymes. The reversibility is achieved in acidic aqueous solutions (pH = 0-6), 1 atm 25% H 2 /Ar, and elevated temperatures (tested from 298 to 348 K) for the glycine (CyGly), arginine (CyArg), and arginine methyl ester (CyArgOMe) derivatives. As expected for a reversible process, the catalytic activity is dependent upon H 2 and proton concentrations. CyArg is significantly faster in both directions (∼300 s −1 H 2 production and 20 s −1 H 2 oxidation; pH = 1, 348 K, 1 atm 25% H 2 /Ar) than the other two derivatives. The slower turnover frequencies for CyArgOMe (35 s −1 production and 7 s −1 oxidation under the same conditions) compared with CyArg suggests an important role for the COOH group during catalysis. That CyArg is faster than CyGly (3 s −1 production and 4 s −1 oxidation) suggests that the additional structural features imparted by the guanidinium groups facilitate fast and reversible H 2 addition/release. These observations demonstrate that outer coordination sphere amino acids work in synergy with the active site and can play an important role for synthetic molecular electrocatalysts, as has been observed for the protein scaffold of redox active enzymes.hydrogenase mimics | reversible catalysis | amino acid catalysts | outer coordination sphere | homogeneous electrocatalysis H ydrogenases are metalloenzymes that interconvert H 2 and protons (Eq. 1), reactions necessary for biological processes within certain organisms to provide energy by splitting hydrogen, as well as to balance the redox potential in the cell (1-4). Their reversible catalytic behavior is a demonstration of their energy efficiency, indicating that they are operating at the equilibrium potential of the H 2 /H + couple. They are also very active under conditions optimized for a particular direction, operating at up to 20,000 s −1 for H 2 production (5) and 10,000 s −1 for H 2 oxidation (6). Under conditions where reversibility is optimized (3, 7-10), net turnover frequencies (TOFs) are typically slower. For hydrogenases attached to electrode surfaces, the TOFs are not always quantitated due to uncertainty in surface coverage (9), but in one example, TOFs for a series of mutants of Desulfovibrio fructosovorans [NiFe]-hydrogenase were reported, ranging from 3 to 500 s −1 for H 2 production and 600 to 1,000 s −1 for H 2 oxidation under one set of conditions (8).The ability of hydrogenases to function reversibly requiring no applied overpotential and with high catalytic TOFs is a direct reflection of their biological optimization, and a hallmark of enzymes. Reversibility requires enzym...

“…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)(3)(4). Another property of some hydrogenases that has attracted considerable interest is their sensitivity (and sometimes their resistance) to O 2 .…”

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confidence: 99%

“…Substitutions of this amino acid have a strong effect on the rates of intramolecular diffusion (23,24) and, consequently, on the Michaelis constant for H 2 (24), on the catalytic bias of the enzyme (2), and on the rates of inhibition by CO and O 2 (23,24). For example, the V74Q, V74M, and V74E mutants are inhibited by O 2 more slowly than the wild-type (WT) enzyme, but this effect on the rate of inhibition is not strong enough to make the enzyme oxygen resistant.…”

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confidence: 99%

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

Abou‐Hamdan

Liebgott

Fourmond

et al. 2012

Self Cite

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...