Cobaloximes are effective electrocatalysts for hydrogen evolution and thus functional models for hydrogenases. Among them, difluoroboryl-bridged complexes appear both to mediate proton electroreduction with low overpotentials and to be quite stable in acidic conditions. We report here a mechanistic study of [Co(dmgBF2)2L] (dmg2- = dimethylglyoximato dianion; L = CH3CN or N,N-dimethylformamide) catalyzed proton electroreduction in organic solvents. Depending on the applied potential and the strength of the acid used, three different pathways for hydrogen production were identified and a unified mechanistic scheme involving cobalt(II) or cobalt(III) hydride species is proposed. As far as working potential and turnover frequency are concerned, [Co(dmgBF2)2(CH3CN)2], in the presence of p-cyanoanilinium cation in acetonitrile, is one of the best synthetic catalysts of the first-row transition-metal series for hydrogen evolution.

In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.

Dedicated to Professor P. Gouzerh on the occasion of his 65th birthday Homogeneous light-driven catalytic systems for hydrogen production and, more generally, efficient photoactivated synthetic multielectron catalysts remain relatively scarce. [1] Such systems [2][3][4] generally consist of 1) a photosensitizer, often based on the ruthenium tris(diimine) moiety, [5] 2) a metal-based catalytic center, and in some cases 3) an additional redox mediator. However, their efficiency remains to be improved in terms of both turnover numbers (stability) and turnover frequencies, and these systems should preferably rely on inexpensive first-row transition-metal catalysts rather than unsustainable noble metals. We and others recently reported that cobaloximes are very efficient and cheap electrocatalysts for hydrogen evolution. [6][7][8][9] We thus decided to couple cobaloximes with ruthenium tris(diimine) moieties in order to make a supramolecular variant of the system previously studied by Lehn et al. for photochemical production of hydrogen. [3] In such a molecular device, the intramolecular electron transfer from the photoactivated center to the catalytic center can potentially be controlled, and the charge-recombination processes limited, to an extent larger than in intermolecular systems, by fine-tuning both the distance between metal centers and the nature of the bridge. [2,10] Such an organized assembly is found in hydrogen-evolving green algae, where the photosystem I is tightly coupled to hydrogenase enzymes. [11] In this paper we describe the synthesis and activity of a series of novel heterodinuclear ruthenium-cobaloxime photocatalysts able to achieve the photochemical production of hydrogen with the highest turnover numbers so far reported for such devices. Compounds 1-3 (Scheme 1) were synthesized in good yields [12] by replacing one axial ligand of cobaloxime moieties with the pyridine residue of the previously reported [(bpy) 2 Ru(l-pyr)] 2+ complex (l-pyr = (4-pyridine)oxazolo-[4,5-f]phenanthroline).[13] NMR measurements and ESI-MS analysis are consistent with the l-pyr ligand connecting the ruthenium and cobalt centers. This was further supported by cyclic voltammetry: [12] in addition to ruthenium-centered processes, which are not significantly modified upon complexation to the cobalt center, cyclic voltammograms of 1-3 show Co II /Co I reversible processes shifted by % 80 mV to more positive potentials relative to the starting cobaloximes, probably because of the overall 2 + charge of the compounds.We checked by cyclic voltammetry that the cobaloxime moieties retain their electrocatalytic properties for hydrogen production in all three heterobinuclear complexes: an electrocatalytic wave corresponding to proton reduction develops at À0.45 V vs. Ag/AgCl upon addition of increasing amounts of p-cyanoanilinium tetrafluoroborate to a solution of 1 in CH 3 CN [12] (electrocatalytic waves are observed at À0.9 V vs.

FeFe hydrogenases are the most efficient H2 producing enzymes, but inactivation by O2 is an obstacle to using them in biotechnological devices. Here we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We find that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of the highly reactive OH radical and hydroxylated cysteine, consistent with recent crystallographic evidence. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species at prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.

Nature is a valuable source of inspiration in the design of catalysts, and various approaches are used to elucidate the mechanism of hydrogenases, the enzymes that oxidize or produce H2. In FeFe hydrogenases, H2 oxidation occurs at the H-cluster, and catalysis involves H2 binding on the vacant coordination site of an iron centre. Here, we show that the reversible oxidative inactivation of this enzyme results from the binding of H2 to coordination positions that are normally blocked by intrinsic CO ligands. This flexibility of the coordination sphere around the reactive iron centre confers on the enzyme the ability to avoid harmful reactions under oxidizing conditions, including exposure to O2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H2 oxidation.

Surprisingly uninhibited: The inhibition of hydrogenases by oxygen is intensely studied because this is the main obstacle to using these enzymes in biofuel cells. The hydrogenase from Clostridium acetobutylicum (see structure) was found to react surprisingly slowly with O2. The inhibition mechanism was elucidated and the kinetics were quantitatively defined. This is a prerequisite for improving the enzyme further by genetic engineering and for assessing its potential in technological devices.

We investigated di-hydrogen transport between the solvent and the active site of FeFe hydrogenases. Substrate channels supposedly exist and serve various functions in certain redox enzymes which use or produce O2, H2, NO, CO, or N2, but the preferred paths have not always been unambiguously identified, and whether a continuous, permanent channel is an absolute requirement for transporting diatomic molecules is unknown. Here, we review the literature on gas channels in proteins and enzymes and we report on the use of site-directed mutagenesis and various kinetic methods, which proved useful for characterizing substrate access to the active site of NiFe hydrogenase to test the putative "static" H2 channel of FeFe hydrogenases. We designed 8 mutations in attempts to interfere with intramolecular diffusion by remodeling this putative route in Clostridium acetobutylicum FeFe hydrogenase, and we observed that none of them has a strong effect on any of the enzyme's kinetic properties. We suggest that H2 may diffuse either via transient cavities, or along a conserved water-filled channel. Nitrogenase sets a precedent for the involvement of a hydrophilic channel to conduct hydrophobic molecules.

Using direct electrochemistry to learn about the mechanism of electrocatalysts and redox enzymes requires that kinetic models be developed. Here we thoroughly discuss the interpretation of electrochemical signals obtained with adsorbed enzymes and molecular catalysts that can reversibly convert their substrate and product. We derive analytical relations between electrochemical observables (overpotentials for catalysis in each direction, positions, and magnitudes of the features of the catalytic wave) and the characteristics of the catalytic cycle (redox properties of the catalytic intermediates, kinetics of intramolecular and interfacial electron transfer, etc.). We discuss whether or not the position of the wave is determined by the redox potential of a redox relay when intramolecular electron transfer is slow. We demonstrate that there is no simple relation between the reduction potential of the active site and the catalytic bias of the enzyme, defined as the ratio of the oxidative and reductive limiting currents; this explains the recent experimental observation that the catalytic bias of NiFe hydrogenase depends on steps of the catalytic cycle that occur far from the active site [Abou Hamdan et al., J. Am. Chem. Soc. 2012, 134, 8368]. On the experimental side, we examine which models can best describe original data obtained with various NiFe and FeFe hydrogenases, and we illustrate how the presence of an intramolecular electron transfer chain affects the voltammetry by comparing the data obtained with the FeFe hydrogenases from Chlamydomonas reinhardtii and Clostridium acetobutylicum, only one of which has a chain of redox relays. The considerations herein will help the interpretation of electrochemical data previously obtained with various other bidirectional oxidoreductases, and, possibly, synthetic inorganic catalysts.