Contents 1. Hydrogen as an Energy Store 4366 1.1. The Hydrogen Half Cell Reaction 4367 1.2. Why Study Hydrogenases on an Electrode? 4368 1.3. Challenges and General Visionary Outlook 4369 2. Hydrogen and Its Redox Chemistry in Biology 4369 2.1. Hydrogen Cycling in Biology 4369 2.2. Types of Hydrogenases 4370 2.3. Redox Partners for [FeFe]-and [NiFe]-Hydrogenases 4372 2.3.1. In vivo Redox Partners 4372 2.3.2. In vitro Redox Partners 4372 2.4. Hydrogenase Structures: Biological Plumbing and Wiring 4373 2.4.1. Electron Relay Centers 4373 2.4.2. Gas Channels May Control the Activity of Hydrogenases 4373 2.5. Complexity of Hydrogenase States 4373 2.5.1. The Role of Protein Film Voltammetry in Navigating between States of Hydrogenases 4374 3. Dynamic Electrochemical Methods for Studying Hydrogenases 4374 3.1. Electrochemical Equipment 4374 3.1.1. The Importance of Controlling Mass Transport 4375 3.1.2. Gas Supply and Gas Purity 4376 3.1.3. Light Intensity 4376 3.2. Methods for Preparing Hydrogenase Films on Electrodes 4376 3.2.1. Direct Adsorption of Hydrogenase onto an Electrode 4377 3.2.2. Strategies for Entrapment and Attachment of Hydrogenases at Electrodes 4378 4. The Study of Enzymes by Protein Film Voltammetry 4379 4.1. How Reactions Are Induced by the Electrode Potential 4379 4.2. What Does Protein Film Voltammetry Reveal? 4379 4.3. Enzymes as Complex Electrocatalysts 4380 4.4. Differences between Characteristic Potential Values Measured by Potentiometry and by Catalytic Voltammetry 4381 5. Electrocatalytic Activity of Hydrogenases 4381 5.1. Ensuring the Electrochemistry Is Not Controlled by Transport of Substrate 4383 5.2. Effects of Interfacial Electron Transfer on the Electrocatalytic Wave Shape 4384 5.3. Catalytic Constants 5.4. Dependence of Activity on pH 5.5. Activity Comparison of Hydrogenases with Pt 5.6. Catalytic Bias: H 2 Oxidation vs H + Reduction 5.7. Rate-Determining Steps 5.7.1. H + /D 2 Exchange Experiments 4366

A new strategy is described for comparing, quantitatively, the ability of hydrogenases to tolerate exposure to O2 and anoxic oxidizing conditions. Using protein film voltammetry, the inherent sensitivities to these challenges (thermodynamic potentials and rates of reactions) have been measured for enzymes from a range of mesophilic microorganisms. In the absence of O2, all the hydrogenases undergo reversible inactivation at various potentials above that of the H+/H2 redox couple, and H2 oxidation activities are thus limited to characteristic "potential windows". Reactions with O2 vary greatly; the [FeFe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757, an anaerobe, is irreversibly damaged by O2, surviving only if exposed to O2 in the anaerobically oxidized state (which therefore affords protection). In contrast, the membrane-bound [NiFe]-hydrogenase from the aerobe, Ralstonia eutropha, reacts reversibly with O2 even during turnover and continues to catalyze H2 oxidation in the presence of O2.

The crystal structure of the membrane-bound O 2 -tolerant [NiFe]-hydrogenase 1 from Escherichia coli (EcHyd-1) has been solved in three different states: as-isolated, H 2 -reduced, and chemically oxidized. As very recently reported for similar enzymes from Ralstonia eutropha and Hydrogenovibrio marinus, two supernumerary Cys residues coordinate the proximal [FeS] cluster in EcHyd-1, which lacks one of the inorganic sulfide ligands. We find that the as-isolated, aerobically purified species contains a mixture of at least two conformations for one of the cluster iron ions and Glu76. In one of them, Glu76 and the iron occupy positions that are similar to those found in O 2 -sensitive [NiFe]-hydrogenases. In the other conformation, this iron binds, besides three sulfur ligands, the amide N from Cys20 and one Oϵ of Glu76. Our calculations show that oxidation of this unique iron generates the high-potential form of the proximal cluster. The structural rearrangement caused by oxidation is confirmed by our H 2 -reduced and oxidized EcHyd-1 structures. Thus, thanks to the peculiar coordination of the unique iron, the proximal cluster can contribute two successive electrons to secure complete reduction of O 2 to H 2 O at the active site. The two observed conformations of Glu76 are consistent with this residue playing the role of a base to deprotonate the amide moiety of Cys20 upon iron binding and transfer the resulting proton away, thus allowing the second oxidation to be electroneutral. The comparison of our structures also shows the existence of a dynamic chain of water molecules, resulting from O 2 reduction, located near the active site.[4Fe-3S] cluster | membrane-bound hydrogenase | Mössbauer spectroscopy | QM/MM | structure/function relationships