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

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Green algae such as Chlamydomonas reinhardtii synthesize an [FeFe] hydrogenase that is highly active in hydrogen evolution. However, the extreme sensitivity of [FeFe] hydrogenases to oxygen presents a major challenge for exploiting these organisms to achieve sustainable photosynthetic hydrogen production. In this study, the mechanism of oxygen inactivation of the [FeFe] hydrogenase CrHydA1 from C. reinhardtii has been investigated. X-ray absorption spectroscopy shows that reaction with oxygen results in destruction of the [4Fe-4S] domain of the active site H-cluster while leaving the di-iron domain (2FeH) essentially intact. By protein film electrochemistry we were able to determine the order of events leading up to this destruction. Carbon monoxide, a competitive inhibitor of CrHydA1 which binds to an Fe atom of the 2FeH domain and is otherwise not known to attack FeS clusters in proteins, reacts nearly two orders of magnitude faster than oxygen and protects the enzyme against oxygen damage. These results therefore show that destruction of the [4Fe-4S] cluster is initiated by binding and reduction of oxygen at the di-iron domain-a key step that is blocked by carbon monoxide. The relatively slow attack by oxygen compared to carbon monoxide suggests that a very high level of discrimination can be achieved by subtle factors such as electronic effects (specific orbital overlap requirements) and steric constraints at the active site.EXAFS ͉ H-cluster ͉ protein film electrochemistry ͉ biological hydrogen production ͉ green algae