Conspectus Many enzymes that produce or transform small molecules such as O2, H2, and CO2 embed inorganic cofactors based on transition metals. Their active site, where the chemical reaction occurs, is buried in and protected by the protein matrix, and connected to the solvent in several ways: chains of redox cofactors mediate long-range electron transfer; static or dynamic tunnels guide the substrate, product and inhibitors; amino acids and water molecules transfer protons. The catalytic mechanism of these enzymes is therefore delocalized over the protein and involves many different steps, some of which determine the response of the enzyme under conditions of stress (extreme redox conditions, presence of inhibitors, light), the catalytic rates in the two directions of the reaction and their ratio (the "catalytic bias"). Understanding all the steps in the catalytic cycle, including those that occur on sites of the protein that are remote from the active site, requires a combination of biochemical, structural, spectroscopic, theoretical, and kinetic methods. Here we argue that kinetics should be used to the fullest extent, by extracting quantitative information from the comparison of data and kinetic models and by exploring the combination of experimental kinetics and theoretical chemistry. In studies of these catalytic mechanisms, direct electrochemistry, the technique which we use and contribute to develop, has become unescapable. It simply consists in monitoring the changes in activity of an enzyme that is wired to an electrode by recording an electric current. We have described kinetic models that can be used to make sense of these data and to learn about various aspects of the mechanism that are difficult to probe using more conventional methods: long-range electron transfer, diffusion along gas channels, redox-driven (in)activations, active site chemistry and photoreactivity under conditions of turnover. In this Account, we highlight a few results that illustrate our approach. We describe how electrochemistry can be used to monitor substrate and inhibitor diffusion along the gas channels of hydrogenases and we discuss how the kinetics of intramolecular diffusion relates to global properties such as resistance to oxygen and catalytic bias. The kinetics and/or thermodynamics of intramolecular electron transfer may also affect the catalytic bias, the catalytic potentials on either side of the equilibrium potential, and the overpotentials for catalysis (defined as the difference between the catalytic potentials and the open circuit potential). This is understood by modeling the shape of the steady-state catalytic response of the enzyme. Other determinants of the catalytic rate, such as domain motions, have been probed by examining the transient catalytic response recorded at fast scan rates. Last, we show that combining electrochemical investigations and MD, DFT, and TD-DFT calculations is an original way of probing the reactivity of the H-cluster of hydrogenase, in particular its reactions with CO, O2, and light. This approach contrasts with the usual strategy which aims at stabilizing species that are presumed to be catalytic intermediates, and determining their structure using spectroscopic or structural methods.
Electrochemical Investigations of Hydrogenases and Other Enzymes That Produce and Use Solar Fuels / Del Barrio, M.; Sensi, M.; Orain, C.; Baffert, C.; Dementin, S.; Fourmond, V.; Leger, C.. - In: ACCOUNTS OF CHEMICAL RESEARCH. - ISSN 0001-4842. - 51:3(2018), pp. 769-777. [10.1021/acs.accounts.7b00622]