Molecular mechanism and reactivities of the complex and highly active Fe-hydrogenase from Clostridium acetobutylicum – CAFE

1) Scientific Background and objectives. Hydrogenases are the enzymes that catalyse the biological conversion between hydrogen and protons, a reaction that is of interest both from a biological and chemical viewpoint. They may be used as catalysts at the anode of bio-fuel cells, or for hydrogen production from water and light by photosynthetic micro-organisms. Hydrogenases are large and complex enzymes: the chemistry occurs at an organo-metallic active site which is buried in the protein and connected to the solvent by several redox cofactors used for transferring electrons, molecular hydrophobic channels involved in intramolecular transport of small molecules (substrate, inhibitors) and machineries for proton transfers. Hydrogenases are classified into two main classes according to the metal content of the active site. Our project deals with the so-called "FeFe-hydrogenases", which have the greater turnover number for hydrogen oxidation. Despite the international competition in this field, their study was long hindered by several obstacles: these enzymes are fragile and produced by micro-organisms which are usually not amenable to genetic engineering. In preliminary experiments described in the proposal, we have addressed major technical hurdles regarding the engineering, production and study of a FeFe-hydrogenase. This made it possible to build an interdisciplinary consortium which unites for the first time three research teams having distinct, complementary expertises in the fields of biochemistry, molecular biology, physical chemistry and modelling. Several other teams assured us of their occasional collaboration on specific aspects of the project. We shall use site-directed mutagenesis and physical techniques on a complex FeFe-hydrogenase to study its mechanism and to identify the structural determinants of reactivity, sensitivity to inhibitors (including O2), and catalytic bias (H2 oxidation vs production). This will provide invaluable guidelines for the ongoing search for the hydrogenase Holy Grail: the highly-active though oxygen-tolerant enzyme 2) Description of the project, methodology. We have selected the FeFe-hydrogenase from the bacterium Clostridium acetobutylicum as a model system for our studies for the following reasons. (1) Team 2 has just developed the biological procedures that will allow us to engineer, selectively modify and purify these proteins to homogeneity. This is a worldwide unique achievement. (2) The molecular structures of two similar enzymes are known, and one of them was actually determined by Team 3. Hence targets for mutagenesis will easily be identified. (3) Team 1 has carried out preliminary experiments using direct electrochemistry, a kinetic technique which proved very useful for investigating the mechanism of this enzyme. Our preliminary data show that this enzyme is extremely active but much less oxygen-sensitive (at least on the time scale of minutes) than was anticipated. This argues against the commonly accepted proposal that the higher reactivity of hydrogenases with respect to their substrate hydrogen is paid for by a greater sensitivity to oxygen. This also suggests that it may be possible to identify or design a hydrogenase with high turnover even in the presence of oxygen. 3) Expected results. Our goal is to study the catalytic mechanism of FeFe-hydrogenases, and to understand how the global properties of the enzyme are tuned at a molecular level, by focussing on all aspects related to function: the structural, electronic and redox properties of the active sites, the dynamics of proton transfers and long-range, intramolecular electron transfers and the kinetics of intramolecular diffusion along the gas channels, in relation to their selectivity with respect to substrate and inhibitors. We will accomplish this using an interdisciplinary approach, by making use of the various biochemical and physical techniques mastered in our groups: genetic engineering, biochemistry, dynamic electrochemistry, and modelling. This should generate new data with a significant impact on the application of hydrogenases for hydrogen production or oxidation, as it demands that the enzymes be optimized to function in a given direction and resist chemical perturbations, including inhibition by O2.