Soutenance de thèse de Christina Felbek

18/11/2021 - 13:30 - GLM
Christina FELBEK, BIP 06

Mechanistic studies of FeFe hydrogenases by electrochemistry: inhibition by cysteine and hydrogen sulfide, intramolecular diffusion and maturation.

FeFe hydrogenases are enzymes that catalyse the oxidation and production of hydrogen. With their high efficiency, these biocatalysts are potent candidates to replace platinum in (bio-)fuel cells to generate the “green” energy carrier hydrogen. The reactivity of these enzymes is in particular linked to a free coordination site (called Fed) on the 2Fe sub-cluster of its active site. In the catalytic cycle, H2 binds to Fed, whereas O2 binding to this site damages the enzyme. The oxygen sensitivity of hydrogenases is a major drawback for any potential application of these enzymes. In this work, we used protein film electrochemistry (PFE) in collaboration with different other techniques to study various aspects of enzyme kinetics in relation to this reactivity in detail. Oxidation in the presence of exogenous sulfide makes the enzyme resistant to O2. Indeed, this leads to an inactive state of the enzyme called Hinact, in which Fed is coordinated by a sulfide ligand; the latter blocks oxygen attack. This oxygen resistant, inactive state can be reactivated by reduction. We describe the mechanism of this reaction in detail by using a combination of PFE studies, MD and DFT calculations. Furthermore, we extensively studied the reversible, sulfide-independant formation of the same Hinact state in the FeFe hydrogenase from Clostridium beijerinckii. Using PFE, IR spectroscopy, XRD crystallography and site-directed mutagenesis, we discovered that Hinact is formed by the coordination of a conserved cysteine sulfide, in a process that is very dependent on the flexibility of the protein backbone. We show that up to 13 Å distant, non-conserved amino acids enable the translocation of the cysteine residue, and thus remotely control active site chemistry. We also characterised the kinetic properties of the bifurcating hydrogenase from Desulfovibrio fructosovorans as well as mutants of the enzyme from C. reinhardtii designed to influence gas diffusion along proposed intramolecular diffusion pathways. We aimed to elucidate how these inhibitors travel to the active site and how this affects the inhibition kinetics. Finally, we probed the last step of the enzyme maturation – the insertion of the 2Fe cluster into the apo-enzyme – in a redox polymer film. In summary, using a multidisciplinary approach that combines state-of-the-art electrochemistry, molecular biology, biochemistry and theoretical methods, we advanced the understanding of oxygen protection mechanisms in FeFe hydrogenases. We also demonstrated the long range influence of several residues on the active site chemistry.

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