Combining Electrochemistry and Protein Engineering to Elucidate Outer-Sphere Effects in Hydrogenase Catalysis

Abstract

Hydrogenases, the bacterial enzymes that oxidize and produce H2, come in two flavors, “NiFe hydrogenases” and “FeFe hydrogenases”, depending on the structure of their active site.In each of these two families of enzymes, the active site and its immediate environment are fully conserved, and yet the investigations of these enzymes using direct electrochemistry have shown that their catalytic properties vary greatly: this is true regarding catalytic bias and reversibility1, resistance to oxygen, and other properties which define whether or not a particular hydrogenase could be used under the operating conditions of a particular device.These functional variations within each family also tell us that structural features that are remote from the active site, the so-called “outer-sphere” effects, define, at least in part, the catalytic properties.2 In the FeFe hydrogenase from Clostridium beijerinckii, we have shown that oxygen resistance is related to a conformational change that depends on non-conserved residues that are up to 18 Å away from the active site. This conformational change occurs in two steps that can be resolved by combining site-directed mutagenesis and detailed protein film voltammetry experiments.3 These remote residues define an emerging family of O2-resistant FeFe hydrogenases. (a) A. Fasano, H. Land, V. Fourmond, G. Berggren, and C. Léger, « Reversible or irreversible catalysis of H+/H2 conversion by FeFe hydrogenases », J. Am. Chem. Soc. 143, 48, 20320-20325 (2021) (b) V. Fourmond, N. Plumeré, C. Léger, « Reversible catalysis », Nature Reviews Chemistry, 5, 348-360 (2021)S. Stripp, B. Duffus, V. Fourmond, C. Léger, S. Leimkühler, S. Hirota, Y. Hu, A. Jasniewski, H. Ogata, M. Ribbe, « Second and Outer Coordination Sphere Effects in Low Valent Metalloenzymes », Chemical Reviews, 122 11900 (2022) (a) M. Winkler, J. Duan, A. Rutz, C. Felbek, L. Scholtysek, O. Lampret, J. Jaenecke, U.-P.r Apfel, G. Gilardi, F. Valetti, V. Fourmond, E. Hofmann, C. Léger, T. Happe « A safety cap protects hydrogenase from oxygen attack » Nature Communications 12, 756 (2021) (b) A. Rutz, C. Das, A. Fasano, J. Jaenecke, S. Yadav, U.P. Apfel, V. Engelbrecht, V. Fourmond, C. Léger, L. Schäfer, T. Happe « Increasing the O2 resistance of the [FeFe]-hydrogenase CbA5H through enhanced protein flexibility », ACS Catalysis 13, 2, 856–865 (2023) Figure 1