Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase

Konstantin Laun; Iuliia Baranova; Jifu Duan; Leonie Kertess; Florian Wittkamp; Ulf-Peter Apfel; Thomas Happe; Moritz Senger; Sven T. Stripp

doi:10.1039/D1DT00110H

Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase†

Konstantin Laun,^ab Iuliia Baranova,^ac Jifu Duan,

^d Leonie Kertess,^d Florian Wittkamp,^e Ulf-Peter Apfel,

^ef Thomas Happe,

^d Moritz Senger

*^ag and Sven T. Stripp

*^a

Author affiliations

* Corresponding authors

^a Department of Physics, Freie Universität Berlin, 14195 Berlin, Germany
E-mail: sven.stripp@fu-berlin

^b Department of Chemistry, Technische Universität Berlin, 10623 Berlin, Germany

^c Faculty of Physics, St. Petersburg State University, 198504 St. Petersburg, Russian Federation

^d Faculty of Biology and Biotechnology, Ruhr-Universität Bochum, 44801 Bochum, Germany

^e Faculty of Chemistry and Biochemistry, Ruhr-Universität Bochum, 44801 Bochum, Germany

^f Fraunhofer UMSICHT, 46047 Oberhausen, Germany

^g Department of Chemistry, Uppsala University, 75120 Uppsala, Sweden
E-mail: moritz.senger@kemi.uu.se

Abstract

Hydrogenases are bidirectional redox enzymes that catalyze hydrogen turnover in archaea, bacteria, and algae. While all types of hydrogenase show H₂ oxidation activity, [FeFe]-hydrogenases are excellent H₂ evolution catalysts as well. Their active site cofactor comprises a [4Fe–4S] cluster covalently linked to a diiron site equipped with carbon monoxide and cyanide ligands. The active site niche is connected with the solvent by two distinct proton transfer pathways. To analyze the catalytic mechanism of [FeFe]-hydrogenase, we employ operando infrared spectroscopy and infrared spectro-electrochemistry. Titrating the pH under H₂ oxidation or H₂ evolution conditions reveals the influence of site-selective protonation on the equilibrium of reduced cofactor states. Governed by pK_a differences across the active site niche and proton transfer pathways, we find that individual electrons are stabilized either at the [4Fe–4S] cluster (alkaline pH values) or at the diiron site (acidic pH values). This observation is discussed in the context of the complex interdependence of hydrogen turnover and bulk pH.

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Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase†

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Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase

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