Accumulating the hydride state in the catalytic cycle of [FeFe]-hydrogenases

Martin Winkler, Moritz Senger, Jifu Duan, Julian Esselborn, Florian Wittkamp, Eckhard Hofmann, Ulf-Peter Apfel, Sven Timo Stripp () and Thomas Happe ()
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Martin Winkler: AG Photobiotechnologie, Lehrstuhl für Biochemie der Pflanzen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum
Moritz Senger: Experimental Molecular Biophysics, Freie Universität Berlin
Jifu Duan: AG Photobiotechnologie, Lehrstuhl für Biochemie der Pflanzen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum
Julian Esselborn: AG Photobiotechnologie, Lehrstuhl für Biochemie der Pflanzen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum
Florian Wittkamp: Fakultät für Chemie und Biochemie, Lehrstuhl für Anorganische Chemie I/Bioanorganische Chemie, Ruhr-Universität Bochum
Eckhard Hofmann: AG Proteinkristallographie, Lehrstuhl für Biophysik, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum
Ulf-Peter Apfel: Fakultät für Chemie und Biochemie, Lehrstuhl für Anorganische Chemie I/Bioanorganische Chemie, Ruhr-Universität Bochum
Sven Timo Stripp: Experimental Molecular Biophysics, Freie Universität Berlin
Thomas Happe: AG Photobiotechnologie, Lehrstuhl für Biochemie der Pflanzen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum

Nature Communications, 2017, vol. 8, issue 1, 1-7

Abstract: Abstract H2 turnover at the [FeFe]-hydrogenase cofactor (H-cluster) is assumed to follow a reversible heterolytic mechanism, first yielding a proton and a hydrido-species which again is double-oxidized to release another proton. Three of the four presumed catalytic intermediates (Hox, Hred/Hred and Hsred) were characterized, using various spectroscopic techniques. However, in catalytically active enzyme, the state containing the hydrido-species, which is eponymous for the proposed heterolytic mechanism, has yet only been speculated about. We use different strategies to trap and spectroscopically characterize this transient hydride state (Hhyd) for three wild-type [FeFe]-hydrogenases. Applying a novel set-up for real-time attenuated total-reflection Fourier-transform infrared spectroscopy, we monitor compositional changes in the state-specific infrared signatures of [FeFe]-hydrogenases, varying buffer pH and gas composition. We selectively enrich the equilibrium concentration of Hhyd, applying Le Chatelier’s principle by simultaneously increasing substrate and product concentrations (H2/H+). Site-directed manipulation, targeting either the proton-transfer pathway or the adt ligand, significantly enhances Hhyd accumulation independent of pH.

Date: 2017
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DOI: 10.1038/ncomms16115

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