Crystallographic and spectroscopic assignment of the proton transfer pathway in [FeFe]-hydrogenases

Jifu Duan, Moritz Senger, Julian Esselborn, Vera Engelbrecht, Florian Wittkamp, Ulf-Peter Apfel, Eckhard Hofmann, Sven T. Stripp, Thomas Happe () and Martin Winkler ()
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Jifu Duan: Ruhr-Universität Bochum
Moritz Senger: Freie Universität Berlin
Julian Esselborn: Ruhr-Universität Bochum
Vera Engelbrecht: Ruhr-Universität Bochum
Florian Wittkamp: Ruhr-Universität Bochum
Ulf-Peter Apfel: Ruhr-Universität Bochum
Eckhard Hofmann: Ruhr-Universität Bochum
Sven T. Stripp: Freie Universität Berlin
Thomas Happe: Ruhr-Universität Bochum
Martin Winkler: Ruhr-Universität Bochum

Nature Communications, 2018, vol. 9, issue 1, 1-11

Abstract: Abstract The unmatched catalytic turnover rates of [FeFe]-hydrogenases require an exceptionally efficient proton-transfer (PT) pathway to shuttle protons as substrates or products between bulk water and catalytic center. For clostridial [FeFe]-hydrogenase CpI such a pathway has been proposed and analyzed, but mainly on a theoretical basis. Here, eleven enzyme variants of two different [FeFe]-hydrogenases (CpI and HydA1) with substitutions in the presumptive PT-pathway are examined kinetically, spectroscopically, and crystallographically to provide solid experimental proof for its role in hydrogen-turnover. Targeting key residues of the PT-pathway by site directed mutagenesis significantly alters the pH-activity profile of these variants and in presence of H2 their cofactor is trapped in an intermediate state indicative of precluded proton-transfer. Furthermore, crystal structures coherently explain the individual levels of residual activity, demonstrating e.g. how trapped H2O molecules rescue the interrupted PT-pathway. These features provide conclusive evidence that the targeted positions are indeed vital for catalytic proton-transfer.

Date: 2018
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DOI: 10.1038/s41467-018-07140-x

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