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

Martin Winkler,Eckhard Hofmann,Julian Esselborn,Thomas Happe,Sven Timo Stripp,Ulf-Peter Apfel,Jifu Duan,Moritz Senger,Florian Wittkamp

doi:10.1038/ncomms16115

Martin Winkler, Eckhard Hofmann + Show 7 more

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https://doi.org/10.1038/ncomms16115

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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.

Highlights

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 doubleoxidized to release another proton
Le Chatelier’s principle was applied to enrich the highly transient H-cluster intermediate Hhyd, which according to the current working model of catalytic H2 turnover is predicted to carry a terminal hydride species
A nearly identical Fourier-transform infrared (FTIR) band pattern was observed in our earlier spectroscopic analysis of the largely inactive HydA1 proton-transfer pathway (PTP) variant C169S

Summary

Introduction

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 doubleoxidized to release another proton. We use different strategies to trap and spectroscopically characterize this transient hydride state (Hhyd) for three wild-type [FeFe]-hydrogenases. According to the current working model for the catalytic mechanism of [FeFe]-hydrogenases[1], binding of H2 to the oxidized active ready state (Hox) results in the heterolytic cleavage of H2, with Hhyd as the first intermediate. A terminal hydride has not been assigned to any of the known redox states[10], nor has another catalytic wild-type state with the postulated t-H À been reported. This implies that the kinetically relevant hydride state is a transient one and difficult to trap under steady-state conditions. Owing to the rules governing steady-state kinetics, which favour thermodynamically stable intermediates in catalysis, the direct characterization of transient states relies on the utilization of time-resolved approaches with stopped-flow or single-turnover set-ups[16]

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