A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

Abbas Abou-Hamdan,Pierre Ceccaldi,Hugo Lebrette,Oscar Gutiérrez-Sanz,Pierre Richaud,Laurent Cournac,Bruno Guigliarelli,Antonio L De Lacey,Christophe Léger,Anne Volbeda,Bénédicte Burlat,Sébastien Dementin

doi:10.1074/jbc.m114.630491

Abstract

The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production.

Highlights

A conserved threonine in [NiFe]-hydrogenases is a putative proton transfer relay
The T18D mutation is less deleterious for H2 production activity than the T18V, T18Q, and T18G mutations, our results show that the alcohol function is important
The data can be analyzed to calculate the rate of H2 diffusion from the active site and the first order rate constant of HD exchange at the active site (k), which is an indicator of PT and H2 splitting recombination at the active site [19, 25]

Summary

Background

A conserved threonine in [NiFe]-hydrogenases is a putative proton transfer relay. Results: Poorly active variants have modified spectroscopic signatures associated with changes in local protein structure. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but noncatalytic conditions. Suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production. In [NiFe]-hydrogenases, the electrons produced at the active site are transferred to the redox partner at the protein surface via a chain of three FeS clusters called proximal, medial, and. The usual experimental approach for identifying proton transfer (PT) relays in [NiFe]-hydrogenases consists of exchanging residues with others whose side chain is not protonatable and measuring the H2 production/oxidation, the para/ortho H2 conversion, and the H/D exchange (HDE) activities of the mutated enzymes [18]. A combination of these three assays has been used to investigate PT in hydrogenase

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION