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J Biol Chem. 2015 Mar 27;290(13):8550-8. doi: 10.1074/jbc.M114.630491. Epub 2015 Feb 9.

A threonine stabilizes the NiC and NiR catalytic intermediates of [NiFe]-hydrogenase.

Author information

1
From the Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, UMR 7281 Aix-Marseille Université/CNRS, 31 Chemin J. Aiguier, 13402 Marseille Cedex 20, France.
2
Metalloproteins Unit, Institut de Biologie Structurale, UMR 5075 Commissariat à l'Energie Atomique/CNRS/Université Joseph Fourier, 6 rue Jules Horowitz, 38000, Grenoble, France.
3
Instituto de Catálisis y Petroleoquímica, Consejo Superior de Investigaciones Científicas, c/Marie Curie 2, L10, 28049 Madrid, Spain.
4
Laboratoire de Bioénergétique et Biotechnologie des Bactéries et Microalgues, Commissariat à l'Energie Atomique, Direction des Sciences du Vivant, Institut de Biologie Environnementale et Biotechnologie, Saint-Paul-lez-Durance, F-13108, France CNRS, UMR 7265 Biologie Végétale et Microbiologie Environnementale (BVME), Saint-Paul-lez-Durance, 13108, France, and Aix-Marseille Université, BVME UMR7265, Marseille F-13284, France.
5
From the Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, UMR 7281 Aix-Marseille Université/CNRS, 31 Chemin J. Aiguier, 13402 Marseille Cedex 20, France, dementin@imm.cnrs.fr.

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.

KEYWORDS:

Electron Paramagnetic Resonance (EPR); Enzyme Kinetics; Fourier Transform IR (FTIR); Hydrogenase; X-ray Crystallography

PMID:
25666617
PMCID:
PMC4375504
DOI:
10.1074/jbc.M114.630491
[Indexed for MEDLINE]
Free PMC Article

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