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PublicationJOURNAL OF BIOLOGICAL CHEMISTRY
Year2015
Volume290 (13)
Pages8550-8558
International

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

Authors:Abbas Abou Hamdan , Gutiérrez-Sanz, Oscar, Pierre Richaud , Laurent Cournac , Bruno Guigliarelli , Antonio López de Lacey, Christophe Leger , Anne Volbeda , Benedicte Burlat , Sebastien Dementin
Groups of research:BioElectroCatalysis
The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H-2 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 noncatalytic 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 (H-2 oxidation, H-2 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 H-2 oxidation and H-2 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 H-2 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 H-2 oxidation/production.
Keywords:ACTIVE-SITE, ACTIVATION, UNREADY OXIDIZED STATES, ESCHERICHIA-COLI, PROTON TRANSPORT PATHWAYS, NIFE HYDROGENASE, DESULFOVIBRIO-GIGAS, ELECTRON TRANSFERS, CLUSTER, CYCLE
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