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Título: | A threonine stabilizes the NiC and NiR catalytic intermediates of [NiFe]-hydrogenase |
Autor: | Abou-Hamdan, A.; Ceccaldi, P.; Lebrette, Hugo; Gutiérrez-Sanz, Óscar CSIC ORCID; Richaud, P.; Cournac, L.; Guigliarelli, Bruno; López de Lacey, Antonio CSIC ORCID ; Léger, Christophe; Volbeda, A.; Burlat, Bénédicte; Dementin, Sébastien | Fecha de publicación: | 27-mar-2015 | Editor: | American Society for Biochemistry and Molecular Biology | Citación: | Journal of Biological Chemistry 290(13): 8550-8558 (2015) | Resumen: | 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. | Descripción: | Esta investigación se publicó originalmente en el Journal of Biological Chemistry. Abou-Hamdan, A.; Ceccaldi, P.; Lebrette, Hugo; Gutiérrez-Sanz, Óscar ; Richaud, P.; Cournac, L.; Guigliarelli, Bruno; López de Lacey, Antonio ; Léger, Christophe; Volbeda, A.; Burlat, Bénédicte; Dementin, Sébastien. A threonine stabilizes the NiC and NiR catalytic intermediates of [NiFe]-hydrogenase. J. Biol. Chem 2015; 290(13): 8550-8558. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. | Versión del editor: | https://doi.org/10.1074/jbc.M114.630491 | URI: | http://hdl.handle.net/10261/189723 | DOI: | 10.1074/jbc.M114.630491 | ISSN: | 0021-9258 | E-ISSN: | 1083-351X |
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