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FTIR spectroelectrochemical study of the activation and inactivation processes of [NiFe] hydrogenases: effects of solvent isotope replacement and site-directed mutagenesis

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Abstract

The kinetics of the activation and anaerobic inactivation processes of Desulfovibrio gigas hydrogenase have been measured in D2O by FTIR spectroelectrochemistry. A primary kinetic solvent isotope effect was observed for the inactivation process but not for the activation step. The kinetics of these processes have been also measured after replacement of a glutamic residue placed near the active site of an analogous [NiFe] hydrogenase from Desulfovibrio fructosovorans. Its replacement by a glutamine affected greatly the kinetics of the inactivation process but only slightly the activation process. The interpretation of the experimental results is that the rate-limiting step for anaerobic inactivation is the formation from water of a μ-OH− bridge at the hydrogenase active site, and that Glu25 has a role in this step.

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Acknowledgements

This work was supported by the Spanish MCYT (project BQU2003-04221) and CAM (project 07N/0068/2002) and by grant G5RD-CT-2002-00750 from the European Commission Competitive and Sustainable Growth Programme. M.R. is “Laboratoire de Recherche Conventionné” with the CEA, no. 25V. We thank Dr A. Volbeda for useful discussions.

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Authors and Affiliations

  1. Instituto de Catálisis, CSIC, Campus de Cantoblanco, 28049 , Madrid, Spain

    Antonio L. De Lacey, Alejandro Pardo & Víctor M. Fernández

  2. Bioenergetique et Ingenerie des Proteins, Institute de Biologie Structurale et Microbiologie, CNRS, Chemin Joseph Aiguier, 13402 Cedex 20, Marseille, France

    Sebastian Dementin, Geraldine Adryanczyk-Perrier, E. Claude Hatchikian & Marc Rousset

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  1. Antonio L. De Lacey
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  2. Alejandro Pardo
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  3. Víctor M. Fernández
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  4. Sebastian Dementin
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  5. Geraldine Adryanczyk-Perrier
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  6. E. Claude Hatchikian
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Correspondence to Antonio L. De Lacey.

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De Lacey, A.L., Pardo, A., Fernández, V.M. et al. FTIR spectroelectrochemical study of the activation and inactivation processes of [NiFe] hydrogenases: effects of solvent isotope replacement and site-directed mutagenesis. J Biol Inorg Chem 9, 636–642 (2004). https://doi.org/10.1007/s00775-004-0559-7

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  • DOI: https://doi.org/10.1007/s00775-004-0559-7

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