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

The kinetics of the activation and anaerobic inactivation processes of Desulfovibrio gigas hydrogenase have been measured in D(2)O by FTIR spectroelectrochemistry. A primary kinetic solvent isotope effect was observed for the inactivation process but not for the activation step. The kinetics of thes...

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Bibliographic Details
Published in:Journal of biological inorganic chemistry 2004-07, Vol.9 (5), p.636-642
Main Authors: De Lacey, Antonio L, Pardo, Alejandro, Fernández, Víctor M, Dementin, Sebastian, Adryanczyk-Perrier, Geraldine, Hatchikian, E Claude, Rousset, Marc
Format: Article
Language:English
Subjects:
Anaerobiosis
Binding Sites
Desulfovibrio gigas - enzymology
Glutamic Acid - chemistry
Hydrogen - chemistry
Hydrogenase - chemistry
Hydrogenase - genetics
Hydrogenase - metabolism
Isotopes
Kinetics
Metalloproteins - chemistry
Metalloproteins - metabolism
Mutagenesis, Site-Directed
Solvents
Spectroscopy, Fourier Transform Infrared - methods
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Summary:The kinetics of the activation and anaerobic inactivation processes of Desulfovibrio gigas hydrogenase have been measured in D(2)O 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 micro-OH(-) bridge at the hydrogenase active site, and that Glu25 has a role in this step.
ISSN:0949-8257
1432-1327
DOI:10.1007/s00775-004-0559-7