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 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.
De Lacey
Antonio L
AL
Instituto de Catálisis, CSIC, Campus de Cantoblanco, 28049 Madrid, Spain. alopez@icp.csic.es
Pardo
Alejandro
A
Fernández
Víctor M
VM
Dementin
Sebastian
S
Adryanczyk-Perrier
Geraldine
G
Hatchikian
E Claude
EC
Rousset
Marc
M
D000693:Anaerobiosis; D001665:Binding Sites; D045286:Desulfovibrio gigas / Q000201:enzymology*; D018698:Glutamic Acid / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007554:Isotopes; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D012997:Solvents; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D007554:Isotopes; D008667:Metalloproteins; D012997:Solvents; D018698:Glutamic Acid; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase