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

被引:44
作者
De Lacey, AL
Pardo, A
Fernández, VM
Dementin, S
Adryanczyk-Perrier, G
Hatchikian, EC
Rousset, M
机构
[1] CSIC, Inst Catalisis, Madrid 28049, Spain
[2] CNRS, Inst Biol Struct & Microbiol, F-13402 Marseille 20, France
来源
JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY | 2004年 / 9卷 / 05期
关键词
Fourier transform infrared spectroelectrochemistry; hydrogen; metalloprotein; site-directed mutagenesis; solvent isotope effect;
D O I
10.1007/s00775-004-0559-7
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 mu-OH- bridge at the hydrogenase active site, and that Glu25 has a role in this step.
引用
收藏
页码:636 / 642
页数:7
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