科研之友
微信
新浪微博
Facebook
分享链接
摘要
The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H-2 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 noncatalytic 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 (H-2 oxidation, H-2 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 H-2 oxidation and H-2 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 H-2 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 H-2 oxidation/production.
- 出版日期2015-3-27
