Enhanced oxygen tolerance of hydrogenase from Klebsiella oxytoca HP1 by Gly-Cys exchanges nearby Fe-S clusters as biocatalysts in biofuel cells or hydrogen production

Abstract Hydrogenase is the key point of H 2 -based biotechnology. However, the O 2 -sensitivity largely hinders its applications in biofuel cells and biological H 2 production. Therefore, substantial breakthrough on understanding the molecular basis of O 2 -sensitivity and developing more O 2 -tolerant hydrogenases are urgently required. In this study, we found adding extra cysteines to the vicinity of the proximal Fe–S cluster to the NiFe active centre could largely enhance oxygen tolerance of hydrogen-evolving hydrogenase 3 from Klebsiella oxytoca HP1 ( Ko Hyd3), through homologous sequence comparison and site-directed mutagenesis. Ratio of aerobic hydrogen yield to anaerobic hydrogen yield (RHH) of Gly47Cys (Gly47 was replaced with Cys47), Gly50Cys, Gly113Cys, Gly120Cys and Gly50Cys–Gly120Cys (double exchange) were increased by 46.99%, 42.15%, 59.19%, 44.74% and 78.72%, respectively, comparing with that of wild type. Moreover, TiO 2 - Ko Hyd3 (Gly47Cys, Gly50Cys, Gly113Cys, Gly120Cys and Gly50Cys–Gly120Cys) particles acted well in UV light-driven H 2 production from water. These results revealed that extra cysteines nearby Fe–S clusters had significant effects on oxygen tolerance of Ko Hyd3. It also provided a promising way to produce O 2 -tolerant hydrogenase as biocatalysts in biofuel cells or H 2 production by photolysis of water.