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 (KoHyd3), 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, TiO2-KoHyd3 (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 KoHyd3. 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. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.