Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O 2 sensitive without affecting its transductory activity

In the photosynthetic bacterium Rhodobacter capsulatus , the synthesis of the energy‐producing hydrogenase, HupSL, is regulated by the substrate H 2 , which is detected by a regulatory hydrogenase, HupUV. The HupUV protein exhibits typical features of [NiFe] hydrogenases but, interestingly, is resistant to inactivation by O 2 . Understanding the O 2 resistance of HupUV will help in the design of hydrogenases with high potential for biotechnological applications. To test whether this property results from O 2 inaccessibility to the active site, we introduced two mutations in order to enlarge the gas access channel in the HupUV protein. We showed that such mutations (Ile65→Val and Phe113→Leu in HupV) rendered HupUV sensitive to O 2 inactivation. Also, in contrast with the wild‐type protein, the mutated protein exhibited an increase in hydrogenase activity after reductive activation in the presence of reduced methyl viologen (up to 30% of the activity of the wild‐type). The H 2 ‐sensing HupUV protein is the first component of the H 2 ‐transduction cascade, which, together with the two‐component system HupT/HupR, regulates HupSL synthesis in response to H 2 availability. In vitro , the purified mutant HupUV protein was able to interact with the histidine kinase HupT. In vivo , the mutant protein exhibited the same hydrogenase activity as the wild‐type enzyme and was equally able to repress HupSL synthesis in the absence of H 2 .