Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O sub(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 sub(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 sub(2). Understanding the O sub(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 sub(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 arrow right Val and Phe113 arrow right Leu in HupV) rendered HupUV sensitive to O sub(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 sub(2)-sensing HupUV protein is the first component of the H sub(2)-transduction cascade, which, together with the two-component system HupT-HupR, regulates HupSL synthesis in response to H sub(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 sub(2).