Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O2 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 H2, which is detected by a regulatory hydrogenase, HupUV. The HupUV protein exhibits typical features of [NiFe] hydrogenases but, interestingly, is resistant to inactivation by O2. Understanding the O2 resistance of HupUV will help in the design of hydrogenases with high potential for biotechnological applications. To test whether this property results from O2 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 O2 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 H2-sensing HupUV protein is the first component of the H2-transduction cascade, which, together with the two-component system HupT/HupR, regulates HupSL synthesis in response to H2 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 H2.
Duché
Ophélie
O
Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (UMR 5092 CNRS-CEA-UJF), Département Réponse et Dynamique Cellulaires, Grenoble, France.
Elsen
Sylvie
S
Cournac
Laurent
L
Colbeau
Annette
A
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D041961:Deuterium Exchange Measurement; D004789:Enzyme Activation / Q000235:genetics; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; C084201:HupT protein, Rhodobacter capsulatus; C101649:HupU protein, Rhodobacter capsulatus; C101650:HupV protein, Rhodobacter capsulatus; D012097:Repressor Proteins; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen