Öztürk, MehmetGürel, EkremWatmough, Nicholas J.Mandacı, Sevnur2021-06-232021-06-2320071225-8687https://hdl.handle.net/20.500.12491/6020https://doi.org/10.5483/BMBRep.2007.40.5.697Cytochrome ebb.; oxidase is a member of the heme-copper oxidase superfamily that catalyses the reduction of molecular oxygen to the water and conserves the liberated energy in the form of a proton gradient. Comparison of the amino acid sequences of subunit I from different classes of heme-copper oxidases showed that transmembrane helix VIII and the loop between transmembrane helices IX and X contain five highly conserved polar residues; Ser333, Ser340, Thr350, Asn390 and Thr394. To determine the relationship between these conserved amino acids and the activity and assembly of the cbb(3); oxidase in Rhodobacter capsulatus, each of these five conserved amino acids was substituted for alanine by site-directed mutagenesis. The effects of these mutations on catalytic activity were determined using a NADI plate assay and by measurements of the rate of oxygen consumption. The consequence of these mutations for the structural integrity of the cbb(3) oxidase was determined by SDS-PAGE analysis of chromatophore membranes followed by TNBZ staining. The results indicate that the Asn390Ala mutation led to a complete loss of enzyme activity and that the Ser333Ala mutation decreased the activity significantly. The remaining mutants cause a partial loss of catalytic activity. All of the mutant enzymes, except Asn390Ala, were apparently correctly assembled and stable in the membrane of the R. capsulatus.eninfo:eu-repo/semantics/openAccessCytochrome cbb(3)-type Oxidase ActivityRespiration and Site-directed MutagenesisRhodobacter CapsulatusArticle40569770717927903N/AWOS:000249823600013Q3