Rhodobacter capsulatus?un cbb3 tipi sitokrom oksidaz enziminin I. alt ünitesindeki korunmus bes amino asitin degistirilmesinin enzime etkiler

The heme-copper oxidases are the terminal elements of the membraneboundrespiratory chains that catalyze the reduction of molecular oxygen to waterand pump protons across the membrane. Analysis of the amino acid sequencesalignment of subunit I from different families of heme-copper oxidases showedthat some of the amino acids, serine 333 (S333), serine 340 (S340), threonine 350(T350), asparagine 390 (N390) and threonine 396 (T396) (R. capsulatusnumbering) are almost strictly conserved. This work was undertaken to determinethe relationships between these amino acids and the activity and assembly of thecbb3 oxidase in R. capsulatus.For this aim, 2.8 kbp fragment of the previously cloned pOX15, containingstructural gene (ccoNOQP) of cytochrome cbb3 oxidase of R. capsulatus, wassubcloned into pBluescript vector. The new clone, pMOZI, was used to substituteconserved amino acids for alanine by site-directed in vitro mutagenesis and theintegrity of each of these mutations was checked by sequencing. The mutatedplasmids each of which carries one of the desired substitutions on structural geneof the cbb3 oxidase were transferred into R. capsulatus strain, GK32. While theeffects of the mutations on cytochrome c oxidase activity were evaluated byNADI plate assays, the structural integrity of the mutant oxidases weredetermined by separation of chromatophore membrane proteins on Schägger-typepoly acrylamide gel stained with 3,3?,5,5?-tetramethylbenzidine (TMBZ).While serine and threonine mutants (S340A, T350A and T396A) had thesame activity with wild-type, serine 333 mutation (S333A) decreased the enzymeactivity and asparagine mutation (N390A) led to a complete loss of the cbb3oxidase activity. When cytochrome c profiles of mutants were examined, it wasfound that cytochrome cbb3 profiles of S333A, S340A, T350A and T396Amutants were the same to that of the wild-type, however the cp subunit of cbb3oxidase was missing and the amount of the co subunit was decreased in N390Amutant. The results indicated that S333 and N390 are very critical residues foractivity or assembly of the cbb3-type cytochrome oxidase in R. capsulatus.Keywords: Rhodobacter capsulatus, respiration, cytochrome cbb3-type oxidaseactivity, site-directed mutagenesis, chromatophore membrane protein isolation,