Molekulare Charakterisierung des Cytochrom -bc1-aa3-Superkomplexes aus Corynebacterium glutamicum

Spectroscopical investigations and inhibitor studies indicated a branched respiratory chain in Corynebacterium glutamicum. Electrons can be transferred to oxygen either via a cytochrome bcl complex and a aa$_{3-}$type cytochrome c oxidase or alternatively via a menaquinol oxidase of the cytochrome bd-type. The aim of this work was a molecular characterization of the postulated bc$_{1}$-aa$_{3}$-branch. The genes of the three subunits of the bcl complex (gcrCAB) and of subunits I and III of cytochrome aa$_{3}$ oxidase (ctaD and ctaE) were cloned and sequenced. Analysis of the deduced protein sequences showed a number of unusual features. The Rieske iron sulfur protein (QcrA) had three putative N-terminal transmembranhelices and cytochrome b (QcrB) possessed a large C-terminal domain consisting of approximately 130 amino acids in addition to the conserved part ofthe protein. Most remarkably, cytochrome c$_{1}$ (QcrC)contained two CXXCH motifs for covalent heme binding indicating that this protein is a diheme-cytochrome c. Inactivation of the bc$_{1}$ complex or the cytochrome aa$_{3}$ oxidase by chromosomal deletion of the qcr genes and the ctaD gene, respectively, led to strongly impaired aerobic growth an glucose minimal medium indicating that the bc$_{1}$-aa$_{3}$-pathway is the main route of respiratoon under these conditions. Cytochrome b (QcrB) and subunit I of cytochrome aa$_{3}$ oxidase (CtaD) were elongated with a C-terminal StrepTag II and purified by affmity chromatography an StrepTactin sepharose. In both cases a bc$_{1}$-aa$_{3}$-supercomplex was obtained which possessed quinol oxidase activity and therefore represented a functional association of bc$_{1}$ complex and cytochrome aa$_{3}$ oxidase. Mutation of the cysteine residues in either heme binding motifs of cytochrome c$_{l}$ (QcrC) led to a similar phenotype like deletion of the qcr genes. No c-type cytochrome could be detected in membranes of both strains and their QcrB content was strongly reduced. Obviously both heme binding motifs of QcrC are essential for activity, assembly and/or stability ofthe bc$_{1}$ complex