Molecular analysis of the cytochrome bc₁-aa₃ branch of the Corynebacterium glutamicum respiratory chain containing an unusual diheme cytochrome c₁

Abstract.

In this work, the genes for cytochrome aa ₃ oxidase and the cytochrome bc ₁ complex in the gram-positive soil bacterium Corynebacterium glutamicum were identified. The monocistronic ctaD gene encoded a 65-kDa protein with all features typical for subunit I of cytochrome aa ₃ oxidases. A ctaD deletion mutant lacked the characteristic 600 nm peak in redox difference spectra, and growth in glucose minimal medium was strongly impaired. The genes encoding subunit III of cytochrome aa ₃ (ctaE) and the three characteristic subunits of the cytochrome bc ₁ complex (qcrABC) were clustered in the order ctaE-qcrCAB. Analysis of the deduced primary structures revealed a number of unusual features: (1) cytochrome c ₁ (QcrC, 30 kDa) contained two Cys-X-X-Cys-His motifs for covalent heme attachment, indicating that it is a di-heme c-type cytochrome; (2) the 'Rieske' iron-sulphur protein (QcrA, 45 kDa) contained three putative transmembrane helices in the N-terminal region rather than only one; and (3) cytochrome b (QcrB, 60 kDa) contained, in addition to the conserved part with eight transmembrane helices, a C-terminal extension of about 120 amino acids, which presumably is located in the cytoplasm. Staining of C. glutamicum proteins for covalently bound heme indicated the presence of a single, membrane-bound c-type cytochrome with an apparent molecular mass of about 31 kDa. Since this protein was missing in a qcrCAB deletion mutant, it most likely corresponds to cytochrome c ₁. Similar to the ΔctaD mutant, the ΔqcrCAB mutant showed strongly impaired growth in glucose minimal medium, which indicates that the bc ₁-aa ₃ pathway is the main route of respiration under these conditions.