A novel hydrophobic diheme c-type cytochrome. Purification from Corynebacterium glutamicum and analysis of the QcrCBA operon encoding three subunit proteins of a putative cytochrome reductase complex

Electrophoresis of a Corynebacterium glutamicum membrane preparation in the presence of sodium dodecyl sulfate, followed by staining for peroxidase ac tivity theme staining), showed only one band at about 28 kDa. This 28 kDa p rotein was purified from C. glutamicum membranes by chromatography in the p resence of decylglucoside using DEAE-Toyopearl and hydroxylapatite columns, as the sole c-type cytochrome in the bacterium. The cytochrome showed an a lpha band at 551 nm, and its E-m,E-7 was about 210 mV. A QcrCAB operon enco ding the subunits of a putative quinol cytochrome c reductase was found 3'- downstream of ctaE encoding subunit III of cytochrome aa(3) in the C. gluta micum genome. The deduced amino acid sequence of qcrC, composed of 283 amin o acid residues, contained two heme C-binding motifs and was in agreement w ith partial peptide sequences obtained from the 28 kDa protein after V8 pro tease digestion. We propose to name this protein cytochrome cc, The presenc e of cytochrome cc is a common feature of high G+C content Gram-positive ba cteria, since we could confirm this protein by electrophoresis; homologous QcrCAB operons are also known in Mycobacterium and Streptomyces. On A and q crB of C, glutamicum encode the Rieske Fe-S protein and cytochrome b, respe ctively, although these proteins were not co-purified with cytochrome cc. T he phylogenetic tree of cytochromes b and bs show that C. glutamicum cytoch rome b, along with those of other bacteria in the high G+C group, is rather different from the Bacillus counterparts, but highly similar to the Deinoc occi and Thermus cytochromes. This indicates that there is a fourth group o f bacteria in addition to the three clades: proteobacterial cytochrome b, c yanobacterial bs and green sulfur-low G+C Grampositive bacteria. (C) 2001 P ublished by Elsevier Science B.V.