Dissecting different modules of cytochrome c maturation in Rhodobacter capsulatus

Cytochrome c maturation (CCM) in Gram-negative bacteria involves at least ten membrane-bound components (CcmABCDEFGHI and CcdA) and the oxidative protein folding pathway (DsbA/DsbB). These components are distributed among three distinct modules: thioredox-loop, heme delivery and heme ligation. In this study, first we focused on the thioredox-loop, by which the heme binding motif CXXCH within the apocyt c is believed to be initially oxidized by DsbA/DsbB and then reduced by the CcdA/CcmG and CcmH. We have previously reported that the cyt c deficiency of CcdA-null mutants can be bypassed by mutations in DsbA and DsbB. Here, we showed that CcdA and CcmG are not essential for the formation of cyt c, but important for the efficiency of CCM in the absence of DsbA. The respective double and triple mutants can produce cyts c, albeit at significantly lower levels, and grow photosynthetically on media supplemented with thiol-reactive reagents. Moreover, CcmG without active site cysteines can facilitate cyt c production in these mutants, suggesting a distinct and previously undefined role of CcmG in addition to its thioredoxin function. Second, we concentrated on the physical interactions among CCM components and on the putative CcmHIF heme ligation complex which catalyzes the thioether bond formation between the heme and the apocyt c. Our co-purification results indicate that CcmH and CcmF form the core complex of CCM. Furthermore, CcmI is in the complex via interaction with CcmH. Gel filtration data shows that CcmF, CcmH and CcmI form a protein complex of about 900-1000 kDa with yet unknown stoichiometry. We report for the first time a CcmHIF containing bacterial heme lyase complex in R. capsulatus. Last, since DsbA-null mutants of R. capsulatus are defective for respiratory growth on enriched medium, we investigated the basis underlying this phenotype by proteomics and molecular biological approaches. We showed that in the absence of DsbA, not only overabundance of major periplasmic protease DegP rendered respiratory growth defective, but also lack of DegP was lethal. Finally, phylogenetic analysis identified two related but distinct bacterial extracytoplasmic protease families