New insights into the cytochrome c biogenesis in Rhodobacter capsulatus

C-type cytochromes are electron carrier proteins that contain a covalently bound heme prosthetic group. The post-translational processes that bring about heme binding to the cytochrome polypeptide are collectively referred to as cytochrome c biogenesis. In Gram-negative bacteria, this process that involves several proteins, is poorly understood. Here, Rhodobacter capsulatus, a Gram-negative facultative phototroph synthesizing various c-type cytochromes, was used as a model organism to advance the current understanding of the cytochrome c biogenesis. Genetic complementation of two c-type cytochrome deficient mutants led to the identification of new components, CycJ and CcdA, of the R. capsulatus cytochrome c biogenesis machinery. R. capsulatus cycJ − strains do not revert suggesting that CycJ performs a unique function. Our results indicated that R. capsulatus CycJ is a 16 kDa membrane-anchored protein homologous to the better characterized E. coli CcmE protein. Striking homology and mutagenesis studies indicated that like CcmE, CycJ also has a histidine residue that is required for its heme-delivery function. Topological analysis of CcdA using gene fusion technique showed that it has six membrane-spanning helices, and its two conserved cysteines are probably membrane embedded. Mutagenesis of these cysteines and phenotypic rescue of the ccdA mutants by thiol compounds suggested that electrons required to reduce the heme-binding cysteines are shuttled across the membrane by CcdA. This demonstrated, for the first time, that CcdA homologs are required for cytochrome c biogenesis in Gram-negative bacteria. Unlike the cycJ mutants, ccdA − strains yielded many revertants. The characterization of one such revertant revealed that a mutation knocking out the periplasmic dithiol:disulfide oxidoreductase, DsbA, suppressed the need for CcdA in cytochrome c biogenesis. Considering that DsbA functions in the oxidative, and CcdA in the reductive pathway of biogenesis, this finding demonstrated the importance of the periplasmic redox homeostasis for successful cytochrome c biogenesis. Many thioredoxins are expected to contribute to the periplasmic redox equilibrium and thus affect cytochrome c biogenesis. The involvement of two thioredoxins in cytochrome c biogenesis was tested to show that the membrane-anchored thioredoxin, TlpA is not required for cytochrome c biogenesis and that the cytoplasmic thioredoxin, TrxA is essential for growth