Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis

Abstract

Cytochromes of the c type contain covalently bound heme. In bacteria, they are located on the outside of the cytoplasmic membrane. Cytochrome c synthesis involves export of heme and apocytochrome across the cytoplasmic membrane followed by ligation of heme to the polypeptide. Using radioactive protoheme IX produced in Escherichia coli, we show that Bacillus subtilis can use heme from the growth medium for cytochrome c synthesis. The B. subtilis ccdA gene encodes a 26-kDa integral membrane protein which is required for cytochrome c synthesis (T. Schiött et al., J. Bacteriol. 179:1962-1973, 1997). In this work, we analyzed the stage at which cytochrome c synthesis is blocked in a ccdA deletion mutant. The following steps were found to be normal in the mutant: (i) transcription and translation of cytochrome c structural genes, (ii) translocation of apocytochrome across the cytoplasmic membrane, and (iii) heme transport from the cytoplasm to cytochrome polypeptide on the outer side of the cytoplasmic membrane. It is concluded that CcdA is required for a late step in the cytochrome c synthesis pathway.

Mechanism of substrate specificity in Bacillus subtilis ResA, a thioredoxin-like protein involved in cytochrome c maturation

“…Additionally, we have shown that ResA red specifically recognizes a model peptide designed to mimic the oxidized form of apocytochrome c. These results suggest a redox cycle in which ResA, the terminal thioredoxin-like protein in B. subtilis CCM, acts as a control point that siphons off the electrons needed for CCM only in response to the direct availability of apocytochrome c. Presumably, ResA ox is regenerated to the reduced state by nonspecific interactions with the integral membrane thiol-disulfide oxidoreductase, CcdA. This idea is further supported by the observation that CcdA regenerates not only ResA, but also StoA, a protein involved in spore coat protein crosslinking (13,16,17,33). ResA red then specifically binds oxidized apocytochrome c, ensuring that electrons are not lost to random disulfides in the extracellular environment.…”

“…This is also true for the thiol-disulfide oxidoreductases responsible for oxidizing reduced apocytochrome c and for shuttling electrons from intracellular thioredoxin through the membrane, thus maintaining the oxidizing environment of the periplasm and extracellular space for protein folding and sporulation (15)(16)(17). However, genetic evidence suggests that the thioredoxin-like proteins DsbE͞CcmG-CcmH or ResA involved in the reverse reaction are specific for CCM (4,14).…”

“…Oxidized apocytochrome c is then reduced by the DsbE͞CcmG-CcmH pair in system I or by ResA in system II before heme insertion (4,14). Electrons transferred through these redox cycles are obtained from cytoplasmic thioredoxin via an integral membrane protein, DsbD͞DipZ (system I) (15) or CcdA (system II) (16,17).…”

Structural Basis of Redox-coupled Protein Substrate Selection by the Cytochrome c Biosynthesis Protein ResA

“…The necessary electrons to do this are probably delivered by the integral membrane protein CcdA, a relative of DsbD (18,39,(41)(42)(43). The role of CcdA in apo-cytochrome disulfide reduction is supported by the fact that inactivating mutations in bdbC and bdbD, which encode orthologs of the E. coli DsbB and DsbA proteins, can complement the cytochrome c-deficient phenotype of a CcdA-inactivated mutant (43).…”

Co-factor Insertion and Disulfide Bond Requirements for Twin-arginine Translocase-dependent Export of the Bacillus subtilis Rieske Protein QcrA

“…Notably, cytochrome bc 1 is a membrane protein complex that faces the extracellular side of the cytoplasmic membrane. The Sec pathway individually translocates the cytochrome b and cytochrome c 1 components across the membrane before they are processed, matured, and combined (32,(35)(36)(37)(38)(39). It is believed that cytochrome b and cytochrome c 1 form a primary protease-resistant complex, where the final step in complex maturation is the incorporation of the Rieske protein (32).…”