Features of Rhodobacter sphaeroides CcmFH

Abstract

In this study, the in vivo function and properties of two cytochrome c maturation proteins, CcmF and CcmH from Rhodobacter sphaeroides, were analyzed. Strains lacking CcmH or both CcmF and CcmH are unable to grow under anaerobic conditions where c-type cytochromes are required, demonstrating their critical role in the assembly of these electron carriers. Consistent with this observation, strains lacking both CcmF and CcmH are deficient in c-type cytochromes when assayed under permissive growth conditions. In contrast, under permissive growth conditions, strains lacking only CcmH contain several soluble and membrane-bound c-type cytochromes, albeit at reduced levels, suggesting that this bacterium has a CcmH-independent route for their maturation. In addition, the function of CcmH that is needed to support anaerobic growth can be replaced by adding cysteine or cystine to growth media. The ability of exogenous thiol compounds to replace CcmH provides the first physiological evidence for a role of this protein in thiol chemistry during c-type cytochrome maturation. The properties of R. sphaeroides cells containing translational fusions between CcmF and CcmH and either Escherichia coli alkaline phosphatase or ␤-galactosidase suggest that they are each integral cytoplasmic membrane proteins with their presumed catalytic domains facing the periplasm. Analysis of CcmH shows that it is synthesized as a higher-molecular-weight precursor protein with an N-terminal signal sequence.

AtCCMH, an essential component of thec-type cytochrome maturation pathway inArabidopsismitochondria, interacts with apocytochromec

“…Therefore, AtCCMH is so far the only component of the putative reducing pathway of system I identified in plant mitochondria. In bacteria, several lines of evidence support a role of CcmH in a redox reaction (17,18,36). We showed that the cysteines of D1-His can form disulfide bonds accessible to different enzymatic reductants in vitro and that a reduced form of D1-His is able to reduce an intra-disulfide bridge in the heme-binding motif of a model peptide for apocytochrome c in vitro.…”

“…CcmG, a thioredoxin-like protein, and CcmH, a putative thiol-disulfide oxidoreductase, reduce intramolecular disulfide bonds in apocytochromes before heme attachment by transferring electrons from the transmembrane disulfide oxidoreductase DsbD (DipZ) (14,15). In bacteria, the loss of CcmH (CycL͞Ccl2) results in either the absence of any detectable c-type cytochromes (2,6) or reduced levels of c-type cytochromes (16,17). CcmH proteins possess an RCXXC motif exposed to the periplasm.…”

CytochromecBiogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

“…Recall that CtaA has a heme b which is involved in electron transfer and the heme o substrate for modification to heme a (76). Based on experimentally determined topological models for CcmF (62,129) and new phoA fusion results (125), a unified topological diagram for CcmF is shown in Fig. 7.…”

Digging deeper: uncovering genetic loci which modulate photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1

“…The Osp protein contains the sequence motif H 92 -C 93 -X-X-X-C 97 at its C terminus and its inverted sequence is identical to the known haem c binding motif found in many cytochrome c-like proteins (C-X 2-3 -C-H) (Rios-Velazquez et al, 2001). To examine whether this motif is important for Osp function, Cys-93 and Cys-97 were individually mutagenized to Ala and the functionality of the mutant forms of Osp was probed by complementation.…”