A thioreduction pathway tethered to the membrane for periplasmic cytochromes c biogenesis; in vitro and in vivo studies.

The c-type cytochromes are distinguished from other heme proteins by the covalent ligation of two heme vinyl groups to two cysteine residues on the apoprotein (at a CXXCH domain). The present study was undertaken to elucidate the roles and topological locations of two of the proteins necessary for cytochrome c biogenesis, the HelX and Ccl2 proteins in the Gram-negative bacteria Rhodobacter capsulatus. From their primary sequence, each of these proteins has a CXXC motif that could be involved in the reduction of the cysteine residues of the apocytochromes c, a prerequisite for covalent ligation to the heme. Results of site-directed mutagenesis of HelX and Ccl2 demonstrate that each cysteine residue is required for the in vivo function of the protein. We demonstrate that the native HelX in R. capsulatus is tethered to the cytoplasmic membrane via its uncleaved signal sequence. Ccl2 is tethered by a single transmembrane domain present in the C terminus with the N-terminal two-thirds of the protein in the periplasm. Thus, both CXXC motifs are exposed to the periplasm. The complete HelX protein and the soluble N-terminal portion of Ccl2 (called Ccl2*) were overproduced and purified from periplasmic fractions. The Ccl2* signal sequence is efficiently processed. In vitro studies with these purified proteins indicate that although neither can reduce insulin, HelX can reduce the Ccl2 cysteine residues and the Ccl2 cysteine residues are oxidized by an apocytochrome c peptide containing the CXXCH domain. Revertants of an helX deletion mutant were isolated that regain the ability to make c-type cytochromes (and thus grow photosynthetically); some of these suppressor strains are enhanced for photosynthetic growth by the addition of thio-reducing agents. In contrast, revertants of a ccl2 deletion strain could not be isolated under any condition. These results suggest that the HelX and Ccl2 proteins form a thioreduction pathway (HelX-->Ccl2-->apocytochrome c) whereby Ccl2 function may be highly specific for apocytochromes c while HelX may act as a more general reductant of proteins with vicinal cysteines. Copyright 1997 Academic Press Limited.