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 t he covalent Ligation of two heme vinyl groups to two cysteine residues on the apoprotein (at a CXXCH domain). The present study was undertak en to elucidate the roles and topological locations of two of the prot eins necessary for cytochrome c biogenesis, the HelX and Ccl2 proteins in the Gram-negative bacteria Rhodobacter capsulatus. From their prim ary sequence, each of these proteins has a CXXC motif that could be in volved 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 r esidue is required for the in vivo function of the protein. We demonst rate that the native HelX in R. capsulatus is tethered to the cytoplas mic membrane via its uncleaved signal sequence. Ccl2 is tethered by a single transmembrane domain present in the C terminus with the N-termi nal two-thirds of the protein in the periplasm. Thus, both CXXC motifs are exposed to the periplasm. The complete HelX protein and the solub le N-terminal portion of Ccl2 (called Ccl2) were overproduced and pur ified from periplasmic fractions. Tile Ccl2 signal sequence is effici ently processed. In vitro studies with these purified proteins indicat e that although neither can reduce insulin, HelX can reduce the Ccl2 c ysteine residues and the Ccl2 cysteine residues are oxidized by an apo cytochrome c peptide containing the CXXCH domain. Revertants of an hel X deletion mutant were isolated that regain the ability to make c-type cytochromes (and thus grow photosynthetically); some of these suppres sor strains are enhanced for photosynthetic growth by the addition of thio-reducing agents. In contrast, revertants of a ccl2 deletion strai n could not be isolated under any condition. These results suggest tha t the HelX and Ccl2 proteins form a thioreduction pathway (HelX --> Cc l2 --> apocytochrome c) whereby Ccl2 function may be highly specific f or apocytochromes c while HelX may act as a more general reductant of proteins with vicinal cysteines. (C) 1997 Academic Press Limited.