Two disulfide-reducing pathways are required for the maturation of plastid c-type cytochromes in Chlamydomonas reinhardtii.

In plastids, conversion of light energy into ATP relies on cytochrome f, a key electron carrier with a heme covalently attached to a CXXCH motif. Covalent heme attachment requires reduction of the disulfide-bonded CXXCH by CCS5 and CCS4. CCS5 receives electrons from the oxidoreductase CCDA, while CCS4 is a protein of unknown function. In Chlamydomonas reinhardtii, loss of CCS4 or CCS5 yields a partial cytochrome f assembly defect. Here, we report that the ccs4ccs5 double mutant displays a synthetic photosynthetic defect characterized by a complete loss of holocytochrome f assembly. This defect is chemically corrected by reducing agents, confirming the placement of CCS4 and CCS5 in a reducing pathway. CCS4-like proteins occur in the green lineage, and we show that HCF153, a distant ortholog from Arabidopsis thaliana, can substitute for Chlamydomonas CCS4. Dominant suppressor mutations mapping to the CCS4 gene were identified in photosynthetic revertants of the ccs4ccs5 mutants. The suppressor mutations yield changes in the stroma-facing domain of CCS4 that restore holocytochrome f assembly above the residual levels detected in ccs5. Because the CCDA protein accumulation is decreased specifically in the ccs4 mutant, we hypothesize the suppressor mutations enhance the supply of reducing power through CCDA in the absence of CCS5. We discuss the operation of a CCS5-dependent and a CCS5-independent pathway controlling the redox status of the heme-binding cysteines of apocytochrome f. Reduction of a disulfide formed between the heme-linking cysteines of apoforms of c-type cytochromes in the thylakoid lumen requires the provision of reducing power from stroma to apocytochrome c via thiol-disulfide exchange. Reducing power is supplied through two different pathways, pathways 1 and 2, with CCDA and CCS4 being common components to both pathways. In pathway 1, CCS5 directly reduces the disulfide-bonded heme-binding site. In pathway 2, one or several yet-to-be identified protein(s) (X) reduce(s) the disulfide. CCS4 functions in stabilizing CCDA. In the absence of CCS5, gain-offunction mutations altering the C terminus of CCS4 enhance the delivery of reducing power via CCDA to the unknown reductase(s) (X), underscoring the functional importance of the soluble domain in CCS4 (indicated by a yellow star). This domain might act by recruiting the stromal reductant to CCDA or control the reactivity of the redox-active cysteines in CCDA by providing an ideal chemical microenvironment for efficient thiol-disulfide exchange. [ABSTRACT FROM AUTHOR]