TWO DISULFIDE-REDUCING PATHWAYS ARE REQUIRED FOR THE MATURATION OF PLASTIDC-TYPE CYTOCHROMES INCHLAMYDOMONAS REINHARDTII

Abstract

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 motif by CCS5 and CCS4, a protein of unknown function. CCS5 receives electrons from the oxido-reductase CCDA at the thylakoid membrane. In Chlamydomonas reinhardtii, loss of CCS4 or CCS5 function yields a partial cytochrome f assembly defect. Here we report that the Δccs4ccs5 double mutant displays a synthetic photosynthetic defect due to a complete loss of holocytochrome f assembly, a phenotype that can be chemically corrected by reducing agents. In Δccs4, the CCDA protein accumulation is decreased, indicating that one function of CCS4 is to stabilize CCDA. Dominant suppressor mutations mapping to the CCS4 gene were identified in photosynthetic revertants of the Δccs4ccs5 mutants. The suppressor mutations correspond to changes in the stroma-facing domain of CCS4 and restore holocytochrome f assembly above the residual levels detected in Δccs5. Because disulfide reduction via CCS5 no longer takes place in Δccs5, we hypothesize the suppressor mutations enhance the supply of reducing power independently of CCS5, uncovering the participation of CCS4 in a distinct redox pathway. CCS4-like proteins occur in the green lineage and are related to mitochondrial COX16, a protein involved in a disulfide reducing pathway. We discuss the operation of two pathways controlling the redox status of the heme-binding cysteines of apocytochrome f and the possible function of CCS4 as a shared component between the two pathways.