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

Abstract

AbstractIn plastids, conversion of light energy into ATP relies on cytochromef, 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. InChlamydomonas reinhardtii, loss of CCS4 or CCS5 function yields a partial cytochromefassembly defect. Here we report that the Δccs4ccs5double mutant displays a synthetic photosynthetic defect due to a complete loss of holocytochromefassembly, 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 theCCS4gene were identified in photosynthetic revertants of the Δccs4ccs5mutants. The suppressor mutations correspond to changes in the stroma-facing domain of CCS4 and restore holocytochromefassembly 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 apocytochromefand the possible function of CCS4 as a shared component between the two pathways.Abstract FigureGraphical abstract.The Δccs4ccs5mutant exhibits a photosynthetic growth defect due to a complete loss of cytochromecassembly.Reduction of apocytochromefin the thylakoid lumen requires the provision of reducing power through two different pathways, pathway 1 and 2. CCDA and CCS5, components of pathway 1, deliver electrons from stroma to apocytochromefvia thiol – disulfide exchange. CCS4 is involved in pathway 1 by stabilizing CCDA, but also functions through a CCS5 – independent pathway (pathway 2). In the absence of CCS5, gain – of – function mutations in the C terminus of CCS4 (indicated by a yellow star) enhance the delivery of reducing power either via CCDA or independently of CCDA to yet-to-be-discovered reductases