Oxidation and Reduction of Heme-Binding Cysteines During Cytochrome c Assembly in the Chloroplast
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Date
2023-03
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Abstract
The c-type cytochromes (cyt c) are metalloproteins with a covalently-bound heme which act as one-electron carriers in energy-converting processes like photosynthesis and respiration. Here, we explored the requirement of disulfide reduction for chloroplast cyt c assembly in the green alga model system, Chlamydomonas reinhardtii. Assembly of holocytochrome c depends on the covalent attachment of a heme group to a heme-binding motif (CXXCH) present in apocytochrome c. This attachment requires the heme-linking cysteines to be maintained in a reduced state by the operation of a disulfide-reducing pathway. In chloroplasts, apocyt c reduction depends on several factors, such as CCS5 and CCS4, that transfer electrons from stroma to the lumen to reduce disulfide-bonded heme-binding cysteines before heme ligation. Both ccs4 and ccs5 mutants are partially deficient for photosynthetic growth due to partial loss of chloroplast cyt c assembly. While CCS4 and CCS5 use electrons conveyed across the thylakoid membrane, the source of electrons in the stroma is still unknown. We hypothesize that stroma-localized thioredoxin-m (TRX-m) is the primary electron donor for apocyt c reduction and tested if overexpression of TRX-m in a ccs4 or a ccs5 mutant rescues their photosynthetic deficiency. Preliminary results show that there is no significant rescue when TRX-m is overproduced in a ccs4 or a ccs5 mutant, but this needs further testing to confirm elevated levels of TRX-m protein in the mutants. We also show that the reducing pathway is necessary to counter oxidation of the CXXCH motif by LTO1, a thylakoid membrane-bound protein. We demonstrate by heme staining that cyt c assembly is restored in a lto1ccs4ccs5 triple mutant, which lacks both oxidizing and reducing pathways. However, there is no rescue of photosynthetic growth, which suggests that loss of both pathways might have additional effects on the photosynthetic machinery besides cyt c assembly. We suggest that heme-binding cysteines are first disulfide bonded by LTO1 in the lumen and subsequently reduced by CCS4 and CCS5 prior to the heme attachment reaction. We continue our analysis in several independent triple mutants to confirm such results and are currently testing protein interactions between LTO1 and cyt c via yeast-two-hybrid assay.
Description
Animal Sciences (The Ohio State University Denman Undergraduate Research Forum)
Keywords
genetics, photosynthesis, chloroplast, redox, cytochrome