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Catalysis of thiol-disulfide chemistry in the context of plastid c-type cytochrome assembly

Das, Ankita
http://rave.ohiolink.edu/etdc/view?acc_num=osu168984637448693

Abstract Details

2023, Doctor of Philosophy, Ohio State University, Molecular Genetics.
The c-type cytochromes or cytochromes c are heme-containing metalloproteins that act as electron carriers in energy transducing membranes involved in processes such as photosynthesis and respiration. The maturation of cytochromes c requires covalent attachment of the heme cofactor to a heme-binding motif (CXXCH)in the apocytochrome c. In chloroplasts, cytochrome c assembly depends on factors known as CCS (Cytochrome c Synthesis), that are essential for the heme attachment reaction taking place in the thylakoid lumen The covalent attachment of heme requires the heme-linking cysteines of apocytochrome c to be maintained in a reduced state (providing free -SH) by the operation of a trans-thylakoid disulfide reducing pathway. CCDA, a polytopic thylakoid membrane oxido-reductase, and CCS5, a thioredoxin-like protein, are two components of a trans-thylakoid disulfide-reducing pathway, which also operates in bacteria This pathway transfers electrons from stroma via signature redox motifs and sequential thiol-disulfide exchanges from CCDA to CCS5 to reduce apocytochrome c disulfide bonded CXXCH in the thylakoid lumen. CCS4, first identified in the unicellular alga Chlamydomonas reinhardtii, is a thylakoid-bound stroma-facing component of this disulfide-reducing pathway with no residue or motif suggestive of a biochemical activity. A ccs4 mutant is partially deficient for photosynthesis (due to a defect in cytochrome c assembly) and can be rescued by application of exogenous thiols or overexpression of CCDA. In this work, we evidenced that CCDA accumulation is decreased in a ccs4 mutant but not in a ccs5 mutant, suggesting a functional interaction between CCS4 and CCDA. Our genetic studies show that CCS4 and CCS5 are partially redundant for the delivery of reducing power for the apocytochrome c CXXCH reduction. We also show that gain-of-function mutations in the CCS4 gene that correspond to changes in the stromal domain of the protein can suppress a ccs5-null mutant. This suggests that activity of the second pathway can be enhanced in the absence of the first, which is CCS5-dependent. CCS4-like proteins occur in the green lineage, and we show that HCF153, a distant ortholog from Arabidopsis thaliana, can substitute for Chlamydomonas CCS4. The mechanism of apocytochrome c CXXCH reduction by CCS4 is unclear but we discuss the possible function of CCS4 in the context of its similarity to COX16, a mitochondrial protein involved in a disulfide reducing pathway. In Chapter 3, we demonstrate that the apocytochrome c, the substrate of the heme attachment reaction, is first enzymatically oxidized by LTO1, a VKOR homolog in plastids via a trans-thylakoid oxidizing pathway, and subsequently reduced by the disulfide-reducing pathway. Characterization of a lto1 mutant shows a photosynthetic growth defect which can be attributed to a specific loss of PSII activity by fluorescence rise and decay kinetics. Holocytochrome c assembly is restored in a ccs4ccs5 double mutant, which is completely blocked, in the presence of the lto1 mutation. However, there is no rescue of the tight photosynthetic growth defect despite the regain of holocytochrome c accumulation in the ccs4 ccs5 lto1 mutant. Other regain of cytochrome c assembly upon LTO1 loss of function is not enough to restore photosynthetic growth in a ccs4ccs5 double mutant, other targets of LTO1 might be required for photosynthesis in the absence of the reducing pathway. It is also possible that CCS4 and CCS5 control other aspects of photosynthesis beside the redox control of the heme attachment reaction that does not depend on LTO1 oxidation. Thus, the defect in the heme attachment step is corrected upon inactivation of LTO1 but any additional function that CCS4 and CCS5 might have, is not. We discuss the analogy between cytochrome c assembly thylakoids and bacteria, where the accepted model postulates that a reducing pathway exists or is required to counter the oxidation of heme-linking cysteines into a disulfide in the apocytochrome c substrate.
Patrice Hamel, Dr. (Advisor)
Natacha Ruiz, Dr. (Committee Member)
Amanda Bird, Dr. (Committee Member)
Iris Meier, Dr. (Committee Member)
219 p.
Biochemistry; Genetics; Microbiology; Molecular Biology
Thiol-disulfide, cytochrome c, redox chemistry, ccs mutants, oxidation and reduction

Recommended Citations

Citations

  • Das, A. (2023). Catalysis of thiol-disulfide chemistry in the context of plastid c-type cytochrome assembly [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu168984637448693

    APA Style (7th edition)

  • Das, Ankita. Catalysis of thiol-disulfide chemistry in the context of plastid c-type cytochrome assembly. 2023. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu168984637448693.

    MLA Style (8th edition)

  • Das, Ankita. "Catalysis of thiol-disulfide chemistry in the context of plastid c-type cytochrome assembly." Doctoral dissertation, Ohio State University, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=osu168984637448693

    Chicago Manual of Style (17th edition)

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