A Homolog of Prokaryotic Thiol Disulfide Transporter CcdA Is Required for the Assembly of the Cytochrome bf Complex in Arabidopsis Chloroplasts

Abstract

The c-type cytochromes are defined by the occurrence of heme covalently linked to the polypeptide via thioether bonds between heme and the cysteine sulfhydryls in the CXXCH motif of apocytochrome. Maintenance of apocytochrome sulfhydryls in a reduced state is a prerequisite for covalent ligation of heme to the CXXCH motif. In bacteria, a thiol disulfide transporter and a thioredoxin are two components in a thio-reduction pathway involved in c-type cytochrome assembly. We have identified in photosynthetic eukaryotes nucleus-encoded homologs of a prokaryotic thiol disulfide transporter, CcdA, which all display an N-terminal extension with respect to their bacterial counterparts. The extension of Arabidopsis CCDA functions as a targeting sequence, suggesting a plastid site of action for CCDA in eukaryotes. Using PhoA and LacZ as topological reporters, we established that Arabidopsis CCDA is a polytopic protein with within-membrane strictly conserved cysteine residues. Insertional mutants in the Arabidopsis CCDA gene were identified, and loss-of-function alleles were shown to impair photosynthesis because of a defect in cytochrome b 6 f accumulation, which we attribute to a block in the maturation of holocytochrome f, whose heme binding domain resides in the thylakoid lumen. We postulate that plastid cytochrome c maturation requires CCDA, thioredoxin HCF164, and other molecules in a membrane-associated trans-thylakoid thiol-reducing pathway.The c-type cytochromes are a virtually ubiquitous yet rather structurally diverse group of hemoproteins that reside on the so-called p-side 1 of the energy-transducing membrane systems where they function typically as electron carriers (1, 2). The c-type cytochromes are defined by the occurrence of heme covalently attached to the polypeptide via two thioether linkages between the vinyl groups of heme and the cysteine sulfhydryls in the apocytochrome (1, 3). A CXXCH 2 sequence in the apocytochrome provides the thiols for formation of the thioether bonds, and the imidazole of the histidine residue serves as one of the axial ligands of the heme. Remarkably, three distinct assembly pathways, referred to as systems I, II, and III, have emerged through genetic analysis of c-type cytochrome maturation in bacteria, chloroplasts, and mitochondria (for review, see Refs. 1 and 4 -7), an unexpected finding for what appears on the surface to be a rather simple chemical reaction (i.e. the addition of apocytochrome cysteine thiols to the vinyls of heme). Each system can be distinguished on the basis of a sequence relationship of specific assembly factors. Yet, there is an underlying assumption that the three systems are united by common biochemical requirements for holocytochrome c biogenesis (for review, see Refs. 2, 4, and 8). For instance, the need for reducing conditions appears to be inherent to the chemistry of thioether bond formation (4). Both substrates, apocytochrome c sulfhydryls and heme, need to be maintained reduced prior to the ligation of heme as indicated from in vitro and in o...

Metal Homeostasis in Cyanobacteria and Chloroplasts. Balancing Benefits and Risks to the Photosynthetic Apparatus

“…Interestingly, deletion mutants in these proteins result in specific inactivation of an Fe-S center containing photosynthetic protein complexes, linking assembly pathways with a single or limited number of targets (Balk and Lobreaux, 2005). A similar pathwaytarget link can be found between the chloroplast cytochrome c assembly pathway and cytochrome b 6 f biogenesis (Page et al, 2004).…”

Activation of the Stt7/STN7 Kinase through Dynamic Interactions with the Cytochrome b6f Complex

“…This configuration of the kinase allows its catalytic domain to act on the substrate sites of the LHCII proteins, which are exposed to the stroma. Although in this model the conserved Cys residues in the lumen are on the opposite side from the stromal thioredoxins, it is possible that thiol-reducing equivalents are transferred across the thylakoid membrane through the CcdA and Hcf164 proteins, which have been shown to operate in this way during heme and Cyt b 6 f assembly (Lennartz et al, 2001;Page et al, 2004) or through the LTO1 protein (Du et al, 2015;Karamoko et al, 2011).…”

Protein kinases and phosphatases involved in the acclimation of the photosynthetic apparatus to a changing light environment

“…At first sight, the fact that the Cys and the thioredoxins are located on opposite sides of the thylakoid membrane seems to exclude such a mechanism. However, a trans-thylakoid trans-membrane thiol pathway has been identified across the thylakoid membrane, which includes the CcdA and Hcf164 proteins, both of which are involved in cyt b 6 f assembly [33,34]. Whether this pathway also operates for the Stt7 kinase is still unknown (figure 2).…”