Essential Histidine and Tryptophan Residues in CcsA, a System II Polytopic Cytochrome c Biogenesis Protein

Abstract

Three distinct systems (I, II, and III) for catalysis of heme attachment to c-type apocytochromes are known. The CcsA and Ccs1 proteins are required in system II for the assembly of bacterial and plastid cytochromes c. A tryptophan-rich signature motif (WWD), also occurring in CcmC and CcmF found in system I, and three histidinyl residues, all strictly conserved in CcsA suggest a function in heme handling. Topological analysis of plastid CcsA in bacteria using the PhoA and LacZ␣ reporters placed the WWD motif, the conserved residues His 212 and His 347 on the lumen side of the membrane, whereas His 309 was assigned a location on the stromal side. Functional analysis of CcsA through site-directed mutagenesis enabled the designation of the initiation codon of the ccsA gene and established the functional importance of the WWD signature motif and the absolute requirement of all three histidines for the assembly of plastid c-type cytochromes. In a ccsA mutant, a 200-kDa Ccs1-containing complex is absent from solubilized thylakoid membranes, suggesting that CcsA operates together with Ccs1. We propose a model where the WWD motif and histidine residues function in relaying heme from stroma to lumen and we postulate the existence of a cytochrome c assembly machinery containing CcsA, Ccs1 and additional components.

PHOTOSYSTEM II PROTEIN33, a Protein Conserved in the Plastid Lineage, Is Associated with the Chloroplast Thylakoid Membrane and Provides Stability to Photosystem II Supercomplexes in Arabidopsis

“…3.63 6 0.97 -3.37 6 0.65 ns 5.64 6 1.00 -5.14 6 0.63 ns 7.04 6 1.18 -6.51 6 0.69 ns 7.64 6 0.86 -7.01 6 0.34 ns DC (170, 670, 1,270, 2,000 PPFD) 1.37 6 0.49 -2.39 6 0.80 ns 2.23 6 0.48 -2.10 6 0.50 ns 0.71 6 0.45 -0.63 6 0.63 ns ---PI 1.43 6 0.09 0.85 6 0.09* 0.62 6 0.09* Oxygen evolution 0.28 6 0.05 0.14 6 0.01* 0.13 6 0.01* which protons accumulate during active electron transport, and between the bacterial cytoplasm negative (n) side and the plastid stroma, which becomes proton deficient. High PhoA activity indicates a p-side location of the reporter, while LacZ activity indicates an n-side location of the reporter (Hamel et al, 2003;Karamoko et al, 2011). As shown in Supplemental Figure S3D, when the PhoA-LacZa reporter is fused to the N-terminal side of the transmembrane region, transformants display predominantly b-galactosidase activity, suggesting that the Rieske-like domain faces the stroma.…”

“…To verify the orientation of PSB33 within the thylakoid membrane, we used a gene fusion, which encodes alkaline phosphatase (PhoA) followed by the alpha peptide of b-galactosidase (LacZa) as a topological reporter in Escherichia coli (Hamel et al, 2003). This approach relies on the bioenergetic analogy both between the bacterial periplasm positive (p) side and the thylakoid lumen, in Table I.…”

Pleiotropic effects of mutations that alter the Sinorhizobium meliloti cytochrome c respiratory system

“…The transposon in mutant W3 was located in ORF SMc00248, which has been annotated as ccsA (Galibert et al, 2001), a gene involved in type II cytochrome c maturation (Beckett et al, 2000;Hamel et al, 2003). However, the protein predicted from SMc00248 showed 49 % identity and 65 % similarity over its entire length to Rhodobacter capsulatus CcdA (accession no.…”

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

“…This approach relies on the topological analogy in a bioenergetic sense between the p-side and n-side compartments in bacteria (periplasm/cytoplasm) and those in plastids (lumen/stroma) and is justified by the previous establishment of its reliability for the analysis of CcsA, a thylakoid membrane polytopic protein (21). Translational fusions between CCDA and PhoA or LacZ were engineered at predicted periplasmic and cytoplasmic loops.…”