The membrane-attached electron carrier cytochrome cy from Rhodobacter sphaeroides is functional in respiratory but not in photosynthetic electron transfer

Abstract

Rhodobacter species are useful model organisms for studying the structure and function of c type cytochromes (Cyt c), which are ubiquitous electron carriers with essential functions in cellular energy and signal transduction. Among these species, Rhodobacter capsulatus has a periplasmic Cyt c2Rc and a membrane-bound bipartite Cyt cyRc. These electron carriers participate in both respiratory and photosynthetic electron-transfer chains. On the other hand, until recently, Rhodobacter sphaeroides was thought to have only one of these two cytochromes, the soluble Cyt c2Rs. Recent work indicated that this species has a gene, cycYRs, that is highly homologous to cycYRc, and in the work presented here, functional properties of its gene product (Cyt cyRs) are defined. It was found that Cyt cyRs is unable to participate in photosynthetic electron transfer, although it is active in respiratory electron transfer, unlike its R. capsulatus counterpart, Cyt cyRc. Chimeric constructs have shown that the photosynthetic incapability of Cyt cyRs is caused, at least in part, by its redox active subdomain, which carries the covalently bound heme. It, therefore, seems that this domain interacts differently with distinct redox partners, like the photochemical reaction center and the Cyt c oxidase, and allows the bacteria to funnel electrons efficiently to various destinations under different growth conditions. These findings raise an intriguing evolutionary issue in regard to cellular apoptosis: why do the mitochondria of higher organisms, unlike their bacterial ancestors, use only one soluble electron carrier in their respiratory electron-transport chains?

Figure 1

(A) Physical and genetic maps of the chromosomal regions surrounding R. sphaeroides and R. capsulatus cycY. Only cycY^Rs is physically linked to the ccoNOQP–ccoGHIS/rdxBHIS cluster required for the biosynthesis of the cbb₃-type Cyt c oxidase in both organisms (9, 30). Arrows and “T” refer to transcription direction and termination signal at the 3′ downstream region of cycY, respectively. (B) Sequence comparison of the 5′ upstream regions of cycY from R. capsulatus (RccycY), R. sphaeroides (RscycY), and P. denitrificans (PdcycM). Putative −35 (TTGAA) and −10 (TAg/cAT) regions for a σ⁷⁰-like promoter are indicated. Additional conserved elements of unknown significance (indicated by arrows) and putative ribosome binding sites rich in adenosine and guanine residues in the vicinity of the boxed translational start (ATG) codons also are shown.

Figure 2

Alignment of the amino acid sequences for horse-heart Cyt c: R. capsulatus (RccycY), R. sphaeroides (RscycY), P. denitrificans (PdcycM), and Bradyrhizobium japonicum (BjcycM) Cyt c_y homologs. Black and gray boxes correspond to identical residues and conserved substitutions, respectively. The Cyt c domains and CXYCH heme binding motifs are indicated by an arrow and asterisks, respectively. Dots represent the key Lys residues known to be important for the interaction of equine Cyt c with its various redox partners (24, 25).

Figure 3

(A) TMBZ-SDS/PAGE analysis of Cyt c of various R. sphaeroides strains. Chromatophore membranes were prepared from cells grown on yeast-extract-casamino acid-enriched medium by respiration, and ≈75 μg of proteins were loaded per lane. Lane 1, Ga (wild type); lane 2, Gadc_y (lacking Cyt c_y^Rs); and lane 3, pHM119/Gadc_y (containing Cyt c_y^Rs-FLAG). The addition of the FLAG epitope changes the molecular mass of Cyt c_y^Rs by ≈1 kDa. (B) Immunoblot analysis of a similar gel with anti-FLAG M2 antibody. Lanes 1 and 2 correspond to pHM119/Gadc_y (Cyt c_y^Rs-FLAG) and Gadc_y, respectively, and contained 25 μg of membrane proteins prepared from cells grown as for A. (C) Immunoblot analysis with anti-FLAG M2 antibody as for B, except that chromatophore membranes (25 μg of total proteins per lane) were obtained from cells of pHM119/Gadc_y (Cyt c_y^Rs-FLAG) grown in yeast-extract-casamino acid-enriched medium in the absence (lane 1) and presence (lane 2) of oxygen.

Figure 4

(A) Schematic representation of the chimeric Cyt c exchange (pHM124/126) and linker-anchor exchange (pHM125/127) cytochromes along the native Cyt c_y^Rs and Cyt c_y^Rc. Control region, Anchor/Linker, and Cytochrome c refer to the various subdomains of these cytochromes. The restriction sites used to create these chimeras are indicated. (B) Immunoblot analysis of various R. capsulatus strains carrying chimeric Cyt c_y-FLAG derivatives with anti-FLAG M2 antibody. Chromatophores were prepared from cells grown in enriched MPYE medium under either Ps (lanes 1–3) or Res (lanes 4–6) growth conditions, and 25 μg membrane proteins were loaded per lane. Lane 1, pHM126/M6G-G4/S4 (Cyt c exchange); lane 2 and 5, pHM127/M6G-G4/S4 (linker-anchor exchange); lane 3, pHM119/M6G-G4/S4 (Cyt c_y^Rs-FLAG); lane 4, pHM126/SL3 (Cyt c exchange); and lane 6, pHM119/SL3 (Cyt c_y^Rs-FLAG).