Regions of Rhodobacter sphaeroides cytochrome c2 required for export, heme attachment, and function.
Cytochrome c2 is a periplasmic redox protein involved in both the aerobic and photosynthetic electron transport chains of Rhodobacter sphaeroides. The process of cytochrome c2 maturation has been analyzed in order to understand the protein sequences involved in attachment of the essential heme moiety to the cytochrome c2 polypeptide and localization of the protein to the periplasm. To accomplish this, five different translational fusions which differ only in the cytochrome c2 fusion junction were constructed between cytochrome c2 and the Escherichia coli periplasmic alkaline phosphatase. All five of the fusion proteins are exported to the periplasmic space. The four fusion proteins that contain the NH2-terminal site of covalent heme attachment to cytochrome c2 are substrates for heme binding, suggesting that the COOH-terminal region of the protein is not required for heme attachment. Three of these hybrids possess heme peroxidase activity, which indicates that they are functional as electron carriers. Biological activity is possessed by one hybrid protein constructed five amino acids before the cytochrome c2 COOH terminus, since synthesis of this protein restores photosynthetic growth to a photosynthetically incompetent cytochrome c2-deficient derivative of R. sphaeroides. Biochemical analysis of these hybrids has confirmed CycA polypeptide sequences sufficient for export of the protein (A. R. Varga and S. Kaplan, J. Bacteriol. 171:5830-5839, 1989), and it has allowed us to identify regions of the protein sufficient for covalent heme attachment, heme peroxidase activity, docking to membrane-bound redox partners, or the capability to function as an electron carrier.
Brandner
J P
JP
Bacteriology Department, University of Wisconsin, Madison 53706.
Stabb
E V
EV
Temme
R
R
Donohue
T J
TJ
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D003578:Cytochrome-c Peroxidase / Q000235:genetics; D045362:Cytochromes c2; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D006418:Heme / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000254:growth & development / Q000502:physiology
D003574:Cytochrome c Group; D011993:Recombinant Fusion Proteins; D006418:Heme; D045362:Cytochromes c2; D003578:Cytochrome-c Peroxidase; D000469:Alkaline Phosphatase