Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Apr;179(8):2623–2631. doi: 10.1128/jb.179.8.2623-2631.1997

Cytochrome c(y) of Rhodobacter capsulatus is attached to the cytoplasmic membrane by an uncleaved signal sequence-like anchor.

H Myllykallio 1, F E Jenney Jr 1, C R Moomaw 1, C A Slaughter 1, F Daldal 1
PMCID: PMC179012  PMID: 9098061

Abstract

During the photosynthetic growth of Rhodobacter capsulatus, electrons are conveyed from the cytochrome (cyt) bc1 complex to the photochemical reaction center by either the periplasmic cyt c2 or the membrane-bound cyt c(y). Cyt c(y) is a member of a recently established subclass of bipartite c-type cytochromes consisting of an amino (N)-terminal domain functioning as a membrane anchor and a carboxyl (C)-terminal domain homologous to cyt c of various sources. Structural homologs of cyt c(y) have now been found in several bacterial species, including Rhodobacter sphaeroides. In this work, a C-terminally epitope-tagged and functional derivative of R. capsulatus cyt c(y) was purified from intracytoplasmic membranes to homogeneity. Analyses of isolated cyt c(y) indicated that its spectral and thermodynamic properties are very similar to those of other c-type cytochromes, in particular to those from bacterial and plant mitochondrial sources. Amino acid sequence determination for purified cyt c(y) revealed that its signal sequence-like N-terminal portion is uncleaved; hence, it is anchored to the membrane. To demonstrate that the N-terminal domain of cyt c(y) is indeed its membrane anchor, this sequence was fused to the N terminus of cyt c2. The resulting hybrid cyt c (MA-c2) remained membrane bound and was able to support photosynthetic growth of R. capsulatus in the absence of the cyt c(y) and c2. Therefore, cyt c2 can support cyclic electron transfer during photosynthetic growth in either a freely diffusible or a membrane-anchored form. These findings should now allow for the first time the comparison of electron transfer properties of a given electron carrier when it is anchored to the membrane or is freely diffusible in the periplasm.

Full Text

The Full Text of this article is available as a PDF (304.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams C. W., Forrest M. E., Cohen S. N., Beatty J. T. Structural and functional analysis of transcriptional control of the Rhodobacter capsulatus puf operon. J Bacteriol. 1989 Jan;171(1):473–482. doi: 10.1128/jb.171.1.473-482.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Berry E. A., Trumpower B. L. Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes. J Biol Chem. 1985 Feb 25;260(4):2458–2467. [PubMed] [Google Scholar]
  4. Bott M., Ritz D., Hennecke H. The Bradyrhizobium japonicum cycM gene encodes a membrane-anchored homolog of mitochondrial cytochrome c. J Bacteriol. 1991 Nov;173(21):6766–6772. doi: 10.1128/jb.173.21.6766-6772.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Caffrey M., Davidson E., Cusanovich M., Daldal F. Cytochrome c2 mutants of Rhodobacter capsulatus. Arch Biochem Biophys. 1992 Feb 1;292(2):419–426. doi: 10.1016/0003-9861(92)90011-k. [DOI] [PubMed] [Google Scholar]
  6. Daldal F., Cheng S., Applebaum J., Davidson E., Prince R. C. Cytochrome c(2) is not essential for photosynthetic growth of Rhodopseudomonas capsulata. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2012–2016. doi: 10.1073/pnas.83.7.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X. W., Finlay D. R., Guiney D., Helinski D. R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. doi: 10.1016/0147-619x(85)90068-x. [DOI] [PubMed] [Google Scholar]
  8. Donohue T. J., McEwan A. G., Van Doren S., Crofts A. R., Kaplan S. Phenotypic and genetic characterization of cytochrome c2 deficient mutants of Rhodobacter sphaeroides. Biochemistry. 1988 Mar 22;27(6):1918–1925. doi: 10.1021/bi00406a018. [DOI] [PubMed] [Google Scholar]
  9. Dutton P. L. Redox potentiometry: determination of midpoint potentials of oxidation-reduction components of biological electron-transfer systems. Methods Enzymol. 1978;54:411–435. doi: 10.1016/s0076-6879(78)54026-3. [DOI] [PubMed] [Google Scholar]
  10. Ferguson S. J. The functions and synthesis of bacterial c-type cytochromes with particular reference to Paracoccus denitrificans and Rhodobacter capsulatus. Biochim Biophys Acta. 1991 May 23;1058(1):17–20. doi: 10.1016/s0005-2728(05)80259-2. [DOI] [PubMed] [Google Scholar]
  11. Fischer R. S., Zhao G., Jensen R. A. Cloning, sequencing, and expression of the P-protein gene (pheA) of Pseudomonas stutzeri in Escherichia coli: implications for evolutionary relationships in phenylalanine biosynthesis. J Gen Microbiol. 1991 Jun;137(6):1293–1301. doi: 10.1099/00221287-137-6-1293. [DOI] [PubMed] [Google Scholar]
  12. Fonstein M., Koshy E. G., Nikolskaya T., Mourachov P., Haselkorn R. Refinement of the high-resolution physical and genetic map of Rhodobacter capsulatus and genome surveys using blots of the cosmid encyclopedia. EMBO J. 1995 Apr 18;14(8):1827–1841. doi: 10.1002/j.1460-2075.1995.tb07171.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fujiwara Y., Oka M., Hamamoto T., Sone N. Cytochrome c-551 of the thermophilic bacterium PS3, DNA sequence and analysis of the mature cytochrome. Biochim Biophys Acta. 1993 Sep 13;1144(2):213–219. doi: 10.1016/0005-2728(93)90175-f. [DOI] [PubMed] [Google Scholar]
  14. Gray K. A., Grooms M., Myllykallio H., Moomaw C., Slaughter C., Daldal F. Rhodobacter capsulatus contains a novel cb-type cytochrome c oxidase without a CuA center. Biochemistry. 1994 Mar 15;33(10):3120–3127. doi: 10.1021/bi00176a047. [DOI] [PubMed] [Google Scholar]
  15. Hochkoeppler A., Jenney F. E., Jr, Lang S. E., Zannoni D., Daldal F. Membrane-associated cytochrome cy of Rhodobacter capsulatus is an electron carrier from the cytochrome bc1 complex to the cytochrome c oxidase during respiration. J Bacteriol. 1995 Feb;177(3):608–613. doi: 10.1128/jb.177.3.608-613.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Iwata S., Ostermeier C., Ludwig B., Michel H. Structure at 2.8 A resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature. 1995 Aug 24;376(6542):660–669. doi: 10.1038/376660a0. [DOI] [PubMed] [Google Scholar]
  17. Jenney F. E., Jr, Daldal F. A novel membrane-associated c-type cytochrome, cyt cy, can mediate the photosynthetic growth of Rhodobacter capsulatus and Rhodobacter sphaeroides. EMBO J. 1993 Apr;12(4):1283–1292. doi: 10.1002/j.1460-2075.1993.tb05773.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jenney F. E., Jr, Prince R. C., Daldal F. Roles of the soluble cytochrome c2 and membrane-associated cytochrome cy of Rhodobacter capsulatus in photosynthetic electron transfer. Biochemistry. 1994 Mar 8;33(9):2496–2502. doi: 10.1021/bi00175a019. [DOI] [PubMed] [Google Scholar]
  19. Jenney F. E., Jr, Prince R. C., Daldal F. The membrane-bound cytochrome cy of Rhodobacter capsulatus can serve as an electron donor to the photosynthetic reaction of Rhodobacter sphaeroides. Biochim Biophys Acta. 1996 Feb 15;1273(2):159–164. doi: 10.1016/0005-2728(95)00137-9. [DOI] [PubMed] [Google Scholar]
  20. Jones M. R., McEwan A. G., Jackson J. B. The role of c-type cytochromes in the photosynthetic electron transport pathway of Rhodobacter capsulatus. Biochim Biophys Acta. 1990 Aug 9;1019(1):59–66. doi: 10.1016/0005-2728(90)90124-m. [DOI] [PubMed] [Google Scholar]
  21. Karrasch S., Bullough P. A., Ghosh R. The 8.5 A projection map of the light-harvesting complex I from Rhodospirillum rubrum reveals a ring composed of 16 subunits. EMBO J. 1995 Feb 15;14(4):631–638. doi: 10.1002/j.1460-2075.1995.tb07041.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  23. MacGregor B. J., Donohue T. J. Evidence for two promoters for the cytochrome c2 gene (cycA) of Rhodobacter sphaeroides. J Bacteriol. 1991 Jul;173(13):3949–3957. doi: 10.1128/jb.173.13.3949-3957.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Manoil C., Beckwith J. A genetic approach to analyzing membrane protein topology. Science. 1986 Sep 26;233(4771):1403–1408. doi: 10.1126/science.3529391. [DOI] [PubMed] [Google Scholar]
  25. Nomoto T., Fukumori Y., Yamanaka T. Membrane-bound cytochrome c is an alternative electron donor for cytochrome aa3 in Nitrobacter winogradskyi. J Bacteriol. 1993 Jul;175(14):4400–4404. doi: 10.1128/jb.175.14.4400-4404.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pearce D. A., Sherman F. Diminished degradation of yeast cytochrome c by interactions with its physiological partners. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3735–3739. doi: 10.1073/pnas.92.9.3735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Prince R. C., Baccarini-Melandri A., Hauska G. A., Melandri B. A., Crofts A. R. Asymmetry of an energy transducing membrane the location of cytochrome c2 in Rhodopseudomonas spheroides and Rhodopseudomonas capsulata. Biochim Biophys Acta. 1975 May 15;387(2):212–227. doi: 10.1016/0005-2728(75)90104-8. [DOI] [PubMed] [Google Scholar]
  28. Prince R. C., Daldal F. Physiological electron donors to the photochemical reaction center of Rhodobacter capsulatus. Biochim Biophys Acta. 1987 Dec 17;894(3):370–378. doi: 10.1016/0005-2728(87)90115-0. [DOI] [PubMed] [Google Scholar]
  29. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Richardson D. J., Bell L. C., McEwan A. G., Jackson J. B., Ferguson S. J. Cytochrome c2 is essential for electron transfer to nitrous oxide reductase from physiological substrates in Rhodobacter capsulatus and can act as an electron donor to the reductase in vitro. Correlation with photoinhibition studies. Eur J Biochem. 1991 Aug 1;199(3):677–683. doi: 10.1111/j.1432-1033.1991.tb16170.x. [DOI] [PubMed] [Google Scholar]
  31. SISTROM W. R. A requirement for sodium in the growth of Rhodopseudomonas spheroides. J Gen Microbiol. 1960 Jun;22:778–785. doi: 10.1099/00221287-22-3-778. [DOI] [PubMed] [Google Scholar]
  32. Schifferli D. M. Use of TnphoA and T7 RNA polymerase to study fimbrial proteins. Methods Enzymol. 1995;253:242–258. doi: 10.1016/s0076-6879(95)53023-1. [DOI] [PubMed] [Google Scholar]
  33. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  34. Scolnik P. A., Walker M. A., Marrs B. L. Biosynthesis of carotenoids derived from neurosporene in Rhodopseudomonas capsulata. J Biol Chem. 1980 Mar 25;255(6):2427–2432. [PubMed] [Google Scholar]
  35. Thomas P. E., Ryan D., Levin W. An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Anal Biochem. 1976 Sep;75(1):168–176. doi: 10.1016/0003-2697(76)90067-1. [DOI] [PubMed] [Google Scholar]
  36. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Turba A., Jetzek M., Ludwig B. Purification of Paracoccus denitrificans cytochrome c552 and sequence analysis of the gene. Eur J Biochem. 1995 Jul 1;231(1):259–265. [PubMed] [Google Scholar]
  38. Umbarger H. E. Amino acid biosynthesis and its regulation. Annu Rev Biochem. 1978;47:532–606. doi: 10.1146/annurev.bi.47.070178.002533. [DOI] [PubMed] [Google Scholar]
  39. Yen H. C., Hu N. T., Marrs B. L. Characterization of the gene transfer agent made by an overproducer mutant of Rhodopseudomonas capsulata. J Mol Biol. 1979 Jun 25;131(2):157–168. doi: 10.1016/0022-2836(79)90071-8. [DOI] [PubMed] [Google Scholar]
  40. Zeilstra-Ryalls J. H., Kaplan S. Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene. J Bacteriol. 1995 Nov;177(22):6422–6431. doi: 10.1128/jb.177.22.6422-6431.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES