Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Jun;179(11):3813–3817. doi: 10.1128/jb.179.11.3813-3817.1997

Formation of pH and potential gradients by the reconstituted Azotobacter vinelandii cytochrome bd respiratory protection oxidase.

J F Kolonay Jr 1, R J Maier 1
PMCID: PMC179186  PMID: 9171438

Abstract

To directly characterize the bioenergetic properties of the cytochrome bd terminating branch of the Azotobacter vinelandii electron transport chain, the purified cytochrome bd oxidase was reconstituted into a phospholipid environment consisting of phosphatidylethanolamine and phosphatidylglycerol (3:1). The average diameter of the proteoliposomes after extrusion through a polycarbonate membrane was 94 +/- 4 nm. Initiation of respiration upon the addition of 20 microM ubiquinone-1 to proteoliposomes loaded with the pH-sensitive dye pyranine resulted in an immediate alkalization of the vesicle lumen by an average pH change of 0.11 unit. This pH gradient was readily collapsed upon the addition of nigericin, carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone, gramicidin, Triton X-100, or 2-heptyl-4-hydroxyquinoline N-oxide (HQNO). Proteoliposomal respiration initiated in the presence of the potentiometric membrane dye rhodamine 123 caused the generation of a transmembrane potential; the potential was collapsed upon the addition of either valinomycin or HQNO. The formation of both pH and potential gradients during turnover demonstrates that the A. vinelandii cytochrome bd oxidase is coupled to energy conservation in vivo.

Full Text

The Full Text of this article is available as a PDF (144.8 KB).

Selected References

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

  1. Ackrell B. A., Jones C. W. The respiratory system of Azotobacter vinelandii. 1. Properties of phosphorylating respiratory membranes. Eur J Biochem. 1971 May 11;20(1):22–28. doi: 10.1111/j.1432-1033.1971.tb01357.x. [DOI] [PubMed] [Google Scholar]
  2. Ackrell B. A., Jones C. W. The respiratory system of Azotobacter vinelandii. 2. Oxygen effects. Eur J Biochem. 1971 May 11;20(1):29–35. doi: 10.1111/j.1432-1033.1971.tb01358.x. [DOI] [PubMed] [Google Scholar]
  3. Dalton H., Postgate J. R. Effect of oxygen on growth of Azotobacter chroococcum in batch and continuous cultures. J Gen Microbiol. 1968 Dec;54(3):463–473. doi: 10.1099/00221287-54-3-463. [DOI] [PubMed] [Google Scholar]
  4. Downs A. J., Jones C. W. Respiration-linked proton translocation in Azotobacter vinelandii. FEBS Lett. 1975 Dec 1;60(1):42–46. doi: 10.1016/0014-5793(75)80414-5. [DOI] [PubMed] [Google Scholar]
  5. Elferink M. G., de Wit J. G., Demel R., Driessen A. J., Konings W. N. Functional reconstitution of membrane proteins in monolayer liposomes from bipolar lipids of Sulfolobus acidocaldarius. J Biol Chem. 1992 Jan 15;267(2):1375–1381. [PubMed] [Google Scholar]
  6. Emaus R. K., Grunwald R., Lemasters J. J. Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Biochim Biophys Acta. 1986 Jul 23;850(3):436–448. doi: 10.1016/0005-2728(86)90112-x. [DOI] [PubMed] [Google Scholar]
  7. Ferber D. M., Maier R. J. Incorporation of a bacterial membrane-bound hydrogenase into proteoliposomes. Anal Biochem. 1992 Jun;203(2):235–244. doi: 10.1016/0003-2697(92)90308-t. [DOI] [PubMed] [Google Scholar]
  8. Gleissner M., Elferink M. G., Driessen A. J., Konings W. N., Anemüller S., Schäfer G. Generation of proton-motive force by an archaeal terminal quinol oxidase from Sulfolobus acidocaldarius. Eur J Biochem. 1994 Sep 15;224(3):983–990. doi: 10.1111/j.1432-1033.1994.00983.x. [DOI] [PubMed] [Google Scholar]
  9. Haddock B. A., Jones C. W. Bacterial respiration. Bacteriol Rev. 1977 Mar;41(1):47–99. doi: 10.1128/br.41.1.47-99.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Henderson P. J. Ion transport by energy-conserving biological membranes. Annu Rev Microbiol. 1971;25:393–428. doi: 10.1146/annurev.mi.25.100171.002141. [DOI] [PubMed] [Google Scholar]
  11. Howitz K. T., McCarty R. E. Measurement of proton-linked transport activities in pyranine loaded chloroplast inner envelope vesicles. Plant Physiol. 1988 Apr;86(4):999–1001. doi: 10.1104/pp.86.4.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Johnson L. V., Walsh M. L., Chen L. B. Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci U S A. 1980 Feb;77(2):990–994. doi: 10.1073/pnas.77.2.990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jones C. W., Redfearn E. R. Electron transport in Azotobacter vinelandii. Biochim Biophys Acta. 1966 Mar 7;113(3):467–481. doi: 10.1016/s0926-6593(66)80005-x. [DOI] [PubMed] [Google Scholar]
  14. Jones C. W., Redfearn E. R. The cytochrome system of Azotobacter vinelandii. Biochim Biophys Acta. 1967 Sep 6;143(2):340–353. doi: 10.1016/0005-2728(67)90088-6. [DOI] [PubMed] [Google Scholar]
  15. Kano K., Fendler J. H. Pyranine as a sensitive pH probe for liposome interiors and surfaces. pH gradients across phospholipid vesicles. Biochim Biophys Acta. 1978 May 18;509(2):289–299. doi: 10.1016/0005-2736(78)90048-2. [DOI] [PubMed] [Google Scholar]
  16. Kelly M. J., Poole R. K., Yates M. G., Kennedy C. Cloning and mutagenesis of genes encoding the cytochrome bd terminal oxidase complex in Azotobacter vinelandii: mutants deficient in the cytochrome d complex are unable to fix nitrogen in air. J Bacteriol. 1990 Oct;172(10):6010–6019. doi: 10.1128/jb.172.10.6010-6019.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kolonay J. F., Jr, Moshiri F., Gennis R. B., Kaysser T. M., Maier R. J. Purification and characterization of the cytochrome bd complex from Azotobacter vinelandii: comparison to the complex from Escherichia coli. J Bacteriol. 1994 Jul;176(13):4177–4181. doi: 10.1128/jb.176.13.4177-4181.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. MacDonald R. C., MacDonald R. I., Menco B. P., Takeshita K., Subbarao N. K., Hu L. R. Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim Biophys Acta. 1991 Jan 30;1061(2):297–303. doi: 10.1016/0005-2736(91)90295-j. [DOI] [PubMed] [Google Scholar]
  19. Matsushita K., Patel L., Kaback H. R. Cytochrome o type oxidase from Escherichia coli. Characterization of the enzyme and mechanism of electrochemical proton gradient generation. Biochemistry. 1984 Sep 25;23(20):4703–4714. doi: 10.1021/bi00315a028. [DOI] [PubMed] [Google Scholar]
  20. Miller M. J., Gennis R. B. The cytochrome d complex is a coupling site in the aerobic respiratory chain of Escherichia coli. J Biol Chem. 1985 Nov 15;260(26):14003–14008. [PubMed] [Google Scholar]
  21. Moshiri F., Kim J. W., Fu C., Maier R. J. The FeSII protein of Azotobacter vinelandii is not essential for aerobic nitrogen fixation, but confers significant protection to oxygen-mediated inactivation of nitrogenase in vitro and in vivo. Mol Microbiol. 1994 Oct;14(1):101–114. doi: 10.1111/j.1365-2958.1994.tb01270.x. [DOI] [PubMed] [Google Scholar]
  22. NEWTON J. W., WILSON P. W., BURRIS R. H. Direct demonstration of ammonia as an intermediate in nitrogen fixation by Azotobacter. J Biol Chem. 1953 Sep;204(1):445–451. [PubMed] [Google Scholar]
  23. Pressman B. C. Biological applications of ionophores. Annu Rev Biochem. 1976;45:501–530. doi: 10.1146/annurev.bi.45.070176.002441. [DOI] [PubMed] [Google Scholar]
  24. Reed P. W. Ionophores. Methods Enzymol. 1979;55:435–454. doi: 10.1016/0076-6879(79)55058-7. [DOI] [PubMed] [Google Scholar]
  25. Reusch R. N., Sadoff H. L. Novel lipid components of the Azotobacter vinelandii cyst membrane. Nature. 1983 Mar 17;302(5905):268–270. doi: 10.1038/302268a0. [DOI] [PubMed] [Google Scholar]
  26. Robson R. L. Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum. Biochem J. 1979 Sep 1;181(3):569–575. doi: 10.1042/bj1810569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Toptygin D., Brand L. Analysis of equilibrium binding data obtained by linear-response spectroscopic techniques. Anal Biochem. 1995 Jan 1;224(1):330–338. doi: 10.1006/abio.1995.1048. [DOI] [PubMed] [Google Scholar]

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

RESOURCES