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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Aug;82(15):4876–4880. doi: 10.1073/pnas.82.15.4876

Kinetics of redox-linked proton pumping activity of native and subunit III-depleted cytochrome c oxidase: a stopped-flow investigation.

P Sarti, M G Jones, G Antonini, F Malatesta, A Colosimo, M T Wilson, M Brunori
PMCID: PMC390460  PMID: 2410909

Abstract

The kinetics of oxidation of reduced cytochrome c by cytochrome c oxidase reconstituted into unilamellar vesicles (COV) has been followed by stopped-flow method in the time range 3 msec-1 sec. In the presence of valinomycin, the oxidation of cytochrome c is linked to proton ejection in the external medium, with an apparent stoichiometry (H+/e-) of 0.93 +/- 0.22, under conditions in which the enzyme is in the more active "pulsed" state (i.e., having undergone oxidation-reduction cycles). The time course of reaction indicates that the conformational change(s) involved in coupling the redox reaction to proton translocation is fast. Similar experiments carried out with cytochrome c oxidase depleted of subunit III show that proton-pumping is maintained, although with a lower efficiency (H+/e- = 0.5). The number of protons ejected per electron appears to be correlated to the value of the respiratory control ratio; although this result is partly due to an increase in the rate of diffusion back into the vesicles, a relationship between the respiratory control ratio and the efficiency of the proton pump may be inferred, suggesting a control of the H+/e-ratio.

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Selected References

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  1. Antonini E., Brunori M., Colosimo A., Greenwood C., Wilson M. T. Oxygen "pulsed" cytochrome c oxidase: functional properties and catalytic relevance. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3128–3132. doi: 10.1073/pnas.74.8.3128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brunori M., Colosimo A., Rainoni G., Wilson M. T., Antonini E. Functional intermediates of cytochrome oxidase. Role of "pulsed" oxidase in the pre-steady state and steady state reactions of the beef enzyme. J Biol Chem. 1979 Nov 10;254(21):10769–10775. [PubMed] [Google Scholar]
  3. Casey R. P., Chappell J. B., Azzi A. Limited-turnover studies on proton translocation in reconstituted cytochrome c oxidase-containing vesicles. Biochem J. 1979 Jul 15;182(1):149–156. doi: 10.1042/bj1820149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casey R. P., Thelen M., Azzi A. Dicyclohexylcarbodiimide binds specifically and covalently to cytochrome c oxidase while inhibiting its H+-translocating activity. J Biol Chem. 1980 May 10;255(9):3994–4000. [PubMed] [Google Scholar]
  5. Hinkle P. C., Kim J. J., Racker E. Ion transport and respiratory control in vesicles formed from cytochrome oxidase and phospholipids. J Biol Chem. 1972 Feb 25;247(4):1338–1339. [PubMed] [Google Scholar]
  6. Ludwig B., Schatz G. A two-subunit cytochrome c oxidase (cytochrome aa3) from Paracoccus dentrificans. Proc Natl Acad Sci U S A. 1980 Jan;77(1):196–200. doi: 10.1073/pnas.77.1.196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Mitchell P., Moyle J. Alternative hypotheses of proton ejection in cytochrome oxidase vesicles. Transmembrane proton pumping or redox-linked deprotonation of phospholipid-cytochrome c complex(es). FEBS Lett. 1983 Jan 24;151(2):167–178. doi: 10.1016/0014-5793(83)80063-5. [DOI] [PubMed] [Google Scholar]
  8. Penttilä T. Properties and reconstitution of a cytochrome oxidase deficient in subunit III. Eur J Biochem. 1983 Jun 15;133(2):355–361. doi: 10.1111/j.1432-1033.1983.tb07470.x. [DOI] [PubMed] [Google Scholar]
  9. Proteau G., Wrigglesworth J. M., Nicholls P. Protonmotive functions of cytochrome c oxidase in reconstituted vesicles. Influence of turnover rate on 'proton translocation'. Biochem J. 1983 Jan 15;210(1):199–205. doi: 10.1042/bj2100199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Puettner I., Carafoli E., Malatesta F. Spectroscopic and functional properties of subunit III-depleted cytochrome oxidase. J Biol Chem. 1985 Mar 25;260(6):3719–3723. [PubMed] [Google Scholar]
  11. Reynafarje B., Alexandre A., Davies P., Lehninger A. L. Proton translocation stoichiometry of cytochrome oxidase: use of a fast-responding oxygen electrode. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7218–7222. doi: 10.1073/pnas.79.23.7218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Saraste M., Penttilä T., Wikström M. Quaternary structure of bovine cytochrome oxidase. Eur J Biochem. 1981 Apr;115(2):261–268. doi: 10.1111/j.1432-1033.1981.tb05232.x. [DOI] [PubMed] [Google Scholar]
  13. Sarti P., Colosimo A., Brunori M., Wilson M. T., Antonini E. Kinetic studies on cytochrome c oxidase inserted into liposomal vesicles. Effect of ionophores. Biochem J. 1983 Jan 1;209(1):81–89. doi: 10.1042/bj2090081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sigel E., Carafoli E. The proton pump of cytochrome c oxidase and its stoichiometry. Eur J Biochem. 1978 Aug 15;89(1):119–123. doi: 10.1111/j.1432-1033.1978.tb20903.x. [DOI] [PubMed] [Google Scholar]
  15. Solioz M., Carafoli E., Ludwig B. The cytochrome c oxidase of Paracoccus denitrificans pumps protons in a reconstituted system. J Biol Chem. 1982 Feb 25;257(4):1579–1582. [PubMed] [Google Scholar]
  16. Wikstrom M. K. Proton pump coupled to cytochrome c oxidase in mitochondria. Nature. 1977 Mar 17;266(5599):271–273. doi: 10.1038/266271a0. [DOI] [PubMed] [Google Scholar]
  17. Wilson M. T., Peterson J., Antonini E., Brunori M., Colosimo A., Wyman J. A plausible two-state model for cytochrome c oxidase. Proc Natl Acad Sci U S A. 1981 Nov;78(11):7115–7118. doi: 10.1073/pnas.78.11.7115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. YONETANI T. Studies on cytochrome oxidase. III. Improved preparation and some properties. J Biol Chem. 1961 Jun;236:1680–1688. [PubMed] [Google Scholar]
  19. Yeagle P. L. 31P nuclear magnetic resonance studies of the phospholipid-protein interface in cell membranes. Biophys J. 1982 Jan;37(1):227–239. doi: 10.1016/S0006-3495(82)84672-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

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