Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1982 Jan;37(1):285–293. doi: 10.1016/S0006-3495(82)84677-8

The active form of cytochrome c oxidase: effects of detergent, the intact membrane, and radiation inactivation.

D A Thompson, M Suárez-Villafañe, S Ferguson-Miller
PMCID: PMC1329139  PMID: 6275925

Abstract

Cytochrome oxidase is a multisubunit, intrinsic membrane protein with a complex function that includes oxidation of cytochrome c, reduction of oxygen and generation of a membrane potential. To clarify the relationship of its normal function to protein and membrane structure, we have examined the kinetic behavior of rat liver cytochrome oxidase in the intact inner mitochondrial membrane and in detergent solubilized states. Dissolution of rat liver mitochondrial membranes alters the kinetic parameters of the oxidase in a manner dependent in part on the dispersing agent, and characterized by a large increase in maximal activity which is not attributable to exposure of more oxidase or diminished affinity for cytochrome c. The most profound effect of solubilization of the membrane is seen on the low affinity reaction of cytochrome c, suggesting that the electron transfer pathway from this site to oxygen is sensitive to alterations in hydrophobic interactions within the oxidase. Purified rat liver and beef heart oxidase exists predominantly in a monodisperse, 300 kilodalton form in laurylmaltoside (Rosevear et al., 1980). However, a smaller, 130 kd species that exhibits high turnover rates equal to the 300 kd form is detected in some beef heart preparations, implying that the dimer may not be essential for high activity. Radiation inactivation studies on purified oxidase reveal a molecular weight for the functional unit of approximately 70 kd. It is concluded that less than a complete set of subunits may be sufficient for both normal binding of cytochrome c and rapid electron transfer to oxygen.

Full text

PDF
285

Selected References

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

  1. Bisson R., Jacobs B., Capaldi R. A. Binding of arylazidocytochrome c derivatives to beef heart cytochrome c oxidase: cross-linking in the high- and low-affinity binding sites. Biochemistry. 1980 Sep 2;19(18):4173–4178. doi: 10.1021/bi00559a006. [DOI] [PubMed] [Google Scholar]
  2. Brautigan D. L., Ferguson-Miller S., Margoliash E. Mitochondrial cytochrome c: preparation and activity of native and chemically modified cytochromes c. Methods Enzymol. 1978;53:128–164. doi: 10.1016/s0076-6879(78)53021-8. [DOI] [PubMed] [Google Scholar]
  3. Briggs M. M., Capaldi R. A. Near-neighbor relationships of the subunits of cytochrome c oxidase. Biochemistry. 1977 Jan 11;16(1):73–77. doi: 10.1021/bi00620a012. [DOI] [PubMed] [Google Scholar]
  4. Chance B., Saronio C., Waring A., Leigh J. S., Jr Cytochrome c-cytochrome oxidase interaction at subzero temperatures. Biochim Biophys Acta. 1978 Jul 6;503(1):37–55. doi: 10.1016/0005-2728(78)90160-3. [DOI] [PubMed] [Google Scholar]
  5. Conrad M. J., Singer S. J. Evidence for a large internal pressure in biological membranes. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5202–5206. doi: 10.1073/pnas.76.10.5202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DAVIES H. C., SMITH L., WASSERMAN A. R. THE INFLUENCE OF IONIC STRENGTH AND POLYCATIONS ON THE OXIDATION OF FERROCYTOCHROME C BY CYTOCHROME C OXIDASE. Biochim Biophys Acta. 1964 May 4;85:238–246. doi: 10.1016/0926-6569(64)90244-5. [DOI] [PubMed] [Google Scholar]
  7. Errede B., Kamen M. D. Comparative kinetic studies of cytochromes c in reactions with mitochondrial cytochrome c oxidase and reductase. Biochemistry. 1978 Mar 21;17(6):1015–1027. doi: 10.1021/bi00599a012. [DOI] [PubMed] [Google Scholar]
  8. Fee J. A., Choc M. G., Findling K. L., Lorence R., Yoshida T. Properties of a copper-containing cytochrome c1aa3 complex: a terminal oxidase of the extreme thermophile Thermus thermophilus HB8. Proc Natl Acad Sci U S A. 1980 Jan;77(1):147–151. doi: 10.1073/pnas.77.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Felgner P. L., Messer J. L., Wilson J. E. Purification of a hexokinase-binding protein from the outer mitochondrial membrane. J Biol Chem. 1979 Jun 25;254(12):4946–4949. [PubMed] [Google Scholar]
  10. Ferguson-Miller S., Brautigan D. L., Margoliash E. Correlation of the kinetics of electron transfer activity of various eukaryotic cytochromes c with binding to mitochondrial cytochrome c oxidase. J Biol Chem. 1976 Feb 25;251(4):1104–1115. [PubMed] [Google Scholar]
  11. Ferguson-Miller S., Brautigan D. L., Margoliash E. Definition of cytochrome c binding domains by chemical modification. III. Kinetics of reaction of carboxydinitrophenyl cytochromes c with cytochrome c oxidase. J Biol Chem. 1978 Jan 10;253(1):149–159. [PubMed] [Google Scholar]
  12. Fluke D. J. Temperature dependence of the direct action of ionizing radiation on beef heart lactate dehydrogenase: enzyme activity, substrate and coenzyme affinities. Radiat Res. 1972 Jul;51(1):56–71. [PubMed] [Google Scholar]
  13. Hartzell C. R., Beinert H. Components of cytochrome c oxidase detectable by EPR spectroscopy. Biochim Biophys Acta. 1974 Dec 19;368(3):318–338. doi: 10.1016/0005-2728(74)90178-9. [DOI] [PubMed] [Google Scholar]
  14. Kagawa Y. Target size of components in oxidative phosphorylation. Studies with a linear accelerator. Biochim Biophys Acta. 1967 May 9;131(3):586–588. doi: 10.1016/0005-2728(67)90019-9. [DOI] [PubMed] [Google Scholar]
  15. Kempner E. S., Schlegel W. Size determination of enzymes by radiation inactivation. Anal Biochem. 1979 Jan 1;92(1):2–10. doi: 10.1016/0003-2697(79)90617-1. [DOI] [PubMed] [Google Scholar]
  16. Kuboyama M., Yong F. C., King T. E. Studies on cytochrome oxidase. 8. Preparation and some properties of cardiac cytochrome oxidase. J Biol Chem. 1972 Oct 25;247(20):6375–6383. [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. NICHOLLS P. OBSERVATIONS ON THE OXIDATION OF CYTOCHROME C. Arch Biochem Biophys. 1964 Jul 20;106:25–48. doi: 10.1016/0003-9861(64)90154-7. [DOI] [PubMed] [Google Scholar]
  20. Nagasawa T., Nagasawa-Fujimori H., Heinrich P. C. Hydrophobic interactions of cytochrome c oxidase. Application to the purification of the enzyme from rat liver mitochondria. Eur J Biochem. 1979 Feb 15;94(1):31–39. doi: 10.1111/j.1432-1033.1979.tb12868.x. [DOI] [PubMed] [Google Scholar]
  21. Nicholls P. Cytochrome c binding to enzymes and membranes. Biochim Biophys Acta. 1974 Dec 30;346(3-4):261–310. doi: 10.1016/0304-4173(74)90003-2. [DOI] [PubMed] [Google Scholar]
  22. Nicholls P., Hildebrandt V., Hill B. C., Nicholls F., Wrigglesworth J. M. Pathways of cytochrome c oxidation by soluble and membrane-bound cytochrome aa3. Can J Biochem. 1980 Oct;58(10):969–977. doi: 10.1139/o80-132. [DOI] [PubMed] [Google Scholar]
  23. Nozaki Y., Schechter N. M., Reynolds J. A., Tanford C. Use of gel chromatography for the determination of the Stokes radii of proteins in the presence and absence of detergents. A reexamination. Biochemistry. 1976 Aug 24;15(17):3884–3890. doi: 10.1021/bi00662a036. [DOI] [PubMed] [Google Scholar]
  24. Robinson N. C., Capaldi R. A. Interaction of detergents with cytochrome c oxidase. Biochemistry. 1977 Feb 8;16(3):375–381. doi: 10.1021/bi00622a006. [DOI] [PubMed] [Google Scholar]
  25. Rosevear P., VanAken T., Baxter J., Ferguson-Miller S. Alkyl glycoside detergents: a simpler synthesis and their effects on kinetic and physical properties of cytochrome c oxidase. Biochemistry. 1980 Aug 19;19(17):4108–4115. doi: 10.1021/bi00558a032. [DOI] [PubMed] [Google Scholar]
  26. SMITH L., CAMERINO P. W. THE REACTION OF PARTICLE-BOUND CYTOCHROME C OXIDASE WITH ENDOGENOUS AND EXOGENOUS CYTOCHROME C. Biochemistry. 1963 Nov-Dec;2:1432–1439. doi: 10.1021/bi00906a040. [DOI] [PubMed] [Google Scholar]
  27. Steffens G. J., Buse G. Studies on cytochrome c oxidase, IV[1--3]. Primary structure and function of subunit II. Hoppe Seylers Z Physiol Chem. 1979 Apr;360(4):613–619. [PubMed] [Google Scholar]
  28. Tanaka M., Haniu M., Zeitlin S., Yasunobu K. T., Yu C. A., Yu L., King T. E. Sequence of the cysteine peptide from the copper-subunit of bovine cardiac cytochrome oxidase. Biochem Biophys Res Commun. 1975 Sep 2;66(1):357–367. doi: 10.1016/s0006-291x(75)80336-6. [DOI] [PubMed] [Google Scholar]
  29. Vik S. B., Capaldi R. A. Lipid requirements for cytochrome c oxidase activity. Biochemistry. 1977 Dec 27;16(26):5755–5759. doi: 10.1021/bi00645a016. [DOI] [PubMed] [Google Scholar]
  30. Vik S. B., Georgevich G., Capaldi R. A. Diphosphatidylglycerol is required for optimal activity of beef heart cytochrome c oxidase. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1456–1460. doi: 10.1073/pnas.78.3.1456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wilson M. T., Greenwood C., Brunori M., Antonini E. Kinetic studies on the reaction between cytochrome c oxidase and ferrocytochrome c. Biochem J. 1975 Apr;147(1):145–153. doi: 10.1042/bj1470145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Winter D. B., Bruyninckx W. J., Foulke F. G., Grinich N. P., Mason H. S. Location of heme a on subunits I and II and copper on subunit II of cytochrome c oxidase. J Biol Chem. 1980 Dec 10;255(23):11408–11414. [PubMed] [Google Scholar]
  33. Yamanaka T., Fujii K., Kamita Y. Subunits of cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis. J Biochem. 1979 Sep;86(3):821–824. doi: 10.1093/oxfordjournals.jbchem.a132590. [DOI] [PubMed] [Google Scholar]
  34. Yu C., Yu L., King T. E. Studies on cytochrome oxidase. Interactions of the cytochrome oxidase protein with phospholipids and cytochrome c. J Biol Chem. 1975 Feb 25;250(4):1383–1392. [PubMed] [Google Scholar]
  35. van Buuren K. J., Nicholis P., van Gelder B. F. Biochemical and biophysical studies on cytochrome aa 3 . VI. Reaction of cyanide with oxidized and reduced enzyme. Biochim Biophys Acta. 1972 Feb 28;256(2):258–276. doi: 10.1016/0005-2728(72)90057-6. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES