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. 1987 Oct 15;247(2):475–484. doi: 10.1042/bj2470475

Zinc cytochrome c fluorescence as a probe for conformational changes in cytochrome c oxidase.

T A Alleyne 1, M T Wilson 1
PMCID: PMC1148432  PMID: 2827630

Abstract

Zinc cytochrome c forms tight 1:1 complexes with a variety of derivatives of cytochrome c oxidase. On complex-formation the fluorescence of zinc cytochrome c is diminished. Titrations of zinc cytochrome c with cytochrome c oxidase, followed through the fluorescence emission of the former, have yielded both binding constants (K approximately 7 x 10(6) M-1 for the fully oxidized and 2 x 10(7) M-1 for the fully reduced enzyme) and distance information. Comparison of steady-state measurements obtained by absorbance and fluorescence spectroscopy in the presence and in the absence of cyanide show that it is the reduction of cytochrome a and/or CuA that triggers a conformational change: this increases the zinc cytochrome c to acceptor (most probably cytochrome a itself) distance by some 0.5 nm. Ligand binding to the fully oxidized or fully reduced enzyme leaves the extent of fluorescence quenching unchanged, whereas binding of cyanide to the half-reduced enzyme (a2+CuA+CuB2+-CN(-)-a3(3+)) enhances fluorescence emission relative to that for the fully reduced enzyme, implying further relative movement of donor and acceptor.

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Selected References

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  1. Azzi A. Cytochrome c oxidase. Towards a clarification of its structure, interactions and mechanism. Biochim Biophys Acta. 1980 Dec;594(4):231–252. doi: 10.1016/0304-4173(80)90002-6. [DOI] [PubMed] [Google Scholar]
  2. Blair D. F., Ellis W. R., Jr, Wang H., Gray H. B., Chan S. I. Spectroelectrochemical study of cytochrome c oxidase: pH and temperature dependences of the cytochrome potentials. Characterization of site-site interactions. J Biol Chem. 1986 Sep 5;261(25):11524–11537. [PubMed] [Google Scholar]
  3. Blair D. F., Gelles J., Chan S. I. Redox-linked proton translocation in cytochrome oxidase: the importance of gating electron flow. The effects of slip in a model transducer. Biophys J. 1986 Oct;50(4):713–733. doi: 10.1016/S0006-3495(86)83511-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brzezinski P., Malmström B. G. Electron-transport-driven proton pumps display nonhyperbolic kinetics: Simulation of the steady-state kinetics of cytochrome c oxidase. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4282–4286. doi: 10.1073/pnas.83.12.4282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Dale R. E., Eisinger J., Blumberg W. E. The orientational freedom of molecular probes. The orientation factor in intramolecular energy transfer. Biophys J. 1979 May;26(2):161–193. doi: 10.1016/S0006-3495(79)85243-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dale R. E., Eisinger J. Intramolecular energy transfer and molecular conformation. Proc Natl Acad Sci U S A. 1976 Feb;73(2):271–273. doi: 10.1073/pnas.73.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dickinson L. C., Chien J. C. Cobalt-cytochrome c. I. Preparation, properties, and enzymic activity. Biochemistry. 1975 Aug 12;14(16):3526–3534. doi: 10.1021/bi00687a003. [DOI] [PubMed] [Google Scholar]
  9. Dockter M. E., Steinemann A., Schatz G. Mapping of yeast cytochrome c oxidase by fluorescence resonance energy transfer. Distances between subunit II, heme a, and cytochrome c bound to subunit III. J Biol Chem. 1978 Jan 10;253(1):311–317. [PubMed] [Google Scholar]
  10. Eisinger J., Dale R. E. Letter: interpretation of intramolecular energy transfer experiments. J Mol Biol. 1974 Apr 25;84(4):643–647. doi: 10.1016/0022-2836(74)90122-3. [DOI] [PubMed] [Google Scholar]
  11. Geren L. M., Millett F. Fluorescence energy transfer studies of the interaction between adrenodoxin and cytochrome c. J Biol Chem. 1981 Oct 25;256(20):10485–10489. [PubMed] [Google Scholar]
  12. Jensen P., Wilson M. T., Aasa R., Malmström B. G. Cyanide inhibition of cytochrome c oxidase. A rapid-freeze e.p.r. investigation. Biochem J. 1984 Dec 15;224(3):829–837. doi: 10.1042/bj2240829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jones M. G., Bickar D., Wilson M. T., Brunori M., Colosimo A., Sarti P. A re-examination of the reactions of cyanide with cytochrome c oxidase. Biochem J. 1984 May 15;220(1):57–66. doi: 10.1042/bj2200057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. King T. E., Bayley P. M., Yong F. C. Optical rotatory dispersion and circular dichroism of cytochrome oxidase. Eur J Biochem. 1971 May 11;20(1):103–110. doi: 10.1111/j.1432-1033.1971.tb01367.x. [DOI] [PubMed] [Google Scholar]
  15. Kornblatt J. A., Luu H. A. The interactions of cytochrome c and porphyrin cytochrome c with cytochrome c oxidase. The resting, reduced and pulsed enzymes. Eur J Biochem. 1986 Sep 1;159(2):407–413. doi: 10.1111/j.1432-1033.1986.tb09883.x. [DOI] [PubMed] [Google Scholar]
  16. Miki M., Iio T. Fluorescence energy transfer measurements between the nucleotide binding site and Cys-373 in actin and their application to the kinetics of actin polymerization. Biochim Biophys Acta. 1984 Nov 9;790(3):201–207. doi: 10.1016/0167-4838(84)90023-2. [DOI] [PubMed] [Google Scholar]
  17. Sarti P., Jones M. G., Antonini G., Malatesta F., Colosimo A., Wilson M. T., Brunori M. Kinetics of redox-linked proton pumping activity of native and subunit III-depleted cytochrome c oxidase: a stopped-flow investigation. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4876–4880. doi: 10.1073/pnas.82.15.4876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Thomson A. J., Greenwood C., Gadsby P. M., Peterson J., Eglinton D. G., Hill B. C., Nicholls P. The structure of the cytochrome a3-CuB site of mammalian cytochrome c oxidase as probed by MCD and EPR spectroscopy. J Inorg Biochem. 1985 Mar-Apr;23(3-4):187–197. doi: 10.1016/0162-0134(85)85025-x. [DOI] [PubMed] [Google Scholar]
  20. Vanderkooi J. M., Adar F., Erecińska M. Metallocytochromes c: characterization of electronic absorption and emission spectra of Sn4+ and Zn2+ cytochromes c. Eur J Biochem. 1976 May 1;64(2):381–387. doi: 10.1111/j.1432-1033.1976.tb10312.x. [DOI] [PubMed] [Google Scholar]
  21. Vanderkooi J. M., Erecińska M. Cytochrome c interaction with membranes. Absorption and emission spectra and binding characteristics of iron-free cytochrome c. Eur J Biochem. 1975 Dec 1;60(1):199–207. doi: 10.1111/j.1432-1033.1975.tb20992.x. [DOI] [PubMed] [Google Scholar]
  22. Vanderkooi J. M., Landesberg R., Hayden G. W., Owen C. S. Metal-free and metal-substituted cytochromes c. Use in characterization of the cytochrome c binding site. Eur J Biochem. 1977 Dec 1;81(2):339–347. doi: 10.1111/j.1432-1033.1977.tb11957.x. [DOI] [PubMed] [Google Scholar]
  23. Wikström K. F., Harmon H. J., Ingledew W. J., Chance B. A re-evaluation of the spectral, potentiometric and energy-linked properties of cytochrome c oxidase in mitochondria. FEBS Lett. 1976 Jun 15;65(3):259–277. doi: 10.1016/0014-5793(76)80127-5. [DOI] [PubMed] [Google Scholar]
  24. YONETANI T. Studies on cytochrome oxidase. I. Absolute and difference absorption spectra. J Biol Chem. 1960 Mar;235:845–852. [PubMed] [Google Scholar]
  25. YONETANI T. Studies on cytochrome oxidase. III. Improved preparation and some properties. J Biol Chem. 1961 Jun;236:1680–1688. [PubMed] [Google Scholar]
  26. van Buuren K. J., Zuurendonk P. F., van Gelder B. F., Muijsers A. O. Biochemical and biophysical studies on cytochrome aa 3 . V. Binding of cyanide to cytochrome aa 3 . Biochim Biophys Acta. 1972 Feb 28;256(2):243–257. doi: 10.1016/0005-2728(72)90056-4. [DOI] [PubMed] [Google Scholar]

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