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. 2001 May;80(5):2039–2045. doi: 10.1016/S0006-3495(01)76177-2

pH-dependent structural changes at the Heme-Copper binuclear center of cytochrome c oxidase.

T K Das 1, F L Tomson 1, R B Gennis 1, M Gordon 1, D L Rousseau 1
PMCID: PMC1301396  PMID: 11325707

Abstract

The resonance Raman spectra of the aa3 cytochrome c oxidase from Rhodobacter sphaeroides reveal pH-dependent structural changes in the binuclear site at room temperature. The binuclear site, which is the catalytic center of the enzyme, possesses two conformations at neutral pH, assessed from their distinctly different Fe-CO stretching modes in the resonance Raman spectra of the CO complex of the fully reduced enzyme. The two conformations (alpha and beta) interconvert reversibly in the pH 6-9 range with a pKa of 7.4, consistent with Fourier transform infrared spectroscopy measurements done at cryogenic temperatures (D.M. Mitchell, J.P. Sapleigh, A.M.Archer, J.O. Alben, and R.B.Gennis, 1996, Biochemistry 35:9446-9450). It is postulated that the different structures result from a change in the position of the Cu(B) atom with respect to the CO due to the presence of one or more ionizable groups in the vicinity of the binuclear center. The conserved tyrosine residue (Tyr-288 in R. sphaeroides, Tyr-244 in the bovine enzyme) that is adjacent to the oxygen-binding pocket or one of the histidines that coordinate Cu(B) are possible candidates. The existence of an equilibrium between the two conformers at physiological pH and room temperature suggests that the conformers may be functionally involved in enzymatic activity.

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Selected References

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  1. Adelroth P., Brzezinski P., Malmström B. G. Internal electron transfer in cytochrome c oxidase from Rhodobacter sphaeroides. Biochemistry. 1995 Mar 7;34(9):2844–2849. doi: 10.1021/bi00009a014. [DOI] [PubMed] [Google Scholar]
  2. Argade P. V., Ching Y. C., Rousseau D. L. Cytochrome a3 structure in carbon monoxide-bound cytochrome oxidase. Science. 1984 Jul 20;225(4659):329–331. doi: 10.1126/science.6330890. [DOI] [PubMed] [Google Scholar]
  3. Babcock G. T., Wikström M. Oxygen activation and the conservation of energy in cell respiration. Nature. 1992 Mar 26;356(6367):301–309. doi: 10.1038/356301a0. [DOI] [PubMed] [Google Scholar]
  4. Behr J., Hellwig P., Mäntele W., Michel H. Redox dependent changes at the heme propionates in cytochrome c oxidase from Paracoccus denitrificans: direct evidence from FTIR difference spectroscopy in combination with heme propionate 13C labeling. Biochemistry. 1998 May 19;37(20):7400–7406. doi: 10.1021/bi9731697. [DOI] [PubMed] [Google Scholar]
  5. Behr J., Michel H., Mäntele W., Hellwig P. Functional properties of the heme propionates in cytochrome c oxidase from Paracoccus denitrificans. Evidence from FTIR difference spectroscopy and site-directed mutagenesis. Biochemistry. 2000 Feb 15;39(6):1356–1363. doi: 10.1021/bi991504g. [DOI] [PubMed] [Google Scholar]
  6. Callahan P. M., Babcock G. T. Origin of the cytochrome a absorption red shift: a pH-dependent interaction between its heme a formyl and protein in cytochrome oxidase. Biochemistry. 1983 Jan 18;22(2):452–461. doi: 10.1021/bi00271a031. [DOI] [PubMed] [Google Scholar]
  7. Das T. K., Friedman J. M., Kloek A. P., Goldberg D. E., Rousseau D. L. Origin of the anomalous Fe-CO stretching mode in the CO complex of Ascaris hemoglobin. Biochemistry. 2000 Feb 1;39(4):837–842. doi: 10.1021/bi9922087. [DOI] [PubMed] [Google Scholar]
  8. Das T. K., Gomes C. M., Teixeira M., Rousseau D. L. Redox-linked transient deprotonation at the binuclear site in the aa(3)-type quinol oxidase from Acidianus ambivalens: implications for proton translocation. Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9591–9596. doi: 10.1073/pnas.96.17.9591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Das T. K., Mazumdar S. Conformational change due to reduction of cytochrome-c oxidase in lauryl maltoside: picosecond time-resolved tryptophan fluorescence studies on the native and heat modified enzyme. Biochim Biophys Acta. 1994 Dec 14;1209(2):227–237. doi: 10.1016/0167-4838(94)90189-9. [DOI] [PubMed] [Google Scholar]
  10. Das T. K., Pecoraro C., Tomson F. L., Gennis R. B., Rousseau D. L. The post-translational modification in cytochrome c oxidase is required to establish a functional environment of the catalytic site. Biochemistry. 1998 Oct 13;37(41):14471–14476. doi: 10.1021/bi981500w. [DOI] [PubMed] [Google Scholar]
  11. Dasgupta S., Rousseau D. L., Anni H., Yonetani T. Structural characterization of cytochrome c peroxidase by resonance Raman scattering. J Biol Chem. 1989 Jan 5;264(1):654–662. [PubMed] [Google Scholar]
  12. Guarrera L., Colotti G., Boffi A., Chiancone E., Das T. K., Rousseau D. L., Gibson Q. H. The apolar distal histidine mutant (His69-->Val) of the homodimeric Scapharca hemoglobin is in an R-like conformation. Biochemistry. 1998 Apr 21;37(16):5608–5615. doi: 10.1021/bi972380f. [DOI] [PubMed] [Google Scholar]
  13. Hallén S., Brzezinski P., Malmström B. G. Internal electron transfer in cytochrome c oxidase is coupled to the protonation of a group close to the bimetallic site. Biochemistry. 1994 Feb 15;33(6):1467–1472. doi: 10.1021/bi00172a024. [DOI] [PubMed] [Google Scholar]
  14. Hallén S., Nilsson T. Proton transfer during the reaction between fully reduced cytochrome c oxidase and dioxygen: pH and deuterium isotope effects. Biochemistry. 1992 Dec 1;31(47):11853–11859. doi: 10.1021/bi00162a025. [DOI] [PubMed] [Google Scholar]
  15. Han S. W., Ching Y. C., Hammes S. L., Rousseau D. L. Vibrational structure of the formyl group on heme a. Implications on the properties of cytochrome c oxidase. Biophys J. 1991 Jul;60(1):45–52. doi: 10.1016/S0006-3495(91)82029-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Harrenga A., Michel H. The cytochrome c oxidase from Paracoccus denitrificans does not change the metal center ligation upon reduction. J Biol Chem. 1999 Nov 19;274(47):33296–33299. doi: 10.1074/jbc.274.47.33296. [DOI] [PubMed] [Google Scholar]
  17. Hu S., Treat R. W., Kincaid J. R. Distinct heme active-site structure in lactoperoxidase revealed by resonance Raman spectroscopy. Biochemistry. 1993 Sep 28;32(38):10125–10130. doi: 10.1021/bi00089a031. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Mitchell D. M., Gennis R. B. Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni(2+)-NTA affinity chromatography. FEBS Lett. 1995 Jul 10;368(1):148–150. doi: 10.1016/0014-5793(95)00626-k. [DOI] [PubMed] [Google Scholar]
  20. Mitchell D. M., Shapleigh J. P., Archer A. M., Alben J. O., Gennis R. B. A pH-dependent polarity change at the binuclear center of reduced cytochrome c oxidase detected by FTIR difference spectroscopy of the CO adduct. Biochemistry. 1996 Jul 23;35(29):9446–9450. doi: 10.1021/bi960392f. [DOI] [PubMed] [Google Scholar]
  21. Mitchell R., Rich P. R. Proton uptake by cytochrome c oxidase on reduction and on ligand binding. Biochim Biophys Acta. 1994 Jun 28;1186(1-2):19–26. doi: 10.1016/0005-2728(94)90130-9. [DOI] [PubMed] [Google Scholar]
  22. Moody A. J., Rich P. R. The effect of pH on redox titrations of haem a in cyanide-liganded cytochrome-c oxidase: experimental and modelling studies. Biochim Biophys Acta. 1990 Feb 2;1015(2):205–215. doi: 10.1016/0005-2728(90)90022-v. [DOI] [PubMed] [Google Scholar]
  23. Mylrajan M., Valli K., Wariishi H., Gold M. H., Loehr T. M. Resonance Raman spectroscopic characterization of compound III of lignin peroxidase. Biochemistry. 1990 Oct 16;29(41):9617–9623. doi: 10.1021/bi00493a016. [DOI] [PubMed] [Google Scholar]
  24. Oliveberg M., Hallén S., Nilsson T. Uptake and release of protons during the reaction between cytochrome c oxidase and molecular oxygen: a flow-flash investigation. Biochemistry. 1991 Jan 15;30(2):436–440. doi: 10.1021/bi00216a019. [DOI] [PubMed] [Google Scholar]
  25. Osborne J. P., Cosper N. J., Stälhandske C. M., Scott R. A., Alben J. O., Gennis R. B. Cu XAS shows a change in the ligation of CuB upon reduction of cytochrome bo3 from Escherichia coli. Biochemistry. 1999 Apr 6;38(14):4526–4532. doi: 10.1021/bi982278y. [DOI] [PubMed] [Google Scholar]
  26. Ostermeier C., Harrenga A., Ermler U., Michel H. Structure at 2.7 A resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody FV fragment. Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10547–10553. doi: 10.1073/pnas.94.20.10547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Proshlyakov D. A., Pressler M. A., DeMaso C., Leykam J. F., DeWitt D. L., Babcock G. T. Oxygen activation and reduction in respiration: involvement of redox-active tyrosine 244. Science. 2000 Nov 24;290(5496):1588–1591. doi: 10.1126/science.290.5496.1588. [DOI] [PubMed] [Google Scholar]
  28. Ralle M., Verkhovskaya M. L., Morgan J. E., Verkhovsky M. I., Wikström M., Blackburn N. J. Coordination of CuB in reduced and CO-liganded states of cytochrome bo3 from Escherichia coli. Is chloride ion a cofactor? Biochemistry. 1999 Jun 1;38(22):7185–7194. doi: 10.1021/bi982885l. [DOI] [PubMed] [Google Scholar]
  29. Rousseau D. L. Bioenergetics. Two phases of proton translocation. Nature. 1999 Jul 29;400(6743):412–413. doi: 10.1038/22664. [DOI] [PubMed] [Google Scholar]
  30. Spiro T. G., Smulevich G., Su C. Probing protein structure and dynamics with resonance Raman spectroscopy: cytochrome c peroxidase and hemoglobin. Biochemistry. 1990 May 15;29(19):4497–4508. doi: 10.1021/bi00471a001. [DOI] [PubMed] [Google Scholar]
  31. Tsukihara T., Aoyama H., Yamashita E., Tomizaki T., Yamaguchi H., Shinzawa-Itoh K., Nakashima R., Yaono R., Yoshikawa S. Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 A. Science. 1995 Aug 25;269(5227):1069–1074. doi: 10.1126/science.7652554. [DOI] [PubMed] [Google Scholar]
  32. Tsukihara T., Aoyama H., Yamashita E., Tomizaki T., Yamaguchi H., Shinzawa-Itoh K., Nakashima R., Yaono R., Yoshikawa S. The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A. Science. 1996 May 24;272(5265):1136–1144. doi: 10.1126/science.272.5265.1136. [DOI] [PubMed] [Google Scholar]
  33. Uno T., Nishimura Y., Tsuboi M., Makino R., Iizuka T., Ishimura Y. Two types of conformers with distinct Fe-C-O configuration in the ferrous CO complex of horseradish peroxidase. Resonance Raman and infarared spectroscopic studies with native and deuteroheme-substituted enzymes. J Biol Chem. 1987 Apr 5;262(10):4549–4556. [PubMed] [Google Scholar]
  34. Verkhovsky M. I., Jasaitis A., Verkhovskaya M. L., Morgan J. E., Wikström M. Proton translocation by cytochrome c oxidase. Nature. 1999 Jul 29;400(6743):480–483. doi: 10.1038/22813. [DOI] [PubMed] [Google Scholar]
  35. Verkhovsky M. I., Morgan J. E., Wikström M. Control of electron delivery to the oxygen reduction site of cytochrome c oxidase: a role for protons. Biochemistry. 1995 Jun 6;34(22):7483–7491. doi: 10.1021/bi00022a023. [DOI] [PubMed] [Google Scholar]
  36. Wang J., Takahashi S., Hosler J. P., Mitchell D. M., Ferguson-Miller S., Gennis R. B., Rousseau D. L. Two conformations of the catalytic site in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides. Biochemistry. 1995 Aug 8;34(31):9819–9825. doi: 10.1021/bi00031a001. [DOI] [PubMed] [Google Scholar]
  37. Wang J., Takahashi S., Rousseau D. L. Identification of the overtone of the Fe-CO stretching mode in heme proteins: a probe of the heme active site. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9402–9406. doi: 10.1073/pnas.92.20.9402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wittung P., Malmström B. G. Redox-linked conformational changes in cytochrome c oxidase. FEBS Lett. 1996 Jun 10;388(1):47–49. doi: 10.1016/0014-5793(96)00513-3. [DOI] [PubMed] [Google Scholar]
  39. Yoshikawa S., Shinzawa-Itoh K., Nakashima R., Yaono R., Yamashita E., Inoue N., Yao M., Fei M. J., Libeu C. P., Mizushima T. Redox-coupled crystal structural changes in bovine heart cytochrome c oxidase. Science. 1998 Jun 12;280(5370):1723–1729. doi: 10.1126/science.280.5370.1723. [DOI] [PubMed] [Google Scholar]
  40. Yoshikawa S., Tera T., Takahashi Y., Tsukihara T., Caughey W. S. Crystalline cytochrome c oxidase of bovine heart mitochondrial membrane: composition and x-ray diffraction studies. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1354–1358. doi: 10.1073/pnas.85.5.1354. [DOI] [PMC free article] [PubMed] [Google Scholar]

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