Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Jan;78(1):439–450. doi: 10.1016/S0006-3495(00)76606-9

Angular dependences of perpendicular and parallel mode electron paramagnetic resonance of oxidized beef heart cytochrome c oxidase.

D J Hunter 1, V S Oganesyan 1, J C Salerno 1, C S Butler 1, W J Ingledew 1, A J Thomson 1
PMCID: PMC1300651  PMID: 10620307

Abstract

Cytochrome c oxidase catalyzes the reduction of oxygen to water with a concomitant conservation of energy in the form of a transmembrane proton gradient. The enzyme has a catalytic site consisting of a binuclear center of a copper ion and a heme group. The spectroscopic parameters of this center are unusual. The origin of broad electron paramagnetic resonance (EPR) signals in the oxidized state at rather low resonant field, the so-called g' = 12 signal, has been a matter of debate for over 30 years. We have studied the angular dependence of this resonance in both parallel and perpendicular mode X-band EPR in oriented multilayers containing cytochrome c oxidase to resolve the assignment. The "slow" form and compounds formed by the addition of formate and fluoride to the oxidized enzyme display these resonances, which result from transitions between states of an integer-spin multiplet arising from magnetic exchange coupling between the five unpaired electrons of high spin Fe(III) heme a(3) and the single unpaired electron of Cu(B). The first successful simulation of similar signals observed in both perpendicular and parallel mode X-band EPR spectra in frozen aqueous solution of the fluoride compound of the closely related enzyme, quinol oxidase or cytochrome bo(3), has been reported recently (Oganesyan et al., 1998, J. Am. Chem. Soc. 120:4232-4233). This suggested that the exchange interaction between the two metal ions of the binuclear center is very weak (|J| approximately 1 cm(-1)), with the axial zero-field splitting (D approximately 5 cm(-1)) of the high-spin heme dominating the form of the ground state. We show that this model accounts well for the angular dependences of the X-band EPR spectra in both perpendicular and parallel modes of oriented multilayers of cytochrome c oxidase derivatives and that the experimental results are inconsistent with earlier schemes that use exchange coupling parameters of several hundred wavenumbers.

Full Text

The Full Text of this article is available as a PDF (164.8 KB).

Selected References

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

  1. Barnes Z. K., Babcock G. T., Dye J. L. Magnetic state of the alpha 3 center of cytochrome c oxidase and some of its derivatives. Biochemistry. 1991 Jul 30;30(30):7597–7603. doi: 10.1021/bi00244a031. [DOI] [PubMed] [Google Scholar]
  2. Blum H., Harmon H. J., Leigh J. S., Salerno J. C., Chance B. The orientation of a heme of cytochrome c oxidase in submitochondrial particles. Biochim Biophys Acta. 1978 Apr 11;502(1):1–10. doi: 10.1016/0005-2728(78)90125-1. [DOI] [PubMed] [Google Scholar]
  3. Brown S., Rumbley J. N., Moody A. J., Thomas J. W., Gennis R. B., Rich P. R. Flash photolysis of the carbon monoxide compounds of wild-type and mutant variants of cytochrome bo from Escherichia coli. Biochim Biophys Acta. 1994 Jan 4;1183(3):521–532. doi: 10.1016/0005-2728(94)90080-9. [DOI] [PubMed] [Google Scholar]
  4. Brudvig G. W., Stevens T. H., Chan S. I. Reactions of nitric oxide with cytochrome c oxidase. Biochemistry. 1980 Nov 11;19(23):5275–5285. doi: 10.1021/bi00564a020. [DOI] [PubMed] [Google Scholar]
  5. Brudvig G. W., Stevens T. H., Morse R. H., Chan S. I. Conformations of oxidized cytochrome c oxidase. Biochemistry. 1981 Jun 23;20(13):3912–3921. doi: 10.1021/bi00516a039. [DOI] [PubMed] [Google Scholar]
  6. Calhoun M. W., Gennis R. B., Ingledew W. J., Salerno J. C. Strong-field and integral spin-ligand complexes of the cytochrome bo quinol oxidase in Escherichia coli membrane preparations. Biochim Biophys Acta. 1994 May 18;1206(1):143–154. doi: 10.1016/0167-4838(94)90083-3. [DOI] [PubMed] [Google Scholar]
  7. Cheesman M. R., Watmough N. J., Gennis R. B., Greenwood C., Thomson A. J. Magnetic-circular-dichroism studies of Escherichia coli cytochrome bo. Identification of high-spin ferric, low-spin ferric and ferryl [Fe(IV)] forms of heme o. Eur J Biochem. 1994 Jan 15;219(1-2):595–602. doi: 10.1111/j.1432-1033.1994.tb19975.x. [DOI] [PubMed] [Google Scholar]
  8. Chepuri V., Gennis R. B. The use of gene fusions to determine the topology of all of the subunits of the cytochrome o terminal oxidase complex of Escherichia coli. J Biol Chem. 1990 Aug 5;265(22):12978–12986. [PubMed] [Google Scholar]
  9. Cooper C. E., Salerno J. C. Characterization of a novel g' = 2.95 EPR signal from the binuclear center of mitochondrial cytochrome c oxidase. J Biol Chem. 1992 Jan 5;267(1):280–285. [PubMed] [Google Scholar]
  10. Day E. P., Peterson J., Sendova M. S., Schoonover J., Palmer G. Magnetization of fast and slow oxidized cytochrome c oxidase. Biochemistry. 1993 Aug 10;32(31):7855–7860. doi: 10.1021/bi00082a003. [DOI] [PubMed] [Google Scholar]
  11. Dunham W. R., Sands R. H., Shaw R. W., Beinert H. Multiple frequency EPR studies on three forms of oxidized cytochrome c oxidase. Biochim Biophys Acta. 1983 Oct 17;748(1):73–85. doi: 10.1016/0167-4838(83)90029-8. [DOI] [PubMed] [Google Scholar]
  12. Greenaway F. T., Chan S. H., Vincow G. An EPR study of the lineshape of copper in cytochrome c oxidase. Biochim Biophys Acta. 1977 Jan 25;490(1):62–78. doi: 10.1016/0005-2795(77)90106-4. [DOI] [PubMed] [Google Scholar]
  13. Hendrich M. P., Debrunner P. G. Integer-spin electron paramagnetic resonance of iron proteins. Biophys J. 1989 Sep;56(3):489–506. doi: 10.1016/S0006-3495(89)82696-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hosler J. P., Ferguson-Miller S., Calhoun M. W., Thomas J. W., Hill J., Lemieux L., Ma J., Georgiou C., Fetter J., Shapleigh J. Insight into the active-site structure and function of cytochrome oxidase by analysis of site-directed mutants of bacterial cytochrome aa3 and cytochrome bo. J Bioenerg Biomembr. 1993 Apr;25(2):121–136. doi: 10.1007/BF00762854. [DOI] [PubMed] [Google Scholar]
  15. Hunter D. J., Moody A. J., Rich P. R., Ingledew W. J. EPR spectroscopy of Escherichia coli cytochrome bo which lacks CuB. FEBS Lett. 1997 Jul 21;412(1):43–47. doi: 10.1016/s0014-5793(97)00735-7. [DOI] [PubMed] [Google Scholar]
  16. Ingledew W. J., Horrocks J., Salerno J. C. Ligand binding to the haem-copper binuclear catalytic site of cytochrome bo, a respiratory quinol oxidase from Escherichia coli. Eur J Biochem. 1993 Mar 15;212(3):657–664. doi: 10.1111/j.1432-1033.1993.tb17703.x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Kauffmann Karl E., Goddard Christine A., Zang Yan, Holm R. H., Münck Eckard. Mössbauer and Magnetization Studies of Heme-Copper-Bridged Assemblies Pertinent to Cytochrome c Oxidase. Inorg Chem. 1997 Mar 12;36(6):985–993. doi: 10.1021/ic960826v. [DOI] [PubMed] [Google Scholar]
  19. Kent T. A., Young L. J., Palmer G., Fee J. A., Münck E. Mössbauer study of beef heart cytochrome oxidase. Comparative study of the bovine enzyme and cytochrome c1aa3 from Thermus thermophilus. J Biol Chem. 1983 Jul 25;258(14):8543–8546. [PubMed] [Google Scholar]
  20. Little R. H., Cheesman M. R., Thomson A. J., Greenwood C., Watmough N. J. Cytochrome bo from Escherichia coli: binding of azide to CuB. Biochemistry. 1996 Oct 29;35(43):13780–13787. doi: 10.1021/bi961221d. [DOI] [PubMed] [Google Scholar]
  21. Rusnak F. M., Münck E., Nitsche C. I., Zimmermann B. H., Fee J. A. Evidence for structural heterogeneities and a study of exchange coupling. Mössbauer studies of cytochrome c1aa3 from Thermus thermophilus. J Biol Chem. 1987 Dec 5;262(34):16328–16332. [PubMed] [Google Scholar]
  22. Salerno J. C., Ingledew W. J. Orientation of the haems of the ubiquinol oxidase:O2 reductase, cytochrome bo of Escherichia coli. Eur J Biochem. 1991 Jun 15;198(3):789–792. doi: 10.1111/j.1432-1033.1991.tb16082.x. [DOI] [PubMed] [Google Scholar]
  23. Schoonover J. R., Palmer G. Reaction of formate with the fast form of cytochrome oxidase: a model for the fast to slow conversion. Biochemistry. 1991 Jul 30;30(30):7541–7550. doi: 10.1021/bi00244a025. [DOI] [PubMed] [Google Scholar]
  24. Thomson A. J., Englinton D. G., Hill B. C., Greenwood C. The nature of haem a3 in the oxidized state of cytochrome c oxidase. Evidence from low-temperature magnetic-circular-dichroism spectroscopy in the near infrared region. Biochem J. 1982 Oct 1;207(1):167–170. doi: 10.1042/bj2070167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Tweedle M. F., Wilson L. J. Electronic state of heme in cytochrome oxidase III. The magnetic susceptibility of beef heart cytochrome oxidase and some of its derivatives from 7-200 K. Direct evidence for an antiferromagnetically coupled Fe (III)/Cu (II) pair. J Biol Chem. 1978 Nov 25;253(22):8065–8071. [PubMed] [Google Scholar]
  28. Tweedle M. F., Wilson L. J. Electronic state of heme in cytochrome oxidase III. The magnetic susceptibility of beef heart cytochrome oxidase and some of its derivatives from 7-200 K. Direct evidence for an antiferromagnetically coupled Fe (III)/Cu (II) pair. J Biol Chem. 1978 Nov 25;253(22):8065–8071. [PubMed] [Google Scholar]
  29. Watmough N. J., Cheesman M. R., Gennis R. B., Greenwood C., Thomson A. J. Distinct forms of the haem o-Cu binuclear site of oxidised cytochrome bo from Escherichia coli. Evidence from optical and EPR spectroscopy. FEBS Lett. 1993 Mar 15;319(1-2):151–154. doi: 10.1016/0014-5793(93)80056-z. [DOI] [PubMed] [Google Scholar]
  30. 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]

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

RESOURCES