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. 2001 Oct;81(4):2331–2338. doi: 10.1016/S0006-3495(01)75879-1

Resolving the individual components of a pH-induced conformational change.

C Blouin 1, J G Guillemette 1, C J Wallace 1
PMCID: PMC1301703  PMID: 11566802

Abstract

This communication introduces a simple method to determine the pKs of microscopic ionizations from complex titration curves. We used this approach to study the alkaline transition (pH-dependent ligand exchange) of mitochondrial cytochrome c. The linearization of titration curves permitted resolution of two to three limiting microscopic ionizations. By combining these data with studies of the temperature dependence of ligand-exchange equilibria, we found evidence that the alkaline transition comprises two chemically distinct processes: the deprotonation of the alternative ligands and the break of the iron-methionine ligation bond. We also noted that, in the horse and untrimethylated S. cerevisiae iso-1 cytochromes c, the permissible deprotonation of the epsilon-amino group of Lys(72) allows formation of an alkaline isomer at lower pH, with lesser stability, which leads to hysteresis in the titration curves. The linearization of the titration curves for different cytochromes c thus brings insight on the microscopic contributions to conformational stability.

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Selected References

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  1. Battistuzzi G., Borsari M., Loschi L., Martinelli A., Sola M. Thermodynamics of the alkaline transition of cytochrome c. Biochemistry. 1999 Jun 22;38(25):7900–7907. doi: 10.1021/bi983060e. [DOI] [PubMed] [Google Scholar]
  2. Berghuis A. M., Guillemette J. G., McLendon G., Sherman F., Smith M., Brayer G. D. The role of a conserved internal water molecule and its associated hydrogen bond network in cytochrome c. J Mol Biol. 1994 Feb 25;236(3):786–799. doi: 10.1006/jmbi.1994.1189. [DOI] [PubMed] [Google Scholar]
  3. Davies A. M., Guillemette J. G., Smith M., Greenwood C., Thurgood A. G., Mauk A. G., Moore G. R. Redesign of the interior hydrophilic region of mitochondrial cytochrome c by site-directed mutagenesis. Biochemistry. 1993 May 25;32(20):5431–5435. doi: 10.1021/bi00071a019. [DOI] [PubMed] [Google Scholar]
  4. Eaton W. A., Hochstrasser R. M. Electronic spectrum of single crystals of ferricytochrome-c. J Chem Phys. 1967 Apr 1;46(7):2533–2539. doi: 10.1063/1.1841081. [DOI] [PubMed] [Google Scholar]
  5. Harbury H. A., Cronin J. R., Fanger M. W., Hettinger T. P., Murphy A. J., Myer Y. P., Vinogradov S. N. Complex formation between methionine and a heme peptide from cytochrome c. Proc Natl Acad Sci U S A. 1965 Dec;54(6):1658–1664. doi: 10.1073/pnas.54.6.1658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hartshorn R. T., Moore G. R. A denaturation-induced proton-uptake study of horse ferricytochrome c. Biochem J. 1989 Mar 1;258(2):595–598. doi: 10.1042/bj2580595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hong X. L., Dixon D. W. NMR study of the alkaline isomerization of ferricytochrome c. FEBS Lett. 1989 Mar 27;246(1-2):105–108. doi: 10.1016/0014-5793(89)80262-5. [DOI] [PubMed] [Google Scholar]
  8. Inglis S. C., Guillemette J. G., Johnson J. A., Smith M. Analysis of the invariant Phe82 residue of yeast iso-1-cytochrome c by site-directed mutagenesis using a phagemid yeast shuttle vector. Protein Eng. 1991 Jun;4(5):569–574. doi: 10.1093/protein/4.5.569. [DOI] [PubMed] [Google Scholar]
  9. Lett C. M., Berghuis A. M., Frey H. E., Lepock J. R., Guillemette J. G. The role of a conserved water molecule in the redox-dependent thermal stability of iso-1-cytochrome c. J Biol Chem. 1996 Nov 15;271(46):29088–29093. doi: 10.1074/jbc.271.46.29088. [DOI] [PubMed] [Google Scholar]
  10. Louie G. V., Brayer G. D. High-resolution refinement of yeast iso-1-cytochrome c and comparisons with other eukaryotic cytochromes c. J Mol Biol. 1990 Jul 20;214(2):527–555. doi: 10.1016/0022-2836(90)90197-T. [DOI] [PubMed] [Google Scholar]
  11. Luntz T. L., Schejter A., Garber E. A., Margoliash E. Structural significance of an internal water molecule studied by site-directed mutagenesis of tyrosine-67 in rat cytochrome c. Proc Natl Acad Sci U S A. 1989 May;86(10):3524–3528. doi: 10.1073/pnas.86.10.3524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Onufriev A., Case D. A., Ullmann G. M. A novel view of pH titration in biomolecules. Biochemistry. 2001 Mar 27;40(12):3413–3419. doi: 10.1021/bi002740q. [DOI] [PubMed] [Google Scholar]
  13. Osheroff N., Borden D., Koppenol W. H., Margoliash E. Electrostatic interactions in cytochrome c. The role of interactions between residues 13 and 90 and residues 79 and 47 in stabilizing the heme crevice structure. J Biol Chem. 1980 Feb 25;255(4):1689–1697. [PubMed] [Google Scholar]
  14. Pearce L. L., Gärtner A. L., Smith M., Mauk A. G. Mutation-induced perturbation of the cytochrome c alkaline transition. Biochemistry. 1989 Apr 18;28(8):3152–3156. doi: 10.1021/bi00434a006. [DOI] [PubMed] [Google Scholar]
  15. Pollock W. B., Rosell F. I., Twitchett M. B., Dumont M. E., Mauk A. G. Bacterial expression of a mitochondrial cytochrome c. Trimethylation of lys72 in yeast iso-1-cytochrome c and the alkaline conformational transition. Biochemistry. 1998 Apr 28;37(17):6124–6131. doi: 10.1021/bi972188d. [DOI] [PubMed] [Google Scholar]
  16. Rosell F. I., Harris T. R., Hildebrand D. P., Döpner S., Hildebrandt P., Mauk A. G. Characterization of an alkaline transition intermediate stabilized in the Phe82Trp variant of yeast iso-1-cytochrome c. Biochemistry. 2000 Aug 1;39(30):9047–9054. doi: 10.1021/bi001095k. [DOI] [PubMed] [Google Scholar]
  17. SCHEJTER A., GEORGE P. THE 695-MMM. BAND OF FERRICYTOCHROME C AND ITS RELATIONSHIP TO PROTEIN CONFORMATION. Biochemistry. 1964 Aug;3:1045–1049. doi: 10.1021/bi00896a006. [DOI] [PubMed] [Google Scholar]
  18. Takano T., Dickerson R. E. Conformation change of cytochrome c. I. Ferrocytochrome c structure refined at 1.5 A resolution. J Mol Biol. 1981 Nov 25;153(1):79–94. doi: 10.1016/0022-2836(81)90528-3. [DOI] [PubMed] [Google Scholar]
  19. Tonge P., Moore G. R., Wharton C. W. Fourier-transform infra-red studies of the alkaline isomerization of mitochondrial cytochrome c and the ionization of carboxylic acids. Biochem J. 1989 Mar 1;258(2):599–605. doi: 10.1042/bj2580599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wallace C. J., Corthésy B. E. Alkylamine derivatives of cytochrome c. Comparison with other lysine-modified analogues illuminates structure/function relations in the protein. Eur J Biochem. 1987 Dec 30;170(1-2):293–298. doi: 10.1111/j.1432-1033.1987.tb13698.x. [DOI] [PubMed] [Google Scholar]
  21. Yang A. S., Honig B. On the pH dependence of protein stability. J Mol Biol. 1993 May 20;231(2):459–474. doi: 10.1006/jmbi.1993.1294. [DOI] [PubMed] [Google Scholar]

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