Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1983 Mar;41(3):233–244. doi: 10.1016/S0006-3495(83)84433-6

Conformational heterogeneity of the copper binding site in azurin. A time-resolved fluorescence study.

A G Szabo, T M Stepanik, D M Wayner, N M Young
PMCID: PMC1329175  PMID: 6404322

Abstract

Comparison of the fluorescence spectra and the effect of temperature on the quantum yields of fluorescence of Azurin (from Pseudomonas fluorescens ATCC-13525-2) and 3-methylindole (in methylcyclohexane solution) provides substantive evidence that the tryptophan residue in azurin is completely inaccessible to solvent molecules. The quantum yields of azurin (CuII), azurin (CuI), and apoazurin (lambda ex = 291 nm) were 0.052, 0.054, and 0.31, respectively. Other evidence indicates that there is no energy transfer from tyrosine to tryptophan in any of these proteins. The fluorescence decay behavior of each of the azurin samples was found to be invariant with emission wavelength. The fluorescences of azurin (CuII) and azurin (CuI) decay with dual exponential kinetics (tau 1 = 4.80 ns, tau 2 = 0.18 ns) while that of apoazurin obeys single exponential decay kinetics (tau = 4.90). The ratio of pre-exponentials of azurin (CuII), alpha 1/alpha 2, is found to be 0.25, and this ratio increases to 0.36 on reduction to azurin (CuI). The results are interpreted as originating from different interactions of the tryptophan with two conformers of the copper-ligand complex in azurin.

Full text

PDF
240

Selected References

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

  1. Adman E. T., Stenkamp R. E., Sieker L. C., Jensen L. H. A crystallographic model for azurin a 3 A resolution. J Mol Biol. 1978 Jul 25;123(1):35–47. doi: 10.1016/0022-2836(78)90375-3. [DOI] [PubMed] [Google Scholar]
  2. Ambler R. P., Brown L. H. The amino acid sequence of Pseudomonas fluorescens azurin. Biochem J. 1967 Sep;104(3):784–825. doi: 10.1042/bj1040784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ambler R. P., Wynn M. The amino acid sequences of cytochromes c-551 from three species of Pseudomonas. Biochem J. 1973 Mar;131(3):485–498. doi: 10.1042/bj1310485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Badea M. G., Brand L. Time-resolved fluorescence measurements. Methods Enzymol. 1979;61:378–425. doi: 10.1016/0076-6879(79)61019-4. [DOI] [PubMed] [Google Scholar]
  5. Brill A. S. Activation of electron transfer reactions of the blue proteins. Biophys J. 1978 Apr;22(1):139–142. doi: 10.1016/S0006-3495(78)85479-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brill A. S., Bryce G. F. Cupric ion in blue proteins. J Chem Phys. 1968 May 15;48(10):4398–4404. doi: 10.1063/1.1668007. [DOI] [PubMed] [Google Scholar]
  7. Brill A. S., Bryce G. F., Maria H. J. Optical and magnetic properties of Pseudomonas azurins. Biochim Biophys Acta. 1968 Feb 19;154(2):342–351. doi: 10.1016/0005-2795(68)90048-2. [DOI] [PubMed] [Google Scholar]
  8. Burstein E. A., Permyakov E. A., Yashin V. A., Burkhanov S. A., Finazzi Agro A. The fine structure of luminescence spectra of azurin. Biochim Biophys Acta. 1977 Mar 28;491(1):155–159. doi: 10.1016/0005-2795(77)90051-4. [DOI] [PubMed] [Google Scholar]
  9. Burstein E. A., Vedenkina N. S., Ivkova M. N. Fluorescence and the location of tryptophan residues in protein molecules. Photochem Photobiol. 1973 Oct;18(4):263–279. doi: 10.1111/j.1751-1097.1973.tb06422.x. [DOI] [PubMed] [Google Scholar]
  10. Cockle S. A., Szabo A. G. Time-resolved fluorescence spectra of tryptophan in monomeric glucagon. Photochem Photobiol. 1981 Jul;34(1):23–27. doi: 10.1111/j.1751-1097.1981.tb08955.x. [DOI] [PubMed] [Google Scholar]
  11. Finazzi-Agrò A., Giovagnoli C., Avigliano L., Rotilio G., Mondovì B. Luminescence quenching in azurin. Eur J Biochem. 1973 Apr 2;34(1):20–24. doi: 10.1111/j.1432-1033.1973.tb02723.x. [DOI] [PubMed] [Google Scholar]
  12. Finazzi-Agrò A., Rotilio G., Avigliano L., Guerrieri P., Boffi V., Mondovì B. Environment of copper in Pseudomonas fluorescens azurin: fluorometric approach. Biochemistry. 1970 Apr 28;9(9):2009–2014. doi: 10.1021/bi00811a023. [DOI] [PubMed] [Google Scholar]
  13. Grinvald A., Schlessinger J., Pecht I., Steinberg I. Z. Homogeneity and variability in the structure of azurin molecules studied by fluorescence decay and circular polarization. Biochemistry. 1975 May 6;14(9):1921–1929. doi: 10.1021/bi00680a018. [DOI] [PubMed] [Google Scholar]
  14. Hershberger M. V., Maki A. H., Galley W. C. Phosphorescence and optically detected magnetic resonance studies of a class of anomalous tryptophan residues in globular proteins. Biochemistry. 1980 May 13;19(10):2204–2209. doi: 10.1021/bi00551a032. [DOI] [PubMed] [Google Scholar]
  15. Hill H. A., Smith B. E. Characteristics of azurin from Pseudomonas aeruginosa via 270-MHz 1H nuclear magnetic resonance spectroscopy. J Inorg Biochem. 1979 Oct;11(2):79–93. doi: 10.1016/s0162-0134(00)80174-9. [DOI] [PubMed] [Google Scholar]
  16. Holwerda R. A., Wherland S., Gray H. B. Electron transfer reactions of copper proteins. Annu Rev Biophys Bioeng. 1976;5:363–396. doi: 10.1146/annurev.bb.05.060176.002051. [DOI] [PubMed] [Google Scholar]
  17. Munro I., Pecht I., Stryer L. Subnanosecond motions of tryptophan residues in proteins. Proc Natl Acad Sci U S A. 1979 Jan;76(1):56–60. doi: 10.1073/pnas.76.1.56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Parr S. R., Barber D., Greenwood C., Brunori M. The electron-transfer reaction between azurin and the cytochrome c oxidase from Pseudomonas aeruginosa. Biochem J. 1977 Nov 1;167(2):447–455. doi: 10.1042/bj1670447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rosen P., Pecht I. Conformational equilibria accompanying the electron transfer between cytochrome c (P551) and azurin from Pseudomonas aeruginosa. Biochemistry. 1976 Feb 24;15(4):775–786. doi: 10.1021/bi00649a008. [DOI] [PubMed] [Google Scholar]
  20. Ryden L., Lundgren J. O. On the evolution of blue proteins. Biochimie. 1979;61(7):781–790. doi: 10.1016/s0300-9084(79)80272-2. [DOI] [PubMed] [Google Scholar]
  21. Ryden L., Lundgren J. Homology relationships among the small blue proteins. Nature. 1976 May 27;261(5558):344–346. doi: 10.1038/261344a0. [DOI] [PubMed] [Google Scholar]
  22. Solomon E. I., Hare J. W., Gray H. B. Spectroscopic studies and a structural model for blue copper centers in proteins. Proc Natl Acad Sci U S A. 1976 May;73(5):1389–1393. doi: 10.1073/pnas.73.5.1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Swank R. T., Munkres K. D. Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal Biochem. 1971 Feb;39(2):462–477. doi: 10.1016/0003-2697(71)90436-2. [DOI] [PubMed] [Google Scholar]
  24. Tullius T. D., Frank P., Hodgson K. O. Characterization of the blue copper site in oxidized azurin by extended x-ray absorption fine structure: Determination of a short Cu-S distance. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4069–4073. doi: 10.1073/pnas.75.9.4069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ugurbil K., Bersohn R. Nuclear magnetic resonance study of exchangeable and nonexchangeable protons in azurin from Pseudomonas aeruginosa. Biochemistry. 1977 Jun 28;16(13):3016–3023. doi: 10.1021/bi00632a032. [DOI] [PubMed] [Google Scholar]
  26. Ugurbil K., Bersohn R. Tyrosine emission in the tryptophanless azurin from Pseudomonas fluorescens. Biochemistry. 1977 Mar 8;16(5):895–901. doi: 10.1021/bi00624a013. [DOI] [PubMed] [Google Scholar]
  27. Ugurbil K., Maki A. H., Bersohn R. Study of the triplet state properties of tyrosines and tryptophan in azuring using optically detected magnetic resonance. Biochemistry. 1977 Mar 8;16(5):901–907. doi: 10.1021/bi00624a014. [DOI] [PubMed] [Google Scholar]
  28. Ugurbil K., Norton R. S., Allerhand A., Bersohn R. Studies of individual carbon sites of azurin from Pseudomonas aeruginosa by natural-abundance carbon-13 nuclear magnetic resonance spectroscopy. Biochemistry. 1977 Mar 8;16(5):886–894. doi: 10.1021/bi00624a012. [DOI] [PubMed] [Google Scholar]
  29. Weinryb I., Steiner R. F. Luminescence of the tryptophan and tyrosine residues of papain in solution. Biochemistry. 1970 Jan 6;9(1):135–146. doi: 10.1021/bi00803a018. [DOI] [PubMed] [Google Scholar]
  30. Wherland S., Pecht I. Protein-protein electron transfer. A Marcus theory analysis of reactions between c type cytochromes and blue copper proteins. Biochemistry. 1978 Jun 27;17(13):2585–2591. doi: 10.1021/bi00606a020. [DOI] [PubMed] [Google Scholar]
  31. Wilson M. T., Greenwood C., Brunori M., Antonini E. Electron transfer between azurin and cytochrone c-551 from Pseudomonas aeruginosa. Biochem J. 1975 Mar;145(3):449–457. doi: 10.1042/bj1450449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wood P. M., Bendall D. S. The kinetics and specificity of electron transfer from cytochromes and copper proteins to P700. Biochim Biophys Acta. 1975 Apr 14;387(1):115–128. doi: 10.1016/0005-2728(75)90056-0. [DOI] [PubMed] [Google Scholar]
  33. YAMANAKA T., KIJIMOTO S., OKUNUKI K. Some properties of Pseudomonas blue protein and its apo-protein. J Biochem. 1963 Mar;53:256–259. doi: 10.1093/oxfordjournals.jbchem.a127689. [DOI] [PubMed] [Google Scholar]

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

RESOURCES