Spectrochemical Studies on the Blue Copper Protein Azurin from Alcaligenes denitrificanst

Eric W. Ainscough, Alistair G. Bingham, Andrew M. Brodie, Walther R. Ellis, Harry B. Gray, Thomas M. Loehr, Jeffrey E. Plowman, Gillian E. Norris, Edward N. Baker

Research output: Contribution to journalArticlepeer-review

94 Scopus citations

Abstract

: Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (c = 5100 M-1cm-1) charge-transfer band at 619 nm, the EPR spectral parameters = 2.059, = 2.255, = 60 X 10-4cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH ~ 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH···S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 °C, give values for and of-55.6 kJ mol-1and -97.0 J K_1mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower E° values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.

Original languageEnglish (US)
Pages (from-to)71-82
Number of pages12
JournalBiochemistry
Volume26
Issue number1
DOIs
StatePublished - 1987
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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