Nuclear Magnetic Resonance Study of Exchangeable and Nonexchangeable Protons in Azurin from Pseudomonas aeruginosa

Kamil Ugurbil, Richard Bersohn

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41 Scopus citations


Proton nuclear magnetic resonance (1H NMR) spectra of the carbon-bound protons in apoazurin and native oxidized, native reduced, and Hg(II)-substituted P. aeruginosa azurin are presented. The pH dependence of chemical shifts is used to assign C(2)-H and C(4)-H protons of a titratable histidine in the fast-exchange domain and another which exhibits slow exchange. The pK* at 24 °C of the histidine with fast-exchange behavior is 7.57 and 7.35 in reduced and oxidized azurin, respectively. Two peaks seen in the reduced but not in the oxidized spectra are assigned to C(2)-H of the two histidines coordinated to copper. Several well-resolved ~3 proton resonances are observed in the methionine S-CH3 region. Difference spectra obtained from oxidized, reduced, and Hg(II)-substituted azurin spectra show that (1) there are local conformational differences between the Cu(I)- and Cu(II)-bound proteins, (2) the Hg(II)-substituted protein is very similar in structure to oxidized azurin, and (3) paramagnetic Cu(II) broadens beyond detection the histidine with the slow-exchange behavior, 2 of the ~3 proton resonances in the Met S-CH3 region, and a large number of resonances which stem from other methyl protons. 1H NMR spectra in H20 exhibit several well-resolved resonances between ‒9.5 and ‒ 12.5 ppm in reduced azurin spectra; the number of detectable resonances in the same chemical-shift region is fewer in the paramagnetic protein. Possible assignments to Trp-48 NH and imidazole NH's are discussed.

Original languageEnglish (US)
Pages (from-to)3016-3023
Number of pages8
Issue number13
StatePublished - Jun 1 1977
Externally publishedYes


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