Studies of individual carbon sites of azurin from Pseudomonas aeruginosa by natural-abundance carbon-13 nuclear magnetic resonance spectroscopy

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Abstract
The environments of the aromatic residues (and of the single arginine residue) of azurin from P. aeruginosa are investigated by natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and C.zeta. of Arg-79) yield narrow resonances. A single-C amide carbonyl resonance with an unusual chemical shift (peak x) is observed. The pH dependence of chemical shifts is used to identify the resonances of C.gamma. of titrating histidines and of C.gamma. and C.zeta. of the 2 tyrosines. The resonances of C.gamma. and C.delta.2 of the single tryptophan residue (and C.zeta. of Arg-79) are identified. The pKa values of the 2 tyrosines are different from each other and higher than typical values of solvent-exposed tyrosine residues. Two of the 4 histidine residues do not titrate (in the pH range 4-11). The resonance of C.gamma. of 1 histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 .+-. 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the N.delta.1-H tautomer. The C.gamma. resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of .apprx. 7. The imidazole form of this histidine is the N.epsilon.2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 C mentioned above yield resonances narrow enough to be detected. Some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information. Peak x arises from an amide group that probably is coordinated to the Cu. The 2 nontitrating histidine residues are probably Cu ligands, with N.delta.1 coordinated to the metal. The side chains of Arg-79 and the 2 tyrosine residues are not coordinated to the Cu, and Trp-48 is probably not a ligand. The .gamma. carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior and 3 or 4 of the 6 phenylalanine residues are sufficiently close to the Cu to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.