Studies of Individual Carbon Sites of Azurin from Pseudomonas aeruginosa by Natural-Abundance Carbon-13 Nuclear Magnetic Resonance Spectroscopy

The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance ¹³C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and C^ζ of Arg-79) yield narrow resonances. Furthermore, a single-carbon 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^γ of titrating histidines, and of C^γ and C^ζ of the two tyrosines. The resonances of C^γ and C^δ₂ of the single tryptophan residue (and C^ζ of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of “solvent-exposed” tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of C^γ of one histidine exhibits a pH titration with fast proton exchange behavior and a pK_a of 7.5 ± 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Nδ₁-H tautomer. The C^γ resonance of the other titrating histidine exhibits slow exchange behavior with a pK_a of about 7. The imidazole form of this histidine is the N^ϵ2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, 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, (i) Peak x arises from an amide group that probably is coordinated to the copper, (ii) The two nontitrating histidine residues are probably copper ligands, with N^δ₁ coordinated to the metal, (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either, (iv) The γ carbons of Trp-48, the tyrosine with the lower pK_a, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.