High-resolution proton nuclear magnetic resonance studies of the nickel(II) derivative of azurin.
High-resolution (360 and 470 MHz) 1H NMR studies of Ni(II) azurin, the nickel(II) derivative of the blue copper protein azurin, are reported. The aliphatic resonances of Ni(II) azurin closely parallel those of apoazurin and Cu(I) azurin and indicate that no major structural changes are associated with the binding of nickel(II). The magnetic moment of Ni(II) azurin (mueff = 3.2 muB) is in keeping with a pseudotetrahedral coordination environment like that of Cu(I) azurin. Resonances of protons from the ligand moieties are shifted as far as 125 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonate and as far as 20 ppm upfield by internal fields due to the nickel center. One of these strongly shifted resonances is assigned to the methyl protons of the methionine ligand. From spectra of Ni(II) azurin as a function of pH, the pKa' values of histidine-35 and histidine-83 have been measured to be approximately 6.0 and 7.5, respectively. Histidine-35 titrates in a discontinuous fashion, and, significantly, so do several of the isotropically shifted ligand protons, also within experimental error with the same pHmid. This result reinforces the suggestion that the conformational change coupled to the protonation of histidine-35 plays an important role in regulating electron transfer reactions of native azurin [Silvestrini, M. C., Brunori, M., Wilson, M. T., & Darley-Usmar, V. M. (1981) J. Inorg. Biochem. 14, 327-338].
D001400:Azurin; D001426:Bacterial Proteins; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D009532:Nickel; D011485:Protein Binding; D011550:Pseudomonas aeruginosa; D013696:Temperature
D001426:Bacterial Proteins; D001400:Azurin; D009532:Nickel