Proton NMR of the histidines of azurin from Alcaligenes faecalis: linkage of histidine-35 with redox kinetics.
On the basis of redox kinetic studies, Rosen and Pecht [Rosen, P. & Pecht, I. (1976) Biochemistry 15, 775-786] postulated a slowly attained (approximately equal to 0.1 sec) conformational equilibrium between two forms of reduced azurin from the bacterium Pseudomonas aeruginosa, one form being faster in electron transfer. NMR investigations have shown that at pH 7 there are two forms of reduced azurin exchanging slowly with each other, differing in the presence or absence of a proton on the imidazole side chain of histidine-35. Rosen et al. [Rosen, P., Segal, M. & Pecht, I. (1981) Eur. J. Biochem. 120, 339-344] observed that the azurin from the bacterium Alcaligenes faecalis shows no such slowly attained equilibrium between two forms. Therefore, a 1H NMR study was carried out on this azurin with emphasis on the downfield region. A resonance was found at 7.95 ppm downfield that does not move with pH, is not seen in the oxidized protein, has the same pseudocontact shift in the Co(II) derivative as the C-2 proton of histidine-35 has in the Co(II) derivative of P. aeruginosa azurin, and, in the apoprotein, exhibits a typical protonation shift downfield at pH less than 5. Therefore, this resonance is assigned to the C-2 proton of histidine-35. The crystal structure of P. aeruginosa azurin shows that at pH 7 the imidazole side chain of histidine-35 is in a crevice within the protein, where its ring is adjacent and parallel to that of histidine-47, a copper ligand. The preceding observations combined with others show that the kinetics of some redox reactions involving azurin depend on the position of histidine-35. The implication is that there is a pathway for electron transport to the copper atom involving passage through histidine-35.
D000421:Alcaligenes / Q000378:metabolism*; D001400:Azurin / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D006639:Histidine / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction
D001426:Bacterial Proteins; D001400:Azurin; D006639:Histidine