Ugurbil K, Norton R S, Allerhand A, Bersohn R
Biochemistry. 1977 Mar 8;16(5):886-94. doi: 10.1021/bi00624a012.
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 Czota of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak chi) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cgamma of titrating histidines, and of Cgamma and Czota of the two tyrosines. The resonances of Cgamma and Cdelta2 of the single tryptophan residue (and Czota 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 Cgamma of one 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 Ndelta1-H tautomer. The Cgamma resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nepsilon2-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 chi arises from an amide group that probably is coordinated to the copper. (ii) The two nontitrating histidine residues are probably copper ligands, with Ndelta1 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 gamma carbons of Trp-48, the tyrosine with the lower pKa, 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.
利用天然丰度的13C傅里叶变换核磁共振光谱法研究了铜绿假单胞菌天青蛋白中芳香族残基（以及单个精氨酸残基）的环境。对于抗磁性的Cu(I)天青蛋白，所有17个非质子化芳香族碳（以及Arg-79的Cζ）产生窄共振。此外，观察到一个具有异常化学位移（峰χ）的单碳酰胺羰基共振。化学位移的pH依赖性用于鉴定滴定组氨酸的Cγ共振，以及两个酪氨酸的Cγ和Cζ共振。还鉴定了单个色氨酸残基的Cγ和Cδ2共振（以及Arg-79的Cζ）。两个酪氨酸的pKa值彼此不同，且高于“溶剂暴露”酪氨酸残基的典型值。四个组氨酸残基中有两个在pH范围4至11内不发生滴定。一个组氨酸的Cγ共振表现出具有快速质子交换行为且pKa为7.5±0.2的pH滴定。滴定位移的方向表明该组氨酸的咪唑形式是Nδ1-H互变异构体。另一个滴定组氨酸的Cγ共振表现出缓慢交换行为，pKa约为7。该组氨酸的咪唑形式是Nε2-H互变异构体。当变为顺磁性的Cu(II)蛋白时，上述19个碳中只有11个产生足够窄以便检测的共振。而且，一些观察到的共振表现出明显的顺磁性展宽。对完全还原的天青蛋白、还原和氧化天青蛋白混合物以及完全氧化天青蛋白的光谱进行比较，得到以下信息。(i)峰χ来自可能与铜配位的酰胺基团。(ii)两个非滴定组氨酸残基可能是铜配体，Nδ1与金属配位。(iii)Arg-79和两个酪氨酸残基的侧链不与铜配位，Trp-48可能也不是配体。(iv)Trp-48的γ碳、pKa较低的酪氨酸、具有缓慢交换行为的滴定组氨酸以及六个苯丙氨酸残基中的三或四个与铜足够接近，以至于在氧化天青蛋白的光谱中会发生明显的顺磁性展宽。
