Strange R W, Murphy L M, Karlsson B G, Reinhammar B, Hasnain S S
Molecular Biophysics Group, CCLRC Daresbury Laboratory, Warrington, Cheshire, U.K.
Biochemistry. 1996 Dec 17;35(50):16391-8. doi: 10.1021/bi961682z.
A pH-dependent X-ray absorption fine structure (XAFS) study has been undertaken to provide a structural interpretation of the spectroscopic properties of the Met121 Glu mutant of azurin from Pseudomonas aeruginosa (Azp). Ligand binding studies have been carried out to investigate the effect of the cavity formed at the Cu site as a result of the mutation. The optical spectrum at pH 4 exhibits an intense band at approximately 600 nm and a weaker band at approximately 450 nm, typical for the blue copper proteins. As the pH is increased, these bands decrease in intensity and shift to 570 and 413 nm, respectively, with the latter becoming the more intense of the two [Karlsson, B.G., et al. (1991) Protein Eng. 4 (3), 343-349]. These changes are accompanied by a change in the EPR spectrum from a rhombic type 1 Cu spectrum at pH 4 to a spectrum with the rhombic splitting decreasing to zero and the hyperfine coupling increasing from 25 to 83 G. X-ray absorption a the Cu K-edge shows that this change results from the lengthening of the Cu-His (by 0.07 A) and Cu-Cys (by 0.06 A) bonds and the coordination of one of the oxygen atoms of the glutamate ligand at pH 8, at a distance as close as 1.90 A. The copper site thus changes from a normal type 1 copper center with three strong bonds at pH 4 to a copper site with four strong bonds at pH 8, with Cu-His distances significantly longer than known distances for type 1 copper centres measured using the XAFS technique. The XAFS of the azide derivative measured at pH 8 shows a similar Cu coordination, with azide replacing glutamate as the fourth ligand. Azide binding at pH 8 is accompanied by a further increase in the EPR hyperfine coupling to 110 G. This structural information when taken together with recent structural sudies on copper proteins points toward the need for a reexamination of the basis on which copper proteins are classified.
已开展一项依赖于pH值的X射线吸收精细结构（XAFS）研究，以对来自铜绿假单胞菌（Azp）的天青蛋白Met121 Glu突变体的光谱性质进行结构解释。已进行配体结合研究，以调查由于突变而在铜位点形成的空腔的影响。pH为4时的光谱在约600 nm处呈现一条强带，在约450 nm处呈现一条较弱的带，这是蓝色铜蛋白的典型特征。随着pH值升高，这些带的强度降低，并分别移至570和413 nm，后者在两者中变得更强[卡尔松，B.G.等人（1991年）《蛋白质工程》4（3），343 - 349]。这些变化伴随着电子顺磁共振（EPR）光谱从pH为4时的菱形1型铜光谱变为菱形分裂减小至零且超精细耦合从25 G增加到83 G的光谱。铜K边的X射线吸收表明，这种变化是由于铜 - 组氨酸（增加0.07 Å）和铜 - 半胱氨酸（增加0.06 Å）键的延长以及在pH为8时谷氨酸配体的一个氧原子配位，距离近至1.90 Å。因此，铜位点从pH为4时具有三个强键的正常1型铜中心变为pH为8时具有四个强键的铜位点，铜 - 组氨酸距离明显长于使用XAFS技术测量的1型铜中心的已知距离。在pH为8时测量的叠氮化物衍生物的XAFS显示出类似的铜配位，叠氮化物取代谷氨酸作为第四个配体。在pH为8时叠氮化物结合伴随着EPR超精细耦合进一步增加至110 G。当将此结构信息与近期关于铜蛋白的结构研究结合起来时，表明需要重新审视对铜蛋白进行分类的基础。
