Basumallick Lipika, Szilagyi Robert K, Zhao Yiwei, Shapleigh James P, Scholes Charles P, Solomon Edward I
Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
J Am Chem Soc. 2003 Dec 3;125(48):14784-92. doi: 10.1021/ja037232t.
A combination of spectroscopic methods and density functional calculations has been used to describe the electronic structure of the axial mutant (Met182Thr) of Rhodobacter sphaeroides nitrite reductase in which the axial methionine has been changed to a threonine. This mutation results in a dramatic change in the geometric and electronic structure of the copper site. The electronic absorption data imply that the type 1 site in the mutant is like a typical blue copper site in contrast to the wild-type site, which is green. Similar ligand field strength in the mutant and the wild type (from MCD spectra) explains the similar EPR parameters for very different electronic structures. Resonance Raman shows that the Cu-S(Cys) bond is stronger in the mutant relative to the wild type. From a combination of absorption, CD, MCD, and EPR data, the loss of the strong axial thioether (present in the wild-type site) results in an increase of the equatorial thiolate-Cu interaction and the site becomes less tetragonal. Spectroscopically calibrated density functional calculations were used to provide additional insight into the role of the axial ligand. The calculations reproduce well the experimental ground-state bonding and the changes in going from a green to a blue site along this coupled distortion coordinate. Geometry optimizations at the weak and strong axial ligand limits show that the bonding of the axial thioether is the key factor in determining the structure of the ground state. A comparison of plastocyanin (blue), wild-type nitrite reductase (green), and the Met182Thr mutant (blue) sites enables evaluation of the role of the axial ligand in the geometric and electronic structure of type 1 copper sites, which can affect the electron-transfer properties of these sites.
已使用光谱方法和密度泛函计算相结合的方式来描述球形红细菌亚硝酸还原酶轴向突变体（Met182Thr）的电子结构，其中轴向甲硫氨酸已被替换为苏氨酸。这种突变导致铜位点的几何结构和电子结构发生显著变化。电子吸收数据表明，与野生型位点（绿色）不同，突变体中的1型位点类似于典型的蓝铜位点。突变体和野生型中相似的配体场强度（来自磁圆二色光谱）解释了具有非常不同电子结构的相似电子顺磁共振参数。共振拉曼光谱表明，相对于野生型，突变体中Cu-S（半胱氨酸）键更强。结合吸收光谱、圆二色光谱、磁圆二色光谱和电子顺磁共振数据可知，野生型位点中存在的强轴向硫醚的缺失导致赤道面硫醇盐-铜相互作用增加，且该位点的四方性降低。使用经过光谱校准的密度泛函计算来进一步深入了解轴向配体的作用。这些计算很好地再现了实验基态键合以及沿着这种耦合畸变坐标从绿色位点转变为蓝色位点时的变化。在弱轴向配体和强轴向配体极限下的几何优化表明，轴向硫醚的键合是决定基态结构的关键因素。通过比较质体蓝素（蓝色）、野生型亚硝酸还原酶（绿色）和Met182Thr突变体（蓝色）位点，可以评估轴向配体在1型铜位点的几何结构和电子结构中的作用，这可能会影响这些位点的电子转移性质。
