Kalverda A P, Salgado J, Dennison C, Canters G W
Leiden Institute of Chemistry, Leiden University, The Netherlands.
Biochemistry. 1996 Mar 5;35(9):3085-92. doi: 10.1021/bi9518508.
Application of the tailored pulse sequences like super-WEFT allows the direct observation of the hyperfine-shifted signals of the paramagnetic Cu(II) forms of blue copper proteins in solution. The signals can be assigned by applying 2D NMR techniques, like EXSY, to solutions containing a mixture of reduced and oxidized species. The Fermi contact shift is separated from the pseudocontact shift on the basis of the known g-tensor anisotropy of the Cu(II) state, allowing the determination of a number of hyperfine-splitting constants between protons on the Cu ligands and the unpaired electron. These results are used to quantify the spin density distribution over the Cu ligands. In amicyanin about 50%-60% of the unpaired electron density is found on the ligands. It appears possible to quantify the Cu-S(Met) interaction on the basis of the NMR results. Application of the technique to the wild type forms of amicyanin and azurin and to two active site mutants of amicyanin (His96Asp and a plastocyanin-amicyanin loop exchange mutant) shows that the Cu-S(Met) interaction parallels the rhombicity and axial distortion of the Cu site.
应用如超WEFT等定制脉冲序列可直接观察溶液中蓝色铜蛋白顺磁性Cu(II)形式的超精细位移信号。通过将二维核磁共振技术（如EXSY）应用于含有还原态和氧化态混合物的溶液中，可对这些信号进行归属。基于Cu(II)态已知的g张量各向异性，费米接触位移与赝接触位移得以分离，从而能够确定Cu配体上质子与未配对电子之间的多个超精细分裂常数。这些结果用于量化Cu配体上的自旋密度分布。在天蓝蛋白中，约50% - 60%的未配对电子密度存在于配体上。似乎可以根据核磁共振结果对Cu - S(Met)相互作用进行量化。将该技术应用于天蓝蛋白和天青蛋白的野生型形式以及天蓝蛋白的两个活性位点突变体（His96Asp和一个质体蓝素 - 天蓝蛋白环交换突变体）表明，Cu - S(Met)相互作用与Cu位点的菱形度和轴向畸变平行。
