Salgado J, Warmerdam G, Bubacco L, Canters G W
Leiden Institute of Chemistry, Leiden University, The Netherlands.
Biochemistry. 1998 May 19;37(20):7378-89. doi: 10.1021/bi9728598.
The soluble domain of the subunit II of cytochrome c oxidase from Paracoccus versutus was cloned, expressed, and studied by 1H NMR at 600 MHz. The properties of the redox-active dinuclear CuA site in the paramagnetic mixed-valence Cu(I)-Cu(II) state were investigated in detail. A group of relatively sharp signals found between 30 and 15 ppm in the 1H NMR spectrum correspond to the imidazole protons of the coordinated histidines (H181 and H224). A second group of broader and farther shifted signals between 50 and 300 ppm are assigned to Hbeta protons of the bridging cysteines (C216 and C220); the protons from the weak M227 and E218 ligands do not shift outside of the diamagnetic envelope. About 40% of the total spin density appears delocalized over the cysteine-bridging ligands while a much smaller amount is delocalized on the two ligand histidines. The latter have similar spin density distributions. Analysis of the pattern of the hyperfine shifts of the Cys H beta protons shows that the ground state bears 2B3u character, in which the sulfur lobes in the singly occupied molecular orbital are aligned with the Cu-Cu axis. Analysis of the temperature dependence of the shifts of the Cys H beta signals leads to the conclusion that the 2B2u excited state is thermally accessible at room temperature (Delta E approximately kT).
克隆、表达了来自嗜甲基副球菌的细胞色素c氧化酶亚基II的可溶性结构域，并在600 MHz下通过1H NMR进行了研究。详细研究了顺磁混合价Cu(I)-Cu(II)态中氧化还原活性双核CuA位点的性质。在1H NMR谱中30至15 ppm之间发现的一组相对尖锐的信号对应于配位组氨酸（H181和H224）的咪唑质子。第二组在50至300 ppm之间更宽且位移更远的信号归属于桥连半胱氨酸（C216和C220）的Hβ质子；来自弱配体M227和E218的质子未在抗磁包络之外发生位移。约40%的总自旋密度似乎离域在半胱氨酸桥连配体上，而在两个配体组氨酸上离域的量要少得多。后者具有相似的自旋密度分布。对Cys Hβ质子超精细位移模式的分析表明，基态具有2B3u特征，其中单占据分子轨道中的硫叶与Cu-Cu轴对齐。对Cys Hβ信号位移的温度依赖性分析得出结论，2B2u激发态在室温下可通过热激发获得（ΔE约为kT）。
