Hough Michael A, Ellis Mark J, Antonyuk Svetlana, Strange Richard W, Sawers Gary, Eady Robert R, Samar Hasnain S
Molecular Biophysics Group, CCLRC Daresbury Laboratory, Warrington WA4 4AD, UK.
J Mol Biol. 2005 Jul 8;350(2):300-9. doi: 10.1016/j.jmb.2005.04.006.
We present high-resolution crystal structures and functional analysis of T1Cu centre mutants of nitrite reductase that perturb the redox potential and the Cys130-His129 "hard-wired" bridge through which electron transfer to the catalytic T2Cu centre occurs. These data provide insight into how activity can be altered through mutational manipulation of the electron delivery centre (T1Cu). The alteration of Cys to Ala results in loss of T1Cu and enzyme inactivation with azurin as electron donor despite the mutant enzyme retaining full nitrite-binding capacity. These data establish unequivocally that no direct transfer of electrons occurs from azurin to the catalytic type 2 Cu centre. The mutation of the axial ligand Met144 to Leu increases both the redox potential and catalytic activity, establishing that the rate-determining step of catalysis is the intermolecular electron transfer from azurin to nitrite reductase.
我们展示了亚硝酸还原酶T1Cu中心突变体的高分辨率晶体结构和功能分析，这些突变体扰乱了氧化还原电位以及Cys130-His129“硬连线”桥，电子通过该桥转移到催化性T2Cu中心。这些数据为如何通过对电子传递中心（T1Cu）进行突变操作来改变活性提供了深入了解。将Cys突变为Ala会导致T1Cu丢失且酶失活，尽管突变酶保留了完整的亚硝酸盐结合能力，但以天青蛋白作为电子供体时仍会如此。这些数据明确表明，电子不会从天青蛋白直接转移到催化性2型Cu中心。轴向配体Met144突变为Leu会增加氧化还原电位和催化活性，这表明催化的速率决定步骤是从天青蛋白到亚硝酸还原酶的分子间电子转移。
