Murphy M E, Turley S, Kukimoto M, Nishiyama M, Horinouchi S, Sasaki H, Tanokura M, Adman E T
Department of Biological Structure, School of Medicine, University of Washington, Seattle 98195-7420, USA.
Biochemistry. 1995 Sep 26;34(38):12107-17. doi: 10.1021/bi00038a003.
The structures at 2.0 and 2.25 A resolution of native and recombinant nitrite reductase from Alcaligenes faecalis show that they are identical to each other and very similar to nitrite reductase from Achromobacter cycloclastes. The crystallographic structure of a mutant, M150E, which unlike the wild-type protein cannot be reduced by pseudoazurin, shows that the glutamate replacement for methionine binds to a metal at the type I Cu site via only one oxygen. Anomalous scattering data collected at wavelengths of 1.040 and 1.377 A reveal that the metal at the type I site is a Zn. No significant differences from the native structure other than local perturbations at the type I site are seen. A local pseudo 2-fold axis relates the two domains of different monomers which form the active site. The two residues, Asp98 and His255, believed to be involved in catalysis are related by this 2-fold. An unusual (+)-(+) charge interaction between Lys269, Glu279, and His100 helps to orient the active site Cu ligand, His100. A number of negatively charged surface residues create an electrostatic field whose shape suggests that it may serve to direct incoming negatively charged nitrite as well as to dock the electron donor partner, pseudoazurin.
粪产碱杆菌天然型和重组型亚硝酸还原酶在分辨率为2.0和2.25埃时的结构表明，它们彼此相同，且与环裂无色杆菌的亚硝酸还原酶非常相似。一种突变体M150E的晶体结构显示，与野生型蛋白不同，它不能被假天青还原，该突变体中用谷氨酸替代甲硫氨酸后，仅通过一个氧原子与I型铜位点的金属结合。在波长为1.040和1.377埃时收集的反常散射数据表明，I型位点的金属是锌。除了I型位点的局部扰动外，未观察到与天然结构的显著差异。一个局部伪二次轴关联形成活性位点的不同单体的两个结构域。据信参与催化的两个残基，天冬氨酸98和组氨酸255，通过这个二次轴相关联。赖氨酸269、谷氨酸279和组氨酸100之间不寻常的（+）-（+）电荷相互作用有助于使活性位点铜配体组氨酸100定向。许多带负电荷的表面残基形成一个静电场，其形状表明它可能用于引导进入的带负电荷的亚硝酸盐，以及对接电子供体伙伴假天青。
