Structure of Alcaligenes faecalis nitrite reductase and a copper site mutant, M150E, that contains zinc.
Structure of Alcaligenes faecalis nitrite reductase and a copper site mutant, M150E, that contains zinc.
复制标题
粪产碱菌亚硝酸还原酶的结构和含锌的铜位点突变体 M150E。
DOI：
10.1021/bi00038a003
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Adman,ET
中科院分区：
文献类型：
--
作者：
Murphy,ME;Turley,S;Kukimoto,M;Nishiyama,M;Horinouchi,S;Sasaki,H;Tanokura,M;Adman,ET
1995年7月11日收到的修订版Mandalpt ®摘要：来自粪产碱杆菌的天然和重组亚硝酸盐还原酶在2.0和2.25 A分辨率下的结构表明它们彼此相同，并且与来自无色杆菌的亚硝酸盐还原酶非常相似。突变体M150 E的晶体结构与野生型蛋白质不同，不能被pseudoazurin还原，表明谷氨酸取代甲硫氨酸仅通过一个氧与I型Cu位点的金属结合。在1.040和1.377 A的波长处收集的异常散射数据表明，在I型位点处的金属是Zn。没有显着的差异，从本地结构以外的thetype I网站的局部扰动。局部假2-折叠轴涉及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Asp 98和His 255通过这2倍相关。Lys 269、Glu 279和His 100之间的不寻常的-I-H电荷相互作用有助于定向活性位点Cu配体His 100。许多带负电荷的表面残基产生静电场，其形状表明其可用于引导进入的带负电荷的亚硝酸盐以及对接电子供体配偶体假天青蛋白。来自厌氧细菌粪产碱杆菌菌株S-6的亚硝酸盐还原酶是一种含铜酶，其在厌氧条件下将亚硝酸盐转化为一氧化氮（NCh-to NO）（Kakutani等人，1981年b）。体内电子供体已经显示为小的蓝色铜蛋白，假天青蛋白（Kakutani等人，1981年a）。当被假天青蛋白还原时，该酶在O2存在下产生H2 O2并被灭活。该酶为绿色，被铜螯合剂强烈抑制，并含有I型和
Revised Manuscript Received July 11, 1995® abstract: 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 thetype 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 thetype 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-I---h charge interaction between Lys269, Glu279, and HislOO helps to orient the active site Cu ligand, HislOO. A number of negatively charged surface residuescreate 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.Nitrite reductase from the denitrifying bacteria Alcaligenes faecalis strain S-6 is a copper-containing enzyme that converts nitrite to nitric oxide (NCh-to NO) under anaerobic conditions (Kakutani et al., 1981b). The in vivo electron donor has been shown to be a small blue copper protein, pseudoazurin (Kakutani et al., 1981a). When reduced by pseudoazurin, the enzyme produces H2O2 in the presence of O2 and is inactivated. The enzyme is green, strongly inhibited by copperchelators, and contains both type I and