Structural insights into the function of a thermostable copper-containing nitrite reductase
Structural insights into the function of a thermostable copper-containing nitrite reductase
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DOI：
10.1093/jb/mvt107
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发表时间：
2014-02-01
影响因子：
2.7
通讯作者：
Inoue, Tsuyoshi
中科院分区：
文献类型：
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作者：
Fukuda, Yohta;Tse, Ka Man;Inoue, Tsuyoshi
含铜亚硝酸盐还原酶（CuNIR）在反硝化过程中催化亚硝酸盐（NO（-）2）还原为一氧化氮（NO）。我们以1.15 A的分辨率确定了来自嗜热革兰氏阳性细菌热脱氮土芽孢杆菌（GtNIR）的氯化物和甲酸盐结合形式的野生型CuNIR的晶体结构，并以1.90 A的分辨率确定了C135 A突变体的亚硝酸盐结合形式。具有亚硝酸根的C135 A的结构在催化铜位点（T2 Cu）处显示出亚硝酸根的独特的η（1）-O配位模式，这在已知的野生型CuNIR中的T2 Cu位点处从未观察到，因为对催化活性至关重要的两个残基Asp 98和His 244的迁移率，在GtNIR中分别被Phe 109限制在Asp 98上方发现的特征环结构上，并被His 244和水之间观察到的异常短的CH-O氢键限制。WT结构与亚硝酸根结合的C135 A结构的详细比较意味着His 244周围的氢键网络的替换，并预测亚硝酸根还原所消耗的质子的流动路径。在此基础上，提出了GtNIR通过eta（1）-O配位的反应机理。
Copper-containing nitrite reductase (CuNIR) catalyzes the reduction of nitrite (NO(-)2)to nitric oxide (NO) during denitrification. We determined the crystal structures of CuNIR from thermophilic gram-positive bacterium, Geobacillus thermodenitrificans (GtNIR) in chloride- and formate-bound forms of wild type at 1.15 A resolution and the nitrite-bound form of the C135A mutant at 1.90 A resolution. The structure of C135A with nitrite displays a unique eta(1)-O coordination mode of nitrite at the catalytic copper site (T2Cu), which has never been observed at the T2Cu site in known wild-type CuNIRs, because the mobility of two residues essential to catalytic activity, Asp98 and His244, are sterically restricted in GtNIR by Phe109 on a characteristic loop structure that is found above Asp98 and by an unusually short CH-O hydrogen bond observed between His244 and water, respectively. A detailed comparison of the WT structure with the nitrite-bound C135A structure implies the replacement of hydrogen-bond networks around His244 and predicts the flow path of protons consumed by nitrite reduction. On the basis of these observations, the reaction mechanism of GtNIR through the eta(1)-O coordination manner is proposed.