Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan; Department of Applied Chemistry, School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Faculty of Life and Environmental Sciences, Department of Environmental Information, Kyoto Prefectural University, Shimogamo-Hanki-cho, Sakyou, Kyoto 606-8522, Japan; Molecular-Recognition Structure Analysis Team, Molecular Profiling Research Center for Drug Discovery (molprof) National Institute of Advanced Industrial Science and Technology (AIST) 2-3-26 Aomi, Koto-ku, Tokyo 135-0064, Japan; Microbial Genome Research Group, Japan Agency of Marine-Earth Science and Technology, 2-15 Natsushima, Yokosuka, Kanagawa 237-0061, Japan; and RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
J Biochem. 2014 Feb;155(2):123-35. doi: 10.1093/jb/mvt107. Epub 2013 Nov 30.
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 Å resolution and the nitrite-bound form of the C135A mutant at 1.90 Å resolution. The structure of C135A with nitrite displays a unique η(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 η(1)-O coordination manner is proposed.
含铜亚硝酸盐还原酶 (CuNIR) 在反硝化过程中催化将亚硝酸盐 (NO(-)2) 还原为一氧化氮 (NO)。我们测定了嗜热革兰氏阳性菌 Geobacillus thermodenitrificans (GtNIR) 中 CuNIR 的晶体结构，分别为野生型在氯和甲酸盐结合形式下的分辨率为 1.15 Å，以及 C135A 突变体在亚硝酸盐结合形式下的分辨率为 1.90 Å。C135A 与亚硝酸盐的结构显示出催化铜位点 (T2Cu) 中亚硝酸盐独特的 η(1)-O 配位模式，这在已知野生型 CuNIRs 的 T2Cu 位点从未观察到过，因为对于催化活性至关重要的两个残基 Asp98 和 His244 的移动性受到限制，在特征环结构上被 Phe109 限制，该环结构位于 Asp98 上方，而 His244 与水之间的 CH-O 氢键也异常短。WT 结构与亚硝酸盐结合的 C135A 结构的详细比较表明 His244 周围氢键网络的替代，并预测了亚硝酸盐还原所消耗质子的流动路径。基于这些观察结果，提出了 GtNIR 通过 η(1)-O 配位方式的反应机制。
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