Atomic resolution structures of resting-state, substrate- and product-complexed Cu-nitrite reductase provide insight into catalytic mechanism
Atomic resolution structures of resting-state, substrate- and product-complexed Cu-nitrite reductase provide insight into catalytic mechanism
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DOI：
10.1073/pnas.0504207102
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发表时间：
2005-08-23
影响因子：
11.1
通讯作者：
Hasnain, SS
中科院分区：
文献类型：
--
作者：
Antonyuk, SV;Strange, RW;Hasnain, SS
含铜亚硝酸盐还原酶催化亚硝酸盐还原为一氧化氮（NO），这是导致陆地氮损失到大气中的反硝化的关键步骤。它们存在于各种土壤和水生生态系统中各种不同生理机能的微生物细菌和真菌中。催化循环中潜在中间体的结构分析是理解酶作用机制的重要目标。使用“晶体收获”和基板浸泡技术，我们已经确定了原子分辨率结构的四种形式的绿色铜亚硝酸还原酶，从土壤细菌无色杆菌cycloclastes。这些结构是酶在0.9埃的静止状态，两种物质在1.10埃和1.15埃显示出与催化型2 Cu结合的亚硝酸盐的不同构象，其中一种是稳定的并且也存在NO，并且在1.12埃分辨率下产物NO侧边结合到催化型2 Cu的稳定形式。这些结构为底物的初始结合、催化前的重新定位、键断裂（O-NO）以及稳定的NO加合物的形成提供了深刻的见解。
Copper-containing nitrite reductases catalyze the reduction of nitrite to nitric oxide (NO), a key step in denitrification that results in the loss of terrestrial nitrogen to the atmosphere. They are found in a wide variety of denitrifying bacteria and fungi of different physiology from a range of soil and aquatic ecosystems. Structural analysis of potential intermediates in the catalytic cycle is an important goal in understanding enzyme mechanism. Using "crystal harvesting" and substrate-soaking techniques, we have determined atomic resolution structures of four forms of the green Cu-nitrite reductase, from the soil bacterium Achromobacter cycloclastes. These structures are the resting state of the enzyme at 0.9 angstrom, two species exhibiting different conformations of nitrite bound at the catalytic type 2 Cu, one of which is stable and also has NO present, at 1.10 angstrom and 1.15 angstrom, and a stable form with the product NO bound side-on to the catalytic type 2 Cu, at 1.12 angstrom resolution. These structures provide incisive insights into the initial binding of substrate, its repositioning before catalysis, bond breakage (O-NO), and the formation of a stable NO adduct.