Antonyuk Svetlana V, Strange Richard W, Sawers Gary, Eady Robert R, Hasnain S Samar
Molecular Biophysics Group, Council for the Central Laboratory of the Research Councils Daresbury Laboratory, Warrington WA4 4AD, United Kingdom.
Proc Natl Acad Sci U S A. 2005 Aug 23;102(34):12041-6. doi: 10.1073/pnas.0504207102. Epub 2005 Aug 10.
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 A, 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 A and 1.15 A, and a stable form with the product NO bound side-on to the catalytic type 2 Cu, at 1.12 A 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.
含铜亚硝酸还原酶催化将亚硝酸盐还原为一氧化氮（NO），这是反硝化作用中的关键步骤，会导致陆地氮素流失到大气中。它们存在于来自一系列土壤和水生生态系统的各种具有不同生理特性的反硝化细菌和真菌中。催化循环中潜在中间体的结构分析是理解酶作用机制的一个重要目标。利用“晶体收集”和底物浸泡技术，我们已经确定了来自土壤细菌环裂无色杆菌的绿色铜亚硝酸还原酶四种形式的原子分辨率结构。这些结构包括处于0.9埃分辨率的酶的静止状态、在催化2型铜位点结合有亚硝酸盐且呈现不同构象的两种状态（其中一种是稳定的且还存在NO，分辨率分别为1.10埃和1.15埃）以及分辨率为1.12埃的一种稳定形式，其中产物NO以侧位结合到催化2型铜上。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。
