Ellis M J, Dodd F E, Strange R W, Prudêncio M, Sawers G, Eady R R, Hasnain S S
Synchrotron Radiation Department, CLRC Daresbury Laboratory, Warrington WA4 4AD, England.
Acta Crystallogr D Biol Crystallogr. 2001 Aug;57(Pt 8):1110-8. doi: 10.1107/s0907444901008654. Epub 2001 Jul 23.
Copper-containing nitrite reductases possess a trimeric structure where the catalytic Cu site, located at the monomer-monomer interface, resembles the catalytic sites of a number of Zn enzymes. Nitrite reductase from Alcaligenes xylosoxidans has optimum activity at pH 5.2 which decreases to a negligible level at pH 8. The structure of this nitrite reductase has previously been determined at pH 4.6. It has now been crystallized under new conditions at pH 8.5. Its crystallographic structure provides a structural explanation for the greatly reduced activity of the enzyme at high pH. Characterization of overexpressed protein in solution by EXAFS suggested that the protein lacked Cu in the catalytic type 2 Cu site and that the site was most probably occupied by Zn. Using the anomalous signals from Cu and Zn, the crystal structure revealed that the expressed protein was devoid of Cu in the catalytic site and that only a trace amount (<10%) of Zn was present at this site in the crystal. Despite the close structural similarity of the catalytic site to a number of Zn enzymes, these data suggest that Zn, if it binds at the catalytic copper site, binds weakly in nitrite reductase.
含铜亚硝酸还原酶具有三聚体结构，其催化铜位点位于单体 - 单体界面，类似于许多锌酶的催化位点。木糖氧化产碱杆菌的亚硝酸还原酶在pH 5.2时具有最佳活性，在pH 8时活性降至可忽略不计的水平。该亚硝酸还原酶的结构先前已在pH 4.6下确定。现在它已在pH 8.5的新条件下结晶。其晶体结构为该酶在高pH下活性大幅降低提供了结构解释。通过扩展X射线吸收精细结构（EXAFS）对溶液中过表达蛋白的表征表明，该蛋白在催化2型铜位点缺乏铜，并且该位点很可能被锌占据。利用铜和锌的异常信号，晶体结构显示表达的蛋白在催化位点没有铜，并且在晶体中该位点仅存在痕量（<10%）的锌。尽管催化位点与许多锌酶在结构上有密切相似性，但这些数据表明，如果锌结合在催化铜位点，它在亚硝酸还原酶中结合较弱。
