MECHANISTIC STUDIES OF A SIDE-ON COPPER NITROSYL COORDINATION BY NITRITE REDUCTA

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。亚硝酸盐还原酶（NiR）是一种含铜酶，它执行细菌异化反硝化的第一步，将亚硝酸盐还原为一氧化氮。粪Alcaligenes faecalis细菌的亚硝酸盐还原酶作为一种高分辨率结构测定的模型系统，提供了对生物学中这种重要酶的深入了解。在SSRL收集的数据产生了cu -亚硝基的高分辨率结构，它显示了意想不到的和前所未有的侧对NO与金属的配位。近红外与底物和产物络合的结构帮助我们修正了亚硝酸盐还原的催化机制，并为生物系统中一氧化氮金属相互作用的世界提供了见解。Cu(II)-NO-的现有结构是通过首先还原晶体并将其暴露于NO中获得的。由于Cu(I)-NO+是该机制中被提议的中间体，因此需要一个与NO相互作用的氧化酶的高分辨率结构来支持我们目前对催化的解释。我们很幸运有一个生物系统，它可以作为铜NO化学的模型，也是反硝化的主要酶。该系统将用于确定反应中间体的高分辨率结构，研究NO结合的pH依赖性，并更好地了解近红外的催化潜力。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Nitrite reductase (NiR) is a copper-containing enzyme, which carries out the first committed step of bacterial dissimilatory denitrification, the reduction of nitrite to nitric oxide. Nitrite reductase from the bacterium Alcaligenes faecalis serves as a model system for determination of high-resolution structures, which provide insight into this vital enzyme in biology. Data collected at SSRL resulted in a high-resolution structure of a Cu-nitrosyl, which showed an unexpected and unprecedented side-on NO coordination to the metal. Structures of NiR complexed with substrate and product have helped us revise the catalytic mechanism of nitrite reduction and have provided insights into the world of nitric oxide metal interactions in a biological system. The current structure of Cu(II)-NO- was achieved by reducing the crystals first and exposing them to NO. Since Cu(I)-NO+ is the proposed intermediate in the mechanism, a high-resolution structure of an oxidized enzyme interacting with NO is needed for supporting our current interpretations of catalysis. We are fortunate to have a biological system, which can serve as a model for copper NO chemistry and is also a major enzyme in denitrification. This system will be used to determine a high-resolution structure of the reaction intermediate, study pH dependence of NO binding and better understand the catalytic potential of NiR.