Nitric oxide reactions in metalloenzymes

描述（申请人提供）：本研究的长期目标是阐明发生在细菌金属酶血红素和非血红素二铁中心的NO还原反应机制。我们的研究将集中在两个酶系统和相关的生物工程或合成模型：1)NO解毒黄二铁蛋白（FDPs）和2)反硝化NO还原酶（denNORs）。已知这两个家族中的所有蛋白质都能将NO还原为不反应的N2O产物，但它们在效率和蛋白质基质结构上存在很大差异。虽然这些蛋白质中的一些已经被x射线晶体学表征，但一氧化氮结合的初始步骤，铁亚硝基还原，以及这些催化事件在系统之间的差异，还没有得到很好的理解。共振拉曼、FTIR和EPR光谱与快速冷冻淬火分析的耦合提供了独特的功能，可以定义双铁簇上NO结合的几何形状，并跟踪N-N键形成、N-O键裂解和质子化步骤，这些步骤必须发生，才能将两个NO分子转化为N2O和H2O。研究多种天然酶和模型将使我们能够比较和对比铁-亚硝基中间体的结构信息以及该反应的还原和质子转移步骤的效率。

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the reaction mechanisms of NO reduction that occur at heme and non-heme diiron centers of bacterial metalloenzymes. Our studies will focus on two enzymatic systems and related bioengineered or synthetic models: 1) NO-detoxifying flavodiiron proteins (FDPs) and 2) denitrifying NO reductases (denNORs). All of the proteins within these two families are known to reduce NO to the unreactive N2O product, but they do so with wide variation in efficiency and protein matrix structure. Although several of these proteins have been characterized by X-ray crystallography, the initial steps of NO binding, iron-nitrosyl reduction, and how these catalytic events differ between systems are not well understood. The coupling of resonance Raman, FTIR, and EPR spectroscopies with rapid-freeze-quench analyses provides unique capabilities to define NO-binding geometries at diiron clusters and to follow the N-N bond formation, N-O bond cleavage, and protonation steps that must take place to convert two NO molecules to N2O and H2O. Studying a diverse group of native enzymes and models will allow us to compare and contrast structural information on iron-nitrosyl intermediates and the efficiency of the reductive and proton transfer steps of this reaction.