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的亚铁血红素和非亚铁血红素中心的NO还原的反应机理。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解毒黄铁蛋白(FDPs)和2)反硝化NO还原酶(DenNORs)。众所周知，这两个家族中的所有蛋白质都能将NO还原为无反应的N2O产物，但它们这样做的效率和蛋白质基质结构的差异很大。虽然这些蛋白质中的几个已经用X射线结晶学进行了表征，但NO结合、铁-亚硝基还原的初始步骤以及这些催化事件在不同系统之间的差异还不是很清楚。将共振拉曼光谱、FTIR光谱和EPR光谱与快速冷冻-猝灭分析相结合，提供了独特的能力来定义双铁原子团簇上的NO结合几何结构，并跟踪N-N键的形成、N-O键的断裂以及将两个NO分子转化为N2O和H2O所必须发生的质子化步骤。研究一组不同的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.