Flavohemoglobin-catalyzed nitric oxide dioxygenation

动植物分泌一氧化氮（NO）来杀死致病微生物。然而，病原体可以解毒一氧化氮并解除这一重要的免疫防御。许多微生物产生黄血红蛋白（flavoHbs），可以代谢NO。黄酮类化合物在NO解毒中的作用是由缺乏黄酮类化合物的微生物对NO的抗生素作用的敏感性和NO诱导黄酮类化合物表达支持的。FlavoHb是广泛分布的o2结合血红蛋白家族（Hbs）的古老成员，似乎具有“氧化”NO形成硝酸盐的内在能力。我们计划验证黄酮类化合物和含有相关黄素还原酶的黄酮类化合物作为高效NO双加氧酶（NODs）的假设。本研究的目的是阐明所提出的NO双加氧酶功能的酶机制，并阐明支持NO双加氧机制和NO解毒功能的黄酮Hbs和带有相关还原酶的Hbs的结构适应性。1)测定了大肠杆菌flavoHb的基本NOD反应速率常数，并与哺乳动物O2转运Hbs的相应值进行了比较。微生物黄酮类化合物、微生物和人类黄酮类化合物及其共表达还原酶正在被检测NOD活性。2)利用停流法、EPR法、红外分光光度法、特异性陷阱和指示剂，对NOD酶机制的中间体进行了阐明。18O同位素标记被用于测试黄氧化血红蛋白和氧化血红蛋白的“双加氧酶”机制，并测量水O原子在硝酸盐形成机制中的贡献。3)利用现有的flavoHb结构提示潜在的机制，并制备了大肠杆菌flavoHb的位点定向突变体，以测试关键保守氨基酸和基序在NOD稳态、配体结合、还原动力学和中间稳定性中的作用。4)制备了多种配体形式的突变型和野生型大肠杆菌flavoHb晶体，用于x射线衍射分析，以阐明结构-功能关系。了解NOD活性的动力学、机制和结构要求将有助于确定家族（flavo） Hbs的NOD功能，也将为抗感染NOD抑制剂的设计提供信息。

Animals and plants secrete nitric oxide (NO ) to kill pathogenic microbes. However, pathogens can detoxify NO and disarm this important immune defense. Many microbes product flavohemoglobins (flavoHbs) that can metabolize NO. A Function for flavoHbs in NO detoxification is supported by the sensitivity of flavoHb-deficient microbes to the antibiotic action of NO and by the induction of flavoHB expression by NO. FlavoHb is an ancient member of the widely-distributed family of O2-binding hemoglobins (Hbs) that appear to bear an intrinsic capacity for 'oxidizing' NO to form nitrate. We plan to tes the hypothesis that flavoHbs, and Hbs with associated flavin-containing reductases, function as efficient NO dioxygenases (NODs). The goals of this proposal are to elucidate the enzyme mechanism of the proposed NO dioxygenase function and to elucidate structural adaptations of flavoHbs, and Hbs with associated reductases, which support a NO dioxygenation mechanism and NO detoxification function. 1) Elementary NOD reaction rate constants for E.coli flavoHb are being measured and compared with the corresponding values for mammalian O2 transport Hbs. Microbial flavoHbs, and microbial and human Hbs with their co-expressed reductases, are being examined for NOD activity. 2) Intermediates of the NOD enzyme mechanism are being elucidated using stopped flow, EPR, and infrared spectrophotometry, specific traps, and indicators. 18O isotope labelling is being used to test the proposed 'dioxygenase' mechanism of flavoHb and oxgenated Hbs and to measure the contribution of water O atoms in the mechanism of nitrate formation. 3) Existing flavoHb structures are being used to suggest potenial mechanisms, and site- directed mutants of E. coli flavoHb are being prepared to test the roles of key conserved amino acids and motifs in NOD steady-state, ligan- binding, reduction kinetics and in intermediate stability. 4) Mutant and wild-type E. coli flavoHb crystals are being prepared in various ligand forms for x-ray diffraction analysis to elucidate structure-function relationships. Knowledge of the kinetic, mechanistic and structural requirements of the NOD activity will help define the NOD function of the family (flavo) Hbs and will also provide information from the design of anti-infective NOD inhibitors.