Flavohemoglobin-catalyzed nitric oxide dioxygenation

动植物分泌一氧化氮(NO)来杀死病原微生物。然而，病原体可以解毒NO并解除这一重要的免疫防御。许多微生物产生能够代谢NO的黄色素。黄曲霉毒素在NO解毒中的作用是由缺乏黄曲霉毒素的微生物对NO的抗生素作用的敏感性和NO诱导黄曲霉毒素表达所支持的。FlavoHb是广泛分布的氧结合血红蛋白(HBs)家族中的一个古老成员，它似乎具有将NO氧化成硝酸盐的内在能力。我们计划验证这样的假设，即黄素Hbs和HbS与相关的含黄素还原酶一样，起着高效的NO双加氧酶(NODS)的作用。这项建议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双氧合机制和NO解毒功能的黄曲霉毒素及其相关还原酶的结构适应性。1)测得了大肠杆菌黄色素Hb的基本节点反应速率常数，并与哺乳动物O2转运HbS的相应值进行了比较。微生物黄曲霉Hbs，以及微生物和人类Hb及其共表达的还原酶，正在被检测NOD活性。2)利用停流、EPR和红外光谱、特定的陷阱和指示剂来阐明NOD酶机制的中间体。18O同位素标记被用来测试黄色素Hb和氧化的HbS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)利用已有的flavHb结构来推测潜在的作用机制，并正在准备E.ColiFlavoHb的定点突变体，以测试关键的保守氨基酸和基序在NOD稳态、配基结合、还原动力学和中间稳定性中的作用。4)突变型和野生型大肠埃希菌核黄素Hb晶体正在以不同的配基形式被制备，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NOD家族的功能，也将从抗感染的NOD抑制剂的设计中提供信息。

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.