Flavohemoglobin-catalyzed nitric oxide dioxygenation

黄素血红蛋白催化的一氧化氮双氧合

• 批准号: 6520595
• 负责人: PAUL R GARDNER
• 金额: $ 19.07万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6520595
• 关键词: Escherichia coli; Saccharomyces cerevisiae; X ray crystallography; electrodes; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; enzyme structure; flash photolysis; flavoproteins; hemoglobin; infrared spectrometry; nitric oxide; oxygen transport; oxygenases; protein structure function; radiotracer; redoxin; site directed mutagenesis; stop flow technique

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.

动物和植物分泌一氧化氮（NO）杀死病原微生物。 然而，病原体可以解毒NO并解除这种重要的免疫防御。 许多微生物产品flavohemoglobins（flavoHbs），可以代谢NO。一个功能flavoHbs在NO解毒支持的敏感性flavoHb缺陷的微生物的抗生素作用的NO和诱导flavoHB的表达NO。FlavoHb是一个古老的成员广泛分布的家庭的O2结合血红蛋白（Hbs），似乎承担的内在能力“氧化”NO形成硝酸盐。 我们计划测试的假设，flavoHbs，与相关的含黄素还原酶，作为有效的NO双加氧酶（NODs）的功能。该建议的目标是阐明所提出的NO双加氧酶功能的酶机制，并阐明flavoHbs和具有相关还原酶的Hbs的结构适应，其支持NO双加氧机制和NO解毒功能。 1)大肠杆菌flavoHb的基本NOD反应速率常数正在测量，并与哺乳动物O2运输血红蛋白的相应值进行比较。 正在检查微生物flavoHbs以及微生物和人类Hbs及其共表达的还原酶的NOD活性。 2)NOD酶机制的中间体正在使用停流、EPR和红外分光光度法、特定陷阱和指示剂来阐明。 18 O同位素标记被用来测试所提出的“双加氧酶”机制的flavoHb和氧合血红蛋白，并测量水O原子的硝酸盐形成的机制中的贡献。 3)现有的flavoHb结构正被用来提出潜在的机制，以及E. coliflavoHb被制备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)突变型和野生型E.大肠杆菌flavoHb晶体被制备成各种配体形式，用于X射线衍射分析以阐明结构-功能关系。 NOD活性的动力学、机制和结构要求的知识将有助于定义家族（flavo）Hbs的NOD功能，并且还将提供来自抗感染NOD抑制剂的设计的信息。