Structural and biochemical characterization of redox reactions within nitric oxid

一氧化氮内氧化还原反应的结构和生化表征

• 批准号: 8287131
• 负责人: Sarah C Hokanson
• 金额: $ 3万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-16 至 2013-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8287131
• 关键词: Active Sites; Affinity; Alzheimer' s Disease; Amino Acid Sequence; Arginine; Bacterial Model; Binding; Binding Sites; Biochemical; Biological Models; Biological Process; Blood Pressure; Blood Vessels; C-terminal; Calmodulin; Cardiovascular Diseases; Catalysis; Cells; Citrulline; Communication; Complex; Crystallization; Drug Design; Electron Transport; Enzymes; Flavins; Genes; Goals; Heme; Hemeproteins; Hypertension; Individual; Isoenzymes; Kinetics; Length; Link; Malignant Neoplasms; Methods; Mixed Function Oxygenases; Modeling; N-terminal; NADP; Nature; Neurodegenerative Disorders; Neurons; Nitric Oxide; Nitric Oxide Synthase; Oxidases; Oxidation-Reduction; Oxidoreductase; Oxygen; Parkinson Disease; Peptide Sequence Determination; Play; Process; Production; Proteins; Pterins; Reaction; Role; Source; Specificity; Spectrum Analysis; Structure; Synechococcus; System; Techniques; Work; angiogenesis; base; cofactor; cold temperature; hormone regulation; interest; novel; pathogen; tetrahydrobiopterin; transmission process

Nitric oxide (NO) is a diffusible, reactive molecule that has many overlapping biological functions, including control of vascular tone and blood pressure, protection against pathogens and cancer, hormone regulation, nerve cell transmission, and angiogenesis. Nitric oxide synthase (NOS) proteins are heme-based monooxygenase enzymes that convert L-arginine to L-citrulline and nitric oxide (NO) by a two-step electron transfer process. Mammalian NOS enzymes are homodimers that contain an N-terminal oxidase domain (NOSox) and C-terminal reductase domain called NOSred. Crosstalk between the two domains is regulated by a calmodulin (CaM)-binding interface. NOSox binds the L-arginine substrate, heme, and the redox-active cofactor 6R-tetrahydrobiopterin (H4B), all of which are required for an active enzyme. NOSred has binding sites for flavin cofactors as well as NADPH, and acts as a source of reducing equivalents for oxygen binding and activation at the heme in NOSox. Controlling the communication between redox-active cofactors in the NOSox and NOSred domains regulates at least two mammalian NOS isozymes, though a structure of the two domains in complex has not yet been achieved. Bacterial NOS enzymes share many similarities to their mammalian counterparts, and because of their stripped-down domain structure and ease of purification, bacterial NOS proteins serve as useful models for investigating the mechanism of NO synthesis. The goal of this proposal is to provide a better understanding about the relationship between NOS structural arrangement, electron transfer and the mechanism of NO production by NOS enzymes. In aim 1, we will study a novel NOS enzyme from S. pcc7335 (spNOS), characterizing its steady state activity and yield of NO synthesis, the reaction kinetics of its NOSox domain, as well as the affinity and specificity of pterin substrates for its redox active site. In aim 2, we will obtain crystal structures of two bacterial NOS enzymes, spNOS and a NOS enzyme from S. cellulosum (scNOS), which contain a fused reductase domain never observed before in bacterial systems. Finally, Aim 3 will target specific redox intermediates in the NOS electron transfer mechanism for structural characterization. Specifically, we will determine detailed structures of two heme-oxy states occurring in G. stearothermophilus NOS (gsNOS).

一氧化氮 (NO) 是一种可扩散的反应性分子，具有许多重叠的生物功能，包括控制血管张力和血压、抵御病原体和癌症、激素调节、神经细胞传递和血管生成。一氧化氮合酶 (NOS) 蛋白是基于血红素的单加氧酶，可通过两步电子转移过程将 L-精氨酸转化为 L-瓜氨酸和一氧化氮 (NO)。哺乳动物 NOS 酶是同二聚体，包含 N 端氧化酶结构域 (NOSox) 和 C 端还原酶结构域（称为 NOSred）。两个结构域之间的串扰由钙调蛋白 (CaM) 结合界面调节。 NOSox 结合 L-精氨酸底物、血红素和氧化还原活性辅因子 6R-四氢生物蝶呤 (H4B)，所有这些都是活性酶所必需的。 NOSred 具有黄素辅因子和 NADPH 的结合位点，并作为 NOSox 中血红素氧结合和活化的还原当量来源。控制 NOSox 和 NOSred 结构域中氧化还原活性辅因子之间的通讯可调节至少两种哺乳动物 NOS 同工酶，尽管这两个结构域的复合体结构尚未实现。 细菌 NOS 酶与其哺乳动物对应物有许多相似之处，并且由于其精简的结构域结构和易于纯化，细菌 NOS 蛋白可作为研究 NO 合成机制的有用模型。该提案的目的是更好地理解 NOS 结构排列、电子转移和 NOS 酶产生 NO 的机制之间的关系。在目标 1 中，我们将研究来自 S. pcc7335 (spNOS) 的一种新型 NOS 酶，表征其稳态活性和 NO 合成产量、其 NOSox 结构域的反应动力学，以及蝶呤底物对其氧化还原活性位点的亲和力和特异性。在目标 2 中，我们将获得两种细菌 NOS 酶 spNOS 和来自纤维链球菌的 NOS 酶 (scNOS) 的晶体结构，其中包含以前在细菌系统中从未观察到的融合还原酶结构域。最后，Aim 3 将针对 NOS 电子转移机制中的特定氧化还原中间体进行结构表征。具体来说，我们将确定嗜热脂肪芽胞杆菌 NOS (gsNOS) 中存在的两种血红素氧态的详细结构。