Structural and biochemical characterization of redox reactions within nitric oxid

描述（由申请人提供）：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学功能，包括控制血管紧张度和血压、防止病原体和癌症、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，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酶是含有N-末端氧化酶结构域（NOSox）和称为NOSred的C-末端还原酶结构域的同源二聚体。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6 R-四氢生物蝶呤（H4 B），所有这些都是活性酶所必需的。NOSred具有黄素辅因子以及NADPH的结合位点，并且作为NOSox中血红素处的氧结合和活化的还原当量的来源。控制NOSox和NOSred结构域中的氧化还原活性辅因子之间的通信调节至少两种哺乳动物NOS同工酶，尽管复合物中的两个结构域的结构尚未实现。细菌NOS酶与它们的哺乳动物对应物有许多相似之处，并且由于它们的剥离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。本研究的目的是为了更好地了解NOS的结构排列、电子传递和NOS酶产生NO的机制之间的关系。目的一是研究一种新的一氧化氮合酶。pc 7335（spNOS）的NO合成的稳态活性和产量，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和一种来自S.纤维素酶（scNOS），其含有之前在细菌系统中从未观察到的融合还原酶结构域。最后，目标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，我们将确定两个血红素氧状态发生在G的详细结构。嗜热脂肪菌NOS（gsNOS）。 公共卫生相关性：一氧化氮合酶（NOS）蛋白将L-精氨酸转化为L-瓜氨酸和一氧化氮（NO）。NO是一种可扩散的反应性分子，其功能是控制血管张力和血压，防止病原体和癌症，激素调节，神经细胞传递和血管生成。细胞中的NO产生是许多不同能力的药物设计的目标，因为NO的过度产生与神经退行性疾病如帕金森病和阿尔茨海默病有关，而NO产生不足与高血压和心血管疾病等病症有关。

DESCRIPTION (provided by applicant): 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). PUBLIC HEALTH RELEVANCE: Nitric oxide synthase (NOS) proteins convert L-arginine to L-citrulline and nitric oxide (NO). NO is a diffusible, reactive molecule that functions to control of vascular tone and blood pressure, protection against pathogens and cancer, hormone regulation, nerve cell transmission, and angiogenesis. NO production in cells is a target for drug design in many different capacities, as overproduction of NO has been linked to neurodegenerative disorders such as Parkinson's and Alzheimer's diseases, and insufficient NO production has been linked to conditions such as hypertension and cardiovascular disease.