Structural and biochemical characterization of redox reactions within nitric oxid

描述（由申请人提供）：一氧化氮（NO）是一种可扩散的活性分子，具有许多重叠的生物学功能，包括控制血管张力和血压，保护病原体和癌症，激素调节，神经细胞传递和血管生成。一氧化氮合酶（NOS）蛋白是一种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端氧化酶结构域（NOSox）和c端还原酶结构域NOSred的同型二聚体。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助因子6r -四氢生物蝶呤（H4B），所有这些都是活性酶所必需的。NOSred具有黄素辅助因子和NADPH的结合位点，是NOSox血红素中氧结合和活化的还原等量物的来源。控制氧化还原活性辅助因子在NOSox和NOSred结构域之间的通讯调节至少两种哺乳动物NOS同工酶，尽管这两个结构域在复合体中的结构尚未实现。细菌的NOS酶与哺乳动物的NOS酶有许多相似之处，由于其结构域结构简化且易于纯化，细菌NOS蛋白可作为研究NO合成机制的有用模型。本文的目的是为了更好地了解NOS的结构排列、电子转移和NOS酶产生NO的机制之间的关系。在目标1中，我们将研究一种来自S. pcc7335的新型NOS酶（spNOS），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酶的晶体结构，spNOS和一种来自S. cellulosum的NOS酶（scNOS），它们包含一个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，我们将确定两种血红素氧状态发生在G.硬脂嗜热菌NOS （gsNOS）的详细结构。

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.