Structural and biochemical characterization of redox reactions within nitric oxid

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，包括控制血管张力和血压、预防病原体和癌症、激素调节、神经细胞传递和血管生成。一氧化氮合酶蛋白是一种以血红素为基础的单加氧酶，通过两步电子转移过程将L精氨酸转化为L精氨酸和一氧化氮。哺乳动物的一氧化氮合酶是一种同源二聚体，含有一个N-末端氧化物区(NOSox)和一个称为NOSred的C-末端还原酶结构域。两个结构域之间的串扰受钙调蛋白(CaM)结合界面的调节。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)，所有这些都是激活酶所必需的。NOSred具有黄素辅因子和NADPH的结合位点，并作为NOSox中血红素氧结合和激活的还原当量的来源。控制NOSox和NOSred结构域中氧化还原活性辅助因子之间的通信调节至少两种哺乳动物的NOS同工酶，尽管这两个结构域在复合体中的结构尚未获得。 细菌一氧化氮合酶与哺乳动物有许多相似之处，由于其结构域结构简单，易于纯化，细菌一氧化氮合酶蛋白可作为研究NO合成机制的有用模型。这一建议的目的是为了更好地理解一氧化氮合酶的结构排列、电子转移和一氧化氮合酶产生NO的机制之间的关系。在目标1中，我们将研究一种新的来自S.pcc7335的一氧化氮合酶(SpNOS)，研究其稳态活性和合成NO的产率，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部位的亲和力和专一性。在目标2中，我们将获得两种细菌一氧化氮合酶的晶体结构，spNOS和一种来自纤维链霉菌的一氧化氮合酶(ScNOS)，它们包含一个以前在细菌系统中从未观察到的融合还原酶域。最后，目标3将针对一氧化氮合酶电子转移机制中的特定氧化还原中间体进行结构表征。具体地说，我们将确定发生在G.stearthermophilus NOS(GsNOS)中的两个血红素氧态的详细结构。

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).