Structural and biochemical characterization of redox reactions within nitric oxid

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，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。

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).