Spectroscopic and Mechanistic Studies of Heme Enzymes

描述（由申请人提供）：紫外-可见和近红外磁圆二色（MCD）光谱和快速扫描停止流光谱将用于研究血红素铁酶及其相关体系。MCD将与共振拉曼光谱一起用于研究胺、羧酸盐或酚酸盐配体连接的肌红蛋白H93G空洞突变体。其他MCD研究将包括血红素蛋白和转录因子NPAS2。对空腔突变体和血红素蛋白maquettes的研究将扩展我们的MCD光谱数据库，用于结构上定义的血红素系统，然后我们将其应用于结构上未表征的血红素铁系统，如NPAS2。轴向配体的鉴定可以深入了解血红素酶的机制。NPAS2蛋白结合两个血红素PAS结构域，似乎调节转录因子的活性。停流研究将通过六种不同的蝶呤和三种不同的底物进行单混合和双混合实验，探索四氢生物蝶呤辅助因子在催化一氧化氮合酶（NOS）中的氧化还原作用。研究NOS的催化作用将有助于更好地理解NOS催化的信号产物一氧化氮（NO）的生物合成和调控。

DESCRIPTION (provided by applicant): UV-Visible and near infrared magnetic circular dichroism (MCD) spectroscopy and rapid scan stopped flow spectroscopy will be used to study heme iron enzymes and related systems. MCD, along with resonance Raman spectroscopy, will be used to study the H93G cavity mutant of myoglobin ligated by amine, carboxylate or phenolate ligands. Additional MCD studies will include heme protein maquettes and the transcription factor NPAS2. Studies of the cavity mutant and the heme protein maquettes will expand our data base of MCD spectra for structurally defined heme systems, which we will then apply to structurally uncharacterized heme iron systems such as NPAS2. Identification of axial ligands can give insight into heme enzyme mechanisms. The NPAS2 protein binds two heme PAS domains that appear to regulate the activity of the transcription factor. Stopped flow studies will explore the redox role of the tetrahydrobiopterin cofactor in the catalysis of nitric oxide synthase (NOS) by conducting single and double mixing experiments with six different pterins and three different substrates. Studying NOS catalysis will allow for a better understanding of the biosynthesis and regulation of nitric oxide gas (NO), the signaling product of NOS catalysis.