Spectroscopic and Mechanistic Studies of Heme Enzymes

描述(申请人提供)：紫外可见和近红外磁性圆二色谱(MCD)光谱和快速扫描停止流动光谱将用于研究血红素铁酶和相关系统。MCD和共振拉曼光谱将用于研究由胺、羧酸盐或酚类配体连接的肌红蛋白的H93G空腔突变。其他的MCD研究将包括血红素蛋白模板和转录因子NPAS2。对空穴突变体和血红素蛋白模板的研究将扩大我们结构定义的血红素系统的MCD光谱数据库，然后我们将应用于结构未表征的血红素铁系统，如NPAS2。轴向配基的鉴定可以深入了解血红素酶的作用机制。NPAS2蛋白与两个血红素PAS结构域结合，这两个结构域似乎调节转录因子的活性。停流研究将通过对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，来探索四氢生物蝶呤辅助因子在一氧化氮合酶(NOS)催化中的氧化还原作用。研究一氧化氮合酶的催化作用有助于更好地理解一氧化氮合酶的信号产物--一氧化氮气体的生物合成和调控。

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.