HEME-IRON MONO-OXYGENASES: SPECTROSCOPY AND MECHANISM

这项建议提出了一套关于卟啉的光谱研究方案。 磁性圆二色谱(MCD)模型络合物的研究 四种氧和过氧化氢的光谱和机理研究 代谢血红素铁蛋白：细胞色素P-450、仲胺 单加氧酶(SAMO)、吲哚胺双加氧酶(IDO)和氯过氧化物酶 (CPO)。总体目标有三个：(1)通过调查模型 确定结构的卟啉和血红素蛋白配位络合物， MCD光谱作为血红素铁电子和电子探针的应用 最终，物理结构将被确定。我们将研究 合成的Fe+3(Pporh)(RS-)(X)和Fe+2(Poph)(X)(CO)配合物 此外，我们还将研究Fe+2/+3(Porph)。 咪唑类配合物与反常六配位高自旋Fe+3卟啉 复合体。用血红素铁络合物进行的实验旨在扩大 每种配体和/或络合物类型的光谱指纹图谱 结构确定。我们还将研究锌的卟啉配合物。 在那里可以更直接地评估轴向结扎的效果。接下来我们 将对IDO、CPO和SAMO及其配体络合物进行研究 以更好地定义它们的协调结构。(2)旨在 确定血红素铁催化的羟基化的结构要求 有机底物活性中心的进一步表征 将实施P-450和SAMO的结构。来自我们的研究 实验室表明，它们具有不同的金属离子环境和 Samo也不同于肌红蛋白。设计的光谱研究 为了更好地定义其法律和环境，将继续进行。的第六个配体 铁P-450可能是氧供体；寻找直接证据表明 对于水配体，我们将研究其在170标记水中的EPR谱。 使用特定的抑制器，我们将尝试捕获密钥 羟化中间体，略高于氧基-P-450。利用核磁共振波谱，我们 还将尝试定义Cnetral铁之间的距离关系 和底物。(3)我们将检验目前关于这一机制的假设 对P-450和SAMO的N-脱烷基进行180标记实验 形成稳定的甲醇胺和相关产品的底物而不是 脱烷基化。然后可以对稳定的产品进行标签测试 从氧气或水中掺入。

This proposal presents a program of spectroscopic studies of porphyrin model complexes with magnetic circular dichroism (MCD) spectroscopy and of spectroscopic and mechanistic studies of four oxygen and peroxide metabolizing heme iron proteins: cytochrome P-450, secondary amine mono-oxygenase (SAMO), indoleamine dioxygenase (IDO) and chloroperoxidase (CPO). There are three overall objectives: (1) By investigating model porphyrin and heme protein ligand complexes of defined structure, the utility of MCD spectroscopy as a probe of heme iron electronic and ultimately, physical structure will be ascertained. We will examine synthetic Fe+3 (porph)(RS-)(X) and Fe+2 (porph)(X)(CO) complexes with variable biomimetic legands, X. In addition, we will study Fe+2/+3 (porph) imidazolate complexes and unusual six-coordinate high-spin Fe+3 porphyrin complexes. Experiments with heme iron complexes are designed to extablish spectroscopic fingerprints for each ligand and/or complex type for use in structure determination. We will also examine zinc porphyrin complexes where the effects of axial ligation can be more directly assessed. Next we will investigate IDO, CPO and SAMO and their ligand complexes in an effort to better define their coordination structure. (2) Studies designed to define the sturctural requirements for heme iron catalyzed hydroxylation of organic substrates by further characterization of the active site structures of P-450 and SAMO will be carried out. Studies from our laboratory indicate that they have different metal ion environments and that SAMO differs from myoglobin as well. Spectroscopic studies designed to better define its legand environment will continue. The sixth ligand to ferric P-450 may be an oxygen donor; to look for direct evidence for a water ligand, we will examine its EPR spectrum in 170 labelled water. Using a specific inhibitor, we will attempt to trap-out the key hydroxylation intermediate just beyond oxy-P-450. With NMR spectroscopy we will also try to define the distance relationship between the cnetral iron and the substrate. (3) We will test current hypotheses about the mechanism of N-dealkylation of P-450 and SAMO by doing 180 labelling experiments with substrates that form stable carbinolamine and related products rather than dealkylating. The stable product can then be tested for label incorporation from either dioxygen or water.