HEME-IRON MONO-OXYGENASES: SPECTROSCOPY AND MECHANISM

本文提出了卟啉的光谱研究方案 用磁性圆二色性（MCD）光谱和 四种氧和过氧化物光谱和机理研究 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：（1）通过调查模型 具有确定结构的卟啉和血红素蛋白配体络合物， MCD光谱作为血红素铁电子探针的应用， 最终将确定物理结构。 我们将研究 合成Fe+3（porph）（RS-）（X）和Fe+2（porph）（X）（CO）配合物， 可变仿生假单胞菌X.此外，我们还将研究Fe ~（2+）/~（3+）（卟啉） 咪唑配合物和罕见六配位高自旋Fe ~（3+）卟啉 配合物 血红素铁络合物的实验旨在建立 每种配体和/或复合物类型的光谱指纹， 结构测定 我们还将研究锌卟啉配合物 其中可以更直接地评估轴向结扎的效果。 接下来我们 将研究IDO，CPO和SAMO及其配体复合物， 以更好地确定其协调结构。 (2)研究旨在 确定了血红素铁催化羟基化的结构要求， 通过进一步表征活性位点的有机基质 P-450和SAMO的结构将被执行。 我们的研究 实验室表明它们具有不同金属离子环境， SAMO也不同于肌红蛋白 设计的光谱研究 以更好地定义其legand环境将继续。 第六配体 铁P-450可能是氧供体;寻找直接证据， 水配体，我们将检查它在170标记水中的EPR谱。 使用一种特殊的抑制剂，我们将尝试捕捉出钥匙 羟基化中间体刚好超过氧-P-450。 通过NMR光谱， 还将试图确定中性铁之间距离关系 和衬底。 (3)我们将测试目前关于该机制的假设 P-450和SAMO的N-脱烷基化，通过用 形成稳定的甲醇胺和相关产物的底物， 脱烷基化。 然后可以对稳定的产品进行标签测试 从分子氧或水引入。

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.