Cytochrome b5--A Case Study in Molecular Recognition

细胞色素b5——分子识别案例研究

• 批准号: 6606890
• 负责人: Mario Rivera
• 金额: $ 24.84万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6606890
• 关键词: NAD(P)H oxidoreductase; X ray crystallography; active sites; biotransformation; chemical binding; cytochrome P450; cytochrome b5 reductase; divalent metal; electrochemistry; electron spin resonance spectroscopy; enzyme activity; ferriheme; heme; heme oxygenase; hydrogen bond; intermolecular interaction; mitochondrial membrane; molecular shape; nuclear magnetic resonance spectroscopy; oxygen transport; protein structure function; site directed mutagenesis; stable isotope; NAD(P)H oxidoreductase; X ray crystallography; active sites; biotransformation; chemical binding; cytochrome P450; cytochrome b5 reductase; divalent metal; electrochemistry; electron spin resonance spectroscopy; enzyme activity; ferriheme; heme; heme oxygenase; hydrogen bond; intermolecular interaction; mitochondrial membrane; molecular shape; nuclear magnetic resonance spectroscopy; oxygen transport; protein structure function; site directed mutagenesis; stable isotope

The degradation of heme in mammalian cells is catalyzed by the enzyme heme oxygenase (HO). The products of HO activity play important physiological functions: CO is believed to play a role similar to that of NO in signal transduction and communication. Biliverdin is the source of bilirubin, a powerful antioxidant. The catalytic cycle of HO parallels that of cyt P450 and other monooxygenases in that both types of enzyme form a ferric hydroperoxide (FeIII-OOH) intermediate. Whereas the FeIII-OOH intermediate in HO attacks the heme in order to form meso-hydroxyheme, the FeIII-OOH intermediate in monooxygenases andperoxidases decays into a ferryl (FeIV=O) Intermeidate. Consequently, the elucidation of the mechanism of action of HO is not only expected to contribute information regarding heme catabolism, but it will also provide information regarding the broader issue of O2 activation at heme centers. The overall objective of the proposed studies is to determine fundamental aspects of hem catabolism with an emphasis on delineating structural and electronic aspects that dictate the reactivity of key intermediates in the pathway of hem degradation. To these ends we will conduct the following research: 1) Conduct 1H and 13C NMR spectroscopic studies in order to determine the electronic configuration of low-spin ferric hemes coordinate by a hydroperoxy axial ligand. 2) Investigate the effect of the electronic structure of the FeIII-OOH intermediate on the regioselectivity of heme oxygenation. 3) Investigate the fundamental aspects underlying the conversion of verdoheme to biliverdin. In order to accomplish this goal we will carry out NMR spectroscopic experiments with the verdoheme complex of hem oxygenase and with the verdoheme complex of a mutant of hem oxygenase not capable of oxidizing verdoheme to biliverdin. Parallel studies willb e carried out with mutants of mitochondrial cytochrome b5 that can and cannot oxidize verdoheme to biliverdin.

哺乳动物细胞中血红素的降解是由血红素氧酶（HO）催化的。 HO活动的产物起着重要的生理功能：CO被认为在信号转导和通信中起着类似于NO的作用。 Biliverdin是胆红素的来源，胆红素是一种强大的抗氧化剂。 HO的催化循环与Cyt P450和其他单加氧酶的催化周期相似，因为两种类型的酶形成了氢过氧化物（FEIIII-OOH）中间体。 feiii-ooh在HO攻击中介导了血红素以形成中羟基血红素，而单加氧酶和过氧化物酶中间的FeIIII-OOH中间又衰减了Ferryl（FEIV = O）互化。 因此，阐明HO的作用机理不仅有望贡献有关血红素分解代谢的信息，而且还将提供有关血红素中心O2激活的更广泛问题的信息。拟议的研究的总体目的是确定下摆分解代谢的基本方面，重点是描述结构和电子方面，这些方面决定了关键中间体在下摆降解途径中的反应性。 为此，我们将进行以下研究：1）进行1H和13C NMR光谱研究，以确定低自旋铁下摆坐标的电子构型通过水上氧基轴向配体坐标。 2）研究FeIII-OOH电子结构中间体对血红素氧合区域选择性的影响。 3）研究Verdoheme转换为Biliverdin的基本方面。 为了实现这一目标，我们将使用HEM氧合酶的Verdoheme复合物以及Hem氧合酶突变体的Verdoheme复合物进行NMR光谱实验，无法氧化Verdoheme至biliverdin。 平行研究将使用线粒体细胞色素B5的突变体进行，该突变体可以并且不能将verdoheme氧化为biliverdin。