Cytochrome b5--A Case Study in Molecular Recognition

血红素加氧酶（HO）催化哺乳动物细胞中血红素的降解。 HO活性产物具有重要的生理功能：CO被认为在信号转导和通讯中起着与NO类似的作用。 胆绿素是胆红素的来源，胆红素是一种强大的抗氧化剂。 HO的催化循环与细胞色素P450和其他单加氧酶的催化循环平行，因为两种类型的酶都形成铁氢过氧化物（FeIII-OOH）中间体。HO中的FeIII-OOH中间体攻击血红素以形成meso-hydroxyheme，而单加氧酶和过氧化物酶中的FeIII-OOH中间体衰变为铁基（FeIV=O）中间体。 因此，HO作用机制的阐明不仅有望提供有关血红素催化剂的信息，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。拟议研究的总体目标是确定hem catalysts的基本方面，重点是描述决定hem降解途径中关键中间体反应性的结构和电子方面。 为此，我们进行了以下研究：1）通过核磁共振氢谱和碳谱研究，确定了低自旋铁血红素与过氧氢轴向配体配位的电子构型。 2)研究FeIII-OOH中间体的电子结构对血红素氧化的区域选择性的影响。 3)研究绿血红素转化为胆绿素的基本方面。 为了实现这一目标，我们将用血红素加氧酶的绿血红素复合物和不能将绿血红素氧化成胆绿素的血红素加氧酶突变体的绿血红素复合物进行NMR光谱实验。 将对线粒体细胞色素b5的突变体进行平行研究，这些突变体能够和不能将绿血红素氧化为胆绿素。

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.