Cytochrome b5--A Case Study in Molecular Recognition

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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.