Reactivity of Transient Cyt P450 Oxygen Intermediates

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): This proposal seeks support for investigations into the generation, characterization and, most importantly, reactivity of the 4 least well-understood cytochrome P450 (and related enzyme) transient intermediates - those involving oxygen: oxy-ferrous, peroxo-ferric, hydroperoxo-ferric, and oxo-iron(IV) [compound I, Cpd I, oxo-iron(IV)porph+]. 2 similar oxo species, compounds II and ES, will also be examined. 3 specific aims will be pursued. First (1), rapid kinetics methods will explore the role of hydrogen bonding in formation of a newly-observed "perturbed" oxy catalytic species by use of mutants with altered proximal and distal hydrogen bonding properties. Parallel studies of oxy NO synthase will use modified tetrahydrobiopterins to probe electron transfer. Second (2), the mechanism whereby peroxo and hydroperoxo P450 may serve as alternate oxidants will be tested using T252A P450-CAM, a mutant that forms those species but almost none of the primary oxidant, Cpd I. One-electron cryoreduction of oxy P450-CAM provides another way to study the peroxo and hydroperoxo states; solvent and substrate isotope effects on the annealing of the hydroperoxo state will reveal important mechanistic information about its reactivity. Both Aim 2 approaches will further test the "two oxidant" hypothesis of P450 reactivity. The third goal (3) is to characterize the properties and reactivities of transient oxo Cpd I (and related) intermediates. Having optimized conditions for P450-CAM Cpd I formation, we will use rapid freeze-quench methods to characterize it spectroscopically. This will establish its electronic properties, and help explain its reactivity. Double-mix stopped-flow experiments will examine Cpd I reactivity and clarify key mechanistic aspects of O atom transfer by peroxidases and P450. With P450-CAM, this will include the first direct reactions of Cpd I with substrates and determination of the intermolecular isotope effect for hydroxylation - a crucial test of the well-accepted "oxygen rebound" mechanism of hydroxylation. The knowledge derived from this work will lead to a more complete understanding of how heme enzymes activate peroxide and dioxygen with important medical implications for human health and disease, especially as described below for P450,. With over 3700 genes, P450 cytochromes are among the most essential and ubiquitous enzymes known. In human health, 57 P450s are responsible for countless critical transformations in steroid, vitamin D, eicosanoid, as well as drug metabolism. In human disease, P450-aromatase is a target for breast cancer chemotherapy owing to its vital role in estrogen hormone biosynthesis and several P450s have been shown to be activators of procarcinogens such as polycyclic aromatic hydrocarbons and nitrosamines. Progress in comprehending the P450 mechanism will promote medical advances to address these health issues.