MOLECULAR PROBES OF THE MECHANISM OF CYTOCHROME P450

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Cytochrome P-450 has become the Rosetta Stone of the heme-containing oxygenases. The isoforms of lung, liver, kidney, and epithelial tissue have been long known to play a central role in carcinogen activation, drug and xenobiotic detoxification and steroid and prostaglandin metabolism. Now, nitric oxide synthase (NOS) has been shown to be a cytochrome P-450 as well, thus opening a new chapter in the study of these enzymes. The goals of this program are to elucidate the organic and inorganic chemistry of the processes mediated by cytochrome P-450. Our two-pronged approach has been (i) to design substrates for cytochrome P-450 and model iron porphyrin complexes which can reveal the nature of unseen intermediates and (ii) to observe reactive intermediates in model systems which can serve as chemical paradigms for these processes. Specific activities proposed for the next grant period are: A. Model Studies of the Mechanism of NO Synthase. The reactivity of oxo- and peroxo-iron porphyrin complexes with compounds related to arginine and N-hydroxy arginine, the natural substrates for nitric oxide synthase (NOS) will be examined. The goals will be: (1) to illuminate the range of possible mechanisms for NOS by looking at iron porphyrin mediated oxidation of these arginine derivatives in well-defined model systems which can allow study of each elementary step of a proposed catalytic cycle and (2) to seek out new and possibly aberrant oxidation processes that may help with the rational design of NOS inhibitors which could have a number of important pharmacological activities. B. Nucleophilic reactions of iron(III) peroxo complexes that have been invoked to explain the unusual carbon-carbon bond scission event in the cytochrome P-450 aromatase and in the oxidation of N-hydroxyarginine to citrulline and nitric oxide by NO synthase will be studied in model complexes designed to illuminate the extent and nature of such processes. C. The reactions of peroxynitrite with water soluble complexes of iron, manganese and copper will be examined in the presence of DNA as a detector for metal-mediated damage. The experiments will be aimed at determining what reactive intermediates are formed and what their biological targets are likely to be. The elaboration of these processes will facilitate the design of metal complexes for the catalytic decomposition of peroxynitrite. Such agents could be of important pharmacological interest for the control of tissue damage from peroxynitrite which have been implicated in such pathological states as ischemia-reperfusion and autoimmune diseases. D. The development of synthetic and semi-synthetic phospholipid assemblies to model and understand the electron transfer events in P-450 action will be extended. Specifically, methods of differentiating the spatial orientation of membrane bound porphyrins and related redox components toward an understanding of vectorial processes in general will be explored and the very recent finding that rates of electron transfer between and among spatially partitioned synthetic redox components of vesicular assemblies afford information about distance between redox centers will be extended.