This proposal seeks support for a program of spectroscopic, physical and mechanistic studies of secondary amine mono-oxygenase and a continued effort to develop the spectroscopic technique known as magnetic circular dichroism for the study of metalloporphyrin-containing enzymes. The study of secondary amine mono-oxygenase will be done in the context of our knowledge of the only other heme-containing mono-oxygenase, cytochrome P- 450, so as to test the relationship between structure and function for the heme iron mono-oxygenase class of enzymes. Efforts will continue to learn about the physical structure of SAMO and to study the electron transfer pathway within the intact protein. Purification of the heme-containing subunit will facilitate the direct study of the heme group. The nature of the heme environment of the enzyme will be examined, the identity of the proximal and distal heme ligands will be assigned in all physiologically relevant protein states, and the extent of exposure of the heme macrocycle within the active site will be probed. Rapid kinetics/freeze quench experiments are planned in order to establish the order of intermediates in the reaction cycle. Determining the source of oxygen incorporated into the product, probing the mechanism for free-radical character, examining non- physiological oxygen and electron donor interaction with SAMO, and measuring the extent of uncoupling and the products of any uncoupling of electron uptake from product formation are important objectives in our plan to elucidate the mechanism of action of SAMO. Demonstrating the utility of magnetic circular dichroism (MCD) spectroscopy in establishing the identity of axial ligands in heme iron and related iron chlorin proteins is the major goal of the spectroscopic portion of this research program. Efforts will be expanded to include studies of structure-dependent charge transfer transitions in the near-IR region (NIR MCD). These approaches will be used to study hemoprotein H-450, site- specific mutants of myoglobin and P-450, guanylate cyclase and E. coli complex II (succinate:ubiquinone oxidoreductase) with UV-Vis and NIR MCD. In addition, further attention will be focused on the unusual heme prosthetic group and spectroscopic properties of myeloperoxidase. Reconstitution of a chlorin into a proximal tyrosine-containing mutant of myoglobin will definitively establish whether tyrosine is the axial ligand in E. coli HPII catalase. Reconstitution of a chlorin into P-450 will reveal the spectral properties and reactivity of a thiolate-ligated iron chlorin. Finally, particular attention will be focused on the properties of the presumed Compound I and II intermediates in chlorin-reconstituted horseradish peroxidase in relationship to the chlorin-containing E. coli heme d oxidase.