The research will apply methods from synthetic and physical inorganic chemistry, as well as more traditional biochemical methods, to selected proteins and enzymes containing transition metals and flavins. The general objective is to understand the relationship between the structure of metal- and flavin-containing systems and their biological function. This will be accomplished by fundamental physical, chemical and biochemical studies of representative members of a novel class of metal-containing hydrolytic enzyme, and by the preparation and study of synthetic systems containing flavins and iron-sulfur clusters as models for complex metalloflavoenzymes. The specific examples to be investigated are members of the novel and biologically widespread class of purple metallophosphatases, an unusual green heme peroxidase from bovine spleen, and models for metal-flavin enzymes. The purple acid phosphatase from bovine spleen has been shown to contain a mixed-valence binuclear iron cluster at the active site, while a similar enzyme from sweet potato is reported to contain manganese. These enzymes, along with a representative purple phosphatase from a microbial source, will be examined by spectroscopic and chemical methods to determine the nature of the metal prosthetic groups and their role in catalyzing a simple hydrolysis reaction. Biochemical and immunological techniques will be used to investigate the physiological function of the bovine spleen enzyme in vivo; preliminary indications are that it is a phosphoprotein phosphatase specific for phosphotyrosine and that it may be involved in regulation of other enzymes in the immune system. The structure and function of the unusual chromophore in the green heme peroxidase from bovine spleen will be examined by resonance Raman and NMR spectroscopy. Electron transfer reactions between iron-sulfur clusters and flavin will be examined, using systems containing flavins covalently attached to iron-sulfur clusters via spacers of different length to determine intramolecular electron transfer rates and the extent of magnetic interactions. Cytochrome c covalently modified with flavin derivatives will be prepared and used to measure flavin-heme electron transfer rates.
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