A study of the biophysical properties of two complex oxidoreductases, xanthine oxidase and trimethylamine dehydrogenase, is proposed. The goal of this work is to fully characterize the several redox-active centers in the proteins from a physical standpoint, with particular regard to their spectroscopic and electron-transfer properties. Xanthine oxidase catalyzes the oxidative hydroxylation of xanthine to form uric acid, a reaction that takes place at the molybdenum center of the enzyme. For a variety of reasons, the enzyme has become the prototypical molybdenum hydroxylase, a small but extremely important group of enzymes that utilize oxygen derived from water rather than dioxygen in the hydroxylation of substrate. Two specific aspects of this enzyme are the focus of the proposed work: (1) the determination of rates of electron transfer among the four redox-active centers of the enzyme and the factors that govern the observed rates (as studied by pulse radiolysis and pH-jump techniques); and (2) the structure of the molybdenum center in a variety of forms, including catalytic intermediates, to be examined by circular dichroism and resonance Raman spectroscopy, with the aim of characterizing the physical and electronic structure of the center. Trimethylamine dehydrogenase catalyzes the oxidative demethylation of trimethylamine to dimethylamine and formaldehyde. The enzyme possesses a covalently linked flavin mononucleotide cofactor and a ferredoxin-type center, and thus represents a useful system to investigate the general applicability of the principles governing the behavior of xanthine oxidase. The proposed studies include: (1) studies of the pH-dependence of electron distribution within partially reduced enzyme, and both pH-jump and pulse radiolysis studies of electron transfer between the two centers; (2) a determination of the spectral contributions and reduction potentials of the flavin and iron-sulfur centers: and (3) rapid and steady-state kinetic studies of the catalytic cycle aimed at elucidating the role of electron transfer in the catalytic cycle of trimethylamine dehydrogenase.
