Free radical reactivity resulting from the ease of making and breaking one electron bonds leads to essential roles for radicals in biological catalysis, providing easy paths for fundamental processes like C-C bond formation and cleavage, atom transfer and redox chemistry. Biology has evolved specialized structures to take advantage of this unique reactivity, but the instability of most biological radicals makes them difficult to study in detail. The Whittaker's research is focused on studies of an unusually stable free radical in the active site of galactose oxidase, a protein radical directly participating in active site redox chemistry as a free radical-coupled copper complex. Galactose oxidase has provided a unique opportunity to study the involvement of free radicals in enzyme catalysis, serving as a paradigm for radical catalysis in other systems where the catalytic free radical is less accessible. The research plan is based on a multidisciplinary approach combining biochemistry, synthesis, spectroscopy, and computational methods that give insight into the biological control of radical reactivity. Mechanistic studies including isotope kinetics will explore the role of protons in radical catalysis. Biochemical characterization of galactose oxidase and two functional variants, glyoxol oxidase and glycerol oxidase, will provide a framework for comparing the structure and function of the radicals in these proteins. The comparative anatomy of glyoxal oxidase will be developed through crystallographic and mechanistic studies on wild type and mutant protein. Spectroscopic studies (absorption, circular dichroism (CD), magnetic circular dichroism (MCD), Stark, and electron paramagnetic resonance (EPR) will extend structural information on the essential radicals to the electronic level that relates directly to the origins of chemical reactivity. Low temperature MCD experiments on the active site cupric ion and inorganic models will extend insight into the role of the metal in catalysis. These multiple experimental approaches will be complemented by theoretical modeling of the spectra and ab initio calculations of the electronic structure of the catalytic radical complex.
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