| The involvement of protein-derived radicals in biological oxidation/reduction reactions has been established in enzymes that include photosystem II, the ribonucleotide reductases and cytochrome C peroxidase. Each of these uses an amino acid residue, together with inorganic cofactor, during catalytic turnover. Described in this thesis are heme and copper-containing enzymes which are proposed to use amino acid radicals or the odd electron forms of post-translationally modified amino acids during catalysis.;Among the amino acid radicals, the tyrosyl radical is perhaps the most commonly found. In an early example, a non-catalytic tyrosyl radical was discovered upon treatment of myoglobin with H2O2. Tyrosine exhibits a relatively low redox potential and pKa compared to other amino acids, explaining its ease of formation. Yet the thermodynamics associated with the tyrosyl radical raises questions as to its usefulness in catalysis, especially in biosynthetic reactions which require the cleavage C--H bonds and in long range electron transfer reactions that occur over several residues and sometimes between proteins.;This dissertation concentrates on protein-derived radicals which utilize O2 in and selective dioxygenase and oxidase mechanisms. The first two chapters include mechanistic investigations of C--H bond oxidations catalyzed by prostaglandin H synthase, otherwise known as cyclooxygenase, and rice alpha-(di)oxygenase. The third chapter focuses on the role of the Cu2+/topaquinone (TPQ) to Cu2+/TPQ semiquinone conversion during molecular oxygen reduction by copper amine oxidase. Studies were conducted to first address kinetic mechanisms and then the underlying reaction chemistry. Experiments that probe the steady-state rates of enzyme turnover as a function of temperature, pH, and solvent viscosity are described. Various kinetic isotope effect and isotopic tracer experiments are also highlighted. The results are assimilated in the interpretation of the protein-derived radical oxidation mechanisms. |
