Dicopper monooxygenases are critical for the biogenesis of catecholamines, and the mechanism by which such enzymes regulate the coupling of C-H and oxygen activation remains unclear. The current proposal aims to improve the understanding of this mechanism by which enzymes like tyramine beta monooxygenase (T[unreadable]M) activate dioxygen/C-H bonds and hydroxylate substrates. The objectives will be accomplished using a workable recombinant system available for T[unreadable]M. Having a T[unreadable]M recombinant system provides an excellent model for dopamine beta monooxygenase (D[unreadable]M) and will improve the understanding of neurotransmission in insects. In addition, the T[unreadable]M expression system makes generating site-specific mutants easier and allows a quick and deliberate approach to address mechanistic questions as they develop. T[unreadable]M likely hydroxylates tyramine by a mechanism similar to dopamine hydroxylation catalyzed by D[unreadable]M. Consequently, a three-fold research approach will be applied to examine the role of individual active site amino acid residues. First, mutants will be generated to test the proposal described herein that communication between the Cu sites is extensive. Second, traditional kinetic parameters (kcat, kcat/Km) will be determined for each mutant and compared to the native enzyme, thus providing important information on how each targeted residue is involved with either electron transfer (detected in kcat) or substrate activation (detected in kcat/Km). Third, substrate hydroxylation is proposed to take place by a hydrogen tunneling event. Thus, the kinetic mechanism will be probed using radioactive labeled substrates to determine the intrinsic kinetic isotope effect (KIE) on the C-H activation step. The effect of mutation on hydrogen tunneling will be examined and this will be manifest in the temperature dependence of the KIE. PUBLIC HEALTH RELEVANCE The enzyme, dopamine beta-monooxygenase (D[unreadable]M), controls the levels of the neurotransmitters, dopamine and norepinephrine. Irregularities in the norepinephrine pathway are linked to hypertension, heart failure, depression, and Parkinson's disease. Specifically, D[unreadable]M inhibitors have been developed as a potential treatment of congestive heart failure. The primary symptoms of Parkinson's disease are the result of insufficient generation and action of dopamine. Using an analogous system, T[unreadable]M, will clarify the mechanism of catalysis for this family of enzymes providing the molecular framework for our understanding of a number of neurological diseases.