The enzyme tyramine β-monooxygenase (TβM) belongs to a small eukaryotic family of physiologically important mononuclear dicopper monooxygenases. The properties of this family include noncoupled mononuclear copper centers ~11 Å apart, with Cu(M) performing C-H and O(2) activation and Cu(H) functioning as an electron storage site [Klinman, J. P. (2006) J. Biol. Chem. 281, 3013-3016]. A conserved tyrosine (Y216 in TβM) is positioned between the copper domains and is associated with Cu(H) (through an interaction with a Cu(H)-coordinating histidine). Mutations at Y216 (to W, I, and A) indicate little or no difference in electron paramagnetic resonance spectra, while X-ray absorption spectroscopy studies show only a very small decrease in distance between Cu(M) and its Met471 ligand in reduced enzyme. High-performance liquid chromatography assays demonstrate that turnover of substrate is complete with Y216W and Y216I, whereas Y216A undergoes a secondary inactivation that is linked to oxidation of ligands at Cu(M). Steady-state kinetic and isotope effect measurements were investigated. The significantly elevated K(m,Tyr) for Y216A, together with a very large (D)(k(cat)/K(m,Tyr)) of ~12, indicates a major impact on the binding of substrate at the Cu(M) site. The kinetic and isotopic parameters lead to estimated rate constants for C-H bond cleavage, dissociation of substrate from the Cu(M) site, and, in the case of Y216A, the rate of electron transfer (ET) from Cu(H) to Cu(M). These studies uncover a rate-limiting ET within the solvent-filled interface and lead to a paradigm shift in our understanding of the mononuclear dicopper monooxygenases.