title: Interdomain long-range electron transfer becomes rate-limiting in the Y216A variant of tyramine β-monooxygenase.
abstract: 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.
authors: Department of Chemistry, University of California, Berkeley, CA 94720, USA.
authors: Robert L
authors: RL
authors: Osborne
authors: Hui
authors: H
authors: Zhu
authors: Anthony T
authors: AT
authors: Iavarone
authors: Ninian J
authors: NJ
authors: Blackburn
authors: Judith P
authors: JP
authors: Klinman
mesh_terms: D000409:Alanine; D000818:Animals; D020134:Catalytic Domain; D003300:Copper / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004789:Enzyme Activation; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D009655:Octopamine / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D014439:Tyramine / Q000378:metabolism; D056928:X-Ray Absorption Spectroscopy
chemical_list: D029721:Drosophila Proteins; D009655:Octopamine; D003300:Copper; D006899:Mixed Function Oxygenases; C527639:tyramine beta-monooxygenase, Drosophila; D000409:Alanine; D014439:Tyramine