Interdomain long-range electron transfer becomes rate-limiting in the Y216A variant of tyramine β-monooxygenase

Robert L. Osborne, Hui Zhu, Anthony T. Iavarone, Ninian J. Blackburn, Judith P. Klinman

Research output: Contribution to journalArticle

13 Scopus citations


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 CuM performing C-H and O2 activation and CuH 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 CuH (through an interaction with a CuH-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 CuM 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 CuM. Steady-state kinetic and isotope effect measurements were investigated. The significantly elevated Km,Tyr for Y216A, together with a very large D(kcat/Km,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 CuH to CuM. 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.

Original languageEnglish (US)
Pages (from-to)1179-1191
Number of pages13
Issue number7
StatePublished - Feb 19 2013

ASJC Scopus subject areas

  • Biochemistry

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