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 Blackburn, Judith P. Klinman

Research output: Contribution to journalArticle

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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 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
JournalBiochemistry
Volume52
Issue number7
DOIs
StatePublished - Feb 19 2013

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Tyramine
Mixed Function Oxygenases
Electrons
Copper
Substrates
X-Ray Absorption Spectroscopy
Ligands
X ray absorption spectroscopy
Kinetics
Electron Spin Resonance Spectroscopy
High performance liquid chromatography
Enzymes
Histidine
Isotopes
Paramagnetic resonance
Tyrosine
Rate constants
Assays
Chemical activation
High Pressure Liquid Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Interdomain long-range electron transfer becomes rate-limiting in the Y216A variant of tyramine β-monooxygenase. / Osborne, Robert L.; Zhu, Hui; Iavarone, Anthony T.; Blackburn, Ninian; Klinman, Judith P.

In: Biochemistry, Vol. 52, No. 7, 19.02.2013, p. 1179-1191.

Research output: Contribution to journalArticle

Osborne, Robert L. ; Zhu, Hui ; Iavarone, Anthony T. ; Blackburn, Ninian ; Klinman, Judith P. / Interdomain long-range electron transfer becomes rate-limiting in the Y216A variant of tyramine β-monooxygenase. In: Biochemistry. 2013 ; Vol. 52, No. 7. pp. 1179-1191.
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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 {\AA} 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.",
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