Interdomain Long-Range Electron Transfer Becomes Rate-Limiting in the Y216A Variant of Tyramine 尾-Monooxygenase

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• 作者：Robert L. Osborne ; Hui Zhu ; Anthony T. Iavarone ; Ninian J. Blackburn ; Judith P. Klinman
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：February 19, 2013
• 年：2013
• 卷：52
• 期：7
• 页码：1179-1191
• 全文大小：564K
• 年卷期：v.52,no.7(February 19, 2013)
• ISSN：1520-4995

文摘

The enzyme tyramine 尾-monooxygenase (T尾M) belongs to a small eukaryotic family of physiologically important mononuclear dicopper monooxygenases. The properties of this family include noncoupled mononuclear copper centers 11 脜 apart, with Cu_M performing C鈥揌 and O₂ activation and Cu_H functioning as an electron storage site [Klinman, J. P. (2006) J. Biol. Chem. 281, 3013鈥?016]. A conserved tyrosine (Y216 in T尾M) 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鈥揌 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.