Shepard Eric M, Okonski Kristina M, Dooley David M
Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, USA.
Biochemistry. 2008 Dec 30;47(52):13907-20. doi: 10.1021/bi8011516.
The role of copper during the reoxidation of substrate-reduced amine oxidases by O(2) has not yet been definitively established. Both outer-sphere and inner-sphere pathways for the reduction of O(2) to H(2)O(2) have been proposed. A key step in the inner-sphere mechanism is the reaction of O(2) directly with the Cu(I) center of a Cu(I)-semiquinone intermediate. To thoroughly examine this possibility, we have measured the spectral changes associated with single-turnover reoxidation by O(2) of substrate-reduced Arthrobacter globiformis amine oxidase (AGAO) under a wide range of conditions. We have previously demonstrated that the internal electron-transfer reaction [Cu(II)-TPQ(AMQ) --> Cu(I)-TPQ(SQ)] (where TPQ(AMQ) is the aminoquinol form of reduced TPQ and TPQ(SQ) is the semiquinone form) occurs at a rate that could permit the reaction of O(2) with both species to be observed on the stopped-flow time scale [Shepard, E. M., and Dooley, D. M. (2006) J. Biol. Inorg. Chem. 11, 1039-1048]. The transient absorption spectra observed for the reaction of O(2) with substrate-reduced AGAO provide compelling support for the reaction of the Cu(I)-TPQ(SQ) form. Further, global analysis of the kinetics and the transient absorption spectra are fully consistent with an inner-sphere reaction of the Cu(I)-semiquinone intermediate with O(2) and are inconsistent with an outer-sphere mechanism for the reaction of the reduced enzyme with O(2).
铜在底物还原型胺氧化酶被O₂再氧化过程中的作用尚未明确确定。已经提出了将O₂还原为H₂O₂的外层和内层途径。内层机制的关键步骤是O₂直接与Cu（I）-半醌中间体的Cu（I）中心反应。为了彻底研究这种可能性，我们在广泛的条件下测量了底物还原型球形节杆菌胺氧化酶（AGAO）被O₂单周转再氧化相关的光谱变化。我们之前已经证明内部电子转移反应[Cu（II）-TPQ（AMQ）→Cu（I）-TPQ（SQ）]（其中TPQ（AMQ）是还原型TPQ的氨基喹啉形式，TPQ（SQ）是半醌形式）的发生速率能够使O₂与这两种物质的反应在停流时间尺度上被观察到[谢泼德，E.M.，和杜利，D.M.（2006年）《生物无机化学杂志》11，1039 - 1048]。观察到的O₂与底物还原型AGAO反应的瞬态吸收光谱为Cu（I）-TPQ（SQ）形式的反应提供了有力支持。此外，对动力学和瞬态吸收光谱的全局分析完全符合Cu（I）-半醌中间体与O₂的内层反应，并且与还原酶与O₂反应的外层机制不一致。
