Kinetics and spectroscopic evidence that the Cu(I)-semiquinone intermediate reduces molecular oxygen in the oxidative half-reaction of Arthrobacter globiformis amine oxidase.

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).