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Article

Kinetics and Spectroscopic Evidence That the Cu(I)−Semiquinone Intermediate Reduces Molecular Oxygen in the Oxidative Half-Reaction of Arthrobacter globiformis Amine Oxidase^†

Supporting Info

Eric M. Shepard, Kristina M. Okonski and David M. Dooley*

Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717

Biochemistry, 2008, 47 (52), pp 13907–13920

DOI: 10.1021/bi8011516

Publication Date (Web): December 3, 2008

†

This research was supported by Grant GM 27659 (D.M.D.) from the National Institutes of Health.

* To whom correspondence should be addressed. E-mail: dmdooley@montana.edu. Telephone: (406) 994-4371. Fax: (406) 994-7989.

Abstract

The role of copper during the reoxidation of substrate-reduced amine oxidases by O₂ has not yet been definitively established. Both outer-sphere and inner-sphere pathways for the reduction of O₂ to H₂O₂ have been proposed. A key step in the inner-sphere mechanism is the reaction of O₂ 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 O₂ 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 O₂ 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 O₂ 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 O₂ and are inconsistent with an outer-sphere mechanism for the reaction of the reduced enzyme with O₂.