Inner-Sphere Mechanism for Molecular Oxygen Reduction Catalyzed by Copper Amine Oxidases

Content Provider	Scilit
Author	Mukherjee, Arnab Smirnov, Valeriy V. Lanci, Michael P. Brown, Doreen E. Shepard, Eric M. Dooley, David M. Roth, Justine P.
Copyright Year	2008
Abstract	Copper and topaquinone (TPQ) containing amine oxidases utilize $O_{2}$ for the metabolism of biogenic amines while concomitantly generating $H_{2}O_{2}$ for use by the cell. The mechanism of $O_{2}$ reduction has been the subject of long-standing debate due to the obscuring influence of a proton-coupled electron transfer between the tyrosine-derived TPQ and copper, a rapidly established equilibrium precluding assignment of the enzyme in its reactive form. Here, we show that substrate-reduced pea seedling amine oxidase (PSAO) exists predominantly in the $Cu^{I}$, TPQ semiquinone state. A new mechanistic proposal for $O_{2}$ reduction is advanced on the basis of thermodynamic considerations together with kinetic studies (at varying pH, temperature, and viscosity), the identification of steady-state intermediates, and the analysis of competitive oxygen kinetic isotope effects,$ ^{18}$O KIEs, [k_{cat}/K_{M}(^{16,16}O_{2})]/[k_{cat}/K_{M}(^{16,18}$O_{2}$)]. The$ ^{18}$O KIE = 1.0136 ± 0.0013 at pH 7.2 is independent of temperature from 5 °C to 47 °C and insignificantly changed to 1.0122 ± 0.0020 upon raising the pH to 9, thus indicating the absence of kinetic complexity. Using density functional methods, the effect is found to be precisely in the range expected for reversible $O_{2}$ binding to $Cu^{I}$ to afford a superoxide, [Cu^{II}(η^{1}-O_{2}$)^{−I}]^{+}$, intermediate. Electron transfer from the TPQ semiquinone follows in the first irreversible step to form a peroxide, Cu^{II}(η^{1}-O_{2}$)^{−II}$, intermediate driving the reduction of $O_{2}$. The similar$ ^{18}$O KIEs reported for copper amine oxidases from other sources raise the possibility that all enzymes react by related inner-sphere mechanisms although additional experiments are needed to test this proposal.
Related Links	http://europepmc.org/articles/pmc2574554?pdf=render https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2574554/pdf
Ending Page	9473
Page Count	15
Starting Page	9459
ISSN	00027863
e-ISSN	15205126
DOI	10.1021/ja801378f
Journal	Journal of the American Chemical Society
Issue Number	29
Volume Number	130
Language	English
Publisher	American Chemical Society (ACS)
Publisher Date	2008-06-27
Access Restriction	Open
Subject Keyword	Journal: Journal of the American Chemical Society Biochemistry and Molecular Biology Oxidation Reduction Oxygen Isotopes Hydrogen Peroxide
Content Type	Text
Resource Type	Article
Subject	Chemistry Colloid and Surface Chemistry Biochemistry Catalysis