The mechanism of O-2 reduction by copper amine oxidase from Arthrobacter globiformus (AGAO) is analyzed in relation to the cobalt-substituted protein. The enzyme utilizes a tyrosine-derived topaquinone cofactor to oxidize primary amines and reduce O-2 to H2O2. Steady-state kinetics indicate that amine-reduced CuAGAO is rewddized by O-2 >10(3) times faster than the CoAGAO analogue. Complementary spectroscopic studies reveal that the difference in the second order rate constant, k(cat)/K-M(O-2), arises from the more negative redox potential of Co-III/II in relation to Cu-II/I. Indistinguishable competitive oxygen-18 kinetic isotope effects are observed for the two enzymes and modeled computationally using a calibrated density functional theory method. The results are consistent with a mechanism where an end-on (eta(1))-metal bound superoxide is reduced to an eta(1)-hydroperoxide in the rate-limiting step.