Experimental and computational evidence of metal-O2 activation and rate-limiting proton-coupled electron transfer in a copper amine oxidase

The mechanism of O2 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 O2 to H2O2. Steady-state kinetics indicate that amine- reduced CuAGAO is reoxidized by O2 >10 3 times faster than the CoAGAO analogue. Complementary spectroscopic studies reveal that the difference in the second order rate constant, kcat/KM(O2), 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 (η 1 )-metal bound superoxide is reduced to an η 1 -hydroperoxide in the rate-limiting step.