Abstract
The mechanism of molecular oxygen activation is the subject of controversy in the copper amine oxidase family. At their active sites, copper amine oxidases contain both a mononuclear copper ion and a protein-derived quinone cofactor. Proposals have been made for the activation of molecular oxygen via both a Cu(II)-aminoquinol catalytic intermediate and a Cu(I)-semiquinone intermediate. Using protein crystallographic freeze-trapping methods under low oxygen conditions combined with single-crystal microspectrophotometry, we have determined structures corresponding to the iminoquinone and semiquinone forms of the enzyme. Methylamine reduction at acidic or neutral pH has revealed protonated and deprotonated forms of the iminoquinone that are accompanied by a bound oxygen species that is likely hydrogen peroxide. However, methylamine reduction at pH 8.5 has revealed a copper-ligated cofactor proposed to be the semiquinone form. A copper-ligated orientation, be it the sole identity of the semiquinone or not, blocks the oxygen-binding site, suggesting that accessibility of Cu(I) may be the basis of partitioning O2 activation between the aminoquinol and Cu(I).
Highlights
Copper amine oxidases activate O2 either at the copper center or aminoquinol cofactor
Methylamine reduction of Hansenula polymorpha amine oxidase 1 (HPAO-1) in an oxygen-limiting atmosphere at pH 6.0, 7.0, and 8.5 has allowed the successful trapping of three distinct species relevant to the oxidative halfreaction. Two of these intermediates correspond to the iminoquinone form of the cofactor, as protonated and deprotonated species
A significant feature of the iminoquinone structures at pH 6.0 and 7.0 is that all of the cofactors in the asymmetric unit are in the off-copper conformation. This is an important observation because the oxidized structure solved in the same space group indicates that four of the six cofactors are in mixed populations of on-copper and off-copper orientations [32]
Summary
Copper amine oxidases activate O2 either at the copper center or aminoquinol cofactor. Stopped-flow kinetics of the oxidative half-reaction in AGAO [7] and kinetic competitive oxygen kinetic isotope effects (KIE) and density functional theory calculations in pea seedling CAO [16] confirmed innersphere electron transfer from Cu(I) to be the likely mechanism of O2 activation in these enzymes. This makes sense in light of their faster electron transfer rates, elevated formation of Cu(I)semiquinone upon anaerobic substrate reduction, and higher kcat values [2].
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