Oxygen activation in a copper-containing amine oxidase

The process by which molecular oxygen is activated to enable it to function as an electron acceptor in biology is poorly understood. The quinoprotein copper-containing amine oxidase (CuAO) catalyses the conversion of primary amines into aldehydes. As well as copper, the enzyme contains an organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ). Following the formation of aldehyde, the enzyme is left as the two-electron reduced aminoquinol form. Reoxidation of the enzyme back to the resting state uses molecular oxygen, which is reduced to H2O2 in the process, with the additional release of NH3. To understand the structural basis of oxygen activation in Escherichia coli CuAO (ECAO), catalytically competent crystals were used to trap catalytic intermediates by exposing then to amine substrate and then freeze-trapping under aerobic and anaerobic conditions. Single-crystal visible microspectrophotometry was used to probe the oxidation state of the quinone in the intermediates, as TPQ exhibits a rich palette of colour changes during catalytic turnover. This review will focus on one of these structures, that of the rate-determining species in the crystal under steady-state conditions. This structure has revealed many details regarding oxygen activation in ECAO, including the site of dioxygen binding, and the proton-transfer pathways involved in H2O2 formation.

Formation in vitro of the 3,4,6-trihydroxyphenylalanine quinone cofactor

An Escherichia coli K-12 2-phenylethylamine oxidase gene with a mutated leader sequence region produced a largely inactive form of the enzyme in the cytoplasm. This form of the enzyme was activated 30-50-fold on incubation at 30 degrees C in the absence of any added cofactors. After activation the enzyme contained a quinone which was not detected in the non-activated form. This is the first report of the formation in vitro of any quinoenzyme cofactor.