Pulse-radiolysis studies on the oxidised form of the multicopper enzyme ascorbate oxidase: evidence for two intramolecular electron-transfer steps

Abstract

Two intramolecular electron-transfer steps have been identified in pulse-radiolysis studies on the multicopper enzyme ascorbate oxidase, which has four Cu atoms in the catalytically active monomer form. The enzyme was initially in the fully oxidised Cu^II₄ state. Pulse radiolysis was carried out at 19 °C, pH 7.0 (40 mM phosphate), l= 0.100 M, in the first instance with formate to generate CO₂˙^– as the only (reducing) radical present. When in addition appropriate amounts of methyl viologen (1,1′-dimethyl-4,4′-bipyridinium, dmbipy²⁺), deazaflavin, or lumiflavin were present the CO₂˙^– was rapidly converted into CO₂ with concomitant formation of the corresponding radical form (e.g. dmbipy˙⁺) as the only reactive species. Reactions of all four radicals with ascorbate oxidase (reactant in excess) give a metastable type 1 copper reduced product. Contrary to earlier reports two intramolecular electron-transfer steps k₁ and k₂ follow in which the colour of the type 1 site is restored. Both are independent of the radical type used. Thus the first stage is assigned as electron transfer from the type 1 Cu^I to the trinuclear combined type 3/type 2 site. Rate constants k₁ and k₂/s^–1 are for CO₂˙^–(120, 2.0), dmbipy˙⁺(127, 2.3), deazaflavin (121, 2.5) and lumiflavin (97, 2.4). Mechanistic assignments for the two stages are considered, and an apparent disagreement with a previous study is explained.