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

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 CuII4 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 CuI 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.