Ascorbate Oxidase

Abstract

Abstract Ascorbate oxidase (AO) is a multicopper oxidase related to laccase and ceruloplasmin. It catalyzes the four‐electron reduction of dioxygen to water with concomitant one‐electron oxidation of the substrate. The most efficient reducing substrate is l‐ascorbate, but several one‐electron reductants may interact with AO. It occurs in higher plants in both vegetative and reproductive organs in the cytoplasm of the cells. The biological function of AO is still unclear. A very attractive function is its involvement in anti‐oxidative defense. AO has been intensively characterized by spectroscopic, kinetic, electrochemical, and X‐ray crystallographic methods. The amino acid sequences have been determined from several plant sources and fungi. The X‐ray structure of AO from zucchini shows that the monomer of 552 residues is built up by three domains arranged sequentially on the polypeptide chain. The folding of all three domains is of a ß‐barrel type. Each monomer has three disulfide bridges. It contains a mononuclear type 1 copper site in domain 3 and a trinuclear type 2/type 3 copper site between domains 1 and 3. The mononuclear copper site has the four canonical type 1 copper ligands (His, Cys, His, Met) also found in plastocyanin and azurin. The trinuclear copper site has eight histidine ligands (symmetrically supplied by domains 1 and 3) and two oxygen ligands. The type 3 copper pair is bridged by an OH − or O 2− . The remaining copper has two histidine ligands and an OH − or H 2 O ligand. The average copper–copper distance in oxidized AO is 3.74 Å. The shortest distance between the type 1 copper center and the trinuclear copper center is 12.2 Å. X‐ray structures of the fully reduced, peroxide and azide forms of AO have been determined. In the fully reduced form, the trinuclear site undergoes strong changes. The bridging oxygen ligand is released and the copper–copper distances increase from an average of 3.7 Å to 5.1 Å for the copper pair and to about 4.2 Å from the paired atoms to the third copper atom. In the peroxide form, the peroxide is bound end‐on to one copper atom of the copper pair while two azide molecules are bound in the same fashion to the same copper atom. Spectroscopic and kinetic studies reveal that the entry site for electrons from the reducing substrate is the mononuclear copper and the dioxygen binding site is the trinuclear copper species. The enzyme acts in a ‘two‐site ping‐pong bi‐bi’ mechanism. A proposal for the catalytic mechanism of AO based on the available data is given. Aspects of intramolecular electron transfer are discussed.