Ascorbate Oxidase from Cucurbita pepo medullosa

Abstract

1. Ascorbate oxidase has been isolated from the green squash Cucurbitapepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20 000 M, could be separated during the purification of the oxidase. The six-step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 f 0.11140000 MI) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 2. The pure enzyme is characterized by the following optical purity indices: A2so/Ablo = 25 +_ 0.5, A330/A610 = 0.65 & 0.05 and A6I0/A500 = 7.0 & 0.25. The molar absorption coefficient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts to 9700 M-' em-'.3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, g , = 2.058, g, = 2.036; A , = 5.0 mT, A , = A , = 0.5 mT, for the type-2 (non-blue) copper gli = 2.242, g l = 2.053; All = 19.0 mT, A1 = 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type-1 class, and one to the type-2 class, as demonstrated by computer simulations of the EPR spectra.4. To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L-ascorbate or reductate must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (%ax = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (l/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm ( A s = 1000 f 50 M-'

Probing the location of the substrate binding site of ascorbate oxidase near type 1 copper: an investigation through spectroscopic, inhibition and docking studies

“…Ascorbate oxidase was isolated and purified from green zucchini squash (Cucurbita pepo) according to the procedure of Marchesini and Kroneck (1979). The protein had optical indices A 280 /A 610 = 24.4 and A 330 /A 610 = 0.8 in 0.1 M phosphate buffer, pH 5.7.…”

Low activation barriers characterize intramolecular electron transfer in ascorbate oxidase.

“…Ascorbate oxidase (AO; L-ascorbate:oxygen oxidoreductase, EC 1.10.3.3) was found to be a 140-kDa protein containing eight copper ions per molecule, which can be classified into the above T1, T2, and T3 by their spectroscopic properties (4).…”

Mavicyanin, a Blue Copper Protein from Cucurbita pepo medullosa

“…Over the past decade a great interest has been directed towards the investigation of the so-called type-1 or blue copper in electron-transferring proteins such as plastocyanin or azurin (the reader is referred to the excellent review articles by Fee [l] and Boulter et al [2]), or in the multicopper oxidases laccase, ceruloplasmin and ascorbate oxidase [3,4]. Within the series of low-molecular-weight copper proteins discovered so far, certainly plastocyanin from different plants and algae [2] and bacterial azurin [l, 5,6] have been studied in great detail with respect to catalytic properties or bioinorganic structure of the active site [7,8].…”