Ascorbate oxidase anaerobic reduction

Figure 6. Anaerobic reduction of ascorbate oxidase by reductate time course of the reaction at 610 (O) and 330 nm (A). Conditions phosphate buffer (pH 7.0), I = 0.20 M, 10.0°C, enzyme 12 fxM, substrate 2.4 mM. The solid Line was obtained by computer simulation.

Figure 9. EPR spectrum of the substrate radical formed during the rapid initial phase of the anaerobic reduction of ascorbate oxidase by reductate. Conditions as in Figure 6. The spectrum was recorded at 77 K, 9.4 GHz, 100 kHz modulation frequency, 0.05 mT modulation amplitude.

In studies on the anaerobic reduction of tree laccase by hydroquinone and ascorbate (49), the existence of a plateau phase at low substrate concentration was reported for the reaction of the type 1 copper. This observation was explained in terms of an intramolecular reoxidation by the type 3 copper pair. A similar plateau phase is a dominant feature of the reduction of both chromophores of ascorbate oxidase by reductate (Figure 7). However, the plateau phase is only observed in the presence of "contaminating dioxygen rigorous removal of these dioxygen traces removes the plateau phase at all wavelengths. The reaction of reduced ascorbate oxidase with dioxygen is very rapid, k = 5 X 10 at pH... 

The anaerobic reduction of the trinuclear copper center for ascorbate oxidase with different substrates presents a distinct picture. The reaction with reductate is monophasic with a unimolecular rate constant of 100 sec (18), independent of pH. Rapid freeze-quench EPR experiments indicate that the type-2 EPR signal vanishes more slowly 18). The pulse radiolysis studies of the radicals of lumiflavin, deazaflavin, CO2 ", and MV at pH 7.0 129,130) showed a biphasic behavior with an initial, faster reaction k = 97-127 sec " ) and a final, slower reaction k 2 sec" ) 129). Different results have been obtained by Farver and Pecht 130) with CO2 " as a substrate. They found a triphasic reaction with unimolecular rate constants k = 201 sec S 2 = 20 sec", and ks = 2.3 sec. The first constant is twice that in a study by Kyritsis et al. 129), whereas the third constant is identical. The second constant was not observed in the study. 

The reaction of nitric oxide with laccase (76) and ascorbate oxidase (147) has been studied as well. Nitric oxide fully reduces fungal and tree laccase when it is added to the oxidized enzyme under anaerobic conditions. In addition the binding of one NO molecule to laccase can be detected. This is characterized by a new EPR signal and has been described as coordinated with the type-2 copper (76). Only the reduction of the type-1 copper has been observed when NO has been added to ascorbate oxidase under anaerobic conditions. 

This chapter summarizes some recent developments in the purification of ascorbate oxidase, the number of copper atoms per active molecule, and the stoichiometry of the different copper sites with reference to the classification introduced by Malkin and Malmstrom (5). Furthermore, physical properties of the metal centers are discussed in relation to other simple copper proteins that have been characterized in recent years. Finally, kinetic investigations of ascorbate oxidase reduction are presented as studied by anaerobic stopped-fiow and rapid-freeze techniques. 

Figure 3. Anaerobic reduction of ascorbate oxidase by i. ascorbate, as followed by fluorescence spectroscopy (A) and by the change in absorbance at 610 nm (B).

Anaerobic Reduction of Ascorbate Oxidase—Number of Redox Equivalents and Midpoint Potentials of the Copper Sites... 

Mechanism of Action of Ascorbate Oxidase—Kinetics of the Anaerobic Reduction by Ascorbate and Reductate... 

Figure 7. Anaerobic reduction of the type 3 Cu of ascorbate oxidase by re-ductate. Conditions as in Figure 6. Curves 1—4 show the effect of stepwise addition of reductate to the enzyme prior to kinetic stopped-fhw runs. Curve 1 represents 6 fxM dioxygen, curves 2—4 demonstrate the successive removal of dioxygen and partial reduction of the enzyme.

Anaerobic reduction of ascorbate oxidase, AOox, by ascorbate ion is an eight-electron process. Initial reduction of the type 1 centre is reversed as the type 3 centre is reduced and it is suggested that type 1 copper is close to the substrate binding site and that type 3 is involved in the interaction with molecular oxygen. Azide, which can bind to type 2 copper, has no effect on the reduction process. A potentially catalytically active intermediate is formed in the reaction between ascorbate oxidase and hydrogen peroxide at pH 7.6, 0.01 M phosphate, and 25 °C ... 

Reducing the 02 concentration around fresh fruits and vegetables reduces their respiration rate to an extent that varies with temperature, commodity, cultivar, and physiological age at harvest. Below a minimum of about 1 to 2% 02, a shift from aerobic to anaerobic respiration occurs with a concomitant increase in CO2 production Q0). The reduction in aerobic metabolism associated with reduced 02 levels is apparently not mediated by cytochrome oxidase, which has a K , value of 10"8 to 10 7 M 02 (11.12). It is more likely that reduction of aerobic respiration results from diminished activity of other oxidases such as ascorbic acid oxidase, polyphenol oxidase (FFO), and glycolic acid oxidase, whose affinities for 02 are 5 to 6 times lower than that of cytochrome oxidase (12). 

The rate of reduction of cytochrome a was examined by addition of various chemical reductants such as p-phenylenediamine, hydro-quinone, and ascorbic acid. These were then tested as substrates for the cytochrome oxidase, the activity of which was measured manometrically. Onlyp-phenylenediamine reduced cytochrome a, while the other reagents tested, except sodium dithionite, had weak reductive activity. In Fig. 11, it is shown that cytochrome a is oxygenated under aerobic conditions and is reduced under anaerobic conditions by addition of 10 Af/>-phenyl-enediamine and the trace of borohydride, used to decrease the concentration of oxygen in the solution in the cuvette. When a trace of oxidized cytochrome c is added to either system, both forms of cytochrome a are instantaneusly oxidized. As shown in Table IX (see Section III.C), the cytochrome a acquires an oxidase activity in cooperation with cytochrome c when the oxygen consumption is measured manometrically in the presence and absence of cytochrome c. 

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