Low activation barriers characterize intramolecular electron transfer in ascorbate oxidase.

Anaerobic reduction kinetics of the zucchini squash ascorbate oxidase (AO; L-ascorbate:oxygen oxidoreductase, EC 1.10.3.3) by pulse radiolytically produced CO2- radical ions were investigated. Changes in the absorption bands of type 1 [Cu(II)] (610 nm) and type 3 [Cu(II)] (330 nm) were monitored over a range of reactant concentrations, pH, and temperature. The direct bimolecular reduction of type 1 [Cu(II)] [(1.2 +/- 0.2) x 10(9) M-1.s-1] was followed by its subsequent reoxidation in three distinct phases, all found to be unimolecular processes with the respective specific rates of 201 +/- 8, 20 +/- 4, and 2.3 +/- 0.2 s-1 at pH 5.5 and 298 K. While at this pH no direct bimolecular reduction was resolved in the 330-nm band, at pH 7.0 such a direct process was observed [(6.5 +/- 1.2) x 10(8) M-1.s-1]. In the same slower time domains where type 1 [Cu(I)] reoxidation was monitored, reduction of type 3 [Cu(II)] was observed, which was also concentration independent and with identical rate constants and amplitudes commensurate with those of type 1 [Cu(II)] reoxidation. These results show that after electron uptake by type 1 [Cu(II)], its reoxidation takes place by intramolecular electron transfer to type 3 [Cu(II)]. The observed specific rates are similar to values reported for the limiting-rate constants of AO reduction by excess substrate, suggesting that internal electron transfer is the rate-determining step of AO activity. The temperature dependence of the intramolecular electron transfer rate constants was measured from 275 to 308 K at pH 5.5 and, from the Eyring plots, low activation enthalpies were calculated--namely, 9.1 +/- 1.1 and 6.8 +/- 1.0 kJ.mol-1 for the fastest and slowest phases, respectively. The activation entropies observed for these respective phases were -170 +/- 9 and -215 +/- 16 J.K-1.mol-1. The exceptionally low enthalpy barriers imply the involvement of highly optimized electron transfer pathways for internal electron transfer.