Laser flash photolysis experiments on the effects of freezing and salt addition on intramolecular electron transfer within one-electron reduced ascorbate oxidase.
Laser flash photolysis has been used to investigate the effects of freezing protein solutions and of adding various salts on the kinetics of one-electron photoreduction by 5-deazariboflavin semiquinone (5-DRFH.) of oxidized ascorbate oxidase (AO) from zucchini in 100 mM phosphate buffer (pH 7.0). The initial reaction between oxidized AO and 5-DRFH. is quite rapid (k approximately 10(8) M-1 s-1) and occurs at the blue Type I Cu center. Subsequent to this, a slower, protein concentration-independent intramolecular reoxidation of the Type I Cu is observed, with kET approximately 150 s-1, resulting in 40-50% reoxidation of the blue Cu center and the establishment of an electron transfer (ET) equilibrium between the various Cu centers in AO. When such a sample of AO was frozen overnight at -30 degrees C, flash photolysis of the thawed sample showed no effect on the kinetics of reduction of the Type I Cu by 5-DRFH. However, the rate constant for intramolecular ET decreased to a value of 2.7 s-1, with only 20% reoxidation of the Type I center. Reduction of the enzyme with ascorbic acid, followed by O2 oxidation, resulted in restoration of rapid intramolecular reoxidation (kET = 130 s-1), with 33% of the Type I Cu reduced by 5-DRFH. being reoxidized. These results are consistent with previous work which showed that samples of AO with initially low activity can be reactivated by ascorbic acid turnover in the presence of O2. When AO was frozen in the presence of ascorbic acid, similar inhibition of intramolecular ET was obtained, whereas upon turnover of this sample by further addition of ascorbic acid and exposure to O2, activity was not restored. The effects of addition of (NH4)2SO4, Na2SO4, NH4Cl, NaCl, KCl, and KF on the kinetics of Type I Cu reduction by 5-deazariboflavin semiquinone and on the subsequent intramolecular ET were also examined. A twofold increase in the bimolecular rate constant for reduction of the Type I Cu was observed for the two sodium salts at high concentrations (500 mM). Intramolecular ET was also significantly affected upon addition of all three chloride salts. Although the intramolecular ET rate constant was not altered, the fraction of reduced Type I Cu reoxidized by the trinuclear cluster decreased with increasing Cl- concentration, regardless of the cation. Total inhibition of intramolecular ET was observed at a significantly lower concentration of KF than observed with the Cl- salts. Sulfate ion had no effect on either parameter. These changes are thus ion specific, suggesting that they are related to ion binding by the protein, possibly at one of the coppers of the trinuclear cluster.