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 fre ezing 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 similar to 10(6) M(-1) s(-1)) and occurs at the blu e Type I Cu center. Subsequent to this, a slower, protein concentratio n-independent intramolecular reoxidation of the Type I Cu is observed, with k(ET) similar to 150 S-1, resulting in 40-50% reoxidation of the blue Cu center and the establishment of an electron transfer (ET) equ ilibrium between the various Cu centers in AO. When such a sample of A O was frozen overnight at -30 degrees C, flash photolysis of the thawe d sample showed no effect on the kinetics of reduction of the Type I C u by 5-DRFH'. However, the rate constant for intramolecular ET decreas ed to a value of 2.7 s(-1), with only 20% reoxidation of the Type I ce nter. Reduction of the enzyme with ascorbic acid, followed by O-2 oxid ation, resulted in restoration of rapid intramolecular reoxidation (k( ET) = 130 S-1) with 33% of the Type I Cu reduced by 5-DRFH' being reox idized. These results are consistent with previous work which showed t hat samples of AO with initially low activity can be reactivated by as corbic acid turnover in the presence of O-2. When AO was frozen in the presence of ascorbic acid, similar inhibition of intramolecular ET wa s obtained, whereas upon turnover of this sample by further addition o f ascorbic acid and exposure to O-2 activity was not restored. The eff ects of addition of (NH4)(2)SO4, Na2SO4, NH4Cl, NaCl, KCl, and KF on t he kinetics of Type I Cu reduction by 5-deazariboflavin semiquinone an d on the subsequent intramolecular ET were also examined. A twofold in crease 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 a ll three chloride salts. Although the intramolecular ET rate constant was not altered, the fraction of reduced Type I Cu reoxidized by the t rinuclear cluster decreased with increasing Cl- concentration, regardl ess of the cation. Total inhibition of intramolecular ET was observed at a significantly lower concentration of RF than observed with the CL (-) salts. Sulfate ion had no effect on either parameter. These change s are thus ion specific, suggesting that they are related to ion bindi ng by the protein, possibly at one of the coppers of the trinuclear cl uster. (C) 1997 Academic Press.