Two substrate binding sites in ascorbate peroxidase: The role of arginine 172

Site-directed mutagenesis has been used to probe the role of Arg172 in asco rbate utilization by ascorbate peroxidase. Arg172 was changed to lysine, gl utamine, and asparagine. Although each of these variants retains the abilit y to utilize guaiacol as a reductant, they exhibit large decreases in their steady-state rates of ascorbate utilization. Spectroscopic, steady-state, and transient-state experiments indicate that these variant proteins are ca pable of reacting with hydrogen peroxide to form Compound I, but their abil ity to oxidize ascorbate to form Compound II, and subsequently the resting state, is severely impeded. Results are presented which highlight the impor tance of Arg172, and a model is proposed to explain its role in ascorbate u tilization.