Control of the transfer of oxidizing equivalents between heme iron and free radical site in yeast cytochrome c peroxidase.

A procedure has been developed for obtaining yeast cytochrome c peroxidase with the heme iron in the Fe(IV) state, without the concomitant formation of the protein free radical that occurs in the ES compound resulting from the oxidation of ferric peroxidase with hydrogen peroxide. In this procedure, ferrous peroxidase, prepared either by photochemical reduction or by trapping the dithionite-reduced enzyme with carbon monoxide, is oxidized with a stoichiometric amount of hydrogen peroxide. The resulting Fe(IV) enzyme oxidizes ferrocyanide monophasically, with a rate constant of 4 x 10(3) M-1 S-1. The optical spectrum of the free radical was obtained as the difference between the spectra of the ES and Fe(IV) compounds. EPR spectra of ES compound prepared with [16O]-and [17O]hydrogen peroxide are identical, demonstrating that no fragment of the oxidant is associated with the free radical. The heme in the Fe(IV) enzyme is stable and does not oxidize the free radical site either intra- or intermolecularly. On the other hand, previous results from the presteady state kinetics of reduction and reductive titrations of the ES compound with ferrocyanide imply that the heme and free radical sites exchange oxidizing equivalents, in particular that the radical site once reduced can be reoxidized, either intra- or intermolecularly, by the ferryl heme. To resolve these contradictions, we propose a catalytic mechanism for cytochrome c peroxidase in which the radical site can exist in two conformations having very different reduction potentials and in which a significant flow of oxidizing equivalents between heme and free radical sites occurs only (i) during the hydrogen peroxide oxidation of the resting Fe(III) enzyme to form compound ES and (ii) within the initial transient intermediate formed upon the one-electron reduction of this oxidized product.