OXIDATION-REDUCTION PROPERTIES OF COMPOUND-I AND COMPOUND-II OF ARTHROMYCES-RAMOSUS PEROXIDASE

At neutral pH, compound I of Arthromyces ramosus peroxidase (ARP) was stable and was reduced to ferric ARP without apparent formation of com pound II upon titration with ascorbate or hydroquinone. In the titrati on experiments, compound II was seen as an intermediate only at alkali ne pH. However, measuring a difference spectrum in the Soret region by a stopped-flow method, we found that compound II was formed during th e catalytic oxidation of ascorbate even at neutral pH. Using an EPR sp ectrometer with a microflow system, we measured the steady-state conce ntration of benzosemiquinone formed in the ARP-catalyzed oxidation of hydroquinone. The results clearly showed that ARP catalyzes the oxidat ion of hydroquinone by a one-electron-transfer mechanism, as does hors eradish peroxidase. These observations led to the conclusion that comp ound I is reduced to compound II through a one-electron reduction by a scorbate or hydroquinone. Therefore, we concluded that ARP compound II is unusually unstable and is rapidly reduced to ferric enzyme without accumulation in the titration experiment. The unusual instability of ARP compound II is explained in terms of the high reduction potential of compound II. The reduction potentials (E(0)') of compounds I and II were measured at several pH values from redox equilibria with potassi um hexachloroiridate on the basis of E(0)' = 0.90 V for the IrCl62--Ir Cl63- couple. These values were determined to be 0.915 and 0.982 V at pH 7, respectively, and decreased with increasing pH. This pH dependen ce was markedly changed by the buffer concentration. The change of E(0 )' associated with buffer concentration had little effect on the pH ac tivity profiles of ARP-catalyzed oxidations of hydroquinone and guaiac ol.