Horseradish peroxidase (HRP) catalyzes the oxidation of a variety of aromatic substrates utilizing hydrogen peroxide, and it is found to be catalytically active in not only aqueous but also nonaqueous media. The recent optical spectroscopic studies on the ferric resting state of polyethylene glycolated HRP (PEG-HRP) suggest that the heme active site remains structurally intact in benzene. However, the reaction intermediates in organic solvents have not been clearly defined. Thus, we report here the UV-visible spectra of compound I and II for HRP dissolved in organic media utilizing rapid scanning spectroscopy.HRP is solubilized in benzene homogeneously by the covalent modification of lysine residues with polyethylene glycol. The addition of a stoichiometric quantity of hydrogen peroxide to polyethylene glycolated horseradish peroxidase (PEG-HRP) in benzene at 5°C gave the compound I chromophore. In the presence of a large excess of guaiacol, compound I is first reduced to compound II, then to the ferric enzyme. Thus, catalytic intermediates in benzene are the ferryl species as established in aqueous buffer. The slower compound I reduction with guaiacol in benzene than in buffer suggests that the accessibility of the active site for the substrates decreases in benzene.Factor analysis of the rapid scan data set reveals that the formation of compound I of PEG-HRP in benzene could consist of two steps. The first step might be the formation of an iron-peroxy complex like compound 0, and the subsequent slow step would be the O-O bond cleavage process. Although compound 0 of PEG-HRP does not seem to be accumulated at -15°C in chlorobenzene, we could significantly stabilize a ferryl species. Compound I could be maintained for more than an hour at -15°C.Our results suggest that the chemical modification of enzymes to dissolve in appropriate organic solvents might be a potentially promising approach to observe transient enzymatic reaction intermediates such as the ferryl species of cytochrome P450 c a m , at the temperature below the freezing point of buffer.