Biochimica et Biophysica Acta, 660 (198l) 1-7 Elsevier/North-Holland Biomedical Press BBA 69297 EVIDENCE FOR HEME rt CATION RADICAL SPECIES IN COMPOUND I OF HORSERADISH PEROXIDASE AND CATALASE WILLIAMR. BROWETTand MARTIN J. STILLMAN *'** Department of Chemistry, The University of Western Ontario, London, Ontario N6A 5B7 {Canada] (Received September 9th, 1980) (Revised manuscript received March 2nd, 1981) Key words: Catalase; Peroxidase; Heme ~r radical; (MCD) Magnetic circular dichroism spectra are reported for the compound I species of beef liver catalase (hydrogen- peroxide: hydrogen-peroxide oxidoreductase, EC 1.11.1.6) and horseradish peroxidase (donor: hydrogen- peroxide oxidoreductase, EC 1.11.1.7) and the n cation radical derivatives of porphydns that have been suggested as models of the electronic configuration of the heme in the compound I species of these enzymes. Comparison of the magnetic circular dichroism spectra of the compound I species with the spectra of [Co(octaethylpor- phydn)]2Br and [Co(octaethylporphyrin)]2CIO4 indicates that in both the intermediate enzyme species the heme has been oxidized to a ~r cation radical. While there is a clear distinction between the magnetic circular dichroism spectra of the 2A2u porphydn, [Co(IlI)octaethylporphydn]2CIO4, and the ~Atu porphyrin, [Co(Ill)- oetaethylporphydn] 2Br, such specific differences are not observed in the spectra of the two enzymes. Analysis of our data suggests that the ground states in the two enzymes are far more similar than the ground states in the two model porphyrins. Introduction During the well known reactions of peroxidases and catalases with peroxide, an intermediate species, compound I, is formed that is two oxidizing equiv- alents above the native heine [1-10]. Two reaction schemes are applicable for these enzyme redox cycles [1,9,10]: the peroxidatic reaction for both peroxi- dases and catalases: (1) ferric enzyme + H202 ~ compound I + H20 (2) compound I + AH2 -~ compound II + AH (3) compound II + AH2 -+ ferric enzyme + AH * To whom correspondence should be addressed. ** Member of the Centre for Chemical Physics at The University of Western Ontario. Abbreviations: MCD, magnetic circular dichroism; OEP, octa- ethylporphyrin. and the catalatic reaction observed, primarily, for catalase: (4) ferric catalase + H202 ~ compound I + H20 (5) compound I+H202-+ferric catalase + 02 + H~O Whilst the stoichiometry of these reactions has been firmly established, the exact location of the oxidizing equivalents in both compounds I and II has not been determined [1-4,11-15]. The results from magnetic susceptibility experi- ments [16,17], and extensive Mossbauer [18-20] and EPR [18,21-23] studies of horseradish perox- idase compound I show that the iron in the heme has an electronic configuration of Fe(IV) with an S = 1 spin state. The second oxidizing equivalent results in a third unpaired electron that the EPR data indicate must be located close to the para- magnetic iron [18,21]. This is quite unlike the case in cytochrome c peroxidase complex ES (the 0 005-2744/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press