MOSSBAUER AND ELECTRON-PARAMAGNETIC RESONANCE STUDIES OF HORSERADISH-PEROXIDASE AND ITS CATALYTIC INTERMEDIATES

The Mossbauer and EPR studies on horseradish peroxidase in the native state and the reaction intermediates with peroxide and chlorite are reported. A detailed analysis of the electronic state of the heme iron is given, and comparisons are drawn with related systems. The native enzyme is high-spin ferric and thus has 3 Kramers doublets. The unusual magnetic properties of the ground doublet and the large energy of the 2, (E2-E1)/k .apprxeq. 41 K, and 3rd doublet, (E3-E1)/k .gtorsim. 170 K, can be modeled with a quartet admixture of .apprx. 11% to the spin sextet. All evidence suggests a ferryl, OFeIV, state of the heme iron in compounds I and II and related complexes. The small isomer shift, .delta.Fe .apprxeq. 0.06 mm/s, the (positive) quadrupole splitting, .DELTA.EQ .apprxeq. 1.4 mm/s, the spin S = 1, and the large positive 0 field splitting, D/k .apprxeq. 35 K, are all characteristic of the ferryl state. In the green compound I the iron weakly couples to a porphyrin radical with spin S'' = 1/2. A phenomenological model with a weak exchange interaction .vector.S.cntdot.J.cntdot..vector.S'', J .ltorsim. 0.1, D, reproduces all Mossbauer and EPR data of compound I, but the structural origin of the exchange and its apparent distribution require further study. Reaction of horseradish peroxidase with chlorite leads to compound X with .delta.Fe = 0.07 mm/s and .DELTA.EQ = 1.53 mm/s, values that are closest to those of compound II. The diamagnetism of compound III and its Mossbauer parameters .delta.Fe = 0.23 mm/s and .DELTA.EQ = -2.31 mm/s at 4.2 K clearly identify it as an oxyheme adduct.