Studies on the Equilibria and Kinetics of the Reactions of Peroxidase with Ligands

Abstract

Abstract Ferrous horseradish peroxidase, when mixed rapidly with oxygen-containing solutions, reacts with 1 molecular equivalent of oxygen to form 1 molecular equivalent of a product, oxyperoxidase. This reaction follows second order kinetics and proceeds without detectable intermediates. Ferrous peroxidase when titrated with oxygen yields 4 moles of ferric peroxidase for every mole of oxygen consumed. We believe that in this reaction the initial product is oxyperoxidase, which subsequently reacts with a further 3 moles of ferroperoxidase. Oxyperoxidase, therfore, retains all 4 oxidizing equivalents of the oxygen. The spectrum of oxyperoxidase closely resembles that of oxyhemoglobin and is very similar to that of the previously described Compound III of peroxidase. On the basis of this and other evidence we suggest that Compound III is actually oxyperoxidase. Our preparations of oxyperoxidase undergo a slow, spontaneous decay to ferric peroxidase; the process follows first order kinetics and no intermediates are detected. We have been unable to show dissociation of oxyperoxidase, even with an intense light pulse. Oxyperoxidase oxidizes dithionite directly, in a reaction not found in other heme proteins. Oxyperoxidase on the one hand shows striking similarities to other oxyheme proteins, and on the other hand may accept electrons, for example, from dithionite or ferroperoxidase. The conjunction of these properties in one molecule makes it a prototype of a terminal oxidase.