Investigation of the Mechanism of Action of Microperoxidase-11, (MP11), a Potential Anti-cataract Agent, with Hydrogen Peroxide and Ascorbate

Abstract

The interaction of hydrogen peroxide, ascorbate and microperoxidase-11 (MP11), a ferriheme undecapeptide derived from cytochrome c, has been investigated using spectrophotometry, oxymetry, electron paramagnetic resonance (EPR), and mass spectroscopy techniques. It is shown that in 50m M phosphate pH 7.0–7.4 in the absence of other reactants H2O2induces a concentration-dependent decrease in absorption at the Soret band (399nm) of the microperoxidase, with concomitant H2O2decomposition and oxygen evolution. The reaction causes irreversible heme degradation, concomitant with loss of enzymatic activity. Ascorbate effectively protects MP11 from degradation and inhibits oxygen evolution. At ascorbate concentrations greater than that of H2O2, microperoxidase degradation is almost completely prevented. Mass spectrometry showed that H2O2oxidizes the microperoxidase to a monooxygenated product, which did not form if ascorbate was included in the reaction system. There appears to be a 1:1 relationship between H2O2degradation and ascorbate oxidation. EPR experiments revealed that an ascorbate radical was formed during the reaction. These reactions may be described by a scheme where a putative ‘compound I’ of the microperoxidase is reduced by ascorbate back to the original redox state (ferric) of the peroxidase in two one-electron steps, concomitantly with oxidation of the ascorbate to an ascorbate radical or in one two-electron transfer step forming dehydroascorbate. In the absence of ascorbate, the ‘compound I’ reacts further with the peroxide causing microperoxidase degradation and partial oxygen evolution. These observations are relevant to the interaction of ferrihemes with H2O2and ascorbic acid and may be pertinent for the potential application of MP11 as an anti-cataract agent.