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 50 m M phosphate pH 7. 0-7.4 in the absence of other reactants H(2)O(2)induces a concentration-dependent decrease in absorption at the Soret band (399 nm) of the microperoxidase, with concomitant H(2)O(2)decomposition 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 H(2)O(2), microperoxidase degradation is almost completely prevented. Mass spectrometry showed that H(2)O(2)oxidizes 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 H(2)O(2)degradation 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 H(2)O(2)and ascorbic acid and may be pertinent for the potential application of MP11 as an anti-cataract agent.
Copyright 2000 Academic Press.