Methionine oxidation by peroxymonocarbonate, a reactive oxygen species formed from CO 2/bicarbonate and hydrogen peroxide

Abstract

Kinetic and thermodynamic evidence is reported for the role of the peroxymonocarbonate ion, HCO 4 −, as a reactive oxygen species in biology. Peroxymonocarbonate results from the equilibrium reaction of hydrogen peroxide with bicarbonate via the perhydration of CO 2. The kinetic parameters for HCO 4 − oxidation of free methionine have been obtained ( k 1 = 0.48 ± 0.08 M −1s −1 by a spectrophotometric initial rate method). At the physiological concentration of bicarbonate in blood (∼25 mM), it is estimated that peroxymonocarbonate formed in equilibrium with hydrogen peroxide will oxidize methionine approximately 2-fold more rapidly than plasma H 2O 2 itself. As an example of methionine oxidation in proteins, the bicarbonate-catalyzed hydrogen peroxide oxidation of α1-proteinase inhibitor (α1-PI) has been investigated via its inhibitory effect on porcine pancreatic elastase activity. The second-order rate constant for HCO 4 − oxidation of α1-PI (0.36 ± 0.06 M −1s −1) is comparable to that of free methionine, suggesting that methionine oxidation is occurring. Further evidence for methionine oxidation, specifically involving Met358 and Met351 of the α1-PI reactive center loop, has been obtained through amino acid analyses and mass spectroscopic analyses of proteolytic digests of the oxidized α1-PI. These results strongly suggest that HCO 4 − should be considered a reactive oxygen species in aerobic metabolism.