Enhanced hyperoxidation of peroxiredoxin 2 and peroxiredoxin 3 in the presence of bicarbonate/CO₂

Hydrogen peroxide undergoes an equilibrium reaction with bicarbonate/CO₂ to produce peroxymonocarbonate (HCO₄^-). Peroxymonocarbonate is more reactive with thiols than H₂O₂ but it makes up only a small fraction of the H₂O₂ in physiological bicarbonate buffers so the increase in rate of oxidation of low molecular weight thiols is modest. However, for some thiol proteins such as protein tyrosine phosphatases, the rate enhancement is very much greater. We have investigated the effect of bicarbonate/CO₂ on the oxidation of peroxiredoxins (Prdxs) 2 and 3. Using an assay in which reduced Prdx2 inhibits oxidation of horseradish peroxidase by H₂O₂, we saw no difference between phosphate and bicarbonate buffers (pH 7.4). However, hyperoxidation of both Prdxs in bicarbonate was considerably enhanced. Hyperoxidation involves the reaction of the sulfenic acid formed at the active site with a second H₂O₂, and prevents its condensation to a disulfide. Using LC/MS analysis, we determined that the presence of 25 mM bicarbonate/CO₂ increased the ratio of hyperoxidation compared with condensation 6-fold for Prdx2 and 11-fold for Prdx3. These results imply that Prdx hyperoxidation will occur more readily under physiological conditions than appreciated from in vitro experiments, which seldom use bicarbonate buffers. They also raise the possibility that variations in bicarbonate concentration could provide a mechanism for regulating the cellular level of active Prdxs.