The bicarbonate/carbon dioxide pair increases hydrogen peroxide-mediated hyperoxidation of human peroxiredoxin 1

Abstract

2-Cys peroxiredoxins (Prxs) rapidly reduce H ₂O ₂, thereby acting as antioxidants and also as sensors and transmitters of H ₂O ₂ signals in cells. Interestingly, eukaryotic 2-Cys Prxs lose their peroxidase activity at high H ₂O ₂ levels. Under these conditions, H ₂O ₂ oxidizes the sulfenic acid derivative of the Prx peroxidatic Cys (C _PSOH) to the sulfinate (C _PSO ₂ ⁻) and sulfonated (C _PSO ₃ ⁻) forms, redirecting the C _PSOH intermediate from the catalytic cycle to the hyperoxidation/inactivation pathway. The susceptibility of 2-Cys Prxs to hyperoxidation varies greatly and depends on structural features that affect the lifetime of the C _PSOH intermediate. Among the human Prxs, Prx1 has an intermediate susceptibility to H ₂O ₂ and was selected here to investigate the effect of a physiological concentration of HCO ₃ ⁻/CO ₂ (25 m m) on its hyperoxidation. Immunoblotting and kinetic and MS/MS experiments revealed that HCO ₃ ⁻/CO ₂ increases Prx1 hyperoxidation and inactivation both in the presence of excess H ₂O ₂ and during enzymatic (NADPH/thioredoxin reductase/thioredoxin) and chemical (DTT) turnover. We hypothesized that the stimulating effect of HCO ₃ ⁻/CO ₂ was due to HCO ₄ ⁻, a peroxide present in equilibrated solutions of H ₂O ₂ and HCO ₃ ⁻/CO ₂. Indeed, additional experiments and calculations uncovered that HCO ₄ ⁻ oxidizes C _PSOH to C _PSO ₂ ⁻ with a second-order rate constant 2 orders of magnitude higher than that of H ₂O ₂ ((1.5 ± 0.1) × 10 ⁵ and (2.9 ± 0.2) × 10 ³ m ⁻¹·s ⁻¹, respectively) and that HCO ₄ ⁻ is 250 times more efficient than H ₂O ₂ at inactivating 1% Prx1 per turnover. The fact that the biologically ubiquitous HCO ₃ ⁻/CO ₂ pair stimulates Prx1 hyperoxidation and inactivation bears relevance to Prx1 functions beyond its antioxidant activity.