CO2 impairs peroxynitrite-mediated inhibition of human caspase-3

Peroxynitrite (ONOO^-) is a transient powerful oxidant produced in vivo as the reaction of nitrogen monoxide (^{{radical dot}}NO) with superoxide (O₂ ^{{radical dot} -}). The peroxynitrite reactivity is modulated by carbon dioxide (CO₂) which enhances the peroxynitrite-mediated nitration of aromatics and partially impairs the oxidation of thiols. Here, the effect of CO₂ on the peroxynitrite-mediated inhibition of human caspase-3, the execution enzyme of the apoptotic cascade, is reported. Peroxynitrite inhibits the catalytic activity of human caspase-3 by oxidizing the Sγ atom of the Cys catalytic residue. In the absence of CO₂, 1.0 equivalent of peroxynitrite inactivates 1.0 equivalent of human caspase-3. In the presence of the physiological concentration of CO₂ (=1.3 × 10^-3 M), 1.0 equivalent of peroxynitrite inactivates only 0.38 equivalents of human caspase-3. Peroxynitrite affects the k_cat value of the human caspase-3 catalyzed hydrolysis of N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin, without altering K_m. Both in the absence and presence of CO₂, the reducing agent dithiothreitol does not prevent human caspase-3 inhibition by peroxynitrite and does not reverse the peroxynitrite-induced inactivation of human caspase-3. These results represent the first evidence for modulation of peroxynitrite-mediated inhibition of cysteine proteinase action by CO₂, supporting the role of CO₂ in fine tuning of cell processes (e.g., apoptosis).