Peroxynitrite (ONOO-) is a transient powerful oxidant produced in vivo as the reaction of nitrogen monoxide ((NO)-N-center dot) with superoxide (O-2(center 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 S gamma 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 x 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-GluVal-Asp-7-amido-4-methylcoumarin, without altering K,,. 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). (c) 2006 Elsevier Inc. All rights reserved.