Carbon dioxide is an omnipresent gas that drives adaptive responses within organisms from all domains of life. The molecular mechanisms by which proteins serve as sensors of CO₂ are, accordingly, of great interest. Because CO₂ is electrophilic, one way it can modulate protein biochemistry is by carboxylation of the amine group of lysine residues. However, the resulting CO₂-carboxylated lysines spontaneously decompose, giving off CO₂, which makes studying this modification difficult. Here we describe a method to stably mimic CO₂-carboxylated lysine residues in proteins. We leverage this method to develop a quantitative approach to identify CO₂-carboxylated lysines of proteins and explore the lysine ‘carboxylome’ of the CO₂-responsive cyanobacterium Synechocystis sp. We uncover one CO₂-carboxylated lysine within the effector binding pocket of the metabolic signaling protein PII. CO₂-carboxylatation of this lysine markedly lowers the affinity of PII for its regulatory effector ligand ATP, illuminating a negative molecular control mechanism mediated by CO₂.