Sulfite salts are widely used as anti-browning agents in food processing. Nevertheless, the exact mechanism by which sulfite prevents enzymatic browning has remained unknown. Here, we show that sodium hydrogen sulfite (NaHSO3 ) irreversibly blocks the active site of tyrosinase from the edible mushroom Agaricus bisporus, and that the competitive inhibitors tropolone and kojic acid protect the enzyme from NaHSO3 inactivation. LC-MS analysis of pepsin digests of NaHSO3 -treated tyrosinase revealed two peptides showing a neutral loss corresponding to the mass of SO3 upon MS(2) fragmentation. These peptides were found to be homologous peptides containing two of the three histidine residues that form the copper-B binding site of mushroom tyrosinase isoforms PPO3 and PPO4, which were both present in the tyrosinase preparation used. Peptides showing this neutral loss behavior were not found in the untreated control. Comparing the effect of NaHSO3 on apo and holo-tyrosinase indicated that inactivation is facilitated by the active site copper ions. These data provide compelling evidence that inactivation of mushroom tyrosinase by NaHSO3 occurs through covalent modification of a single amino acid residue, likely via addition of HSO3 (-) to one of the copper-coordinating histidines in the copper-B site of the enzyme. This article is protected by copyright. All rights reserved.