Inhibitory mechanism of tyrosinase-induced browning in Agaricus bisporus by garlic-derived H2S: Nucleophilic attack leading to catalytic-cu flexible loop collapse and thiol-persulfidation

Tyrosinase (TY) drives melanin accumulation and enzymatic browning in produce. This study explored the inhibition mechanism of garlic-derived H₂S on TY and its feasibility in preventing Agaricus bisporus browning. Garlic sulfides, including diallyl trisulfide (DATS), diallyl disulfide (DADS), and diallylthiosulfinate (Allicin), all inhibited TY activity via reduced glutathione (GSH)-induced H₂S release, with DATS exhibiting the strongest effect due to its high Sn⁰ content. H₂S bound TY at a single site, disrupted hydrogen bonds and hydrophobic interactions, reducing α-helix content, weakening van der Waals forces, inducing Cys83 persulfidation, destabilized the flexible loop, and increased the CuA-CuB distance. The “Nucleophilic attack leading to flexible loop collapse and persulfidation” mechanism enriches the biochemical theory of H₂S as enzyme inhibitors. Additionally, garlic-derived H₂S effectively reduced browning, malondialdehyde levels, PPO/POD activity, and volatile flavor deterioration in Agaricus bisporus homogenate, providing a novel strategy for natural TY inhibitors and fresh-produce preservation.