Probing the mechanism of flavin action in the oxidative decarboxylation catalyzed by salicylate hydroxylase.

Salicylate hydroxylase (NahG) is a FAD-dependent monooxygenase in which the reduced flavin activates O2 coupled to the oxidative decarboxylation of salicylate to catechol or uncoupled from substrate oxidation to afford H2O2. This chapter presents different methodologies in equilibrium studies, steady-state kinetics, and identification of reaction products, which were important to understand the SEAr mechanism of catalysis in NahG, the role of the different FAD parts for ligand binding, the extent of uncoupled reaction, and the catalysis of salicylate's oxidative decarboxylation. These features are likely familiar to many other FAD-dependent monooxygenases and offer a potential asset for developing new tools and strategies in catalysis.