The family of FMN-dependent, a-hydroxy acid-oxidizing enzymes catalyzes substrate dehydrogenation by a mechanism the first step of which is abstraction of the substrate a-proton (so-called carbanion mechanism). For flavocytochrome b2 and lactate oxidase, it was shown that once on the enzyme this proton is lost only slowly to the solvent (Lederer F, 1984, In: Bray RC, Engel PC, Mayhew SG, eds, Flavins &flavoproteins, Berlin: Walter de Gruyter & Co., pp 513-526; Urban P, , J Biol Chem 260: 11 115-1 1122). This suggested the occurrence of a pK, increase of the catalytic histidine upon enzyme reduction by substrate. For flavocytochrome b2, the crystal structure indicated 2 possible origins for the stabilization of the imidazolium form of His 373: either a network of hydrogen bonds involving His 373, Tyr 254, flavin N5 and 0 4 , a heme propionate, and solvent molecules, and/or electrostatic interactions with Asp 282 and with the reduced cofactor N1 anion. In this work, we probe the effect of the hydrogen bond network at the active site by studying proton exchange with solvent for 2 mutants: Y254F and the recombinant flavodehydrogenase domain, in which this network should be disrupted. The rate of proton exchange, as determined by intermolecular hydrogen transfer experiments, appears identical in the flavodehydrogenase domain and the wild-type enzyme, whereas it is about 3-fold faster in the Y254F mutant. It thus appears that specific hydrogen bonds to the solvent do not play a major role in stabilizing the acid form of His 373 in reduced flavocytochrome b2. Removal of the Y254 phenol group induces a pK, drop of about half a p H unit for His 373 in the reduced enzyme. Even then, the rate of exchange of the imidazolium proton with solvent is still lower by several orders of magnitude than that of a normally ionizing histidine. Other factors must then also contribute to the pK, increase, such as the electrostatic interactions with D282 and the anionic reduced cofactor, as suggested by the crystal structure.