The FMN prosthetic group of flavocytochrome b2 or L-lactate dehydrogenase oxidizes lactate to pyruvate. The reducing equivalents are then transferred one by one, intramolecularly, to heme b2 and then to external acceptors. Substrate oxidation is thought to begin with abstraction of the substrate alpha-hydrogen as a proton by an enzyme base. It has been proposed that this role is played by His373, which lies close to the flavin in the crystal structure and interacts with Asp282. It has also been shown before, using hydrogen exchange measurements, that the pKa of His373 is substantially increased in the wild-type reduced enzyme compared to that in the oxidized state. We report here the enzymatic properties of the D282N mutant flavocytochrome b2. Steady-state rate measurements with [2-1H]lactate and [2-2H]-lactate indicate that, as predicted, the Michaelis complex stability is hardly affected, whereas the transition state for proton abstraction increases in energy by 2.8 kcal/mol. Steady-state inhibition studies were conducted with a number of active-site ligands: sulfite, D-lactate, pyruvate, and oxalate. Binding was found to be most affected for oxalate, but kinetic patterns indicated oxalate and pyruvate were still capable of binding to the enzyme both at the oxidized and semiquinone stages, whereas inhibition by excess substrate, due to lactate binding at the semiquinone stage, was lost. Finally, analysis of the intermolecular hydrogen transfer catalyzed by the enzyme between [2-3H]lactate and fluoropyruvate indicated that the substitution with asparagine facilitates exchange of the histidine-bound proton and hence induces a decrease in the pKa value of H373 in the reduced enzyme of about 1.4 pH units. Nevertheless, the rate constant value for exchange with the solvent of the enzyme-bound substrate alpha-proton indicates that H373 is still protonated in the reduced mutant enzyme at neutral pH. Thus, the D282N mutation destabilizes the transition state for proton abstraction and decreases the pKa of H373 in the reduced enzyme but is insufficient to bring it back to a normal value.