Chemical modification of L-lactate 2-monooxygenase with fluorodinitrobenzene: evidence for two essential histidine residues

The modification of L-lactate 2-monooxygenase (lactate oxidase) with radioactively labeled fluorodinitrobenzene in 50 mM tris(hydroxymethyl)aminomethane (Tris)-acetate, pH 6.0, resulted in complete inactivation with the incorporation of 1 mol of dinitrophenyl per catalytic site. Analysis of acid hydrolysates using high-pressure liquid chromatography and an amino acid analyzer showed that Nim-(dinitrophenyl)histidine accounted for 95% of the incorporated label. Approximately 50% of the flavin in the modified enzyme was reduced by lactate, formed a sulfite complex, and underwent a photochemical reaction with oxalate. These results suggest that each active site in lactate oxidase contains two essential histidine residues that are modified in a mutually exclusive manner.