Interaction of two arginine residues in lactate oxidase with the enzyme flavin: Conversion of FMN to 8-formyl-FMN

Two arginine residues, Arg-181 and Arg-268, are conserved throughout the kn own family of FMN-containing enzymes that catalyze the oxidation of ru-hydr oxyacids. In the lactate oxidase from Aerococcus viridans, these residues h ave been changed to lysine in two single mutations and in a double mutant f orm. In addition, Arg-181 has been replaced by methionine to determine the effect of removing the positive charge on the residue. The effects of these replacements on the kinetic and thermodynamic properties are reported. Wit h ail mutant forms, there are only small effects on the reactivity of the r educed flavin with oxygen. On the other hand, the efficiency of reduction o f the oxidized flavin by L-lactate is greatly reduced, particularly with th e R268K mutant forms. The results demonstrate the importance of the two arg inine residues in the binding of substrate and its interaction with the fla vin, and are consistent with a previous hypothesis that they also play a ro le of charge neutralization in the transition state of substrate dehydrogen ation. The replacement of Arg-268 by lysine also results in a slow conversi on of the 8-CH3- substituent of FMN to yield 8-formyl-FMN, still tightly bo und to the enzyme, and with significantly different physical and chemical p roperties from those of the FMN-enzyme.