Lactate monooxygenase utilizes oxygen in the conversion of L-lactate to acetate, CO2, and water. The gene for the enzyme from Mycobacterium smegmatis had been cloned into Escherichia coli (Giegel, D. A., Williams, C. H., Jr., and Massey, V. (1990) J. Biol. Chem. 265, 6626-6632) and the derived amino acid sequence compared to glycolate oxidase and flavocytochrome b2, enzymes of known three-dimensional structure (Lindqvist, Y., and Brändén, C. I. (1989) J. Biol. Chem. 264, 3624-3628; Xia, Z. X., and Mathews, S. F. (1990) J. Mol. Biol. 212, 837-863). There is strong homology, especially around residues in the active site. The mechanism proposed for lactate monooxygenase involves an intermediate having a negative charge at the N(1)-position of the FMN. Based on the homology, lysine 266 is the residue suggested to neutralize that charge. Wild type enzyme and several forms of the enzyme altered at active site residues by site-directed mutagenesis have been expressed in E. coli and purification procedures developed. The properties determined for the recombinant wild type enzyme were, in every case, the same as those previously determined for the enzyme isolated from M. smegmatis. Mutation of lysine 266 to a methionine created K266M. The semiquinone showed spectral features different from those found in the wild type enzyme and was no longer thermodynamically stable. This indicates a redox potential for the enzyme-bound semiquinone/reduced flavin couple that is higher than the midpoint potential for the oxidized flavin/semiquinone couple. The two-electron redox potential was determined to be -180 mV at 25 degrees C, pH 7.0. In wild type enzyme, attack of the flavin ring by sulfite creates a negative charge at the FMN N(1)-position. In K266M, the stabilization of the sulfite adduct was 17,000-fold weaker (Kd approximately 10(-3) M) than in the wild type enzyme, with a rate of association that is lowered by 10,000-fold (kon = 1.2 M-1 s-1). The rate of reduction with L-lactate is significantly decreased in K266M. Unexpectedly, binding of substrate and inhibitors is significantly weaker in K266M than in the wild type enzyme. In all properties involving a negative charge at position N(1) of the FMN, K266M is distinctly different from wild type enzyme. This makes it quite likely that lysine 266 serves the postulated role of interacting with this negative charge.