Lactate monooxygenase (LMO) catalyzes the conversion of L-lactate to acetate, CO(2), and water with the incorporation of molecular oxygen. Arginine 187 of LMO is highly conserved within the family of L-alpha-hydroxyacid oxidizing enzymes (Lê, K. H. D., and Lederer, F. (1991) J. Biol. Chem. 266, 20877-20881). By comparison with the equivalent residue in flavocytochrome b(2) from Saccharomyces cerevisiae (Pike, A. D., Chapman, S. K, Manson, F. D. C,. Reid, G. A. , Gondry, M., and Lederer, F. (1996) in Flavins and Flavoproteins (Stevenson, K. J., Massey, V., and Williams, C. H., Jr., eds) pp. 571-574, University of Calgary Press, Calgary, AB, Canada), arginine 187 might be expected to have an important role in catalytic efficiency and substrate binding in LMO. Histidine 240 is predicted to be close to the substrate binding site of LMO, although it is not conserved within the enzyme family. Arginine 187 has been replaced with methionine (R187M), and histidine 240 has been replaced with glutamine (H240Q). L-Lactate oxidation by R187M is very slow. The binding of L-lactate to the mutant enzyme appears to be very weak, as is the binding of oxalate, a transition state analogue. The binding of pyruvate to the reduced enzyme is also very weak, resulting in complete uncoupling of enzyme turnover, with H(2)O(2) and pyruvate as the final products. In addition, anionic forms of the flavin are unstable. The K(d) for sulfite is increased nearly 400-fold by this mutation. The semiquinone form of R187M is also thermodynamically unstable, although the overall midpoint potential for the two-electron reduction of R187M is only 34 mV lower than for the wild-type enzyme. H240Q more closely resembles the wild-type enzyme. The steady-state activity of H240Q is completely coupled. The k(cat) is similar to that for the wild-type enzyme.