Stopped flow studies on the nonenzymatic reduction of methemoglobin by reduced flavin mononucleotide

The nonenzymatic reduction of methemoglobin by the reduced form of flavin mononucleotide was studied under various conditions by following the reaction with a stopped flow apparatus. The reaction was very fast, compared with the reduction of the flavin by NADPH-flavin reductase of human erythrocytes, and followed a second order rate law: the rate constant (K) for the reduction of methemoglobin by reduced flavin mononucleotide was determined to be 5.5 X 10(6) M-1 S-1 in 50 mM phosphate buffer (pH 7.0) at 25 degrees C. The reaction was not influenced by changing phosphate buffer concentration from 10 to 100 mM. The rate of reduction at the physiological pH, 7.0, was about 95% of the maximal value that observed at around pH 6.4. Formation of deoxyhemoglobin and oxidized form of flavin mononucleotide by the reaction proceeded stoichiometrically in a ratio of unity. These results apparently indicate that the limiting step for the reduction of methemoglobin by the NADPH-flavin reductase system in human erythrocytes is the enzymatic reduction of flavin.