Many flavoenzymes--oxidases and monooxygenases--react faster with oxygen than free flavins do. There are many ideas on how enzymes cause this. Recent work has focused on the importance of a positive charge near N5 of the reduced flavin. Fructosamine oxidase has a lysine near N5 of its flavin. We measured a rate constant of 1.6 × 10(5) M(-1) s(-1) for its reaction with oxygen. The Lys276Met mutant reacted with a rate constant of 291 M(-1) s(-1), suggesting an important role for this lysine in oxygen activation. The dihydroorotate dehydrogenases from E. coli and L. lactis also have a lysine near N5 of the flavin. They react with O(2) with rate constants of 6.2 × 10(4) and 3.0 × 10(3) M(-1) s(-1), respectively. The Lys66Met and Lys43Met mutant enzymes react with rate constants that are nearly the same as those for the wild-type enzymes, demonstrating that simply placing a positive charge near N5 of the flavin does not guarantee increased oxygen reactivity. Our results show that the lysine near N5 does not exert an effect without an appropriate context; evolution did not find only one mechanism for activating the reaction of flavins with O(2).