AbstractRiboflavin synthase catalyses a mechanistically complex dismutation affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione from 6,7-dimethyl-8-ribityllumazine. A pentacyclic adduct (compound2) of two substrate molecules was used as substrate for pre-steady-state kinetic analysis. Whereas the wild-type enzyme catalyses the decomposition of compound2into a mixture of riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, as well as into two equivalents of 6,7-dimethyl-8-ribityllumazine, a H102Q mutant enzyme predominantly catalyses the former reaction. Stopped-flow experiments with this mutant enzyme failed to identify a reaction intermediate between compound2and riboflavin. However, the apparent rate constants for the formation of riboflavin as observed by stopped-flow and quenched-flow experiments were significantly different, thus suggesting that the reaction proceeds via a significantly populated intermediate, the absorbance of which is similar to that of compound2. An F2A mutant enzyme converts compound2predominantly into 6,7-dimethyl-8-ribityllumazine. Stopped-flow experiments using compound2as substrate indicated a slight and rapid initial increase in absorbance at 310 nm, followed by a slower decrease. This finding, in conjunction with different apparent rates for the formation of 6,7-dimethyl-8-ribityllumazine, suggests the involvement of a significantly populated intermediate in the transition between compound2and 6,7-dimethyl-8-ribityllumazine, the optical spectrum of which is similar to that of compound1.