Unifying concepts in flavin-dependent catalysis.

Flavin-dependent proteins can be involved in a wide variety of biochemical reactions. Among these reactions are electron transfer, oxidase activity, dehydroxygenase activity and mono- and dioxygenations. Furthermore, the oxygenation steps may proceed by either electrophilic or nucleophilic reaction mechanisms. The present paper focuses on aromatic hydroxylation by a nucleophilic C(4a) hydroperoxyflavin cofactor. The reactivity of this intermediate is highly dependent on its actual protonation state as well as on the (de)protonation of the substrate to be converted. Electrophilic attacks by the peroxyflavin intermediate on the substrate and favoured by a protonated C(4a)-hydroperoxyflavin and a (partially) deprotonated hydroxyl moiety in an aromatic substrate. Thus, the actual mechanism of enzyme catalysis is basically that the enzyme provides possibilities for the coexistence of (de)protonation states of cofactor and substrate that would normally not be possible in one solution at one pH value. Another concept presented in this paper, originating from results obtained in molecular orbital studies on flavin-dependent mono-oxygenation reaction by phenol hydroxylase and 4-hydroxybenzoate-3-hydroxylase, questions the actual unifying concept found in some biochemical textbooks that proposes that enzymes accelerate chemical reactions by stabilization of the transition state only. The results of the present study demonstrate that increasing the reactivity of the substrate and/or cofactor may be another important mechanism to decrease the activation barrier of a chemical reaction.