4a-Peroxyflavin

Flavin-dependent monooxygenases function through a reaction cycle that is schematically presented in Fig. 4.79. These enzymes perform their catalytic reaction through the generation of a reactive form of the flavin cofactor, formed by 2-electron reduction of the flavin followed by its reaction with molecular oxygen. As a result, the so-called C(4a)-peroxyflavin intermediate is formed, whose electrophilic reactivity is further increased by protonation of the distal oxygen of the peroxide to yield the C(4a)-hydroperoxyflavin form of the cofactor (Fig. 4.80). 

Bacterial luciferase catalyzes the reaction of reduced flavin mononucleotide (FMNH2) with O2 to form a 4a-peroxyflavin derivative that reacts with a... 

Several unique features of the catalytic cycle of the FMOs are important for understanding the mechanism by which they oxidize xenobiotics. The catalytic mechanism for the FMO has been shown to involve the formation of an enzyme bound 4a-hydroperoxyl-flavin (Figure 10.6) in an NAD PH and 02 dependent reaction. Reduction of the flavin by NAD PH occurs before binding of oxygen can occur, and activation of oxygen by the enzyme occurs in the absence of substrate by oxidizing NADPH to form NADP and peroxide. Finally, addition of the substrate to the peroxyflavin complex is the last step prior to oxygenation. This is in contrast to the CYP catalytic cycle in which the substrate binds to the oxidized enzyme which is subsequently reduced. 

Fig. 4.80. Structure of the activated C(4a)-(hyclro)peroxyflavin formed upon 2-electron reduction of the oxidized flavin and its subsequent reaction with molecular oxygen.

However, the activated, protonated C(4a)-hydroperoxyflavin is not by itself a powerful oxidizing species. From detailed biochemical studies on the catalytic mechanism of such enzymes, especially para-hydroxybenzoate hydroxylase (PHBH) and phenol hydroxylase (PH), it appears that not only the peroxyflavin needs to be activated through protonation of the distal oxygen, but also the phenolic substrates require activation in order to obtain substrate conversion. This substrate activation is achieved through deprotonation of the hydroxyl moiety of the phenolic substrate. The active site of PHBH, for example, shows a tyrosine network consisting of tyrosines 385 and 201 (Fig. 4.82) responsible for this deprotonation and activation of the substrate. 

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