Flavin electrophilic centers

The formation of a covalent bond between NADH and FAD has been suggested and previous studies indicated that a preequilibrium complex is probably formed with flavins. Second, theoretical calculations have shown that position N-5 should be a good electrophilic center. Therefore, Bruice (287) argued that changing that position for a carbon atom should also yield a strong electrophilic center with the advantage that the new C—H bond formed will be stable and not exchange with the solvent. 

Binding of 4-hydroxybenzoate (S) in the phenolate form facilitates flavin reduction by NADPH. After NADP+ release, the flavin hydroquinone reacts with molecular oxygen to yield the flavin C4a-hydroperoxide oxygenation species. Protonation of the distal oxygen of the peroxflavin facilitates the electrophilic attack on the nucleophilic carbon center of the substrate phenolate. After monooxygenation, the resulting hydroxyflavin is... 

Obviously, more work is required to further substantiate the presence of the proposed radical intermediates in the p-hydroxybenzoate hydroxylase reaction, possibly via EPR and spin-trapping studies. Studies by Detmer and Massey 247) on phenol hydroxylase have indicated that the reaction rate constants for the conversion of meta-substituted substrates plotted versus the Hammett parameters yield a straight line of slope equal to 0.5. This is consistent with the mechanism proposed by Anderson, as the negative slope is expected for an electrophilic aromatic substitution reaction, while the small magnitude of the slope may be indicative of a radical mechanism. Furthermore, recent work by Massey and co-workers on p-hydroxybenzoate hydroxylase utilizing 6-hydroxy-FAD as cofactor and p-aminobenzoate as substrate indicated that the absorption spectrum of intermediate 67 exhibited a satellite band at 440 nm 248). Anderson et al. suggest that the satellite band may result from the formation of an aromatic phenoxyl radical at the C-6 position of the isoalloxazine ring of the flavin 244). This species would result from a shift of the initial peroxyl radical center from C(4a) to C-6 via N(5) 245). 

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