Enzyme-bound flavin

Enzyme-Bound Flavin Semiquinones which are not Catalytic... 

Enzyme-Bound Flavin Semiquinone which are not Catalytic Intermediates... 

Further transfer of the acyl group to coenzyme A is catalyzed by the same enzyme. This displacement reaction produces reduced lipoic acid. A third enzyme, dihydrolipoyl dehydrogenase, catalyzes oxidation of this product back to the disulfide form. The electrons lost in that oxidation are transferred first to an enzyme-bound flavin (not shown in the figure) and then to NAD +. ... 

Baeyer-ViUiger oxidation involves NADPH and flavin (FAD) as cofactors and was originally proposed by Walsh et al. based on data obtained from cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus (Fig. 24) [156]. In a first step, enzyme-bound flavin is reduced, followed by the addition of oxygen yielding a hydroperoxide anion. Reaction with the ketone substrate gives a Criegee intermediate, which is then converted into the product under dissociation of water. The cofactor FAD is recovered via oxidation with NADP+. 

Fw. 18. Anerobic titration of NADPH-cytochrome P-450 reductase with NADPH. Curve 1, oxidized enzyme curves 2-6 after the addition of 0.16, 054, 0.49, 058, and 1.4 moles of NADPH per mole of total enzyme-bound flavin. The inset, B, shows the changes at 455 and 586 nm as a function of the NADPH added (405)-... 

Glucose oxidase (GOX) oxidizes /3-o-glucose to D-glucono- -lactone (Equation (5)) with the concomitant reduction of the enzyme-bound flavin. 

The scheme is undoubtedly oversimplified. It is possible, for example, that reduction of the enzyme by ascorbate involves the formation of semidehydroascorbate although dehydroascorbate is the only product that has so far been detected. It is also possible that Reaction 3, the combination of oxygen with the enzyme-bound copper, does not occur in the absence of substrate. It is of interest in this regard that the reduction of the enzyme by ascorbate occurs rapidly in the absence of substrate. This is in contrast to results obtained with salicylate hydroxylase, where rapid reduction of the enzyme-bound flavin by DPNH requires the presence of substrate (9). 

Adenosine phosphosulfate — AMP -p enzyme-bound-flavin-sulfate... 

Scheme 9.105. A representation of the oxidation of succinate (1,4-butanedioic acid) to fum-erate (1,4-butenedioic acid) with the concomitant reduction of enzyme bound flavin adenine...

The authors argue that flavin nitroxyl radical may play a role in flavo-enzyme chemistry and suggest that the nitroxyl radical intermediate may be derived from enzyme-bound flavin 4a-hydroperoxide. 

Binding of fully oxidized FMN to enzyme was considerably enhanced in the presence of substrate, while a smaller enhancement was observed with product, leading the authors to suggest that a ternary enzyme-FMN-(substrate/product) complex is formed. Reduction of FMN in the presence of enzyme resulted in the formation of fully reduced, enzyme-bound, flavin. When FMN was reduced in the presence of both enzyme and the substrate analogue (6R)-6-fluoro-5-enol-pyruvylshikimate-3-phosphate a neutral flavin radical was formed. The radical was also formed to a lesser extent in the presence of 5-enolpyruvylshikimate-3-phosphate (40%) and chorismate (14%). 

Fig. 37. Mechanism for oxidation of substrate (SHi) by flavoprotein (36). Y represents the yellow enzyme, fatty acyl CoA dehydrogenase FAD and FADH2 represent the oxidized and reduced forms, respectively, of enzyme-bound flavin and S and SHt represent the oxidized and reduced forms of substrate.

A related observation provides more information and virtually confirms that these compounds do not provide good models for the enzymic reaction. To obtain model peroxides in this series it is necessary to use alkyl flavinium salts to prevent the elimination of H2O2 which occurs in all but enzyme-bound flavin peroxides. The 5-alkyl salts provide the exact counterpart of the 4 a-peroxide believed to occur on the enzyme. However the 10 a-peroxide gives the only unambiguously identified fluorescent product. In the case of 4 a-substitution the product is not fluorescent. 

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