Reduced flavins, reactions with oxygen

Reduced flavins, reactions with oxygen 794 Reducing equivalents from citric acid cycle 515... 

After the oxidized product has left the enzyme, the reduced flavin reacts with oxygen to form, initially, the flavin semiquinone radical and superoxide. These undergo the sequence of rapid reactions shown in Table 7.3, ultimately resulting in reoxidation of the flavin and formation of hydrogen peroxide. 

Gutfreund, H. (1959b). The reactions of reduced flavine nucleotides with oxygen. BiochemicalJournal, 74, lip. 

PHBH is the protype of the flavoprotein aromatic hydroxylases. Each subunit of this dimeric enzyme contains two active sites which, during catalysis, are alternately visited by the isoalloxazine ring of the FAD cofactor (31). Catalysis is iiutiated by reduction of the flavin in the exterior active site. The reduced flavin then moves to the interior active site where the reactions with oxygen occur. A similar conformational flexibility of the FAD cofactor has been observed in the crystal structures of phenol hydroxylase (EC 1.14.13.7) and 3-hydroxybenzoate 4-hydroxylase (EC 1.14.13.23). PHBH obeys the following kinetic mechanism ... 

Experimental evidence for the participation of the flavin moiety in Oi- formation in nonenzymic reactions was presented by Ballou et al. (151). During the oxidation of different reduced flavins by molecular oxygen substantial yields of Oi- were obtained. Tetra-acetyl riboflavin proved most appropriate as a model compound (153). The respective EPR parameters for Oi- were in close agreement with those obtained in the KIO4—H2O2 system, in the xanthine oxidase reaction (149), and in the oxidation of the reduced tetra-acetyl riboflavin. They also agree well with the EPR data obtained earlier (154—156). The time course of the appearance and decay of Oi- produced by the reaction of tetra-acetyl riboflavin is shown in Fig. 24. 

Bacterial concentrations have also been determined by using the enzyme-catalyzed chemiluminescent reaction of reduced flavin mononucleotide (FMN) with oxygen and aldehydes. The detection limit was reported to be 10 ceUs of E. coli, which contains 7 x 10 g of FMN per ceU (303). 

Reduction by sodium dithionite. A small amount of sodium dithionite, solid or in solution, is added to a luciferase solution made with 50 mM phosphate, pH 7.0, containing 50 pM FMN. The amount of dithionite used should be minimal but sufficient to remove oxygen in the solution and to fully reduce the flavin. The solution made is injected into an air-equilibrated buffer solution containing a long-chain aldehyde and luciferase to initiate the luminescence reaction. With this method, the reaction mixture will be contaminated by bisulfite and bisulfate ions derived from dithionite. 

V. Massey, S. Strickland, S.G. Mayhew, L.G. Howell, P.C. Engel, R.G. Matthews, M. Schuman, and P.A. Sullivan, Production of superoxide anion radicals in the reaction of reduced flavins and flavopro-teins with molecular oxygen. Biochem. Biophys. Res. Commun. 36, 891-897 (1969). 

Riboflavin is the redox component of flavin adenine dinucleotide FAD. It is derived from FAD by hydrolysis of a phosphate ester link. The fully oxidised form of FAD is involved in many dehydrogenaze reactions during which it is converted to the fully reduced form. The fully oxidised state is restored either by another redox enzyme or by interaction with oxygen and hydrogen peroxide is liberated. The one-electron reduced, semiquinone form of FAD, is involved in some electron transfer steps. 

Reduction of flavin by two electrons yields the 1,5-dihydroflavin (Scheme 3), often called reduced flavin . Since isomeric two-electron reduced flavin structures are known (cf. below), the term reduced flavin should be avoided unless defined to prevent misunderstanding. From all flavin species possible in a redox reaction the solution of 1,5-dihydroflavin is, in contrast to that of some isomeric compounds, devoid of a stront colour but not colourless as indicated by the term leucoflavin , which is still used (Table 4). The only true colourless species is (77). Because of the very high oxygen-sensitivity of 1,5-dihydroflavin its chemical and physical properties were investigated only recently Long before crystallographic data on flavins were available, conclusions were drawn from the molar extinction coefficient at 450 nm of 1,5-dihydroflavins with respect to the planarity of the molecules. From the data presented in Table 5 it was proposed that anionic... 

Massey, V., Palmer, G., Ballou, D. P. On the reaction of reduced flavins with molecular oxygen. In Oxidases and related redox systems (King, T. E., Mason, H. S., Morrison, M. eds.) pp. 25-49. Baltimore, Univ. Park Press 1973... 

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