Flavin Oxidases

Flavin oxidases include d- and l-amino acid oxidases, and some amine oxidases, although others are quinoproteins (Section 9.8.3). In these enzymes, the flavin is reduced by dehydrogenation of the substrate, byway of an intermediate substrate-flavin adduct, as occurs in the dehydrogenases (Section 7.3.3). 

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. 

X-H2 + flavin - X + flavin-H2, flavin-H2 + O2 flavin + H2O2 Fully reduced flavin-Fl2 reacts with oxygen to form the flavin semiquinone radical and superoxide 

Flavin semiquinone and superoxide react to form flavin hydroperoxide flavin-Fl + 02 - flavin-HOOH 

Flavin hydroperoxide slowly breaks down to yield flavin semiquinone and perhydroxyl flavin-HOOH flavin-H -1- OjH Perhydroxyl decays to superoxide plus a proton 

X—Fi2 + flavin - X + fiavin-Fi2, fiavin-Fi2 + 02- - flavin + Fi202 Fuiiy reduced fiavin-Fi2 reacts with oxygen to form the fiavin semiquinone radicai and superoxide 

Fiavin hydroperoxide siowiy breaks down to yieid fiavin semiquinone and perhydroxyi fiavin-FiOOFi fiavin-Fi -i- 02Fi Perhydroxyi decays to superoxide pius a proton 

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These include the mitochondrial respiratory chain, key enzymes in fatty acid and amino acid oxidation, and the citric acid cycle. Reoxidation of the reduced flavin in oxygenases and mixed-function oxidases proceeds by way of formation of the flavin radical and flavin hydroperoxide, with the intermediate generation of superoxide and perhydroxyl radicals and hydrogen peroxide. Because of this, flavin oxidases make a significant contribution to the total oxidant stress of the body. 

In contrast to the flavin oxidases, flavin dehydrogenases pass electrons to carriers within electron transport chains and the flavin does not react with 02. Examples include a bacterial trimethylamine dehydrogenase (Fig. 15-9) which contains an iron-sulfur duster that serves as the immediate electron acceptor167 169 and yeast flavocytochrome b2, a lactate dehydrogenase that passes electrons to a built-in heme group which can then pass the electrons to an external acceptor, another heme in cytochrome c.170-173 Like glycolate oxidase, these enzymes bind their flavin coenzyme at the ends of 8-stranded a(i barrels similar... 

Formation of H202 by flavin oxidases can occur via elimination of a peroxide anion HOO from the adduct of Eq. 15-31 with regeneration of the oxidized flavin. 

Superoxide is also a product of various enzyme reactions catalyzed by the flavin oxidases (e.g., xanthine oxidase and monoamine oxidase). In addition, 07 is a product of the noncatalytic oxidation of oxyhemoglobin, of which about 3% is converted each day to methemoglobin. Moreover, 02 is readily formed in phagocytic cells (i.e., neutrophils and monocytes) during the respiratory burst. Furthermore, in addition to the Fenton reaction, the Haber-Weiss reaction results in the conversion of 02 to the potent HO via the following reactions (H3) ... 

Figure 5 Illustration of possible partial reaction cycles of some copper- and flavin-dependent oxidase enzymes, (a) Copper amine oxidase 30, 31 (b) galactose oxidase (32) (c) catechol oxidase (10) (d) multicopper oxidases (10) (e) flavin oxidases (30) (f) cytochrome c oxidase (38).

Rice bran contains active enzymes (30). Germ and the outer layers of the caryopsis have higher enzyme activities. Some enzymes that are present include a-amylase, p-amylase, ascorbic acid oxidase, catalase, cytochrome oxidase, dehydrogenase, deoxyribonuclease, esterase, flavin oxidase, a and p-glycosidase, invertase, lecithi-nase, lipase, lipoxygenase, pectinase, peroxidase, phosphatase, phytase, proteinase, and succinate dehydrogenase. 

The second initiation way for the lipoperoxidation in the organism can be defined as semi-enzymatic or quasi-enzymatic. During this mechanism the O " radicals are generated by enzymes including NAD(P)H-dependent oxidases of mitochondrial and microsomal electron transport chaines, NADPH-dependent oxidase of phagocytes, xanthine oxidase and other flavine oxidases. After the HO formation the oxidation process develops in non-enzymatic way. 

To understand the carbanion mechanism in flavocytochrome 62 it is useful to first consider work carried out on related flavoenzymes. An investigation into o-amino acid oxidase by Walsh et al. 107), revealed that pyruvate was produced as a by-product of the oxidation of )8-chloroalanine to chloropyruvate. This observation was interpreted as evidence for a mechanism in which the initial step was C -H abstraction to form a carbanion intermediate. This intermediate would then be oxidized to form chloropyruvate or would undergo halogen elimination to form an enamine with subsequent ketonization to yield pyruvate. The analogous reaction of lactate oxidase with jS-chlorolactate gave similar results 108) and it was proposed that these flavoenzymes worked by a common mechanism. Further evidence consistent with these proposals was obtained by inactivation studies of flavin oxidases with acetylenic substrates, wherein the carbanion intermediate can lead to an allenic carbanion, which can then form a stable covalent adduct with the flavin group 109). Finally, it was noted that preformed nitroalkane carbanions, such as ethane nitronate, acted as substrates of D-amino acid oxidase 110). Thus three lines of experimental evidence were consistent with a carbanion mechanism in flavoenzymes such as D-amino acid oxidase. 

Superoxide can also be produced by other enzymes, such as the range of flavin oxidases located in peroxisomes, and by oxidation of certain compounds including ascorbic acid, thiols, and adrenaline in the presence of transition metal ions. The autoxidation of reduced transition metal can also generate the superoxide... 

Oi-, OH or OH+), a similar mode of action can be taken into consideration for the native flavoproteins. The specific action of different flavin oxidases, dehydrogenases and hydroxylases can most likely be attributed to specific proteins which are bound with the flavin moiety and are in a position to dictate the particular breakdown of HFIOOH. For example, the oxidases were thought to react specifically, yielding oxidized flavin and H2O2, while the dehydrogenases were supposed to yield both flavin and superoxide radicals (HF1- + HO 2)- The hydroxylases were assumed to account for the reaction of one of the activated oxygen intermediates with the respective substrate. 

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