Flavin:NADH reductase

Reduced flavins (FADH2, FMNH2, and riboflavin) generated by flavin-dependent reductases have been hypothesized to reduce azo dyes in a nonspecific chemical reaction, and flavin reductases have been revealed to be indeed anaerobic azoreductases. Other reduced enzyme cofactors, for example, NADH, NADH, NADPH, and an NADPH-generating system, have also been reported to reduce azo dyes. Except for enzyme cofactors, different artificial redox mediating compounds, especially such as quinines, are important redox mediators of azo dye anaerobic reduction (Table 1). 

Flavin-dependent reductases use hydrogen atoms from NADH or NADPH to reduce many specific substances or classes of compounds. The FAD-containing ferredoxin ... 

Thus for dehydrogenase partial activities of NR, the 28kDa FAD domain alone is a NADH ferricyanide reductase (NADH FR) and the FAD domain linked to the haem domain (40kDa) is a NA DH cytochrome c reductase (NADH CR) as well as a NADH dichloro-phenolindophenol reductase (NADH.DR). As for the nitrate-reducing activities, the methylviologen nitrate reductase (MV NR) and the reduced flavin nitrate reductase (FMN NR) activities involve the MoCo domain (75 kDa) linked to the haem domain, whereas reduced brom-... 

They all have a flavin-containing reductase subunit that uses NADPH, and not NADH, as a substrate. 

B) They all contain a flavin-containing reductase unit that uses NADH and not NADPH as a source of electrons. 

As its name implies, this complex transfers a pair of electrons from NADH to coenzyme Q a small, hydrophobic, yellow compound. Another common name for this enzyme complex is NADH dehydrogenase. The complex (with an estimated mass of 850 kD) involves more than 30 polypeptide chains, one molecule of flavin mononucleotide (FMN), and as many as seven Fe-S clusters, together containing a total of 20 to 26 iron atoms (Table 21.2). By virtue of its dependence on FMN, NADH-UQ reductase is a jlavoprotein. 

If the luciferase sample solution contains a flavin-reductase, luciferase activity can be measured by the addition of FMN and NADH, instead of FMNH2. In this case, the turnover of luciferase takes place repeatedly using the FMNH2 that is enzymatically generated thus, the luminescence reaction continues until aldehyde or NADH is exhausted. A crude luciferase extracted from luminous bacteria usually contains a flavin-reductase. 

Under conditions of copper deficiency, some methanotrophs can express a cytosolic, soluble form of MMO (sMMO) (20-23), the properties of which form the focus of the present review. The sMMO system comprises three separate protein components which have all been purified to homogeneity (24,25). The hydroxylase component, a 251 kD protein, contains two copies each of three subunits in an a 82y2 configuration. The a subunit of the hydroxylase houses the dinuclear iron center (26) responsible for dioxygen activation and for substrate hydroxylation (27). The 38.6 kD reductase contains flavin adenine dinucleotide (FAD) and Fe2S2 cofactors (28), which enable it to relay electrons from reduced nicotinamide adenine dinucleotide (NADH) to the diiron center in the... 

The flavin reductase has been purified by several researchers. This enzyme from R. erythropolis IGTS8 was partially purified by Ohshiro et al., and reported to have an optimum pH and temperature of 6.0 and 35°C, respectively [153], The DszD enzyme from IGTS8 was also purified [53] and reported to be of 25 kDa size however, no kinetic details related to DszD were reported. This enzyme couples with FMN with NADH to produce reduced flavin required for DszC and DszA catalyzed reactions. 

The report of Basran et al. (entry 5 of Table 2) contains two studies involving hydride transfer with nicotinamide cofactors. In morphinone-reductase catalyzed reduction by NADH of the flavin cofactor FMN (schematic mechanism in Fig. 5), the primary isotope effects are modest (around 4 for H/D), but exhibit a small value of Ajj/Aq (0.13) and an exalted isotopic difference in energies of activation (8.2kJ/mol) that alone would have generated an isotope effect around 30. The enthalpies of activation are in the range of 35-45 kJ/mol. This is behavior typical of Bell tunneling as discussed above. It can also be reproduced by more complex models, as will be discussed in later parts of this review. 

Examination of one real-life case may benefit the reader s understanding. Strittmatter studied the primary kinetic isotope effects arising in the NADH-dependent cytochrome bs reductase (EC 1.6.2.2). The oxidation of NADH and subsequent reduction of cytochrome bs is facilitated by the enzyme-bound FAD group, and the kinetics of the direct transfer of a hydrogen from the A-face (or pro-R) of NADH to the flavin can be monitored by the loss of the 340 run absorbance of the NADH s dihydropyridine ring. Using deuterated isotopic isomers of NADH and several related compounds, Strittmatter obtained the primary kinetic isotope effect data compiled in the table below. 

Pyridine nucleotide-dependent flavoenzyme catalyzed reactions are known for the external monooxygenase and the disulfide oxidoreductases However, no evidence for the direct participation of the flavin semiquinone as an intermediate in catalysis has been found in these systems. In contrast, flavin semiquinones are necessary intermediates in those pyridine nucleotide-dependent enzymes in which electron transfer from the flavin involves an obligate 1-electron acceptor such as a heme or an iron-sulfur center. Examples of such enzymes include NADPH-cytochrome P4S0 reductase, NADH-cytochrome bs reductase, ferredoxin — NADP reductase, adrenodoxin reductase as well as more complex enzymes such as the mitochondrial NADH dehydrogenase and xanthine dehydrogenase. 

Hyde, G.E. Campbell, W.H. (1990). High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH-nitrate, reductase and its comparison to human NADH-cytochrome b5 reductase. Biochemical and Biophysical Research Communications 168, 1285-91. 

Enantioselective oxidation of racemic alcohols as well as reduction of racemic ketones and aldehydes have been widely applied to obtain optically active alcohols.25 27 The enzymes catalyzing these reactions are alcohol dehydrogenase, oxidases, and reductases etc. Coenzymes (NADH, NADPH, flavine etc) are usually necessary for theses enzymes. For example, for the oxidation of alcohols, NAD(P)+ are used. The hydride removed from the substrate is transferred to the coenzyme bound in the enzyme, as shown in Figure 24. There are four stereochemical patterns, but only three types of the enzymes are known. 

Nitroreductase activity has been demonstrated in liver homogenates as well as in the soluble fraction, whereas other studies have reported that nitroreductase activity has been found in all liver fractions evaluated. The reductase appears to be distributed in liver, kidney, lung, heart, and brain. The reaction utilizes both NADPH and NADH and requires anaerobic conditions. The reaction can be inhibited by the addition of oxygen. The reaction is stimulated by FMN and FAD, and at high flavin concentrations they can act simply as nonenzymatic electron donors. The reduction... 

Recently, the first asymmetric cell-free application of styrene monooxygenase (StyAB) from Pseudomonas sp. VLB 120 was reported [294]. StyAB catalyses the enantiospecific epoxidation of styrene-type substrates and requires the presence of flavin and NADH as cofactor. This two-component system enzyme consists of the actual oxygenase subunit (StyA) and a reductase (StyB). In this case, the reaction could be made catalytic with respect to NADH when formate together with oxygen were used as the actual oxidant and sacrificial reductant respectively. The whole sequence is shown in Fig. 4.106. The total turnover number on StyA enzyme was around 2000, whereas the turnover number relative to NADH ranged from 66 to 87. Results for individual substrates are also given in Fig. 4.106. Excellent enantioselectivities are obtained for a- and -styrene derivatives. 

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