Cytochrome reductase, flavoprotein

Microsomes contain, in addition to the two cytochrome reductases just discussed, a flavoprotein which catalyzes the mixed function oxidation of secondary and tertiary amines to the hydroxylamines and amine oxides, respectively (333, 334). This flavoprotein, which contains about 2 moles of phospholipid and 1 mole of FAD per 70,000 g of protein, is specific for NADPH (333, 334) The enzyme is also able to catalyze the further oxidation of the hydroxylamines to nitrones (336). The reactions... 

Two dehydrogenases and a cytochrome carry the electrons from cellular NADPH and NADH to the terminal oxidase, cytochrome P-450. This cytochrome catalyzes the hydroxylation of a substrate. The reaction scheme has been described by Peterson et al. (1973). Fpj and Fpa (see Fig. 28) are the two flavoproteins NADPH cytochrome P-450 reductase and NADH cytochrome reductase. 

Methemoglobinemia has long been associated with the absence of an NADH-linked diaphorase (363, 363). However, flavoproteins isolated from the red cell were never sufficiently active to account for the methemoglobin reductase activity calculated to be necessary. It has now been shown that a methemoglobin reductase system of high activity is composed of soluble forms of cytochrome reductase and cytochrome bs... 

The two-domain, structural motif in FNR represents a common structural feature in a large class of enzymes that catalyze electron transfer between a nicotinamide dinucleotide molecule and a one-electron carrier. Beside the photosynthetic electron-transfer enzyme, others non-photosynthetic ones include flavodoxin reductase, sulfite reductase, nitrate reductase, cytochrome reductase, and NADPH-cyto-chrome P450 reductase. FNR belongs to the group of so-called dehydrogenases-electron transferases, i.e., flavoproteins that catalyze electron transfer from two, one-electron donor molecules to a single two-electron acceptor molecule. 

Stearoyl-CoA desaturase (SCD) resides in the ER where it catalyzes the biosynthesis of MUFA from SFA that are either synthesized de novo or derived from the diet (Fig. 2). In conjunction with NADH, the flavoprotein cytochrome reductase, the electron acceptor cytochrome b, and molecular oxygen, SCD introduces a single double bond into a spectrum of methylene-intemipted fatty acyl-CoA substrates (Fig. 3). 

Fisher, C.W., M.S. Shet, D.L. Caudle, C.A. Martin-Wixtrom, and R.W. Estabrook (1992). High-level expression in Escherichia coli of enzymatically active fusion proteins containing the domains of mammalian cytochromes P450 and NADPH-P450 reductase flavoprotein. Proc. Natl. Acad. Sci. USA 89, 10817-10821. 

In all these reactions, the flavin nucleotide combines with a protein to form a flavoprotein. In the reactions involved in electron transport, the flavoprotein acts as a hydrogen carrier and plays an important role in the transfer of electrons from pyridine nucleotides to cytochromes. Among the flavoproteins of interest in the electron transport chain are (1) coenzyme 1-diaphorase, (2) the Warburg flavoprotein, (3) NADH cytochrome c reductase, (4) NADPH cytochrome c reductase, (5) NADH cytochrome reductase, (6) NADH 2-methyl-l,4maphthoquinone reductase, and (7) succinic cytochrome c reductase. The most important among these are succinic cytochrome c reductase and NADH cytochrome c reductase. The following discussion of these two flavoproteins may serve as an example for the others. 

The structure of calf liver cytochrome 65 has been determined at 2.8 A and more recently at 2 A resolution. This protein has a molecular weight of 11 000 and contains 93 amino-acids. It interacts specifically with a flavoprotein, cytochrome reductase, which catalyses the transfer of electrons from NADH to the haem iron of the cytochrome. 

Cytochrome Reductase of Yeast. A third yeast flavoprotein was isolated by Haas and co-workers. This was called cytochrome reductase because its rate of reaction with cytochrome c is over 150,000 times as great as the rate of cytochrome c reduction by old yellow enzyme. Its rate of reaction with oxygen is only 8 per cent that of old yellow enzyme. The prosthetic group is FMN. Reduction occurs only with TPNH. Another enzyme, DPN-cytochrome reductase, was partially purified in Hogness laboratory. 

Diaphorase and Cytochrome Reductase. Enzymes have been isolated from animal sources that have many of the properties of the various yeast enzymes. The first, and simplest, was liberated from particulate structures by Straub, who employed dilute ethanol and ammonium sulfate at 43 C. The enzyme could then be purified and was named a diaphorase. Diaphorase was coined to identify a widespread group of enzymes that transfer electrons from DPNH to dyes. Many of the purified flavoproteins have been found to oxidize pyridine nucleotides, and almost all of the flavoproteins can reduce dyes. Straub s diaphorase reduces methylene blue but not cytochrome c. Slight modification of the isolation procedure was found by workers at the Enzyme Institute of the University of Wisconsin to yield a cytochrome reductase. The relation between these preparations is not known, but it is possible that one is derived from the other. Cytochrome reductase contains 4 atoms of iron for each flavin, whereas Straub s preparation contains little iron. Both proteins have molecular weights around 75,000, and contain 1 equivalent of FAD. 

Metallo-Flavoproteins. As was mentioned in the case of cytochrome reductase, enzymes are known that contain metal cofactors in addition to flavin. These are called metallo-flavoproteins. The presence of metals introduces complexity into the reaction, since the metals involved, iron, molybdenum, copper, and manganese, all exist in at least two valence states and can participate in oxidation-reduction reactions. The enzymes known to be metallo-flavoproteins include xanthine oxidase, aldehyde oxidase, nitrate reductase, succinic dehydrogenase, fatty acyl CoA dehydrogenases, hydrogenase, and cytochrome reductases. Before these are discussed in detail some physical properties of flavin will be presented. 

Both the oxidation-reduction potential and the fluorescence of flavin nucleotides are modified profoundly by attachment of the nucleotide to various proteins. Flavin enzymes have been reported to have oxidation-reduction potentials at pH 7 ranging from —0.4 to 0.187. The combination to proteins also results in shifts of the absorption maxima. The 450 m u band is found at 451 mju in Straub s diaphorase and at 455 m/t in Haas yellow enzyme, while the 375 m/t band appears at 359 m/t and 377 m/t in these preparations. Most flavin enzymes do not fluoresce, and it is assumed that the quenching of fluorescence implies binding of the flavin to the enzyme through N-3. Straub s diaphorase, unlike most other flavoproteins, does fluoresce. This may be evidence that this diaphorase is a partially degraded cytochrome reductase. 

NADH-cytochrome reductase is a flavoprotein of molecular weight 43 kDa, while cytochrome is a heme-containing protein of molecular weight 16.7 kDa. Cytochrome bj displays a catalytic hydrophilic region with 85-amino acid residues, including the NH -terminal, and the protein terminates in a hydrophobic COOH-terminal tail of approximately 40 amino acids. The latter connects the protein to the membrane. 

Both NADH-cytochrome reductase (a flavoprotein) and cytochrome 5 bind to preformed phospholipid vesicles and microsomes. 

Figure 1. Electron transport chains of the endoplasmic reticulum. (A) Microsomal acyl-Co A desaturation system composed of NADH-cytochrome reductase, cytochrome fos (a flavoprotein), and fatty acyl-CoA desaturase. (B) Microsomal hydroxylase system depicting participation of the NADPH-cytochrome P-450 reductase (a flavoprotein), cytochrome P-450, and phosphatidylcholine. The role of the phospholipid appears to be in enhancing interaction of the proteins. The reduced form of the hemoprotein cytochrome P-450, on addition of carbon monoxide, envinces a Soret maximum at 450 nm, accounting for its designation. There is evidence that these two systems (A and B) interact in the membrane.

To summarize, squalene epoxidase is a flavoprotein capable of catalyzing the insertion of oxygen into the 2,3-double bond of squalene to give 2,3-oxidosqualene, with the second oxygen atom from 02 being reduced to water. The reducing equivalents necessary for this transformation are relayed from NADPH through NADPH-cytochrome c reductase to the flavin cofactor of the epoxidase. 

Another pathway is the L-glycerol 3-phosphate shuttle (Figure 11). Cytosolic dihydroxyacetone phosphate is reduced by NADFl to s.n-glycerol 3-phosphate, catalyzed by s,n-glycerol 3-phosphate dehydrogenase, and this is then oxidized by s,n-glycerol 3-phosphate ubiquinone oxidoreductase to dihydroxyacetone phosphate, which is a flavoprotein on the outer surface of the inner membrane. By this route electrons enter the respiratory chain.from cytosolic NADH at the level of complex III. Less well defined is the possibility that cytosolic NADH is oxidized by cytochrome bs reductase in the outer mitochondrial membrane and that electrons are transferred via cytochrome b5 in the endoplasmic reticulum to the respiratory chain at the level of cytochrome c (Fischer et al., 1985). 

H)2-D3 is a weak agonist and must be modified by hydroxylation at position Cj for full biologic activity. This is accomplished in mitochondria of the renal proximal convoluted tubule by a three-component monooxygenase reaction that requires NADPFl, Mg, molecular oxygen, and at least three enzymes (1) a flavoprotein, renal ferredoxin reductase (2) an iron sulfur protein, renal ferredoxin and (3) cytochrome P450. This system produces l,25(OH)2-D3, which is the most potent namrally occurring metabolite of vitamin D. 

Although reduction of chromate Cr to Cr has been observed in a number of bacteria, these are not necessarily associated with chromate resistance. For example, reduction of chromate has been observed with cytochrome Cj in Desulfovibrio vulgaris (Lovley and Phillips 1994), soluble chromate reductase has been purified from Pseudomonas putida (Park et al. 2000), and a membrane-bound reductase has been purified from Enterobacter cloacae (Wang et al. 1990). The flavoprotein reductases from Pseudomonas putida (ChrR) and Escherichia coli (YieF) have been purified and can reduce Cr(VI) to Cr(III) (Ackerley et al. 2004). Whereas ChrR generated a semi-quinone and reactive oxygen species, YieR yielded no semiquinone, and is apparently an obligate four-electron reductant. It could therefore present a suitable enzyme for bioremediation. 

Yamazaki, H., Ueng, Y.F., Shimada, T. and Guengerich, F.P. (1995) Roles of divalent metal ions in oxidations catalyzed by recombinant cytochrome P450 3A4 and replacement of NADPH-cytochrome P450 reductase with other flavoproteins, ferredoxin, and oxygen surrogates. Biochemistry, 34, 8380—8389. 

The primary function of flavoprotein NADPH-cytochrome P-450 reductase is the hydro-xylation of various substrates, which occurs during electron transfer from NADPH to cytochrome P-450 [1] ... 

While cytochrome P-450 catalyzes the interaction with substrates, a final step of microsomal enzymatic system, flavoprotein NADPH-cytochrome P-450 reductase catalyzes the electron transfer from NADPH to cytochrome P-450. As is seen from Reaction (1), this enzyme contains one molecule of each of FMN and FAD. It has been suggested [4] that these flavins play different roles in catalysis FAD reacts with NADPH while FMN mediates electron... 

Defects of complex II. These have not been fully characterized in the few reported patients, and the diagnosis has often been based solely on a decrease of succinate-cytochrome c reductase activity (Fig. 42-3). However, partial complex II deficiency was documented in muscle and cultured fibroblasts from two sisters with clinical and neuroradiological evidence of Leigh s syndrome, and molecular genetic analysis showed that both patients were homozygous for a point mutation in the flavoprotein subunit of the complex [17]. This was the first documentation of a molecular defect in the nuclear genome associated with a respiratory chain disorder. 

It has been well recognized that the mixed-function oxidase system of Bacillus megaterium is involved in steroid hydroxylation (, as already described above. This enzyme system is composed of a NADPH-specific FMN flavoprotein (megaredoxin reductase), an iron-sulfur protein (megaredoxin) and cytochrome P cn. The megaredoxin protein plays an important role as an intermediate component of electron transfer from reduced flavoprotein to cytochrome P en. 

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