Studies on the microsomal mixed-function oxidase system: mechanism of action of hepatic NADPH-cytochrome P-450 reductase

Iyanagi, Takashi; Makino, Ryu; Fk, Anan

doi:10.1021/bi00510a004

Cited by 98 publications

(45 citation statements)

References 30 publications

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“…Typically, this second group can undergo only a one-electron change between its oxidized and reduced forms (22). This second prosthetic group may reside on a separate polypeptide, as in the toluene dioxygenase (17,33,34), w-hydroxylase (36,37), and the spinach ferredoxin (15,35) (5,6), and the NADPHcytochrome c (P450) systems (21,38). In this arrangement, the reductase components are capable of reducing cytochrome c directly in the presence of NAD(P)H. Loss of cytochrome c reductase activity upon removal of the ironsulfur clusters from 4-methoxybenzoate-O-demethylase (6) and benzoate-1,2-dioxygenase (43) indicates that the ironsulfur center is involved in electron transfer to cytochrome c. FerredoxinNAp reductase catalyzes cytochrome c reduction independently, which suggests that it contains a second prosthetic group.…”

Section: Resultsmentioning

confidence: 99%

Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816

1990

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Cells of Pseudomonas sp. strain NCIB 9816, after growth with naphthalene or salicylate, contain a multicomponent enzyme system that oxidizes naphthalene to cis-(lR,2S)-dihydroxy-1,2-dihydronaphthalene. We purified one of these components to homogeneity and found it to be an iron-sulfur flavoprotein that loses the flavin cofactor during purification. Dialysis against flavin adenine dinucleotide (FAD) showed that the enzyme bound 1 mol of FAD per mol of enzyme protein. The enzyme consisted of a single polypeptide with an apparent molecular weight of 36,300. The purified protein contained 1.8 g-atoms of iron and 2.0 g-atoms of acid-labile sulfur and showed absorption maxima at 278, 340, 420, and 460 nm, with a broad shoulder at 540 nm. The purified enzyme catalyzed the reduction of cytochrome c, dichlorophenolindophenol, Nitro Blue Tetrazolium, and ferricyanide. These activities were enhanced in the presence of added FAD. The ability of the enzyme to catalyze the reduction of the ferredoxin involved in naphthalene reduction and other electron acceptors indicates that it functions as an NAD(P)H-oxidoreductase in the naphthalene dioxygenase system. The results suggest that naphthalene dioxygenase requires two proteins with three redox groups to transfer electrons from NADH to the terminal oxygenase.Naphthalene dioxygenase, a multicomponent enzyme system which oxidizes naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene, is induced in Pseudomonas sp. strain NCIB 9816 during growth on naphthalene or salicylate (2,12,20). After growth on naphthalene, the organism also oxidizes acenaphthalene (31), indole (13), and indan (39). Indan is apparently oxidized by naphthalene dioxygenase in a monooxygenation reaction similar to that described for toluene dioxygenase (39). This suggests that naphthalene dioxygenase can function as either a monooxygenase or a dioxygenase, depending on the substrate.Naphthalene dioxygenase consists of a terminal ironsulfur-containing oxygenase (ISPNAP) and two other proteins which function as a short electron transfer chain (11,12). Thus, naphthalene dioxygenase is similar to the threecomponent enzyme systems involved in the oxidation of benzene (1, 16), toluene (32-34), and pyrazon (29

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Section: Resultsmentioning

confidence: 99%

Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816

1990

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“…The NADPH-cytochrome P450 reductase was purified from phenobarbital-induced rat liver microsomes with DEAE-Toyopearl and 2Ј, 5Ј-ADP-Sepharose 4B column chromatographies as previously described (29,30). Concentration of the reductase was determined from the absorption spectrum of the oxidized form using ⑀ 457 nm ϭ 10.7 mM Ϫ1 cm Ϫ1 (29,30).…”

Section: Nadph-cytochrome P450 Reductasementioning

confidence: 99%

Interactions between the Isolated Oxygenase and Reductase Domains of Neuronal Nitric-oxide Synthase

Rozhkova¹,

Fujimoto²,

Sagami³

et al. 2002

Journal of Biological Chemistry

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Nitric-oxide synthase (NOS) is a fusion protein composed of an oxygenase domain with a heme-active site and a reductase domain with an NADPH binding site and requires Ca 2؉ /calmodulin (CaM) for NO formation activity. We studied NO formation activity in reconstituted systems consisting of the isolated oxygenase and reductase domains of neuronal NOS with and without the CaM binding site. Reductase domains with 33-amino acid C-terminal truncations were also examined. These were shown to have faster cytochrome c reduction rates in the absence of CaM. N G -hydroxy-L-Arg, an intermediate in the physiological NO synthesis reaction, was found to be a viable substrate. Turnover rates for N Ghydroxy-L-Arg in the absence of Ca 2؉ /CaM in most of the reconstituted systems were 2.3-3.1 min ؊1 . Surprisingly, the NO formation activities with CaM binding sites on either reductase or oxygenase domains were decreased dramatically on addition of Ca 2؉ /CaM. However, NADPH oxidation and cytochrome c reduction rates were increased by the same procedure. Activation of the reductase domains by CaM addition or by C-terminal deletion failed to increase the rate of NO synthesis. Therefore, both mechanisms appear to be less important than the domain-domain interaction, which is controlled by CaM binding in wild-type neuronal NOS, but not in the reconstituted systems. The well known fusion protein, cytochrome P450BM3, is composed of a P450 oxygenase domain and a CPR domain and is also driven via intramolecular electron transfer. However, NOS is reported to have a unique intermolecular/intersubunit electron transfer system in which electrons from the reductase domain of one subunit transfer crosswise to the oxygenase domain of the other subunit in the homodimeric enzyme (9 -12). The crucial role of (6R)-5,6,7,8-tetrahydro-L-biopterin (H4B) in catalysis with NOS, probably associated with redox function and/or electron transfer, is also noted (13-15).The oxygenase domain of NOS can be efficiently expressed heterogeneously in Escherichia coli (Refs. 1-15 and references therein). It is stable, easily handled, and purified as a homodimer in the presence of H4B. However, for simplicity, this has not usually included the CaM binding site. Therefore, to further examine the role of CaM binding in catalysis, particularly its effect on the oxygenase domain, it was thought necessary to examine the catalytic properties of an oxygenase domain mutant including the CaM binding site. Conversely, most of the isolated reductase domains so far studied include the CaM-binding site to study the effect of CaM on the intramolecular electron transfer from FAD to . Intriguingly, the C termini of the reductase domains of inducible and constitute full-length NOSs to attenuate electron flow through the flavine and heme domains (19,20). The isolated reductase domain without the CaM-binding site and isolated C-terminal truncated reductase domains are also examined in this paper.* This work was supported in part by Japan Society for the Promotion of Science Fellowship 9...

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“…Heterologous expression of the full-length P450 reductase followed by trypsinolysis to remove the 6-kDa Nterminal membrane-binding domain yields a soluble protein whose crystal structure has been determined (22). The electron flow in this protein has been established by studies with cytochrome c and cytochrome P450 as electron acceptors to proceed from the NADPH to the FAD and finally to the FMN prior to transfer to the acceptor heme group (23)(24)(25). A role for charge pairing in formation of transient electron transfer complexes between cytochrome P450 enzymes and P450 reductase is supported by site-directed mutagenesis studies (26,30).…”

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confidence: 99%

The Binding Sites on Human Heme Oxygenase-1 for Cytochrome P450 Reductase and Biliverdin Reductase

Wang

Montellano

2003

Journal of Biological Chemistry

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Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000 Heme oxygenase oxidizes heme, 1 a pro-oxidant and toxic species, to biliverdin, CO, and free iron (1). Each of the three metabolites formed in the reaction is thought to be physiologically important. Biliverdin is converted by biliverdin reductase to bilirubin, which must then be conjugated with glucuronic acid to be excreted (2). However, bilirubin, albeit toxic at high concentrations, has potent antioxidant properties that may contribute to the anti-inflammatory activity of the heme oxygenases (3, 4). Although still controversial, CO appears to behave as a gaseous signaling molecule akin to nitric oxide (5-7). Finally, the iron released from heme is largely recycled and is critical for iron homeostasis, since less than 3% of the body's daily iron needs are met by absorption from the diet (8). As a result of these activities, heme oxygenase has been shown to have, inter alia, important anti-inflammatory (9) and antiatherosclerotic (10) functions.Human heme oxygenase-1 (hHO-1) is a 33-kDa membranebound protein of 288 amino acid residues that binds heme and uses the bound entity as both the prosthetic group and substrate. A soluble form of hHO-1 (hHO-1 265 ) has been obtained by heterologous expression in E. coli of a truncated version of 265 amino acids that lacks the membrane binding domain (11). The crystal structures of hHO-1 truncated to a length of 233 amino acids (12), a rat homologue truncated to a length of 267 residues (13), and an inherently soluble full-length microbial heme oxygenase (14) have been determined. As recently reviewed, mechanistic studies have established that the transformation of heme to biliverdin is a three-step process that consumes three molecules of oxygen and seven electrons (15, 16). In the first step, ferric heme is oxidized to ␣-meso-hydroxyheme in a reaction that consumes one molecule of oxygen and two electrons. The ferric ␣-meso-hydroxyheme is then converted to ferrous verdoheme in a reaction that consumes a further molecule of oxygen and two electrons and releases CO. In the final stage of the reaction sequence, the verdoheme is converted to ferrous biliverdin with the consumption of a further molecule of oxygen and three electrons. The ferrous iron is then released, followed by dissociation of the biliverdin. Single turnover studies have shown that the rate-limiting step is the release of biliverdin, but in the presence of biliverdin reductase this step is accelerated, and one of the electron transfer steps becomes rate-limiting (17).The electrons required for the catalytic turnover of heme oxygenase are provided by NADPH-cytochrome P450 reductase (1,19,20), a 78-kDa membrane-bound flavoprotein that incorpo...

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Studies on the microsomal mixed-function oxidase system: mechanism of action of hepatic NADPH-cytochrome P-450 reductase

Cited by 98 publications

References 30 publications

Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816

Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816

Interactions between the Isolated Oxygenase and Reductase Domains of Neuronal Nitric-oxide Synthase

The Binding Sites on Human Heme Oxygenase-1 for Cytochrome P450 Reductase and Biliverdin Reductase

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