Redox properties of the reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 and reduced nicotinamide adenine dinucleotide-cytochrome b<sub>5</sub> reductases

Iyanagi, Takashi; Makino, Nobuo; Mason, Howard S.

doi:10.1021/bi00705a023

Cited by 211 publications

(148 citation statements)

References 35 publications

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“…Addition of NADPH under aerobic conditions resulted in the appearance of a long wavelength absorption band with a peak at 585 nm and a shoulder at 640 nm. This is consistent with the formation of an air stable semiquinone as seen in P-450 reductase (22,23) (26), respectively, and that from an equimolar mixture of the independently expressed flavin containing domains of human cytochrome P-450 reductase (27).…”

Section: Purification Of Recombinant Human Methionine Synthasesupporting

confidence: 83%

Human Methionine Synthase Reductase, a Soluble P-450 Reductase-like Dual Flavoprotein, Is Sufficient for NADPH-dependent Methionine Synthase Activation

Olteanu

Banerjee

2001

Journal of Biological Chemistry

169

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Methionine synthase is a key enzyme in the methionine cycle that catalyzes the transmethylation of homocysteine to methionine in a cobalamin-dependent reaction that utilizes methyltetrahydrofolate as a methyl group donor. Cob(I)alamin, a supernucleophilic form of the cofactor, is an intermediate in this reaction, and its reactivity renders the enzyme susceptible to oxidative inactivation. In bacteria, an NADPH-dependent twoprotein system comprising flavodoxin reductase and flavodoxin, transfers electrons during reactivation of methionine synthase. Until recently, the physiological reducing system in mammals was unknown. Identification of mutations in the gene encoding a putative methionine synthase reductase in the cblE class of patients with an isolated functional deficiency of methionine synthase suggested a role for this protein in activation In this study, we have cloned and expressed the cDNA encoding human methionine synthase reductase and demonstrate that it is sufficient for supporting NADPH-dependent activity of methionine synthase at a level that is comparable with that seen in the in vitro assay that utilizes artificial reductants. Methionine synthase reductase is a soluble, monomeric protein with a molecular mass of 78 kDa. It is a member of the family of dual flavoproteins and is isolated with an equimolar concentration of FAD and FMN. Reduction by NADPH results in the formation of an air stable semiquinone similar to that observed with cytochrome P-450 reductase. Methionine synthase reductase reduces cytochrome c in an NADPH-dependent reaction at a rate (0.44 mol min ؊1 mg ؊1 at 25°C) that is comparable with that reported for NR1, a soluble dual flavoprotein of unknown function, but is ϳ100-fold slower than that of P-450 reductase. The K m for NADPH is 2.6 ؎ 0.5 M, and the K act for methionine synthase reductase is 80.7 ؎ 13.7 nM for NADPH-dependent activity of methionine synthase.Homocysteine is a key junction metabolite in the methionine cycle that is formed by the hydrolysis of S-adenosylhomocysteine, the product of S-adenosylmethionine-dependent methylation reactions. Elevated levels of homocysteine are correlated with cardiovascular diseases, neural tube defects, and Alzheimer's disease (1-4). Intracellular levels of homocysteine are kept low by the action of two classes of reactions. The transmethylation reactions catalyzed by methionine synthase or betaine homocysteine methyltransferase salvage homocysteine back to the methionine cycle. Alternatively, the ␤-replacement reaction catalyzed by cystathionine ␤-synthase commits homocysteine to the trans sulfuration pathway and provides a mechanism for generating cysteine from the essential amino acid, methionine. Of the two transmethylases, betaine homocysteine methyltransferase has a relatively restricted tissue distribution, whereas methionine synthase is believed to be ubiquitous (5).The mammalian methionine synthase is a methylcobalamindependent enzyme that catalyzes the successive transfer of a methyl group from CH 3 -H 4 folate 1 to the cob...

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Section: Purification Of Recombinant Human Methionine Synthasesupporting

confidence: 83%

Human Methionine Synthase Reductase, a Soluble P-450 Reductase-like Dual Flavoprotein, Is Sufficient for NADPH-dependent Methionine Synthase Activation

Olteanu

Banerjee

2001

Journal of Biological Chemistry

169

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“…The effects of the mutations on the binding affinity of hHO-1 265 for P450 reductase were then determined (Table II). These mutagenesis studies identified the positively charged residues Lys 18 , Lys 22 23 , and Glu 201 , had little of no effect on the binding of P450 reductase. In accord with the negative charged surface of P450 reductase in the vicinity of the FMN group, from which electrons are transferred to the heme group of acceptor proteins, most of the mutations that significantly decreased P450 reductase binding involved conversion of a positively charged side chain on hHO-1 265 to a neutral side chain.…”

Section: Discussionmentioning

confidence: 97%

“…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).…”

mentioning

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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“…This fitting procedure only allowed access to the relative values for redox potentials, absolute values for ATR1 were positioned assuming the same value for the FAD oxidized/reduced redox potential as for the rat enzyme (Ϫ327 mV). Values for rat and BM3 CPRs are from literature [35,36,39,43,44]. Experimental anaerobic titration was performed for ATR1 and SCR1 (the two central panels).…”

Section: Analysis Of Flavin Redox Potentials By Dithionite Titrationmentioning

confidence: 99%

“…Fully reduced flavin (᭹) was deduced from the cofactor balance. Full lines represent calculated curves; for these, flavin repartition between the different redox states was calculated for a virtual titration experiment based on redox potentials described on the top panel and the Nernst equation, assuming thermodynamic equilibrium between all flavin redox state, as described previously [39] and in Experimental Procedures. In the case of rat and BM3 curves, points are not experimental, but calculated, values for equal potential steps.…”

Section: Analysis Of Flavin Redox Potentials By Dithionite Titrationmentioning

confidence: 99%

Differential redox and electron‐transfer properties of purified yeast, plant and human NADPH‐cytochrome P‐450 reductases highly modulate cytochrome P‐450 activities

Louérat-Oriou

Perret

Pompon

1998

European Journal of Biochemistry

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Saccharomyces, human and two Arabidopsis (ATR1 and ATR2) NADPH-P-450 reductases were expressed in yeast, purified to homogeneity and used to raise antibodies. Among the P-450-reductases, ATR2 contrasted by its very low FMN affinity and required a thiol-reducing agent for efficient cofactor binding to the FMN-depleted enzyme. Analysis of reductase kinetic properties using artificial acceptors and different salt conditions suggested marked differences between reductases in their FAD and FMN environments and confirmed the unusual properties of the ATR2 FMN-binding domain. Courses of flavin reductions by NADPH were analysed by rapid kinetic studies. The human enzyme was characterized by a FAD reduction rate sixfold to tenfold slower than values for the three other reductases. Following the fast phase of reduction, expected accumulation of flavin semiquinone was observed for the human and ATR1 but not for ATR2 and the yeast reductases. Consistently, redox potential for the FMN semiquinone/ reduced couple in the yeast enzyme was found to be more positive than the value for the FMN oxidized/ semiquinone couple. This situation was reminiscent of similar inversion observed in bacterial P-450 BM3 reductase. Affinities of reductases for rabbit P-450 2B4 and supported monooxygenase activities in reconstituted systems highly depended on the reductase source. The human enzyme exhibited the highest affinity but supported the lowest k cat whereas the yeast reductase gave the best k cat but with the lowest affinity. ATR1 exhibited both high affinity and efficiency. No simple relation was found between reductase activies with artificial and natural (P-450) acceptors. Thus marked differences in kinetic and redox parameters between reductases dramatically affect their respective abilities to to support P-450 functions.Keywords : NADPH-cytochrome P-450 reductase; redox potential ; kinetic property; P-450 interaction.NADPH-P-450 reductase (CPR; NADPH : ferrihemoprotein reductase) is a membrane-anchored protein that catalyzes electron transfers from NADPH to cytochrome P-450 (P-450) [1Ϫ4], heme oxygenase, cytochrome b 5 and squalene epoxidase [5,6]. The various members of the P-450 superfamily [7] are involved in CPR-supported-oxidative metabolism of a wide range of xenobiotics and endogenous compounds [8]. Contrasting with the large variety of P-450s, a single CPR is generally present in each organism [9,10]. Unlike animals and yeast, higher plants express multiple forms of CPRs as inferred from western blot analysis of purified materials [11,12]. Durst et al.[13] have isolated two distinct partial length cDNA from Helianthus tuberosus, corresponding to distinct mRNA species. The two CPRs isolated from parsley were 80% identical in amino acid sequence and a distinct metabolic role was clearly assigned to each other [14]. In Arabidopsis thaliana, two distantly related Enzymes. NADPH-cytochrome P-450 reductase (EC 1.6.2.4); cytochrome P-450 (EC 1.14.14.1).reductases (ATR1 and ATR2, 64% amino acid sequence identity) were cloned and may ...

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Redox properties of the reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 and reduced nicotinamide adenine dinucleotide-cytochrome b₅ reductases

Cited by 211 publications

References 35 publications

Human Methionine Synthase Reductase, a Soluble P-450 Reductase-like Dual Flavoprotein, Is Sufficient for NADPH-dependent Methionine Synthase Activation

Human Methionine Synthase Reductase, a Soluble P-450 Reductase-like Dual Flavoprotein, Is Sufficient for NADPH-dependent Methionine Synthase Activation

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

Differential redox and electron‐transfer properties of purified yeast, plant and human NADPH‐cytochrome P‐450 reductases highly modulate cytochrome P‐450 activities

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Redox properties of the reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 and reduced nicotinamide adenine dinucleotide-cytochrome b5 reductases

Cited by 211 publications

References 35 publications

Human Methionine Synthase Reductase, a Soluble P-450 Reductase-like Dual Flavoprotein, Is Sufficient for NADPH-dependent Methionine Synthase Activation

Human Methionine Synthase Reductase, a Soluble P-450 Reductase-like Dual Flavoprotein, Is Sufficient for NADPH-dependent Methionine Synthase Activation

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

Differential redox and electron‐transfer properties of purified yeast, plant and human NADPH‐cytochrome P‐450 reductases highly modulate cytochrome P‐450 activities

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Redox properties of the reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 and reduced nicotinamide adenine dinucleotide-cytochrome b₅ reductases