Crystal Structure of NADH-Cytochrome b5 Reductase from Pig Liver at 2.4 .ANG. Resolution

Nishida, Hirokazu; Inaka, Koji; Yamanaka, Masaki; Kaida, Satoshi; Kobayashi, Kazuo; Miki, Kunio

doi:10.1021/bi00009a004

Cited by 92 publications

(72 citation statements)

References 26 publications

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“…with other members of the ferredoxin reductase family of enzymes, including spinach ferredoxin-NADP + oxidoreductase (FNR) [10], corn nitrate reductase [11] and porcine cytochrome b & reductase [12]. The FLDR structure reveals an unusual flavinbinding conformation, with a hydrogen bond between the adenine and isoalloxazine rings, possibly explaining the unusual visible spectrum of the bound flavin [7].…”

Section: Introductionmentioning

confidence: 99%

Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

Leadbeater

McIver

Campopiano

et al. 2000

Biochemical Journal

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The structure of the Escherichia coli flavodoxin NADP + oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6 min V " ; R174A, 132.1 min V " ; R184A, 305.5 min V " versus wild type, 338.9 min V ") and increased K m for NADPH (R144A, 5.3 µM ; R174A, 20.2 µM ; R184A, 54.4 µM versus wild type, 3.9 µM). The k cat value for NADH-dependent cytochrome c reduction was increased for R174A (42.3 min V ") and R184A (50.4 min V ") compared with the wild type (33.0 min V "), consistent with roles for R174 and R184 in discriminating between NADPH\NADH by interaction with the adenosine ribose 2h-phosphate. Stopped-flow studies indicated that affinity (K d ) for NADPH was markedly reduced in mutants R144A (635 µM) and R184A (2.3 mM) compared with the wild

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Section: Introductionmentioning

confidence: 99%

Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

Leadbeater

McIver

Campopiano

et al. 2000

Biochemical Journal

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“…The preliminary tertiary structure of human erythrocyte b5R (15,16), and the detailed tertiary structures of porcine and rat liver b5Rs at 2.1 Å resolution have been determined by x-ray crystallography (17)(18)(19)(20)(21). These structural studies revealed that NADH-cytochrome b 5 reductase belongs to the structurally related so-called "ferredoxin reductase family" (22,23) together with other flavoenzymes such as ferredoxin-NADP ϩ oxidoreductase (FNR) (22), phthalate dioxygenase reductase (24), flavodoxin reductase (25), NADPH-cytochrome P-450 reductase (26), and the cytochrome b reductase domain of nitrate reductase (27).…”

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confidence: 99%

Role of Thr66 in Porcine NADH-cytochromeb 5 Reductase in Catalysis and Control of the Rate-limiting Step in Electron Transfer

Kimura

Kawamura

Iyanagi

2003

Journal of Biological Chemistry

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Site-directed mutagenesis of Thr 66 in porcine liver NADH-cytochrome b 5 reductase demonstrated that this residue modulates the semiquinone form of FAD and the rate-limiting step in the catalytic sequence of electron transfer. The absorption spectrum of the T66V mutant showed a typical neutral blue semiquinone intermediate during turnover in the electron transfer from NADH to ferricyanide but showed an anionic red semiquinone form during anaerobic photoreduction. The apparent k cat values of this mutant were ϳ10% of that of the wild type enzyme (WT). These data suggest that the T66V mutation stabilizes the neutral blue semiquinone and that the conversion of the neutral blue to the anionic red semiquinone form is the rate-limiting step. In the WT, the value of the rate constant of FAD reduction (k red ) was consistent with the k cat values, and the oxidized enzyme-NADH complex was observed during the turnover with ferricyanide. This indicates that the reduction of FAD by NADH in the WT-NADH complex is the ratelimiting step. In the T66A mutant, the k red value was larger than the k cat values, but the k red value in the presence of NAD ؉ was consistent with the k cat values. The spectral shape of this mutant observed during turnover was similar to that during the reduction with NADH in the presence of NAD ؉ . These data suggest that the oxidized T66A-NADH-NAD ؉ ternary complex is a major intermediate in the turnover and that the release of NAD ؉ from this complex is the rate-limiting step. These results substantiate the important role of Thr 66 in the one-electron transfer reaction catalyzed by this enzyme. On the basis of these data, we present a new kinetic scheme to explain the mechanism of electron transfer from NADH to one-electron acceptors including cytochrome b 5 .NADH-cytochrome b 5 reductase (EC 1.6.2.2) is a member of the large family of flavin-dependent oxidoreductases that transfer an electron from two-electron carriers of nicotinamide dinucleotides to one-electron carriers such as heme proteins and ferredoxins. This enzyme catalyzes the electron transfer from NADH to cytochrome b 5 (b5) 1 (1-3), and participates in fatty acid synthesis (4, 5), cholesterol synthesis (6), and xenobiotic oxidation (7) as a member of the electron transport chain on the endoplasmic reticulum. In erythrocytes, this enzyme participates in the reduction of methemoglobin (8).The outline of the catalytic cycle of the solubilized catalytic domain of NADH-cytochrome b 5 reductase (b5R) is understood as follows (3) (Scheme I). At first, two electrons are transferred from NADH to FAD by hydride (H Ϫ ) transfer. Then the twoelectron reduced enzyme-NAD ϩ complex (E-FADH Ϫ -NAD ϩ ) transfers two electrons to two one-electron acceptors one by one via the anionic red semiquinone form (E-FAD⅐ Ϫ -NAD ϩ ), and the reduced enzyme returns to the oxidized state. Strittmatter (9 -11) suggested that the reduction of FAD by NADH is the rate-limiting step in electron transfer catalyzed by b5R. Iyanagi et al. (3,12) found that the anionic red sem...

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“…The molecule of NADH-cytochrome b5 reductase is composed of two domains, the FAD binding domain (residues 2-118) and the NADH domain (residues 119-272) (Nishida et al, 1995). The highest scored docking pose for inhibitors (luteolin, quercetin and taxifolin) revealed that all the inhibitors occupied the NADH binding domain (Fig 1).…”

Section: Resultsmentioning

confidence: 99%

Molecular construction of NADH-cytochrome b5 reductase inhibition by flavonoids and chemical basis of difference in inhibition potential: Molecular dynamics simulation study

Verma¹

2012

J. App. Pharm. Sci.

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NADH-cytochrome b5 reductase, a flavoprotein, plays a central role in many diverse metabolic reactions. NADH-cytochrome b5 reductase has been shown to be responsible for the generation of free radicals from heterocyclic amines. Flavonoids compounds share remarkable similarity in structure but showed differences in their cytochrome b5 reductase inhibition pattern. Our molecular dynamics simulation studies revealed that the difference in substitution at C3 position of ring C may lead to difference in interaction with enzyme. Absence of hydroxyl group substitution at C3 in luteolin facilitates the strong cation-π interaction between Lys185 and ring A, and C and π-π between Phe92 and ring A, and C along with h-bonding between Lys185 and oxo group. Ring B of luteolin showed strong π-π interaction with FAD. These interactions were found absent in quercetin and taxifolin. These results suggest that absence of hydroxyl group substitution at C3 increases the potency of flavonoid inhibitors for cytochrome b5 reductase.

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Crystal Structure of NADH-Cytochrome b5 Reductase from Pig Liver at 2.4 .ANG. Resolution

Cited by 92 publications

References 26 publications

Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

Role of Thr66 in Porcine NADH-cytochromeb 5 Reductase in Catalysis and Control of the Rate-limiting Step in Electron Transfer

Molecular construction of NADH-cytochrome b5 reductase inhibition by flavonoids and chemical basis of difference in inhibition potential: Molecular dynamics simulation study

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