Determination of the Rate of Reduction of Oxyferrous Cytochrome P450 2B4 by 5-Deazariboflavin Adenine Dinucleotide T491V Cytochrome P450 Reductase

Zhang, Haoming; Gruenke, Larry D.; Arscott, Dave; Shen, Anna L.; Kasper, Charles B.; Harris, Danni L.; Glavanovich, Michael; Johnson, R. Jeremy; Waskell, Lucy

doi:10.1021/bi034968u

Cited by 45 publications

(76 citation statements)

References 39 publications

(62 reference statements)

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“…However, the lack of any significant change in the rate of the first electron transfer observed in the absence of benzphetamine suggests that any modulation in the redox potential is relatively insignificant with respect to its effect on the electron transfer rate. The substantial increase in the rate of the electron transfer to the unmodified protein in the presence of 1 mM benzphetamine can be attributed to an increase in the redox potential of P450 2B4, given that it has been reported that benzphetamine binding to P450 2B4 raises the redox potential by ϳ85 mV, leading to a ϳ10-fold increase in the rate of the electron transfer from CPR to the ferric P450 2B4 (Zhang et al, 2003). This substantial stimulation of the rate of electron transfer is absent for the tBPA-modified P450 2B4.…”

Section: Discussionmentioning

confidence: 99%

tert-Butylphenylacetylene Is a Potent Mechanism-Based Inactivator of Cytochrome P450 2B4: Inhibition of Cytochrome P450 Catalysis by Steric Hindrance

et al. 2009

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We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH-and time-dependent manner with a K I of 0.44 M and k inact of 0.12 min Ϫ1 . The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 Å away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by ϳ20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4.

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Section: Discussionmentioning

confidence: 99%

tert-Butylphenylacetylene Is a Potent Mechanism-Based Inactivator of Cytochrome P450 2B4: Inhibition of Cytochrome P450 Catalysis by Steric Hindrance

et al. 2009

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“…The rate of product formation was measured in a rapid chemical quench apparatus by mixing a 1:1 complex of the preformed dithionite reduced Fe 2ϩ cyt P450:2-electron reduced ⌬TGEE with oxygen (49,28). The reaction was quenched, and the product was quantified by high-pressure liquid chromatography-mass spectrometry.…”

Section: Two-electron Reduced ⌬Tgee Catalyzes Product Formation By Cymentioning

confidence: 99%

“…The reduction of cyt P450 by the 2-residue deletion mutant was slightly slower than that of wild-type (6.7 s Ϫ1 versus 4.2 s Ϫ1 , Table 4) and occurred with a rate constant similar to the rate of electron transfer from FAD to FMN. Cyt P450 reduction was faster in the presence of benzphetamine, because the substrate raises the redox potential of the heme, which increases the thermodynamic driving force of the reaction (49). Fig.…”

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confidence: 94%

Structure and Function of an NADPH-Cytochrome P450 Oxidoreductase in an Open Conformation Capable of Reducing Cytochrome P450

Hamdane

Xia

et al. 2009

Journal of Biological Chemistry

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NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner. NADPH-cytochrome P450 oxidoreductase (CYPOR)4 is a ϳ78-kDa, multidomain, microsomal diflavin protein that shuttles electrons from NADPH 3 FAD 3 FMN to members of the ubiquitous cytochrome P450 superfamily (1, 2). In humans, the cytochromes P450 (cyt P450) are one of the most important families of proteins involved in the biosynthesis and degradation of a vast number of endogenous compounds and the detoxification and biodegradation of most foreign compounds. CYPOR also donates electrons to heme oxygenase (3), cytochrome b 5 (4), and cytochrome c (5).The FAD receives a hydride anion from the obligate two electron donor NADPH and passes the electrons one at a time to FMN. The FMN then donates electrons to the redox partners of CYPOR, again one electron at a time. Cyt P450 accepts electrons at two different steps in its complex reaction cycle. Ferric cyt P450 is reduced to the ferrous protein, and oxyferrous cyt P450 receives the second of the two electrons to form the peroxo (Fe ϩ3 OO) 2Ϫ cyt P450 intermediate (6). In vivo, CYPOR cycles between the one-and three-electron reduced forms (7,8). Although the one-electron reduced form is an air-stable, neutral blue semiquinone (FMN ox/sq , Ϫ110 mV), it is the FMN hydroquinone (FMN sq/hq , Ϫ270 mV), not the semiquinone, that donates an electron to its redox partners (8 -11). CYPOR is the prototype of the mammalian diflavin-containing enzyme family, which includes nitric-oxide synthase (12), methionine synthase reductase (13,14), and a novel reductase expressed in the cytoplasm of certain cancer cells (15). CYPOR is also a target for anticancer therapy, because it reductively activates anticancer prodrugs (16).CYPOR consists of an N-terminal single ␣-helical transmembrane anchor (ϳ6 kDa) responsible for its local...

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“…The slower oxidation of cyt P450-CO by ferric cyt b 5 may result from the increased redox potential of cyt P450-CO or the slow dissociation of CO from cyt P450-CO and subsequent reduction of cyt b 5 by ferrous cyt P450. It has been estimated that the binding of CO to ferrous cyt P450 raises the mid-point potential by Х180 mV versus NHE (36) relative to the ferric-ferrous couple in the presence of 1 mM benzphetamine (Х Ϫ245 mV versus NHE) (37). Increasing the potential would reduce the driving force for electron transfer compared with the ferrous protein.…”

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confidence: 99%

Cytochrome b5 Inhibits Electron Transfer from NADPH-Cytochrome P450 Reductase to Ferric Cytochrome P450 2B4

Zhang

Hamdane²,

Im³

et al. 2008

Journal of Biological Chemistry

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Microsomal cytochromes (cyt) 3 P450, functioning as monooxygenases, catalyze the oxidative biotransformation of numerous pharmaceuticals, carcinogens, pro-carcinogens, and endogenous compounds like fatty acids and steroids. Cyts P450 require two electrons and two protons to carry out catalysis that leads to insertion of a single oxygen atom into the substrate. In the mammalian microsomal cyt P450 system, the two electrons are delivered to cyt P450 by NADPH-dependent cytochrome P450 reductase (CPR). Like cyt P450, CPR is membrane-bound and located in the membrane of the endoplasmic reticulum. CPR contains two flavin molecules, FMN and FAD. The diflavin moiety of CPR is essential for sequential electron transfer to cyt P450 as it permits CPR to accept two electrons from NADPH and transfer one electron at a time to cyt P450. The first electron from CPR reduces ferric cyt P450 to ferrous cyt P450, which rapidly binds oxygen to form oxyferrous cyt P450. The second electron is then delivered to oxyferrous cyt P450. This is followed by protonation of the reduced oxyferrous intermediate leading to heterolytic cleavage of the oxygen bond to form water and an oxyferryl intermediate, the putative, active, oxidizing species of cyt P450. An oxygen atom is inserted into the substrate, and the more hydrophilic product dissociates from the enzyme. Readers are referred to a recent review for further details about the cyt P450 reaction cycle (1).An alternative electron donor to cyt P450 is cyt b 5 , another microsomal hemoprotein also located in the endoplasmic reticulum membrane. Because of its relatively high mid-point redox potential (ϩ25 mV versus NHE), cyt b 5 can deliver only the second electron to oxyferrous cyt P450 but not the first electron to ferric cyt P450. It has been recognized for 3 decades that cyt b 5 may either increase, decrease, or not alter the activity of selected cyts P450 (2, 3). Cyt b 5 has been reported to affect the catalytic activity of more than 20 cyt P450 isoforms, including the majority of the human drug-metabolizing cyt P450 isoforms like cyt P450 3A4, 2B6, 2C9, 2C19, and 2E1 (1, 4 -8). The effect of cyt b 5 has also been shown to depend on the cyt P450 isozyme and substrate (2, 9). In the case of cyt P450 2B4 and 2E1, the electron donating properties of cyt b 5 are required for its stimulatory activity (6, 7, 9 -11), although some studies suggest that apo-cyt b 5 can stimulate the activity of cyt P450 3A4 via an allosteric effect (12). At present, the ability of apo-cyt b 5 to stimulate cyt P450 3A4 is controversial (13). Experiments performed in the reconstituted system with purified proteins have demonstrated that ferrous cyt b 5 can rapidly reduce oxyferrous cyt P450 2B4 (14,15).It is known that cyt P450 2B4 forms a 1:1 complex with CPR and with cyt b 5 in a purified, reconstituted system (16,17). A site-directed mutagenesis study of the interactions of cyt P450 2B4 with CPR and cyt b 5 has identified residues, primarily in the * This work was supported by National Institutes of Health Grant ...

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Determination of the Rate of Reduction of Oxyferrous Cytochrome P450 2B4 by 5-Deazariboflavin Adenine Dinucleotide T491V Cytochrome P450 Reductase

Cited by 45 publications

References 39 publications

tert-Butylphenylacetylene Is a Potent Mechanism-Based Inactivator of Cytochrome P450 2B4: Inhibition of Cytochrome P450 Catalysis by Steric Hindrance

tert-Butylphenylacetylene Is a Potent Mechanism-Based Inactivator of Cytochrome P450 2B4: Inhibition of Cytochrome P450 Catalysis by Steric Hindrance

Structure and Function of an NADPH-Cytochrome P450 Oxidoreductase in an Open Conformation Capable of Reducing Cytochrome P450

Cytochrome b5 Inhibits Electron Transfer from NADPH-Cytochrome P450 Reductase to Ferric Cytochrome P450 2B4

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