Reduction kinetics of purified rat liver cytochrome P-450. Evidence for a sequential reaction mechanism dependent on the hemoprotein spin state

Tamburini, Paul P.; Gibson, Gordon; Backes, Wayne L.; Sligar, Stephen G.; Schenkman, John B.

doi:10.1021/bi00315a004

Cited by 46 publications

(28 citation statements)

References 32 publications

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“…Furthermore, a conversion from the hexa-coordinated low-spin to the penta-coordinated high-spin state, which is triggered by reduction of the reductase flavins may have an important impact on the catalytic efficiency and coupling of the P450. This transition is known to increase the rate of the electron transfer [2, 4, 5, 7] due to a considerable decrease in the reorganization energy associated with the reduction process [67, 68] along with a positive displacement of the redox potential of cytochromes P450 [69, 70], and 3A4 [71] in particular. At the same time, the modulation of the spin state of cytochromes P450 in response to the changes in the redox state of the reductase may explain an apparent lack of correlation between the rate of NADPH-dependent reduction or the rate of substrate turnover and the spin-state for certain microsomal cytochromes P450 [8], which was considered as a disproof of the theory of the spin-state control of the rate of electron transfer.…”

Section: Discussionmentioning

confidence: 99%

“…Although there is no commonly accepted hypothesis on the mechanism of this biphasicity, its close connection to the position of P450 spin equilibrium is beyond doubt. In most cases, displacement of the spin equilibrium towards the high spin state caused by addition of Type I substrates increases the fraction of the fast phase [2, 4, 5, 7]. Furthermore, in some cases, the first exponential phase of reduction was shown to represent solely the reduction of the high-spin heme protein [5, 6].…”

Section: Introductionmentioning

confidence: 99%

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Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: The effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium

Davydov

Sineva

Sistla

et al. 2010

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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We studied the kinetics of NADPH-dependent reduction of human CYP3A4 incorporated into Nanodiscs (CYP3A4-ND) and proteoliposomes in order to probe the effect of P450 oligomerization on its reduction. The flavin domain of cytochrome P450-BM3 (BMR) was used as a model electron donor partner. Unlike CYP3A4 oligomers, where only 50% of the enzyme was shown to be reducible by BMR, CYP3A4-ND could be reduced almost completely. High reducibility was also observed in proteoliposomes with a high lipid-to-protein ratio (L/P=910), where the oligomerization equilibrium is displaced towards monomers. In contrast, the reducibililty in proteoliposomes with L/P=76 did not exceed 55 ± 6%. The effect of the surface density of CYP3A4 in proteoliposomes on the oligomerization equilibrium was confirmed with a FRET-based assay employing a cysteine-depleted mutant labeled on Cys-468 with BODIPY iodoacetamide. These results confirm a pivotal role of CYP3A4 oligomerization in its functional heterogeneity. Furthermore, the investigation of the initial phase of the kinetics of CYP3A4 reduction showed that the addition of NADPH causes a rapid low-to-high spin transition in the CYP3A4-BMR complex, which is followed by a partial slower reversal. This observation reveals a mechanism whereby the CYP3A4 spin equilibrium is modulated by the redox state of the bound flavoprotein.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: The effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium

Davydov

Sineva

Sistla

et al. 2010

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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“…Those results reveal a strict correlation between the rate of substrate N -demethylation and its coupling to NADPH oxidation with the amplitude of the substrate-induced spin shift. Furthermore, the low-to-high spin transition is known to increase the rate of electron transfer to cytochromes P450 (Tamburini et al , 1984; Backes et al , 1985; Backes and Eyer, 1989) due to a considerable decrease in the reorganization energy associated with the reduction process (Honeychurch et al ., 1999; Denisov et al ., 2005) along with a positive displacement of the P450 redox potential (Fisher and Sligar, 1985). This has been demonstrated with CYP3A4 (Das et al , 2007) in particular.…”

Section: Discussionmentioning

confidence: 99%

Multiple substrate-binding sites are retained in cytochrome P450 3A4 mutants with decreased cooperativity

Fernando¹,

Rumfeldt²,

Davydova³

et al. 2010

Xenobiotica

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Abstract1. The basis of decreased cooperativity in substrate binding in the cytochrome P450 3A4 mutants F213W, F304W and L211F/D214E was studied with fluorescence resonance energy transfer (FRET) and absorbance spectroscopy.2. Whereas in the wild type enzyme the absorbance changes reflecting the interactions with 1-pyrenebutanol exhibit a Hill coefficient (n H ) around 1.7 (S 50 = 11.7 μM), the mutants showed no cooperativity (n H ≤ 1.1) with unchanged S 50 values.3. Contrary to the premise that the mutants lack one of the two binding sites, the mutants exhibited at least two substrate binding events. The high affinity interaction is characterized by a dissociation constant (K D ) ≤ 1.0 μM, whereas the K D of the second binding has the same magnitude as the S 50 .4. Theoretical analysis of a two-step binding model suggests that n H values may vary from 1.1 to 2.2 depending on the amplitude of the spin shift caused by the first binding event.5. Alteration of cooperativity in the mutants is caused by a partial displacement of the "spinshifting" step. Whereas in the wild type the spin shift occurs in the ternary complex only, the mutants exhibit some spin shift upon binding of the first substrate molecule.

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“…Some earlier studies suggested that the heme iron spin state may serve as a surrogate marker (Sligar et al, 1979;Tamburini et al, 1984;Backes et al, 1985). Although not applicable for every P450 isoform (Eyer and Backes, 1992) as a result of differences in reduction potentials (Ost et al, 2003), CYP2C9 is reduced much faster in the presence of substrate even though the substrate oxidation rate may be slow (Guengerich and Johnson, 1997).…”

Section: Discussionmentioning

confidence: 99%

Polymorphic Variants of CYP2C9: Mechanisms Involved in Reduced Catalytic Activity

Lian

Locuson²,

Tracy³

2007

Mol Pharmacol

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CYP2C9 catalyzes the demethylation of the biphasic kinetics substrate (S)-naproxen, and the CYP2C9*2 (R144C) and CYP2C9*3 (I359L) variants are associated with lower rates of (S)-naproxen demethylation. To assess the reasons for these reductions in catalytic activity of the two variants and potential substrate concentration-dependent differences in a biphasic kinetics substrate, cytochrome P450 (P450) cycle coupling and uncoupling were monitored during coincubation of (S)-naproxen and CYP2C9 over a range of P450 reductase concentrations. Coupling was greatest in the CYP2C9.1 enzyme, followed by CYP2C9.2, and then CYP2C9.3. Uncoupling in CYP2C9.1 and CYP2C9.3 was primarily to H 2 O 2 . In contrast, CYP2C9.2 uncoupled to excess water preferentially. The conversion of enzyme to the high spin state was similar in CYP2C9.1 and CYP2C9.2, but lower in CYP2C9.3. It is noteworthy that neither altered substrate binding nor altered interaction with reductase seemed to be involved in reduced catalysis. These results suggest that in addition to coupling differences, differential uncoupling to shunt products and differences in spin state help explain the reduced catalytic activity in these enzymes.The cytochrome P450 (P450) enzymes are responsible for the biotransformation of a wide range of exogenous and endogenous substrates (Wrighton and Stevens, 1992). P450-mediated metabolism occurs via a catalytic cycle (Scheme 1) that involves several steps: 1) substrate binding; 2) oneelectron addition to the substrate-P450 complex by CPR; 3) oxygen binding to the ferrous P450; 4) transfer of a second electron by either CPR or cytochrome b 5 and protonation of the resulting iron-peroxo anion intermediate; 5) cleavage of the O-O bond to generate H 2 O; 6, 7) oxidation of the substrate; and 8) and release of product with subsequent

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Reduction kinetics of purified rat liver cytochrome P-450. Evidence for a sequential reaction mechanism dependent on the hemoprotein spin state

Cited by 46 publications

References 32 publications

Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: The effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium

Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: The effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium

Multiple substrate-binding sites are retained in cytochrome P450 3A4 mutants with decreased cooperativity

Polymorphic Variants of CYP2C9: Mechanisms Involved in Reduced Catalytic Activity

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