Membrane topology of microsomal cytochrome P-450: Saturation transfer EPR and freeze-fracture electron microscopy studies

doi:10.1016/0006-291X(90)91373-Z

Biochemical and Biophysical Research Communications

Volume 171, Issue 1, 31 August 1990, Pages 175-181

https://doi.org/10.1016/0006-291X(90)91373-Z Get rights and content

Abstract

The rotation of cytochrome P-450 LM2 (CYPIIB4) incorporated into large microsomal-like lipid vesicles was investigated by saturation transfer EPR using ¹⁵N- and ²H-substituted spin labels. In combination with rotational diffusion, the distribution and size of protein particles in the bilayer were studied by freeze-fracture electron microscopy. The data from both methods suggest an oligomeric and membrane-spanning aggregate for the topology of microsomal cytochrome P-450.

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Assembling the P450 puzzle: on the sources of nonadditivity in drug metabolism
2021, Trends in Pharmacological Sciences
There is an increasing number of indications of an oversimplification in the premise that the cumulative properties of the human drug-metabolizing ensemble represent a simple aggregate of the properties of the constituting enzymes. Recent studies of the functional effects of hetero-association of multiple cytochrome P450 species and their interactions with metabolically related enzymes revealed a tight integration in the drug-metabolizing ensemble. In our opinion, the sources of interindividual variability in drug metabolism can be elucidated only when considering this ensemble as a multienzyme system, the functional parameters of which are determined by interactions between its constituents. In this article, we present a conceptual model providing a mechanistic explanation for the functional effects of the interactions between multiple P450 species and propose a clue to understanding the nonadditive behavior of the drug-metabolizing ensemble.
Interactions among cytochromes P450 in microsomal membranes: Oligomerization of cytochromes P4503A4,3A5, and 2E1 and its functional consequences
2015, Journal of Biological Chemistry
Citation Excerpt :
Competition of different P450 species for a limited amount of CPR, which has been observed by numerous investigators (1–6), constitutes an important element in the inter-P450 cross-talk. However, the integrative connections in microsomal monooxygenase go far beyond that and involve functional effects of physical interactions of multiple P450 molecules that result in oligomerization (7–17). Besides evidence of homo-oligomerization of various P450 species (10, 12, 15, 17–23), there are also multiple indications of interactions between dissimilar P450s in membranes of proteoliposomes (13), microsomes (14), and living cells (16).
The body of evidence of physiologically relevant P450-P450 interactions in microsomal membranes continues to grow. Here we probe oligomerization of human CYP3A4, CYP3A5, and CYP2E1 in microsomal membranes. Using a technique based on luminescence resonance energy transfer, we demonstrate that all three proteins are subject to a concentration-dependent equilibrium between the monomeric and oligomeric states. We also observed the formation of mixed oligomers in CYP3A4/CYP3A5, CYP3A4/CYP2E1, and CYP3A5/CYP2E1 pairs and demonstrated that the association of either CYP3A4 or CYP3A5 with CYP2E1 causes activation of the latter enzyme. Earlier we hypothesized that the intersubunit interface in CYP3A4 oligomers is similar to that observed in the crystallographic dimers of some microsomal drug-metabolizing cytochromes P450 (Davydov, D. R., Davydova, N. Y., Sineva, E. V., Kufareva, I., and Halpert, J. R. (2013) Pivotal role of P450-P450 interactions in CYP3A4 allostery: the case of α-naphthoflavone. Biochem. J. 453, 219–230). Here we report the results of intermolecular cross-linking of CYP3A4 oligomers with thiol-reactive bifunctional reagents as well as the luminescence resonance energy transfer measurements of interprobe distances in the oligomers of labeled CYP3A4 single-cysteine mutants. The results provide compelling support for the physiological relevance of the dimer-specific peripheral ligand-binding site observed in certain CYP3A4 structures. According to our interpretation, these results reveal an important general mechanism that regulates the activity and substrate specificity of the cytochrome P450 ensemble through interactions between multiple P450 species. As a result of P450-P450 cross-talk, the catalytic properties of the cytochrome P450 ensemble cannot be predicted by simple summation of the properties of the individual P450 species.
Background: There are multiple cytochrome P450 species co-localized in the endoplasmic reticulum.
Results: Membrane-bound cytochromes P450 3A4, 3A5, and 2E1 associate into heteromeric complexes, where the properties of the individual enzymes are considerably modified.
Conclusion: The properties of the P450 ensemble cannot be predicted by summation of the properties of the individual enzymes.
Significance: We disclose a mechanism of regulatory cross-talk between multiple P450 species through hetero-oligomerization.
Formation of P450 • P450 complexes and their effect on P450 function
2012, Pharmacology and Therapeutics
Citation Excerpt :
This finding also provides a perspective to interpret metabolism studies of P450–P450 interactions when CPR concentrations are in excess to those of the P450. The EPR analysis performed in microsomes (described above) also was used to study the rotation and mobility of spin-labeled CYP2B4 in lipid vesicles having a phospholipid composition similar to that of microsomes (Schwarz et al., 1990). Again by comparing the measured rotational correlation times to the theoretical rotation of a sphere roughly the size of a CYP2B4 monomer, it was postulated that the enzyme rotated as an aggregate (probably hexameric) both in solution and when the P450 was integrally incorporated in a lipid bilayer.
Cytochromes P450 (P450) are membrane-bound enzymes that catalyze the monooxygenation of a diverse array of xenobiotic and endogenous compounds. The P450s responsible for foreign compound metabolism generally are localized in the endoplasmic reticulum of the liver, lung and small intestine. P450 enzymes do not act alone but require an interaction with other electron transfer proteins such as NADPH–cytochrome P450 reductase (CPR) and cytochrome b₅. Because P450s are localized in the endoplasmic reticulum with these and other ER-resident proteins, there is a potential for protein–protein interactions to influence P450 function. There has been increasing evidence that P450 enzymes form complexes in the ER, with compelling support that formation of P450 · P450 complexes can significantly influence their function. Our goal is to review the research supporting the formation of P450 · P450 complexes, their specificity, and how drug metabolism may be affected. This review describes the potential mechanisms by which P450s may interact, and provides evidence to support each of the possible mechanisms. Additionally, evidence for the formation of both heteromeric and homomeric P450 complexes are reviewed. Finally, direct physical evidence for P450 complex formation in solution and in membranes is summarized, and questions directing the future research of functional P450 interactions are discussed with respect to their potential impact on drug metabolism.
Cytochromes P450 in nanodiscs
2011, Biochimica et Biophysica Acta - Proteins and Proteomics
Nanodiscs have proven to be a versatile tool for the study all types of membrane proteins, including receptors, transporters, enzymes and viral antigens. The self-assembled Nanodisc system provides a robust and common means for rendering these targets soluble in aqueous media while providing a native like bilayer environment that maintains functional activity. This system has thus provided a means for studying the extensive collection of membrane bound cytochromes P450 with the same biochemical and biophysical tools that have been previously limited to use with the soluble P450s. These include a plethora of spectroscopic, kinetic and surface based methods. Significant improvements in homogeneity and stability of these preparations open new possibilities for detailed analysis of equilibrium and steady-state kinetic characteristics of catalytic mechanisms of human cytochromes P450 involved in xenobiotic metabolism and in steroid biosynthesis. The experimental methods developed for physico-chemical and functional studies of membrane cytochromes P450 incorporated in Nanodiscs allow for more detailed understanding of the scientific questions along the lines pioneered by Professor Klaus Ruckpaul and his array of colleagues and collaborators.
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
2010, Biochimica et Biophysica Acta - Bioenergetics
Citation Excerpt :
Numerous experimental observations reveal a considerable degree of homo- and hetero-oligomerization of cytochromes P450 in the endoplasmic reticulum (see [16] for review). In particular, oligomerization of cytochromes P450 in membranes was evidenced by rotational diffusion measurements in various implementations [17–22], freeze-fracture electron microscopy [20], immunochemical cross-linking [23], and inter-molecular FRET studies employing the P450 enzymes fused with fluorescent proteins [24]. Homo- and hetero-oligomerization of cytochromes P450 in the membrane may play an important functional role (see [14,16,25] for review).
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.
Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4
2008, Archives of Biochemistry and Biophysics
Glutathione (GSH) exerted a profound effect on the oxidation of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and 7-benzyloxyquinoline (BQ) by human liver microsomes as well as by CYP3A4-containing insect cell microsomes (Baculosomes). The cooperativity in O-debenzylation of both substrates is eliminated in the presence of 1–4 mM GSH. Addition of GSH also increased the amplitude of the 1-PB induced spin shift with purified CYP3A4 and abolished the cooperativity of 1-PB or BFC binding. Changes in fluorescence of 6-bromoacetyl-2-dimethylaminonaphthalene attached to the cysteine-depleted mutant CYP3A4(C58,C64) suggest a GSH-induced conformational changes in proximity of α-helix A. Importantly, the K_S value for formation of the GSH complex and the concentrations in which GSH decreases CYP3A4 cooperativity are consistent with the physiological concentrations of GSH in hepatocytes. Therefore, the allosteric effect of GSH on CYP3A4 may play an important role in regulation of microsomal monooxygenase activity in vivo.

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Membrane topology of microsomal cytochrome P-450: Saturation transfer EPR and freeze-fracture electron microscopy studies

Abstract

J. Biol. Chem

J. Biol. Chem

Arch. Biochem. Biophys

J. Biol. Chem

Biochem. Biophys. Res. Commun

J. Magn. Reson

Biophys. J

J. Magn. Reson

J. Biol. Chem

J. Biol. Chem