Studies on the stimulation of cytochrome P-450-dependent monooxygenase activity by dilauroylphosphatidylcholine

doi:10.1016/0003-9861(81)90477-X

Archives of Biochemistry and Biophysics

Volume 211, Issue 1, 1 October 1981, Pages 454-458

https://doi.org/10.1016/0003-9861(81)90477-X Get rights and content

Abstract

Steady-state kinetic and deuterium isotope effect studies have been conducted to determine the influence of the phospholipid dilauroylphosphatidylcholine on the catalytic activity of a reconstituted monooxygenase system composed of cytochrome P-450 and NADPH-cytochrome c (P-450) reductase. Addition of this lipid up to a concentration equivalent to its CMC resulted in an increase in V for benzphetamine N-demethylation. Above the CMC, no further change in V was observed. In contrast, the K_m was not affected throughout the entire lipid concentration range. Furthermore, the deuterium isotope effect for 7-ethoxycoumarin O-deethylation was not affected by the lipid concentration indicating that the contribution of the carbon-hydrogen bond cleavage step to V was also not affected. These data are consistent with the mass-action model proposed earlier (G. T. Miwa, S. B. West, M. T. Huang, and A. Y. H. Lu, (1979), J. Biol. Chem.254, 5695–5700) for cytochrome P-450 and NADPH-cytochrome c (P-450) reductase association during catalysis. The lipid concentration, below its CMC, appears to decrease the apparent dissociation constant for the cytochrome P-450-reductase complex thus causing an increase in the steady-state concentration of this catalytically active complex.

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Effects of membrane mimetics on cytochrome P450-cytochrome b5 interactions characterized by NMR spectroscopy
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This implies the ability of lipids to facilitate tighter binding between wt-CYP2B4 and cytb5. Our results are in agreement with the previous findings that phospholipids enhance the interactions between P450s and the other redox partner, CPR (27–30). In order to investigate the influence of different membrane mimetic environments on CYP2B4-cytb5 interaction, two-dimensional 15N/1H TROSY-HSQC NMR spectra were recorded to monitor the interaction between 15N-labeled cytb5 and unlabeled wt-CYP2B4 in lipid-free solution, DLPC/DHPC isotropic bicelles, and DPC micelles.
Mammalian cytochrome P450 (P450) is a membrane-bound monooxygenase whose catalytic activities require two electrons to be sequentially delivered from its redox partners: cytochrome b₅ (cytb₅) and cytochrome P450 reductase, both of which are membrane proteins. Although P450 functional activities are known to be affected by lipids, experimental evidence to reveal the effect of membrane on P450-cytb₅ interactions is still lacking. Here, we present evidence for the influence of phospholipid bilayers on complex formation between rabbit P450 2B4 (CYP2B4) and rabbit cytb₅ at the atomic level, utilizing NMR techniques. General line broadening and modest chemical shift perturbations of cytb₅ resonances characterize CYP2B4-cytb₅ interactions on the intermediate time scale. More significant intensity attenuation and a more specific protein-protein binding interface are observed in bicelles as compared with lipid-free solution, highlighting the importance of the lipid bilayer in stabilizing stronger and more specific interactions between CYP2B4 and cytb₅, which may lead to a more efficient electron transfer. Similar results observed for the interactions between CYP2B4 lacking the transmembrane domain (tr-CYP2B4) and cytb₅ imply interactions between tr-CYP2B4 and the membrane surface, which might assist in CYP2B4-cytb₅ complex formation by orienting tr-CYP2B4 for efficient contact with cytb₅. Furthermore, the observation of weak and nonspecific interactions between CYP2B4 and cytb₅ in micelles suggests that lipid bilayer structures and low curvature membrane surface are preferable for CYP2B4-cytb₅ complex formation. Results presented in this study provide structural insights into the mechanism behind the important role that the lipid bilayer plays in the interactions between P450s and their redox partners.
Two surfaces of cytochrome b<inf>5</inf> with major and minor contributions to CYP3A4-catalyzed steroid and nifedipine oxygenation chemistries
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The phospholipid composition markedly influences P450 turnover and b5 stimulation [30,31], and CYP3A family activities are highest when reconstituted with acidic phospholipids such as PS [22,32]. PS with unsaturated fatty acids improves interactions between the P450 and POR [32–34] and binds to CYP3Al, which alters its conformation [35]. In our assay system, CYP3A4-catalyzed testosterone 6β-hydroxylation decreases with phospholipids in the order PS > PE > PC.
Conserved human cytochrome b₅ (b₅) residues D58 and D65 are critical for interactions with CYP2E1 and CYP2C19, whereas E48 and E49 are essential for stimulating the 17,20-lyase activity of CYP17A1. Here, we show that b₅ mutations E48G, E49G, D58G, and D65G have reduced capacity to stimulate CYP3A4-catalyzed progesterone and testosterone 6β-hydroxylation or nifedipine oxidation. The b₅ double mutation D58G/D65G fails to stimulate these reactions, similar to CYP2E1 and CYP2C19, whereas mutation E48G/E49G retains 23–42% of wild-type stimulation. Neither mutation impairs the activity stimulation of wild-type b₅, nor does mutation D58G/D65G impair the partial stimulation of mutations E48G or E48G/E49G. For assays reconstituted with a single phospholipid, phosphatidyl serine afforded the highest testosterone 6β-hydroxylase activity with wild-type b₅ but the poorest activity with b₅ mutation E48G/E49G, and the activity stimulation of mutation E48G/E49G was lost at [NaCl] > 50 mM. Cross-linking of CYP3A4 and b₅ decreased in the order wild-type > E48G/E49G > D58G/D65G and varied with phospholipid. We conclude that two b₅ acidic surfaces, primarily the domain including residues D58–D65, participate in the stimulation of CYP3A4 activities. Our data suggest that a minor population of CYP3A4 molecules remains sensitive to b₅ mutation E48G/E49G, consistent with phospholipid-dependent conformational heterogeneity of CYP3A4.
Beta sheet 2-alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners
2010, Biochimica et Biophysica Acta - Proteins and Proteomics
As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein–protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1•CPR complex and inactive (CYP2B1)₂•CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge–charge interactions between CPR and complex partners.
Electrochemical characterisation of the human cytochrome P450 CYP2C9
2005, Biochemical Pharmacology
Citation Excerpt :
Thus, without any other contributing factors like substrate or even oxygen, a lipid environment may be able to alter the electron transfer event from a thermodynamically unfavourable process (positive ΔG) to a favourable, spontaneous process (negative ΔG). This point is further emphasised by the fact that a lipid-like environment is required for catalytic activity [12,50–52] and numerous other structural and electronic properties [20–27] for many P450 enzymes. We have expressed and purified CYP2C9, and characterised the enzyme by direct electrochemistry.
The electrochemistry of human cytochrome P4502C9 (CYP2C9) was characterised using purified His-tagged enzyme. The His-tagged enzyme was shown to have similar functional characteristics to native CYP2C9 heterologously expressed in Escherichia coli and to the CYP2C9 activity of human liver microsomes. Evidence was observed for a reversible one-electron transfer between the P450 heme and the electrode. Both pH and ionic strength influenced the electrochemical behaviour of CYP2C9. A range of substrates was investigated to determine the effect of the heme–substrate interaction on CYP2C9 redox potential. In the absence of oxygen, tolbutamide, diclofenac, warfarin and sulfaphenazole did not alter the redox potential of the iron heme. In contrast, torsemide, carbon monoxide and oxygen led to an anodic shift in redox potential. These results suggest alternative mechanisms by which CYP2C9 (and by inference other P450 enzymes) may alter redox potential to facilitate electron delivery from physiological donors.
Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b<inf>5</inf> enzymes
2005, Archives of Biochemistry and Biophysics
Citation Excerpt :
Both proteins aggregate in solution [59], and the addition of non-ionic detergents to favor monomers will either alter the apparent binding parameters, due to facilitated interaction in vesicles, or disrupt interaction of the P450s with the other proteins [60,61]. Although P450 · NPR complexes have been discussed in the literature [56–66], estimates of dissociation constants are limited. French et al. [63] estimated Kd values for 0.04–0.12 μM for the P450 2B4 · NPR complex, depending upon conditions, utilizing spectral perturbation of the P450 heme by NPR.
An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b₅ (b₅) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin–peroxidase complex was added, and protein–protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein–protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r² = 0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K_d value for the Pdx · PdR complex was 0.054 μM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K_d values (with the reductase) ranged between 0.005 and 0.1 μM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b₅ or apo-b₅ (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b₅, although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b₅, with P450 3A4 yielding the lowest K_d values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b₅ or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450 · substrate K_d values estimated from substrate-induced UV–visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b₅, with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.
Conformational change and activation of cytochrome P450 2B1 induced by salt and phospholipid
1998, Archives of Biochemistry and Biophysics
A stimulatory effect of increased salt concentration on the enzymatic activity of rat liver microsomes and a reconstituted system containing cytochrome P450 (P450) 2B1 and NADPH-P450 reductase was seen. Structural change of P450 2B1 accompanying the salt-induced increase in its enzyme activity was investigated by circular dichroism, fluorescence spectroscopy, and absorption spectroscopy. It was found that the salt increased α-helix content of P450 2B1 in the presence as well as in the absence of a phospholipid. Intrinsic fluorescence emissions also increased with increasing salt concentration. The low-spin iron configuration of P450 2B1 shifted toward the high-spin configuration in response to the increased salt concentration. It was found that the activity increase of P450 coincides with the raised α-helix content. The presence of phospholipid magnified this effect. It is proposed that the interaction with salts and phospholipid molecules surrounding P450 2B1 in the endoplasmic reticulum is important for a functional conformation of P450 2B1 in a monooxygenase system including NADPH-P450 reductase.

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^☆: Preliminary results of this work were presented at the 4th International Symposium on Microsomes and Drug Oxidations held at Ann Arbor, Mich, in July 1979.

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Studies on the stimulation of cytochrome P-450-dependent monooxygenase activity by dilauroylphosphatidylcholine☆

Abstract

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