Evidence That Nonbilayer Phase Propensity of the Membrane Is Important for the Side Chain Cleavage Activity of Cytochrome P450SCC (CYP11A1)

Schwarz, Dieter; Kisselev, Pyotr; Pfeil, Wolfgang; Pisch, Sandra; Bornscheuer, Uwe T.; Schmid, Rolf D.

doi:10.1021/bi9714262

Cited by 16 publications

(36 citation statements)

References 42 publications

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“…the potency to stimulate the activity of P450SCC is correlated with the enhancement in the fraction of the enzyme in the high spin form. The results suggest our previous conclusion that the hydrophobic volume of the branched PCs is an important determinant of the P450SCC-lipid interaction (8,9).…”

Section: Table II Stopped Flow Kinetics Of P450scc Incorporation Intosupporting

confidence: 84%

“…Particularly the latter point must be considered as a possible explanation because recent calorimetric investigations of the thermotropic behavior of DMPC/PC(14,10) dispersions demonstrated a significantly destabilized bilayer structure of the membrane and existence of a second phase at higher PC (14,10) contents. This might be related to separated membrane domains enriched in PC (14,10) and/or CHL (9).…”

Section: Table II Stopped Flow Kinetics Of P450scc Incorporation Intomentioning

confidence: 97%

“…By systematic variation of the lengths of both the branched and main chains of these lipids and systematic introduction of unsaturation, we found a relationship between the potency of activation of these PCs and their capacity to increase the propensity of the membrane to form nonbilayer phases. As a major determinant of the potency of activation, we suggested the hydrophobic volume and/or headgroup spacing rather than a specific P450SCC-lipid interaction (9).…”

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confidence: 89%

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Branched Phosphatidylcholines Stimulate Activity of Cytochrome P450SCC (CYP11A1) in Phospholipid Vesicles by Enhancing Cholesterol Binding, Membrane Incorporation, and Protein Exchange

Kisselev

Wessel

Pisch

et al. 1998

Journal of Biological Chemistry

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Phosphatidylcholines (PCs) with branched fatty acyl chains substituted in the two positions of the main chains (branched PCs) have been shown to be potent activators of the side chain cleavage activity of cytochrome P450SCC (CYP11A1) (Schwarz, D., Kisselev, P., Wessel, R., Jueptner, O., and Schmid, R. D. (1996) J. Biol. Chem. 271, 12840 -12846). The present study reports on the effect of a series of branched PC on cholesterol binding, membrane integration, and protein exchange in large unilamellar vesicles prepared by an extrusion technique. Enzyme kinetics using vesicles as well as optical titration using a micelle system with the detergent Tween 20 demonstrate that activation is correlated with the fraction of P450SCC in the high spin form. The potency of branched PCs both to activate the enzyme and to induce spin state changes increases with increasing lengths of both the branched and main fatty acyl chains. We found that the extent as well as the rate of integration of P450SCC into vesicle membranes studied by gel chromatography and stopped flow kinetics were increased by branched PC. Finally, it is demonstrated by measurement of the enzymatic activity in primary and secondary vesicles that branched PCs are potent in retaining a very rapid exchange of P450SCC between vesicles, in contrast to cardiolipin, that partially inhibits this exchange process. The data suggest that different properties of P450SCC in membrane systems including cholesterol binding, membrane integration, and protein exchange are affected by branched PCs and probably by other phospholipids, too, and therefore must be considered in an explanation of the observed high stimulation of activity.Cytochrome P450SCC (CYP11A1) is an integral mitochondrial enzyme that is located on the matrix face of the inner membrane of mitochondria. It catalyzes the side chain cleavage of cholesterol to yield pregnenolone, the common precursor of steroid hormones. Enzymatic studies employing both solution (micelle) and membrane (vesicle) systems demonstrated stimulation of activity to varying extent by phospholipids depending on the phospholipid used, the kind of reconstitution, size of vesicles, and small amounts of impurities (for a review see Ref. and references therein and Refs. 2-4). Under the lipids used in reconstitution CL1 played a special role mainly because (i) it is a typical mitochondrial lipid and (ii) it represents the most potent activator of P450SCC activity (5-7). With regard to the mechanism of activation, CL is of further interest because of the exceptional structure of its hydrophobic part, i.e. consisting of four fatty acyl chains instead of two of most other phospholipids.It has been recently reported by us that certain branched PCs that in the structure of their hydrophobic part are similar to CL but have fully saturated acyl chains cause potent activation of P450SCC comparable only with those by CL (8). By systematic variation of the lengths of both the branched and main chains of these lipids and systematic introduction of unsaturation,...

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Section: Table II Stopped Flow Kinetics Of P450scc Incorporation Intosupporting

confidence: 84%

Section: Table II Stopped Flow Kinetics Of P450scc Incorporation Intomentioning

confidence: 97%

mentioning

confidence: 89%

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Branched Phosphatidylcholines Stimulate Activity of Cytochrome P450SCC (CYP11A1) in Phospholipid Vesicles by Enhancing Cholesterol Binding, Membrane Incorporation, and Protein Exchange

Kisselev

Wessel

Pisch

et al. 1998

Journal of Biological Chemistry

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“…The activity of several membrane enzymes, among which are protein kinase C, mitochondrial reductases and ATPases, mitochondrial cytochrome P450 (CYP11A1), and others [18,36,37], is increased by nonbilayer lipids. There is some evidence that the latter influence the conformation of membrane-bound proteins by changing membrane properties, e.g.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, the presence of specific lipids of the nonbilayer class was found to be essential for optimal activity of microsomal and mitochondrial P450s. For instance, rabbit liver CYP2B4 requires phosphatidylethanolamine (PtdEtn) [12], and bovine adrenal CYP11A1 requires cardiolipin, PtdEtn or branched phosphatidylcholine (PtdCho) [17][18][19] for optimal activity as well as for membrane reconstitution. Reconstitution of purified human CYP1A1 using phospholipid vesicles with CYP1A1 and P450-reductase incorporated into the membrane has not been reported to our knowledge.…”

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confidence: 99%

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane

Kisselev¹,

Schwarz²,

Platt³

et al. 2002

Eur J Biochem

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Human cytochrome P4501A1 (CYP1A1) is one of the key enzymes in the bioactivation of environmental pollutants such as benzo [a]pyrene (B[a]P) and other polycyclic aromatic hydrocarbons. To evaluate the effect of membrane properties and distinct phospholipids on the activity of human CYP1A1 purified insect cell-expressed human CYP1A1 and of human NADPH-P450 reductase were reconstituted into phospholipid vesicle membranes. Conversion rates of up to 36 pmolAEmin )1 AEpmol )1 CYP1A1 of the enantiomeric promutagens (-)-and (+)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P (7,8-diol) to the genotoxic diolepoxides were achieved. The highest rates were obtained when negatively charged lipids such as phosphatidylserine and phosphatidylinositol and/or nonbilayer phospholipids such as phosphatidylethanolamine were present in the membrane together with neutral lipids. Both V max and K m values were changed. This suggests a rather complex mechanism of stimulation which might include altered substrate binding as well as more effective interaction between CYP1A1 and NADPH-P450 reductase. Furthermore, the ratio of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE2) to r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE1) formed from (-)-7,8-diol was significantly increased by the introduction of anionic lipids, but not by that of nonbilayer lipids. Thus, charged lipids affect the stereoselectivity of the epoxidation by leading to the formation of a larger amount of the ultimate mutagen DE2 than of DE1, which is far less carcinogenic. These data suggest that membrane properties such as negative charge and nonbilayer phase propensity are important for the efficiency and selectivity of enzymatic function of human CYP1A1. ((-)-7,8-diol) and then metabolized by CYP1A1 to the ultimately genotoxic r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (so-called diolepoxide-2 or antidiolepoxide, DE2) [1][2][3]. The last reaction appeared to be highly stereoselective as no or much less of the less carcinogenic r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydo-B[a]P (diolepoxide-1 or syn-diolepoxide, DE1) was produced from (-)-7,8-diol [4,5]. Racemic (+/-)-7,8-diol is mainly converted by human CYP1A1 to the DE2 [6,7].CYP1A1-dependent activity can be reconstituted by mixing the basic components of the monooxygenase system, i.e. purified CYP1A1, NADPH-cytochrome P450 reductase, which transfers electrons from NADPH to P450, and dilaurylglycerophosphocholine (Lau 2 PtdCho) [8][9][10]. However, this micellar system is not appropriate for studying the interactions between the components of the system as some of its properties are unlike those of the endoplasmic reticulum membrane, the natural environment of the microsomal monooxygenase system. This membrane is a bilayer, and it is highly probable that CYP1A1 and NADPH-cytochrome P450 reductase exhibit other important protein-lipid and protein-protein interactions there. Indeed, reconstitution systems using bilayer vesicles yielded higher rates of activity with rabbit liver CYP...

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Membrane‐Mediated Protein–Protein Interactions of Cholesterol Side‐Chain Cleavage Cytochrome P450 with its Associated Electron Transport Proteins

et al. 2016

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Cytochrome P450scc (P450scc or CYP11A1) catalyses the first enzymatic step of steroid biosynthesis, the cleavage of the side chain of cholesterol to produce pregnenolone in the mitochondrion. The activity of P450scc is dependent upon electron delivery from NADPH‐dependent adrenodoxin reductase (AdR), via adrenodoxin (Adx), to the P450scc. However, despite the structural and kinetic data that supports the mechanism by which Adx shuttles electrons one at a time between AdR and the P450scc, there are limited data available on the influence of the lipid membrane on these essential interactions. In this paper, the protein–membrane interactions between P450scc and its redox partners were examined on 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC) membranes containing cholesterol (20 %), using a quartz crystal microbalance with dissipation monitoring. P450scc showed strong binding to these membranes, whereas AdR and Adx both showed weaker association. If pre‐mixed, all three proteins bound independently to the membrane layer in a distinctive two‐stage process, as observed by frequency changes upon binding. Concomitant changes in the dissipation revealed specific protein–protein interaction occurs upon reaching a critical concentration of proteins in the membrane layer. These changes were specific for the binding of the three pre‐mixed proteins and were not observed for a binary mixture of P450 and Adx, or sequential binding of the three proteins. A simple model was developed for the binding of all three proteins in a 1:1:1 mixture to the membrane and reproduces the experimental data describing the interaction of P450scc with the other proteins (AdR and Adx) after initial binding of the individual proteins. Thus, we conclude that the lipid membrane assists in the assembly of electron transport proteins and the activity of P450scc by providing a surface for the localised concentration of proteins, enabling them to act together as a metabolon.

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Evidence That Nonbilayer Phase Propensity of the Membrane Is Important for the Side Chain Cleavage Activity of Cytochrome P450SCC (CYP11A1)

Cited by 16 publications

References 42 publications

Branched Phosphatidylcholines Stimulate Activity of Cytochrome P450SCC (CYP11A1) in Phospholipid Vesicles by Enhancing Cholesterol Binding, Membrane Incorporation, and Protein Exchange

Branched Phosphatidylcholines Stimulate Activity of Cytochrome P450SCC (CYP11A1) in Phospholipid Vesicles by Enhancing Cholesterol Binding, Membrane Incorporation, and Protein Exchange

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane

Membrane‐Mediated Protein–Protein Interactions of Cholesterol Side‐Chain Cleavage Cytochrome P450 with its Associated Electron Transport Proteins

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