Phosphatidylcholine vesicle reconstituted cytochrome P-450scc. Role of the membrane in control of activity and spin state of the cytochrome.

Cytochrome P-450scc from bovine adrenal cortex mitochondria was purified and reconstituted into phosphatidylcholine vesicles which varied in both cholesterol content and in the fatty acyl composition of the phospholipid. Under conditions of optimal ionic strength, pH, and excess adrenodoxin and adrenodoxin reductase, it was found that at a constant cholesterol: phospholipid ratio, the membrane composition had large effects on the rate of hemoprotein-catalyzed side chain cleavage of cholesterol. Rate effects were due to phospholipid-induced changes in the enzyme's Km for cholesterol, and not due to Vmax effects. Binding of cholesterol to cytochrome P-450 could also be monitored optically by measuring the fraction of enzyme in the high spin form. Dissociation constants determined in this manner for cholesterol binding in phospholipid of differing fatty acyl composition showed an excellent inverse correlation with the rates of pregnenolone formation in the same lipids (at constant cholesterol concentration) (see Fig. 6); thus, phospholipid exerts its rate effects by modulating the binding of cholesterol to the cytochrome. The membrane-mediated effects on spin state and activity mimic closely the effects seen in mitochondria isolated from adrenocorticotropic hormone-treated versus control adrenal cells. This behavior suggests to us that acute steroidogenic action of adrenocorticotropic hormone may be mediated through changes in the composition of the inner mitochondrial membrane in which cytochrome P-45scc is embedded.

Cytochrome P-450,,, from bovine adrenal cortex mitochondria was purified and reconstituted into phosphatidylcholine vesicles which varied in both cholesterol content and in the fatty acyl composition of the phospholipid. Under conditions of optimal ionic strength, pH, and excess adrenodoxin and adrenodoxin reductase, it was found that at a constant cholesterol: phospholipid ratio, the membrane composition had large effects on the rate of hemoprotein-catalyzed side chain cleavage of cholesterol. Rate effects were due to phospholipid-induced changes in the enzyme's K,,, for cholesterol, and not due to V,, effects. Binding of cholesterol to cytochrome P-450 could also be monitored optically by measuring the fraction of enzyme in the high spin form. Dissociation constants determined in this manner for cholesterol binding in phospholipid of differing fatty acyl composition showed an excellent inverse correlation with the rates of pregnenolone formation in the same lipids (at constant cholesterol concentration) (see Fig. 6); thus, phospholipid exerts its rate effects by modulating the binding of cholesterol to the cytochrome. The membrane-mediated effects on spin state and activity mimic closely the effects seen in mitochondria isolated from adrenocorticotropic hormone-treated uersus control adrenal cells. This behavior suggests to us that acute steroidogenic action of adrenocorticotropic hormone may be mediated through changes in the composition of the inner mitochondrial membrane in which cytochrome P-450,,, is embedded.
Cytochrome P-450,,, from bovine adrenal cortex mitochondria is an inner membrane-associated hemoprotein which can function, together with its soluble electron transport components adrenodoxin (a ferredoxin-type protein) and NADPHadrenodoxin reductase (a flavoprotein), in the enzymatic side chain cleavage of cholesterol to yield pregnenolone, the common precursor of the adrenal steroid hormones (1,2). Cleavage of the 20, 22 carbon-carbon bond of cholesterol occurs in three steps utilizing three molecules of oxygen and a total of six electrons (1, 3). We have studied the mechanism of electron * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. +Recipient of Grant GM07403 from the National Institutes of Health. Present address, Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30332. 5 Recipient of Grant GM21226 from the National Institutes of Health. transfer in this system and find that adrenodoxin acts as a mobile one-electron shuttle between adrenodoxin reductase and cytochrome P-450,,, (4)(5)(6); the adrenodoxin must interact with an aqeuous-exposed site on the cytochrome once during each of its electron shuttles.' Our recent successful reconstitution of cytochrome P-450,, into phosphatidylcholine vesicles (5) has permitted us to begin studies on the interaction of cholesterol and phospholipid with cytochrome P-450,,,.. We find that the cholesterol binding site of the cytochrome is in communication with the hydrophobic milieu of the phospholipid membrane (5). When cholesterol is incorporated into the phospholipid membrane, binding of cholesterol to the cytochrome occurs rapidly and is accompanied by a conversion of the optical spectrum from a low spin or L-type spectrum to a high spin or H-type spectrum (9). The fraction of hemoprotein in the high spin form is a function of the cholestero1:phospholipid ratio rather than the total cholesterol concentration expressed in terms of the aqueous volume; we have therefore adopted the convention of expressing cholesterol concentration as a cholestero1:phospholipid molar ratio. The present studies have used the reconstitution technique (5) to investigate further the interactions among cholesterol, phospholipid, and cytochrome P-45OS,,; activity and spectral properties of the cytochrome were studied in reconstituted systems which varied in cholesterol content and in the fatty acyl side chain composition of phosphatidylcholine.
Results of the present studies may be useful in interpreting the mechanism by which adrenocorticotropic hormone controls the production of glucocorticoid hormones in the adrenal cortex. The rate of cholesterol side chain cleavage has been shown to be limiting in steroid hormone production (lo), and is stimulated by ACTH' by some as yet poorly understood mechanism.:' Thus, data pertaining to the rate-limiting step in the catalytic cycle of adrenodoxin reductase:adrenodoxin:cytochrome P-45OS,,.-catalyzed side chain cleavage is of particular interest. In a recent study of the effects of ions on the rates of adrenodoxin reduction, 1 lp-hydroxylation, and cholesterol side chain cleavage (using purified lipid-reconstituted com-' Recent work by Hanukoglu and Jefcoate (7) using an independent approach has confumed that adrenodoxin functions as an electron shuttle. We have found no evidence to support a recent proposal (8) that the system operates as a ternary complex among the three protein components.
,'The early steps in the control mechanism are relatively well understood. ACTH binds to a cell surface receptor and activates adenylate cyclase to form cyclic AMP (11,12). This, in turn, stimulates the cytosolic translation of a "labile protein" factor, which then acts by some unknown mechanism to stimulate the side chain cleavage reaction (13,14).

Vesicle Reconstituted
Cytochrome P-450,,, 8283 ponents) (4), we found that when very low or very high concentrations of salts were used and when the ratio of adrenodoxin reductase to adrenodoxin was low (-l:lO), the rate of reduction of adrenodoxin was limiting in cytochrome P-450-dependent hydroxylations. However, when intermediate ion concentrations were used in assays, a step or steps other than adrenodoxin reduction became rate-determining in cholesterol side chain cleavage. In uivo studies have demonstrated that the rate of adrenodoxin reduction is not limiting either with or without ACTH stimulation (15), so that some other rate-controlling step must be considered. In uiuo and in vitro studies have also shown that the ACTH-regulated ratelimiting step is intramitochondrial (16), that the rate stimulation is accompanied by an increase in the fraction of cytochrome P-450,, in the high spin form in isolated mitochondria (17), but that the spin state changes are not correlated with changes in the total mitochondrial cholesterol content (16,18). Such findings have often been interpreted in terms of ACTH control of cholesterol "pools" within the mitochondrion (18,19). In the present studies we used optimal ion concentrations and high ratios of adrenodoxin reductase to adrenodoxin to insure that reduction of adrenodoxin was not rate-limiting; under these conditions we find that at all achievable ratios of cholesterol to phospholipid, the system operates at below saturating levels of cholesterol. Furthermore, effects of changes in the composition of the lipid phase mimic closely the effects of ACTH (17); changes in phospholipid composition resulted in parallel changes in both the cholesterol side chain cleavage activity and the spin state of cytochrome P-

Phosphatidylcholine Vesicle Reconstituted Cytochrome P-450,,,
phospholipid in a total of 0.5 ml of buffer, 37"C, 24 h) and separated on DEAE-cellulose columns (0.8 X 1 cm) with 2.5 m~ increments of KPi buffer. Donor vesicles were eluted while acceptor vesicles were retained on the column. Exchange of cholesterol was measured from the "C to '"H ratio before and after separation of vesicles.

RESULTS
Effect of Saturation of Fatty Acyl Chains of Egg Phosphatidylcholine on Side Chain Cleavage Actiuity-We have reported previously that cytochrome P-45OS,, is relatively active when reconstituted into purified egg phosphatidylcholine or synthetic dioleoylphosphatidylcholine, but is less active in synthetic dimyristoyl-or dipalmitoylphosphatidylcholine (5).
Purified egg phosphatidylcholine has been demonstrated to contain 57% unsaturated fatty acyl chains, primarily Clx (35%) monounsaturated chains.6 Fig. 1 c o n f i i s t h a t t h e side chain cleavage enzyme is relatively active in native egg phosphatidylcholine, and demonstrates that hydrogenation of this phospholipid results in a reconstituted system with less than 8% of the original activity. Thus, the saturation state of the fatty acyl chains of phosphatidylcholine can exert a major effect on the activity of the side chain cleavage system. The mechanism by which this activity is controlled is considered below.
Effect of Cholesterol to Phospholipid Ratio on Cholesterol Side Chain Cleavage Actiuity-As discussed previously the activity of cytochrome P-450,,.,., under optimal conditions of ionic strength (4) and pH (25), appears to be governed by the cholesterol to phospholipid ratio (see Fig. 2 and Ref. 5), rather than the absolute cholesterol concentration expressed in terms of the aqeuous volume. Such a finding is consistent with our proposal that cholesterol interacts with the cytochrome via the hydrophobic phospholipid milieu rather than through the aqueous environment (5,6). Using our previous assay system, the double label system of Doering (27), it was difficult to measure rates at high cholesterol to phospholipid ratios since the fractional change in the 'H to I4C ratio became smaller than the "noise level" of the assay. The use of the radioimmunoassay for pregnenolone allows accurate measurement at high cholesterol to phospholipid ratios, and has permitted us to determine K,,, and Ifmax values more accurately. Fig. 2 shows the effect of varying the cholesterol to phospholipid ratio upon activity. The V,,,, value calculated from the y intercept is 21.3 mol of pregnenolone produced/min/mol of cytochrome P-450,,,.. Since each pregnenolone produced requires three hydroxylations, this corresponds to a turnover number/hydroxylation of 65 min", assuming there is no accumulation of hydroxycholesterol intermediates. This is compared to turnover of 30 min-' obtained by us for Ilp-hydroxylation by cytochrome P-45011,j (4). Thus, when suffkient substrate is supplied to the side chain cleavage enzyme, it can operate at a rate similar to that of the llp-hydroxylase. Fig. 2 also allows calculation of a K,,, for cholesterol of 0.22 (expressed in terms of a cholesterol to phospholipid molar ratio, since phospholipid is the solvent in which cholesterol is "dissolved"). Since the maximum achievable ratio of cholesterol to phospholipid is -1:l (28), the relatively high K,,, of 0.22 indicates that the system operates at subsaturating levels of cholesterol under all assay conditions. It is likely that this is also the case in uiuo; adrenal cortex mitochondrial cholesterol content has been measured under a variety of conditions, and found to be low (10 to 100 nmol/mg of mitochondrial protein' (29, 30)). I' Avanti Biochemicals catalog, 1978, product specifications.
' Assumnrg that the mitochondrial membrane is -50% phospholipid by weight, and assuming an average molecular weight of 760/ phospholipid, this represents a cholesterol:phospholipid ratio of 0.01 to 0.10, values at least 2 to 20 times lower than the lowest K,, value determined in this study (in diphytanoylphosphatidylcholine).

Rate of Side Chain Cleavage in Diphytanoylphosphatidyl-
choline-The rate of side chain cleavage of cholesterol was measured in diphytanoylphosphatidylcholine at various cholesterol to phospholipid ratios. Comparison of Figs. 1 and 3 reveal that the requirement for unsaturated lipid can be filled by the fully saturated diphytanoylphosphatidylcholine which contains a methyl group at every 5th position on its C:16 fatty acid chains. Activity (corrected for differences in cytochrome concentration) at the highest cholesterol to phospholipid ratio was 33% higher in the latter lipid than in egg lipid. In experiments (not shown) it was also determined that there was a 30 to 40% loss in heme absorbance and cytochrome P-450 content (as judged by the CO difference spectrum) when the cytochrome was incubated overnight at 4°C with the egg lipid, whereas there was no detectable loss with diphytanoyl lipid.
Thus, diphytanoylphosphatidylcholine appears to be the lipid of choice in reconstitution studies requiring long incubations with the cytochrome. A double reciprocal plot of activity uersus cholesterol to phospholipid ratio as in Fig. 2  Effects of Various Lipids on Cholesterol Side Chain Cleavage Activity a n d on the K , for Cholesterol-V,,, and K,,, values determined above, as well as those for synthetic dioleoylphosphatidylcholine determined by the same method, are summarized in Table I. Also shown are turnover numbers (pregnenolone production) in a variety of phosphatidylcholines at a cholesterol to phospholipid ratio of 0.5 mol/mol. As can be seen, there is an almost 20-fold range of turnover numbers depending upon the phospholipid used. For the three lipids in which V, , , and K,,, could be determined (see Table   I), the extrapolated maximal velocities were the same within 15% and the small differences seen could not account for the 2.1-fold difference in rates. The K, range of 0.02 to 0.36, however, can account for a 1.9-fold range of rates. Thus, phospholipid appears to exert its effect by changing the enzyme's K,,, for cholesterol, a t least for the three phospholipids tested. For phospholipids with lower rates, K,,, and VmaX values could not be determined. 8 We will show in subsequent sections, however, that the rate in these phospholipids is also determined by cholesterol binding to cytochrome.
Table I also demonstrates that there is no simple correlation of activity with the degree of unsaturation of the phospholipid. The enzyme is least active in fully saturated lipids. A single double bond per acyl chain results in an approximately %fold increase in activity, while a second double bond again decreases activity to near that of the fully saturated lipid. Three double bonds per chain again results in an increase in activity (14-fold over saturated phospholipid). Possible explanations for this perplexing series will be discussed below (see "Discussion"). Regardless of the mechanism for these effects, a large range of activities are achievable when the fatty acyl chains are varied while keeping phospholipid head group the same (see Table I).
Effects of Phospholipid Acyl Chains on the Cholesterolinduced High Spin Conversion of Cytochrome P-450se,-Our preparation of purified cytochrome P-450,,, contains between 0.5 and 1 mol of cholesterol/mol of cytochrome (4,5) and exists at pH 7 as a mixture of H-and L-type hemoprotein (always greater than 70% H-type spectrum). In previous studies (4,5), we showed that reconstitution of cytochrome p-" Approach to saturation could not be achieved since higher than 1:1 cholesterol to phospholipid ratios would be required, and since experimental error at lower ratios made the long extrapolations necessary to determine these values impossible.   mitoyl-and hydrogenated egg phosphatidylcholine, respect i~e l y .~ Thus, we suggest that the nonideal behavior of the latter lipids may result either from poor reconstitution of the cytochrome or from unavailability of substrate for binding to cytochrome in gel state lipids.

Phosphatidylcholine Vesicle Reconstituted
When unsaturated or branched chain (saturated) phosphatidylcholines were used, however, linear plots were obtained (see Fig. 5). Dissociation constants obtained from these plots indicate that cholesterol binding to cytochrome P-45OS,,. is highly dependent upon the fatty acyl portion of the phospholipid used, with the affinity in the following order: diphytanoyl-> egg > dioleoyl-> dilinoleoyl-> dimyristoylphosphatidylcholine." This order is identical with that seen for activity at a cholesterol to phospholipid ratio of 0.5 (see Table I). This correlation is demonstrated quantitatively in Fig. 6 Cholesterol, at high concentrations, appears to have a "fluidizing" effect on phospholipids below their phase transitions (see, for example, Ref. 31) and may account for the more ideal binding and activity measurements at very high cholesterol concentration in dipalmitoyland hydrogenated egg phosphatidylcholine.
A cholesterol binding constant in dilinoleoylphosphatidylcholine could not be determined due to extensive development of turbidity during the reconstitution.
" Free fatty acid alone, although a high spin inducer, acts as an inhibitor of enzyme activity. In contrast to the acylated form, its effects are independent of unsaturation. The mechanism of these effects is under study (Lambeth, unpublished). where L = lipid and C = cholesterol. A third possibility and the one which we prefer, is that the phospholipid affects cholesterol binding by interacting with the cytochrome itself. The fiist and third possibilities are considered in the "Discussion," while the second is explored below.
If the mechanism represented by Equation 1 were operating, then the order of affinity of various phospholipids for cholesterol should be inversely correlated with the affinity of cytochrome P-450 for cholesterol in the respective phospholipid. The turnover of cytochrome P-450,, (pregnenolone formation) was determined at a f i e d cholesterolphospholipid ratio (see Table I)  was used as the donor phospholipid, and our results, as well as those of Nakagawa et al. (26), are summarized in Table 11. Inspection of the data reveal an affinity order for cholesterol as follows: where DMPC is dimyristoylphosphatidylcholine and PC is phosphatidylcholine. A striking feature of this series is the decrease in cholesterol affinity with increased amount of fatty acyl chain unsaturation. Although this order is of interest in its own right, the inverse correlation with cholesterol binding to enzyme is poor, indicating that some other mechanism controls the cholesterol-cytochrome binding equilibrium.

DISCUSSION
Phospholipid has been shown to affect the activity of a variety of membrane-associated enzymes and transport proteins (see Ref. 32 for a review). In many cases, a relatively nonspecific requirement for lipid is seen, while a more specific role has been proposed for a few enzymes. Liver microsomal cytochrome P-450 together with its flavoprotein reductase have been shown to require phospholipid for activity in hydroxylation reactions (33). Cytochrome oxidase isolated by a variety of procedures contained 1 mol/mol of tightly bound cardiolipin which appeared to be important for catalytic activity (34, 35). /3-Hydroxybutyric dehydrogenase requires phosphatidylcholine, and highest activity was seen when the lipid contained unsaturated fatty acyl chains (36). We have previously demonstrated an effect of phospholipid on the activity of purified cytochrome P-450,, from beef adrenal cortex mitochondria (5). An activity difference dependent upon the nature of the fatty acyl side chain was found; little or no activity was seen with fully saturated chains, while activity was high when the phospholipid contained unsaturated chains.
In the present investigation, the role of phospholipid in the cholesterol side chain cleavage system has been investigated further. The cytochrome has been reconstituted into phosphatidylcholine vesicles in which the fatty acyl groups of the phospholipid were varied. Phosphatidylcholines produce stable lamellar or vesicular structures in the presence of excess water, and use of this head group eliminated possible artifacts due to the nonbilayer arrangements of hydrated lipids seen with some other head groups (37). Large acyl chain-dependent rate effects were seen in the reconstituted system (see Table   I); this finding is consistent with our previous proposal of a membrane phase interaction of cholesterol with the cytochrome (5,6). The effects occur only when the fatty acids were part of a phospholipid molecule, since free fatty acids, whether saturated or unsaturated, were potent inhibitors in the vesicle reconstituted system.12 There appears to be no simple relationship between activity and the degree of chain unsaturation; although activity was relatively low in straight chain saturated phosphatidylcholines, another saturated lipid, diphytanoylphosphatidylcholine, gave the highest activity. Similarly, among unsaturated lipids, the activity order (dilinolenoylphosphatidylcholine (C18:3) > dioleoylphosphatidylcholine (C18:l) > dilinoleoylphosphatidylcholine (C18:2), see Table 11) failed to correlate with unsaturation.
While the molecular explanation for the activity order with various fatty acyl groups is not yet fully understood (see below), it is clear from the present studies that these groups exert their effect by modulating the binding of cholesterol to the active site of cytochrome P-450,,.,.; in Fig. 6 the dissociation constants for binding of cholesterol to hemoprotein in the various lipids show an excellent correlation with the activities in these lipids (at a constant cholestero1:phospholipid ratio). Table I demonstrates that this lipid effect on cholesterol binding is also seen in the catalytic K,, for cholesterol, and little or no effect on theV,,., is seen.
The molecular mechanism by which phospholipid acyl chains exert their effect is of particular interest. The present studies, while they do not explain the details of the molecular interactions involved, allow us to choose among various general types of mechanisms. First, as has been suggested for several enzymes and transport proteins, the activity might be regulated by the physical properties of the lipid phase. For example, sugar transport in fatty acid auxotrophs of Escherichia coli is apparently dependent upon the fluidity of the phospholipid membrane (38). Such a mechanism is not supported for the side chain cleavage system. Cornwell and Patil (39) have pointed out that the physical properties of phospholipids vary with unsaturation as a step function rather than a continuous function; major changes occur with the introduction of a one double bond, and additional unsaturation results in only minor variations in physical properties. This is in contrast to the activity order seen in Table I for side chain cleavage, where introduction of additional double bonds produces large effects on activity. Similarly, lateral diffusion in lipid bilayers above their transition temperatures is rapid (40), and we have provided evidence that the rate of association of cholesterol with cytochrome P-450,,.,. is far more rapid than turnover (9). Thus our data do not support a rate effect due to known physical properties of the lipid phase itself. A second possible mechanism, that lipid effects cholesterol binding to cytochrome competitively due to its own affinity for cholesterol (see Equation l ) , is discussed above under "Results." Comparison of Table I and Fig. 6 with Table I1 reveals no positive or negative correlation of activity of Kd with the affinity of a given lipid for cholesterol, thus indicating that this simple mechanism alone does not modulate the cholesterol binding equilibrium. A third possibility, that the binding effects are due to a direct interaction of lipid with the enzyme, is most consistent '' D. Lambeth, unpublished observations. with the available data. Thus, the structural specificity of a lipid binding site(s) on the enzyme could explain the observed activity order in Table I. The lipid could, for example, either stabilize or destabilize a cholesterol-binding confirmation of the enzyme, thus affecting binding by interacting with a site physically distinct from the cholesterol binding site. Adrenodoxin, which forms a 1:l complex with cytochrome P-450,,, by binding at an aqeuous phase site, facilitates the binding of cholesterol by such a mechanism (9). Another possible mechanism of this general class is suggested by the observation that the cholesterol binding site on cytochrome P-450,,.,. is in communication with the hydrophobic phospholipid milieu ( 5 ) : the fatty acyl chains themselves might participate in the binding of cholesterol to its enzyme binding site. Thus, a thermodynamically stable complex among phospholipid, cholesterol, and enzyme might be formed. The present data suggest strongly that the lipid exerts its effect by a direct interaction with the enzyme, but do not distinguish between these latter two possibilities.
Finally the present studies are of particular interest regarding the short term response (-15 min) of the adrenal cortex to ACTH. This pituitary peptide hormone binds to a specific cell surface receptor and, via a series of intracellular steps (see Footnote 1 and Ref. 41 for a review), acts ultimately within the mitochondrion by an unknown mechanism to stimulate the rate of side chain cleavage of cholesterol (10). A hallmark of the steroidogenic effect of ACTH is an increase in the fraction of the mitochondrial cytochrome P-450,,, the high spin form (17) with no short term change in the total content of cytochrome P-450. The spin state change has been interpreted as reflecting cholesterol binding to cytochrome, but no correlation of spin state with total mitochondrial cholesterol content is seen (16); the results are therefore often explained by proposing ACTH control of cholesterol pools within the mitochondrion. From the present studies it is clear that the parallel changes in spin sate and activity seen with ACTH treatment are consistent with an ACTH-mediated decrease in the cholesterol K,,, of cytochrome P-450,,,.. Variations in the hydrophobic composition of the phospholipid in the reconstituted system induce spin state and activity changes which mimic those seen in mitochondria isolated following ACTH treatment of cells or tissues, and we suggest the possibility that these ACTH-induced changes could be mediated by some membrane phase component or components. In support of this working hypothesis, ACTH has been demonstrated to exert a variety of effects on adrenal phospholipid metabolism; ACTH causes an increased turnover of both phosphatidic acid and phosphatidylinositol within 2 min (42, 44) and an increased production of polyphosphoinositides within 15 min (43). Although the subcellular location and catalytic significance of these ATCH-controlled changes in lipid metabolism have not been reported, the present studies suggest that changes in the composition of the inner mitochondrial membrane in which cytochrome P-450,,,. is embedded could mediate the steroidogenic effects of ACTH.