Electron paramagnetic resonance study of ferrous cytochrome P-450scc-nitric oxide complexes: effects of 20(R),22(R)-dihydroxycholesterol and reduced adrenodoxin

Electron paramagnetic resonance (EPR) spectra of ferrous-nitric oxide (14NO and 15NO) cytochrome P-450scc complexed with 20(R),22(R)-dihydroxycholesterol were measured at 77 K with X-band (9.35 GHz) microwave frequency. The EPR spectra clearly showed the spin system to have rhombic symmetry (gx = 2.068, gz = 2.001, gy = 1.961, and Az = 1.89 mT for 14NO) and were distinct from those of 20(S)-hydroxycholesterol complexes. The unique nature of the 20(S)-hydroxycholesterol complexes indicates that 20(S)-hydroxycholesterol is not a proper intermediate in the cholesterol side-chain cleavage reaction. In addition, among various steroid complexes of ferrous-NO species having rhombic symmetry, the EPR spectra of 20(R),22(R)-dihydroxycholesterol complexes were significantly different from those of 22(R)-hydroxycholesterol complexes, suggesting that upon 20S-hydroxylation of 22(R)-hydroxycholesterol the conformation of the active site changes so as to facilitate subsequent cleavage of the C20-C22 bond of the cholesterol side chain. Addition of reduced adrenodoxin to the ferrous-NO cytochrome P-450scc complex in the presence of cholesterol caused a complete shift of the gx = 2.070 signal to gx = 2.075, indicating a reorientation of cholesterol in the substrate-binding site of the enzyme upon adrenodoxin binding. Without reduced adrenodoxin, the process of reorientation of cholesterol in the substrate-binding site was very slow, requiring more than 50 h of incubation at 0 degrees C. The present observations suggest that adrenodoxin may have another positive role in the cholesterol side-chain cleavage reaction, in addition to transferring an electron to the heme of cytochrome P-450scc.     

Adrenodoxin with a COOH-terminal deletion (des 116-128) exhibits enhanced activity

Adrenodoxin, purified from bovine adrenal cortex, was subjected to trypsin cleavage to yield a trypsin-resistant form, designated TT-adrenodoxin. Sequencing with carboxypeptidase Y identified the trypsin cleavage site as Arg-115, while Edman degradation indicated no NH2-terminal cleavage. Native adrenodoxin and TT-adrenodoxin exhibited similar affinity for adrenodoxin reductase as determined in cytochrome c reductase assays. In side chain cleavage assays using cytochrome P-450scc, however, TT-adrenodoxin demonstrated greater activity than adrenodoxin with cholesterol, (22R)-22-hydroxycholesterol, or (20R,22R)-20,22-dihydroxycholesterol as substrate. This enhanced activity is due to increased affinity of TT-adrenodoxin for cytochrome P-450scc; TT-adrenodoxin exhibits a 3.8-fold lower apparent Km for the conversion of cholesterol to pregnenolone. TT-Adrenodoxin was also more effective in coupling with cytochrome P-450(11) beta, exhibiting a 3.5-fold lower apparent Km for the 11 beta-hydroxylation of deoxycorticosterone. In the presence of partially saturating cholesterol, TT-adrenodoxin elicited a type I spectral shift with cytochrome P-450scc similar to that induced by adrenodoxin, and spectral titrations showed that oxidized TT-adrenodoxin exhibited a 1.5-fold higher affinity for cytochrome P-450scc. These results establish that COOH-terminal residues 116-128 are not essential for the electron transfer activity of bovine adrenodoxin, and the differential effects of truncation at Arg-115 on interactions with adrenodoxin reductase and cytochromes P-450 suggest that the residues involved in the interactions are not identical.     

Effects of cholesterol analogues and inhibitors on the heme moiety of cytochrome P-450scc: a resonance Raman study

Interactions of cholesterol analogues and inhibitors with the heme moiety of cytochrome P-450scc were examined by resonance Raman spectroscopy. The Raman spectra of ferric cytochrome P-450scc complexed with inhibitors such as cyanide, phenyl isocyanide, aminoglutethimide, and metyrapone were characteristic of low-spin state and were very similar. However, the effect of exchange of the sixth ligand from the oxygen atom (ferric low-spin state) to the nitrogen atom upon aminoglutethimide and metyrapone binding was seen as down-frequency shifts of the v3 band from 1503 to 1501 and 1502 cm-1, respectively, while cyanide and phenyl isocyanide binding caused an up-frequency shift of the v3 band to 1505 cm-1. The effects of cholesterol analogues [22(R)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22-ketocholesterol, 20(S)-hydroxycholesterol, and 25-hydroxycholesterol] on a Fe2+-CO stretching frequency of cytochrome P-450scc in ferrous CO form were examined. The 22(R)-hydroxycholesterol complex could not give a clear Fe2+-CO stretching Raman band due to a strong photodissociability. 22(S)-Hydroxycholesterol and 25-hydroxycholesterol complexes gave the Raman bands at 487 and 483 cm-1, respectively, whereas 20(S)-hydroxycholesterol and 22-ketocholesterol complexes gave Fe2+-CO stretching frequencies (478 cm-1) almost identical with that without substrate (477 cm-1). These findings suggest the existence of the following physiologically important natures of the cytochrome P-450scc active site: (1) there is a strong steric interaction between heme-bound carbon monoxide and the 22(R)-hydroxyl group or the 22(R)-hydrogen of the steroid side chain and (2) the hydroxylation at the 20S position may cause a conformational change of the side-chain group relative to the heme.     

Cytochrome P-450 of adrenal mitochondria. In vitro and in vivo changes in spin states.

Steroid-induced difference spectra have been used to examine the combination of cholesterol with adrenal mitochondrial cytochrome P-450 which participates in cholesterol side chain cleavage (P-450scc) and the depletion of cholesterol from the cytochrome which results from turnover of the enzyme system. Type I difference spectra-induced by cholest-5-ene-3beta, 25-diol (25-hydroxycholesterol) and cholest-5-ene-3beta, 20 alpha, 22R-triol (20alpha, 22R dihydroxycholesterol) have been used to quantitate binding of cholesterol to two sites (I and II) on cytochrome P-450scc. The action of adrenocorticotropic hormone (ACTH) in vivo and the action of calcium or phosphate ions on isolated mitochondria stimulate the combination of cholesterol with site I but not site II. Cholesterol derived from lecithin-cholesterol micelles, however, binds to both sites. Malate-induced cholesterol depletion occurred at a comparable rate to the transfer of cholesterol from lecithin-cholesterol micelles. However, a residual proportion of cholesterol-cytochrome P-450scc complexes remained, even after 10 min of exposure to malate, and was of similar magnitude in mitochondria from both cycloheximide-treated and stressed rats. It is suggested that this reflects a less reactive form of cholesterol-cytochrome complex. Steroid-induced difference spectra indicate that sites I and II on cytochrome P-450scc are similarly depleted after metabolism of mitochondrial cholesterol in vitro and after inhibition of the action of ACTH in vivo. Anaerobiosis of adrenal cells after excision of the accumulation of cholesterol at cytochrome P-450cc. When anaerobiosis was prevented, cytochrome P-450scc in the freshly isolated mitochondria was apparently essentially free of complexed cholesterol, irrespective of the extent of ACTH action. For 30 min after suspension of the mitochondria in 0.25 M sucrose at 4 degrees, cholesterol combines with cytochrome P-450scc. The extent of this process was not affected by the presence of cycloheximide during ether stress treatment of the rats. It is concluded that there are at least two pools of mitochondrial cholesterol with access to cytochrome P-450scc but that ACTH stimulates only the pool which most readily interacts with the cytochrome.     

Electron paramagnetic resonance study of ferrous cytochrome P-450scc-nitric oxide complexes: effects of cholesterol and its analogues

Electron paramagnetic resonance (EPR) spectra of nitric oxide (NO) complexes of ferrous cytochrome P-450scc were measured at 77 K for the first time without using the rapid-mixing and freeze-quenching technique. Without substrate the EPR spectra were very similar to those of cytochrome P-450cam (from Pseudomonas putida) and cytochrome P-450LM (from rat liver microsomes) with rhombic symmetry; gx = 2.071, gz = 2.001, gy = 1.962, and Az = 2.2 mT for 14NO complexes. Upon addition of substrates [such as cholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, 25-hydroxycholesterol, and 22-ketocholesterol], the EPR spectra exhibited many variations having rhombic symmetry in the major component and an additional minor component with less rhombic symmetry. Furthermore, addition of 20(S)-hydroxycholesterol caused a striking change in the EPR spectrum. The component with rhombic symmetry disappeared completely, and the component with less rhombic symmetry dominated (gx = 2.027, gz = 2.007, gy = 1.984, and Az = 1.76 mT for 14NO complexes). These observations suggest the existence of the following physiologically important natures: (1) the conformational flexibility of the active site of the enzyme due to the steric interaction between the substrate and the heme-bound ligand molecule and (2) the importance of the hydroxylation of the cholesterol side chain at the 20S position to proceed the side-chain cleavage reaction in cytochrome P-450scc.     

Discriminatory inhibition of adrenocortical 17 alpha-hydroxylase activity by inhibitors of cholesterol side chain cleavage cytochrome P-450

Two inhibitors of the cholesterol side chain cleavage reaction were tested for their ability to inhibit bovine adrenocortical 17 alpha-hydroxylase and 21-hydroxylase activities. One inhibitor, 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC), was found to be a potent inhibitor of 17 alpha-hydroxylation of either progesterone or pregnenolone but was inactive on 21-hydroxylase activity. 22-ABC was found to be a competitive inhibitor of 17 alpha-hydroxylase (cytochrome P-45017 alpha) activity, having an apparent inhibitor constant of 29 nM when using pregnenolone as the substrate. Spectral binding studies showed that 22-ABC produces a type II difference spectrum when added to a bovine adrenocortical microsomal preparation, due presumably to a coordination of its amine nitrogen atom to the heme-iron of cytochrome P-45017 alpha. The second cholesterol side chain cleavage inhibitor tested, (20R)-20-phenyl-5-pregnene-3 beta,20-diol (20-PPD), was found not to inhibit either the 21- or 17 alpha-hydroxylase activities. It is proposed that the phenyl group projecting from C-20 of 20-PPD prevents this steroid from binding to cytochrome P-45017 alpha. The discriminatory interaction of these two steroids with adrenocortical cytochromes P-450 provides some insight with respect to possible structural features of the active-site regions of these enzymes.     

Pregnenolone synthesis from cholesterol and hydroxycholesterols by mitochondria from ovaries following the stimulation of immature rats with pregnant mare's serum gonadotropin and human choriogonadotropin

The rate of pregnenolone synthesis by cytochrome P-450scc was measured in mitochondria isolated from ovaries of immature rats treated with pregnant mare's serum gonadotropin and human choriogonadotropin. Using cholesterol, 25-hydroxycholesterol, 20 alpha-hydroxycholesterol, (22R)-22-hydroxycholesterol and (22R)-20 alpha,22-dihydroxycholesterol as substrates, we have determined that the first hydroxylation of cholesterol, in the 22R position, is rate limiting in pregnenolone synthesis. It proceeds at only 22% of the rate of either of the subsequent two hydroxylations. 25-Hydroxycholesterol proved to be a suitable substrate for determining the maximum rate of pregnenolone synthesis by cytochrome P-450scc in isolated mitochondria. The maximum rate was 13 mol steroid.min-1.mol cytochrome P-450scc-1 and did not change after the follicles in the immature ovary had been stimulated to mature and luteinize with gonadotropin. Using endogenous cholesterol in isolated mitochondria as substrate, the time course of pregnenolone synthesis was the same during the follicular phase as in the luteal stage of gonadotropin-induced development. We conclude that during the artificial induced development of follicles in the immature ovary, the major cause of the increase in the rate of pregnenolone synthesis is the increase in the cytochrome P-450scc content of the mitochondria, rather than changes in the catalytic activity of cytochrome P-450scc or the cholesterol availability to the cytochrome.     

Fluorescein isothiocyanate specifically modifies lysine 338 of cytochrome P-450scc and inhibits adrenodoxin binding

Treatment of cytochrome P-450scc with fluorescein isothiocyanate (FITC) resulted in covalent labeling with 1.0 +/- 0.1 eq of FITC. Reverse-phase high performance liquid chromatography of tryptic and chymotryptic digests of the labeled protein revealed that a single FITC-labeled peptide accounted for 75% of the label. This peptide was found to be specifically labeled at lysine 338 by amino acid sequencing. The modification of lysine 338 with FITC resulted in 85 +/- 15% inhibition of adrenodoxin binding to cytochrome P-450scc. In a complementary experiment it was found that if a complex between adrenodoxin and native cytochrome P-450scc was formed in the presence of cholesterol and then treated with FITC, there was almost no labeling of lysine 338. The modification of lysine 338 by FITC was not inhibited by 22(R)-hydroxycholesterol, the first intermediate in the side chain cleavage reaction which binds to the active site 300 times more tightly than cholesterol itself. These experiments suggest that lysine 338 is located at the binding site for adrenodoxin and electrostatically interacts with one of the carboxylate groups on adrenodoxin that has been implicated in binding. The fluorescence emission of the FITC label on cytochrome P-450scc was only 14% as large as that of an equivalent concentration of FITC-labeled bovine serum albumin, suggesting that it was quenched by Forster energy transfer to the heme group.     

Cytochrome P-450 of adrenal mitochondria. Spin states as detected by difference spectroscopy.

Adrenal mitochondrial cytochrome P-450 which functions in cholesterol side chain cleavage (P-450scc) exhibited type I (lambdamax 385, lambdamin 420 nm) and inverse type I (lambdamin 385, lambdamax 420 nm) difference spectra with several steroids. The magnitude and type of response were dependent on the particular steroid and on the extent to which cholesterol was bound to the cytochrome in the intact mitochondrion. the inverse type I difference spectrum induced by 3beta-hydroxy-pregn-5-ene-20-one (pregnenolone) was dependent on the proportion of high spin cholesterol-cytochrome P-450scc complexes. With rat adrenal mitochondria cholest-5-ene-3beta, 20alpha-diol (20alpha-hydroxycholesterol) invariably induced a smaller inverse type I response and, under conditions where cytochrome P-450scc was nearly free of cholesterol, even produced a small type I response. Two distinct steroid binding sites on cytochrome P-450scc were detected by, respectively, the slow type I response to cholest-5-ene-3beta, 25-diol (25-hydroxycholesterol) and the rapid type I response to a subsequent addition of cholest-5-ene-3beta, 20alpha, 22 R-triol (20alpha, 22R-dihydroxycholesterol). The relative proportions of the spectral responses to these steroids were dependent on the previous extent of adrenal activation by adrenocorticotropic hormone (ACTH), because this stimulatory process altered the combination of mitochondrial cholesterol with cytochrome P-450scc. It is proposed that the two steroid binding sites on cytochrome P-450scc interact with steroids in the following way: site I binds cholesterol, 25-hydroxycholesterol, and 20alpha, 22R-dihydroxycholesterol with formation of a partially high spin cytochrome; site II binds both pregnenolone and 20alpha-OH cholesterol resulting in a low spin cytochrome. Interactions between sites I and II are not competitive, and occupancy of site II ensures a low spin state irrespective of the occupancy of site I. A second mode of interaction by 20alpha, 22R-dihydroxycholesterol stabilizes a high spin cytochrome and is competitive with site II binding by 20alpha-hydroxycholesterol or pregnenolone. Formation of a maximally high spin cytochrome follows occupancy by 20alpha, 22R-dihydroxycholesterol at both sites.     

Amino-steroids as inhibitors and probes of the active site of cytochrome P-450scc. Effects on the enzyme from different sources

A series of analogues of cholesterol, each having a primary amine attached to a shortened side chain, were tested for their effects on cytochrome P-450scc from several different sources. Reconstituted enzyme systems using disrupted mitochondria from bovine adrenal and placenta, adult human adrenal and placenta, neonatal human adrenal, and rat adrenal and testis were used to assay for inhibitory effects on the side chain cleavage of cholesterol to pregnenolone. Two of the derivatives tested, 22-amino-23,24-bisnor-5-cholen-3 beta-ol and 23-amino-24-nor-5-cholen-3 beta-ol, were found to be potent inhibitors of this reaction; the derivatives in which the amine was attached closer to or further from the steroid ring, (20 R and S)-20-amino-5-pregnen-3 beta-ol and 24-amino-5-cholen-3 beta-ol, were much weaker inhibitors. In addition, spectral studies with rat adrenal mitochondria and a soluble preparation of human placental cytochrome P-450scc showed that binding of the 22-amine derivative to the enzyme produces difference spectra characteristic of nitrogen bonding to the heme; this indicates that the heme is positioned close to C-22 in the steroid-enzyme complex. These findings on the relative effectiveness of the amino-steroid inhibitors and the type of complex formed are similar to results obtained with purified bovine adrenocortical cytochrome P-450scc. This establishes that the proximity of the substrate binding site and the heme-iron catalytic site is a feature common to the enzyme from several sources and is therefore likely to be a necessary property of the active site structure.     

NMR studies of cytochrome P-450scc. Effects of steroid binding on water proton access to the active site of the ferric enzyme

Water proton relaxation rates of various complexes of cholesterol side chain cleavage cytochrome P-450 (-450scc) were investigated to gain information about the structure and dynamics of the steroid binding site. In all cases bulk water protons were found to be in rapid exchange with protons near the paramagnetic Fe3+ center, and the long electron spin relaxation time of the heme iron, tau s approximately 0.3 ns, resulted in fast relaxation rates. For the steroid-free enzyme, the closest approach of exchangeable protons is approximately 2.5 A, a distance consistent with a water molecule binding directly to the heme iron or rapidly exchanging with a coordinated ligand. When cholesterol was bound, the distance increased to approximately 4 A, indicative of displacement of water from the immediate coordination sphere of the heme but still in close proximity to the active site. For the complex with (22R)-22-hydroxycholesterol, a distance of approximately 2.7 A is observed, suggesting a reorganization of the active site when this intermediate is formed from cholesterol. Complexes of P-450scc with the competitive inhibitors (22R)-22-aminocholesterol, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, or (20R)-20-phenyl-5-pregnene-3 beta,20-diol, also yielded distances of approximately 2.5 A and reveal no effect of side chain size on access of protons to the heme. In the nitrogen-coordinated amino-steroid complexes, the distances observed indicate solvent proton exchange with the heme-bound nitrogen ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

The enzyme activity of bovine adrenocortical cytochrome P-450 producing pregnenolone from cholesterol: Kinetic and electrophoretic studies on the reactivity of hydroxycholesterol intermediates

Electrofocusing of a highly-purified preparation of bovine adrenocortical cytochrome P-450(scc) showed a single peak of enzyme activity at pH 6.8, when either cholesterol, [20S]-20-hydroxycholesterol, [22R]-22-hydroxycholesterol or [20R, 22R]-20, 22-dihydroxycholesterol was used as the substrate for the side chain cleavage reaction. The formation of pregnenolone from these hydroxycholesterols was inhibited by [20R, 22S]-20, 22-epoxycholesterol similarly in a competitive manner and the Ki value for the epoxide was found to be 12–15 μM for all these substrates. When one of the above mentioned substrates was incubated in a concentration sufficient for maximal reaction velocity, the addition of another hydroxycholesterol did not result in further increase of pregnenolone production. These results support the assumption that a single species of enzyme catalyzes all the three steps of the reaction, i.e., 20-hydroxylation, 22-hydroxylation and cleavage of carbon chain between carbon-20 and carbon-22.     

Participation of the membrane in the side chain cleavage of cholesterol. Reconstitution of cytochrome P-450scc into phospholipid vesicles.

Cytochrome P-450scc can be reconstituted into a phospholipid bilayer in the absence of added detergent by incubation of purified hemoprotein with preformed phosphatidylcholine vesicles. Salt effects demonstrate that the primary interaction between the cytochrome and phospholipid vesicles is hydrophobic rather than ionic; in contrast, neither adrenodoxin reductase nor adrenodoxin will bind to phosphatidylcholine vesicles by hydrophobic interactions. Insertion of cytochrome P-450scc into a phospholipid bilayer results in conversion of the optical spectrum to a low spin type, but this transition is markedly diminished if cholesterol is incorporated within the bilayer. Vesicle-reconstituted cytochrome P-450scc metabolizes cholesterol within the bilayer (turnover = 13 nmol/min/nmol of cytochrome P-450scc); virtually all (greater than 94%) of the cholesterol within the vesicle is accessible to the enzyme. "Dilution" of cholesterol within the bilayer by increasing the phospholipid/cholesterol ratio at a constant amount of cholesterol and cytochrome P-450scc results in a decreased rate of side chain cleavage, and cytochrome P-450scc incorporated into a cholesterol-free vesicle cannot metabolize cholesterol within a separate vesicle. In addition, activity of the reconstituted hemoprotein is sensitive to the fatty acid composition of the phospholipid. These results indicate that the cholesterol binding site on vesicle-reconstituted cytochrome P-450scc is in communication with the hydrophobic bilayer of the membrane. The reducibility of vesicle-reconstituted cytochrome P-450scc as well as spectrophotometric and activity titration experiments show that all of the reconstituted cytochrome P-450scc molecules possess an adrenodoxin binding site which is accessible from the exterior of the vesicle. Activity titrations with adrenodoxin reductase also demonstrate that a ternary or quaternary complex among adrenodoxin reductase, adrenodoxin, and cytochrome P-450scc is not required for catalysis, a finding consistent with our proposed mechanism of steroidogenic electron transport in which adrenodoxin acts as a mobile electron shuttle between adrenodoxin reductase and cytochrome P-450 (Lambeth, J.D., Seybert, D.W., and Kamin, H. (1979) J. Biol. Chem. 254, 7255-7264.     

Active site-directed inhibitors of cytochrome P-450scc. Structural and mechanistic implications of a side chain-substituted series of amino-steroids

A series of analogues of cholesterol, each having a shortened side chain and a primary amine group, were prepared and tested for their effects on bovine adrenocortical cholesterol side chain cleavage cytochrome P-450 (P-450scc). A previous study had shown that one derivative, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, is a potent competitive inhibitor of the enzyme and forms a complex in which the steroid ring binds to the cholesterol site and the side chain amine forms a bond with the heme iron (Sheets, J. J., and Vickery, L. E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5773-5777). In the studies reported here, the 23-amine derivative, 23-amino-24-nor-5-cholen-3 beta-ol, was found to be an equally potent inhibitor and to be competitive with respect to cholesterol (Ki = 38 nM). Binding of the 23-amine to P-450scc also caused formation of a low spin complex with an absorption maximum at 422 nm, indicative of a nitrogen-donor ligand. Other derivatives in which the side chain amine was linked closer to the steroid, 17 beta-amino-5-androsten-3 beta-ol and (20 R + S)-20-amino-5-pregnen-3 beta-ol, were found to be only very weak inhibitors (I50 greater than 100 microM) and did not produce the 422 nm spectral form when bound. Derivatives in which the amine was attached a greater distance from the steroid ring, 24-amino-5-cholen-3 beta-ol and 25-amino-26,27-bisnor-5-cholesten-3 beta-ol, caused a progressive decrease in inhibitory potency and a failure to produce the 422 nm form on binding. The dependence of the type of interaction of these amino-steroids with P-450scc upon the amine position establishes that the steroid binding site and the heme catalytic site of the enzyme are fixed within a specific distance of one another. The heme appears to be located sufficiently close to the position that the side chain of cholesterol would occupy to allow for direct attack of an iron-bound oxidant to occur during hydroxylation and side chain cleavage.     

Polyphosphoinositide activation of cholesterol side chain cleavage with purified cytochrome P-450scc

Highly purified beef adrenal cytochrome P-450 specific for cholesterol side chain cleavage (P-450-scc) has been reconstituted with sonicated vesicles containing cholesterol and either dimyristoyl phosphatidylcholine (DMPC) or dioleoyl phosphatidylcholine (DOPC). When cholesterol was present in DMPC vesicles at 1:15 molar ratio, cardiolipin and L-alpha-phosphatidylinositol 4-monophosphate (DPI) increased side chain cleavage by at least 5-fold (0.7 min-1-3.5 min-1). In DOPC vesicles, a smaller increase was observed (2.8 min-1-5.0 min-1). Activator phospholipids increased the rate of transference of cholesterol both to and from the cytochrome when, respectively, cholesterol-free P-450scc and cholesterol-P-450scc complex are combined with either DMPC or DOPC vesicles. Transfer of cholesterol to and from cytochrome P-450 occurred with similar first order rate constants and was also independent of the concentrations of cholesterol vesicles and P-450. It is suggested that transfer in both directions is limited by the rate of insertion of P-450scc into the membrane. Phospholipid stimulatory effects for both cholesterol transfer and for activation of side chain cleavage occurred with the same ranking, even though cholesterol transfer, following reconstitution, was 5-10 times slower than the turnover of side chain cleavage. DPI increased Vmax for side chain cleavage in both DMPC and DOPC vesicles to the same rate (12 min-1) without effect on the Km for cholesterol, while cardiolipin both produced a similar increase in Vmax and decreased Km (cholesterol). This activation by DPI is attributed to more favorable incorporation of P-450scc in these membranes and is consistent with previously reported effects of acidic phospholipids on other mitochondrial proteins.     

Evidence for the presence of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin in fresh granulosa cells. Effects of follicle-stimulating hormone and cyclic AMP on cholesterol side chain cleavage cytochrome P-450 synthesis and activity

The synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and adrenodoxin was studied both in freshly harvested bovine granulosa cells and in granulosa cells maintained in primary monolayer culture. In addition, the action of follicle-stimulating hormone (FSH) and cyclic AMP analogs to stimulate the synthesis of cytochrome P-450scc was investigated in cultured cells. Precursor forms of cytochrome P-450scc and adrenodoxin were immunoisolated from a cell-free translation system directed by RNA prepared from freshly obtained granulosa cells that were not luteinized. Furthermore, the presence of cytochrome P-450scc in lysates of granulosa cells freshly obtained from very small follicles (containing less than 0.1 ml of follicular fluid) and in mitochondria of freshly obtained granulosa cells was demonstrated by using an immunoblotting technique. Continuous treatment of cultured granulosa cells with FSH or with cyclic AMP analogs (dibutyryl cyclic AMP or 8-bromo cyclic AMP) for 72 h increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc. Moreover, FSH, dibutyryl cyclic AMP, and 8-bromo cyclic AMP stimulated pregnenolone production by cultured granulosa cells (2.3-, 4.0-, and 7.5-fold increase over control, respectively), indicative of an increase in cholesterol side chain cleavage activity. The results of this study demonstrate for the first time the presence of two components of the cholesterol side chain cleavage system in freshly obtained granulosa cells, and provide direct evidence for the trophic effect of FSH and its presumed mediator, cyclic AMP, on the synthesis of cytochrome P-450scc in granulosa cells.     

Interaction of a spin-labelled cholesterol derivative with the cytochrome P-450scc active site

The cholesterol analogue 25-doxyl-27-nor-cholesterol (CNO), was found to be a substrate for cytochrome P-450scc. Upon incubation with the cytochrome P-450scc electron transfer system, CNO is transformed to pregnenolone (Km = 33 microM, Vmax = 0.32 min-1). The pregnenolone formation from endogenous cholesterol is strongly inhibited by CNO (50% at 5 microM). It binds tightly to cytochrome P-450scc as evidenced by a reversed type I spectral absorbance change (Kd = 5.9 microM) which is paralleled by a greater hyperfine splitting of the room-temperature CNO ESR spectrum due to an enhanced probe immobilization (Kd = 1.9 microM). This finding is in accord with a rotational correlation time of about 10(-7) s, which is close to the tumbling rate of the protein. At 110 K the CNO-bound cytochrome P-450scc displays the ESR g-values gx = 2.404/2.456, gy = 2.245 and gz = 1.916; these are different from those of cholesterol-liganded cytochrome P-450scc and may thus serve as a marker for cytochrome P-450scc. Our data indicate that the stereospecificity of the cytochrome P-450scc side-chain-cleaving activity is not dependent on the nature of the cholesterol side-chain termination (C25 to C27). The substrate binding site is however rather sensitive to a modification of the side chain. The doxyl ring confers a stronger affinity of the substrate to the enzyme. Upon binding it becomes embedded in the protein matrix, and we estimate that its final position is 0.6-1.0 nm from the heme moiety.     

Catalytic properties of cytochrome P-450scc from bovine and porcine adrenocortical mitochondria: effect of Tween20 concentration

Cholesterol side-chain cleavage activities of cytochrome P-450ssc purified from bovine adrenocortical mitochondria were measured for various substrates, including cholesterol, 20[S]-hydroxycholesterol, 22[R]-hydroxycholesterol and 20[R], [R]-dihydroxycholesterol, in the reconstituted enzyme system at various Tween20 concentrations. The side-chain cleavage activity for cholesterol showed more than 10-fold enhancement upon addition of 0.1% Tween20, compared with that without the detergent. Addition of Tween20 did not cause any enhancement of the side-chain cleavage activities for 20[S]-hydroxycholesterol and 22[R]-hydroxycholesterol; rather, it resulted in an inhibition of the activities. The side-chain cleavage activity for 20[R],22[R]-dihydroxycholesterol showed a very high value even without the detergent. As the stimulatory effect of Tween20 was only specific for cholesterol, Tween20 seemed to enhance the rate of access of cholesterol to cytochrome P-450scc. These results are consistent with the suggestion that a transfer of substrate, cholesterol, in mitochondrial inner membrane, to the substrate-binding site of cytochrome P-450scc is the rate-limiting step in the cholesterol side-chain cleavage reaction.     

Electronic and stereochemical characterizations of intermediates in the photolysis of ferric cytochrome P450scc nitrosyl complexes. Effects of cholesterol and its analogues on ligand binding structures.

Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric cytochrome P450scc was examined by EPR spectroscopy to elucidate the stereochemical interaction between heme-bound ligand and side-chain of cholesterol or its hydroxylated analogues at the substrate-binding site. The photoproducts of the NO complexes trapped at 5 K exhibited new EPR absorptions providing information on the steric crowding of the distal heme moiety. Without substrate, the photoproduct exhibited a broad EPR absorption at g-8 due to magnetic dipole-dipole interaction between the photo-dissociated NO (S = 1/2) and the ferric iron (S = 5/2). This indicates that the photo-dissociated NO can move far away from the heme iron in the less restricted distal heme moiety of the substrate-free cytochrome P450scc. In the presence of substrates, such as cholesterol, 20(S)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 25-hydroxycholesterol, the EPR spectra of the photoproducts exhibited many variations having broad g-8 absorptions and/or the widespread signals together with zero-field absorption. Among the steroid complexes used, 20(S)-hydroxycholesterol complex exhibited a conspicuously widespread EPR signal with a distinct zero-field absorption due to a spin-coupled interaction between the ferric iron (S = 5/2) and the photolyzed NO (S = 1/2). These results indicate that the 20(S)-hydroxycholesterol complex has restricted substrate-binding structure and that the hydroxylation of the cholesterol side-chain at the 22R position is necessary to proceed the side-chain cleavage reaction properly in cytochrome P450scc.     

Active site of bovine adrenocortical cytochrome P-450(11) beta studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450scc

Cytochrome P-45011 beta was purified as the 11-deoxycorticosterone-bound form from bovine adrenocortical mitochondria and its active site was investigated by resonance Raman and EPR spectroscopies. Resonance Raman spectra of the purified sample revealed that the heme iron adopts the pure pentacoordinated ferric high-spin state on the basis of the nu 10 (1629cm-1) and nu 3 (1490 cm-1) mode frequencies, which are higher than those of the hexacoordinated ferric high-spin cytochrome P-450scc-substrate complexes. In the ferrous-CO state, a Fe2(+)-CO stretching mode was identified at 481.5 cm-1 on the basis of an isotopic substitution technique; this frequency is very close to that of cytochrome P-450scc in the cholesterol-complexed state (483 cm-1). The EPR spectra of the purified sample at 4.2 K showed ferric high-spin signals (at g = 7.98, 3.65, and 1.71) that were clearly distinct from the cytochrome P-450scc ferric high-spin signals (g = 8.06, 3.55, and 1.68) and confirmed previous assignments of ferric high-spin signals in adrenocortical mitochondria. The EPR spectra of the nitric oxide (NO) complex of ferrous cytochrome P-45011 beta showed EPR signals with rhombic symmetry (gx = 2.068, gz = 2.001, and gy = 1.961) very similar to those of the ferrous cytochrome P-450scc-NO complex in the presence of 22(S)-hydroxycholesterol and 20(R),22-(R)-dihydroxycholesterol at 77 K.(ABSTRACT TRUNCATED AT 250 WORDS)