The oxyferro complex of adrenal cytochrome P-450scc. Effect of cholesterol and intermediates on its stability and optical characteristics.

The binding of O2 to reduced cytochrome P-450 is the initial step in the activation of oxygen; subsequent addition of a second electron leads to substrate hydroxylation. Without the second electron, the complex between reduced cytochrome and O2 (oxyferro) undergoes internal electron transfer to regenerate the oxidized enzyme and, presumably, superoxide. We have used 38% ethylene glycol and subzero temperatures to stabilize the oxyferro complex of cytochrome P-450scc and have examined the effect of cholesterol, and hydroxycholesterol intermediates in the conversion of cholesterol to pregnenolone, on the complex. The binding of cholesterol or the intermediates 20 alpha-hydroxycholesterol, 22R-hydroxycholesterol, and 20 alpha, 22R-dihydroxycholesterol to the cytochrome perturbed the optical spectra of the oxyferro complex with Soret maximums varying from 416 to 423 nm. Activation energies for the autooxidation of each of these sterol-oxyferro complexes were similar (approximately 22 kcal/mol). The half-time for autooxidation of the oxyferro complex was increased 15-fold by cholesterol over substrate-free cytochrome, and the hydroxycholesterols caused a further 3-17-fold increase in the stability of the oxyferro complex over that observed for cholesterol, the stability increasing with the number of hydroxyl groups on the cholesterol side chain. This was observed in both 38% ethylene glyco at -17 degrees C and dioleoyl phosphatidylcholine vesicles at 2 degrees C. The data indicate that the 1-electron-reduced-oxygenated complex of cytochrome P-450scc is kinetically stabilized by the binding of the reaction intermediates, preserving the complex for the arrival of the second electron.

The binding of O2 to reduced cytochrome P-450 is the initial step in the activation of oxygen; subsequent addition of a second electron leads to substrate hydroxylation. Without the second electron, the complex between reduced cytochrome and O2 (oxyferro) undergoes internal electron transfer to regenerate the oxidized enzyme and, presumably, superoxide. We have used 38% ethylene glycol and subzero temperatures to stabilize the oxyferro complex of cytochrome P-450,,, and have examined the effect of cholesterol, and hydroxycholesterol intermediates in the conversion of cholesterol to pregnenolone, on the complex. The binding of cholesterol or the intermediates 20a-hydroxychalesterol, 22?2-hydroxycholesterol, and 20cu,22R-dihydroxycholesterol to the cytochrome perturbed the optical spectra of the oxyferro complex with Soret maximums varying from 416 to 423 nm. Activation energies for the autooxidation of each of these sterol-oxyferro complexes were similar (approximately 22 kcal/mol). The half-time for autooxidation of the oxyferro complex was increased 15-fold by cholesterol over substratefree cytochrome, and the hydroxycholesterols caused a further 3-17-fold increase in the stability of the oxyferro compIex over that observed for cholesterol, the stability increasing with the number of hydroxyl groups on the cholesterol side chain. This was observed in both 388 ethylene glyco at -17 "C and dioleoyl phosphatidylcholine vesicles at 2 "C. The data indicate that the 1-electron-reduced-oxygenated complex of cytochrome P-450,,, is kinetically stabilized by the binding of the reaction intermediates, preserving the complex for the arrival of the second electron.
Cytochrome P-450,,, from the mitochondria of the adrenal cortex catalyzes the three hydroxylations necessary for the cleavage of the side chain of cholesterol to produce pregnenolone (1-3). The reaction occurs in the inner mitochondrial membrane (4, 5), is rate limiting in the synthesis of the glucocorticoids, and is regulated by adrenocorticotropin (6-8).
The first hydroxylation occurs in the 22R position, the second in the 20a position, while the third results in cleavage of the C2O-C22 bond (2, 9, 10). Initial hydroxylation in the 2Oa position rather than the 22R position may contribute a minor pathway in the conversion of cholesterol to pregnenolone (9, 11). We have previously shown, using phosphatidylcholine reconstituted cytochrome P-450,,, that the hydroxycholes-teroI intermediates bind 100-300 times tighter than choles-terol to the cytochrome (12). We have also shown, using these intermediates, that below 37 "C the first hydroxylation in the 22 position is rate limiting while above 37 "C each hydroxylation occurs at approximately the same rate (12).
In the present study, we have used low temperature to stabilize the 02-bound form of reduced cytochrome P-450,,,. We have examined the effects of substrate and hydroxycholesterol intermediates on the spectral characteristics of the oxyferro complex and present spectra of the substrate and intermediate-bound forms of the complex. Results on the autooxidation rate of the oxyferro complex indicate that the 1-electron-reduced-oxygenated form of cytochrome P-450,,, is kinetically stabilized by complexation with the hydroxycholesterols, while waiting for the second electron to complete the hydroxylation.

EXPERIMENTAL PROCEDURES
Materials-Cholesterol and 22-ketocholesterol were purchased from Sigma Chemical Co. 20a,22R-Dihydroxycholesterol was the generous gift of Dr. Enrico Forcieili of Syntex Research. Analytical reagent grade ethylene glycol was from Mallinckrodt. The sources of other materials used in this study have been previously reported (12).
Purification of Cytochrome P-450,,,-Cytochrome P-450,, was purified from bovine adrenal cortex mitochondria by cholate extraction, ammonium sulfate fractionation, and hexyl agarose chromatography as described previously (24). The purified cytochrome was stored under liquid N2 and retained essentially all its activity after thawing. Using an extinction coeffkient of 91 mM" cm" for A4:* minus A490 in the reduced-CO minus reduced difference spectrum (25,26), the extinction coefficient of dithionite-reduced cytochrome P-450,, at 411 nM was determined to be 103 f 1 (mean 2 S.D., n = 4) mM" cm". This value was used to calculate the concentration of the cytochrome used in the formation of the oxyferro complex.
Depletion of Substrate from the Cytochrome-Cholesterol associated with the purified cytochrome was removed by incubation of the cytochrome under conditions where catalytic conversion of the cholesterol to pregnenolone could occur. A previously described proce-

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The Oxyferro Complex of Adrenal Cytochrome P-45OS,, dure (27) was modified as follows. The incubation of the purified cytochrome (36 p~) , adrenodoxin (8 p~) , adrenodoxin reductase (0.5 pM), NADP' (50 pM), glucose 6-phosphate (2 mM), and glucose-6phosphate dehydrogenase (2 units/ml) was carried out overnight a t room temperature in 20 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.0, 100 mM NaC1, 0.1 mM dithiothreitol, 0.1 mM EDTA. Glycerol was replaced by ethylene glycol (20% by volume) in the subsequent separation of the cytochrome from the other components by hexyl agarose chromatography. The spectrum of the resulting cytochrome P-450,,, was predominantly low spin with the Soret maximum a t 416 nm. A shoulder at 492 nm (see "Results") suggested that a small amount of cholesterol remained associated with the cytochrome. No cytochrome P-420 absorbance was apparent in the reduced-CO minus reduced difference spectra of the substrate-depleted cytochrome.
Formation. of Oxyferro Complex-The instability of the complex between reduced cytochrome and 0, necessitated that most determinations be done below 0 "C. Ethylene glycol (38% by volume) was therefore added to the buffer (100 mM phosphate, pH 7.2, 0.1 mM dithiothreitol, 0.1 mM EDTA) to prevent freezing. The pH of such solvent mixtures has been reported to vary only slightly with temperature from the pH of the initial aqueous buffer (29). Hydroxycholesterols were added to the cytochrome in 10 pl of ethanol to give a final sterol concentration of 13 p~. Solutions of cytochrome P-450,,, (3-5 p~) in buffer (plus ethylene glycol as required) were made anaerobic by repeated evacuation and flushing with 02-free argon gas. The cytochrome was reduced a t room temperature (22-25 "C) by adding sodium dithionite to a final concentration of 74 p~, and then cooled to the required temperature in the spectrophotometer cuvette holder. The temperature of the cuvette holder was contolled by circulation of alcohol from an American Instruments alcohol bath. The lowest stable temperature obtainable with this apparatus was -17 "C. A gentle stream of Ni gas through the cuvette compartment prevented condensation from occurring. The oxyferro complex of the cytochrome was formed by bubbling with 10-15 ml of cold air for 5 s. The addition of more air did not increase the amount of Fez+. O2 complex formed, indicating that saturation of the cytochrome with 0 2 was obtained under the conditions employed.
Recording a n d Analysis of Spectra-Spectra were recorded on a Varian 219 spectrophotometer interfaced to a MINC-11 Digital computer. The autooxidation of the Fe2+. 0 2 complex to the oxidized cytochrome was followed by repetitive scanning (see Fig. 1). The rate of decay was found to be independent of the wavelength used in the analysis and was routinely determined at the wavelength giving the maximum absorbance change (430-440 nm, depending on the sterol bound to the cytochrome). The MINC-11 computer was used to average spectra, calculate difference spectra, and correct spectra for any dilutions as required. It was also used to correct spectra of the Fe'+.O, complex recorded a t some time after its formation, to zero time, using the first order rate constant for the autooxidation established in experiments such as Fig. 2. Spectra from the computer were plotted using a Hiplot digital plotter from Houston Instruments.

RESULTS
Effect of Ethylene Glycol on the Spectra of Cytochrome P-450,v,,-Purified cytochrome P-45OS,, in aqueous solution is predominantly in the high spin form due to the binding of its substrate, cholesterol, which co-purifies with the cytochrome (11, 24, 30, 31). The addition of 38% ethylene glycol to the cytochrome a t room temperature caused conversion of the cytochrome to the low spin form. This conversion was slowly reversed on cooling the cytochrome to generate the high spin (cholesterol-bound) form and was usually complete within 3 h at -17 "C. Subsequent warming of the sample to room temperature resulted in regeneration of the low spin form, indicating the transition is reversible.
The Oxyferro Complex of Substrate-bound Cytochrome P-450,,,-The addition of O2 to reduced cytochrome P-450,,, at -17 "C resulted in the formation of a new spectral form of the cytochrome with absorption maxima at 423 and 553 nm (Fig.  1). The spectral characteristics of this species are similar to those reported for the oxyferro complex of cytochrome P-450,,, (161, cytochrome P-450,,, (17, 18), and cytochrome P-4501, (22) formed under similar conditions, indicating that this is indeed the species observed. Also, the addition of CO to the cytochrome-Fe2+. O2 complex resulted in formation of the Fe2+. CO complex, as evidenced by a shift in the Soret absorption peak from 423 to 445 nm.
The oxyferro complex of the cholesterol-bound cytochrome decayed by a first order monophasic process to yield the initial high spin oxidized form of the cytochrome (Fig. l ) , with a half-time of 38 min (Fig. 2). The autooxidation of the complex to yield the oxidized cytochrome (high spin) occurred with clear isosbestic points at 352, 410, 493, 534, and 590 nm (Fig.   1). Reduction of the cytochrome with dithionite after completion of the autooxidation showed that less than 4% of the original absorbance of the reduced cytochrome was lost during the process and no cytochrome P-420 absorbance was observed in the reduced CO minus reduced difference spectrum. The Oxyferro Complex of Substrate-depleted Cytochrome-Substrate associated with the purified cytochrome was removed by overnight metabolic depletion (see "Experimental Procedures"). The computer-corrected spectrum of the oxyferro complex of the substrate-depleted cytochrome, recorded at -17 "C, had absorption maxima a t 420 and 555 nm (Fig. 3). The formation and autooxidation of the Fe2+. O2 complex was accompanied by a loss of 10-1576 of the heme absorbance. The oxidation of Fez+ to Fe3+ was biphasic; the initial rapid phase had a half-time of 2.6 min and represented more than 80% of the reaction (Fig. 4). The half-time for the slow phase (35 min) is close to that observed for cholesterol-bound cytochrome (38-43 min) and may be due to incomplete removal of substrate. The spectrum of the oxidized cytochrome was not completely low spin as judged from the 392 nm absorbance (Fig. 3) which also suggests that some cholesterol may have remained associated with the cytochrome. Analysis of the autooxidation of the Fez+. 0 2 complex of cytochrome for which the metabolic depletion was carried out for 1 h rather than overnight supported the interpretation that the slow phase was due to cholesterol. The spectra designating the spin state of this cytochrome indicated it still had 50-60% of its endogenous cholesterol remaining, and the Fe".02 complex also decayed biphasically at -17 "C (not shown). Here, the initial phase had a half-time of 3.0 min and represented 42% of the reaction while the slow phase had a half-time of 38 min.
The Effect of Binding of Intermediates on the Optical Spectra of the Oxyferro Complex-Since the cholesterol side chain cleavage reaction intermediates 20a-hydroxycholesterol, 22R-hydroxycho1estero1, and 20a,22R-dihydroxycholesterol bind to cytochrome P-450,,,. more tightly than does cholesterol (12), it was not necessary to remove the endogenous cholesterol from the purified cytochrome. At the concentration used (13 p~) , the hydroxycholesterols completely displaced cholesterol from the active site of the cytochrome as judged from the complete conversion of the cytochrome from the high spin form (cholesterol-bound) to the low spin form by 20a-hydroxycholesterol and 22R-hydroxycholesterol, both low spin inducers (31). Like the oxidized enzyme, the Fe2+.02 complex formed from each of the hydroxycholesterol-bound forms of cytochrome P-45OS,, had its The Fe".O, complex was formed by adding O2 (air) to the reduced cytochrome (4.7 p~) a t -17 "C in 38% ethylene glycol and its spectrum was recorded 10 s later a t 5 nm/s. Also shown is the spectrum of the Fez+. O2 complex, computer-corrected to the time of Or addition, for the amount of autooxidation due to the fast phase (see Fig. 4). The spectrum of the oxidized enzyme was recorded 4.5 h after the addition of 0 2 . own individual spectal characteristics (Fig. 5). The Soret absorbance maximum varied from 416 to 423 nm for the absolute spectra (Fig. 5A) and from 430 to 437 nm in the Fez+. Oe minus reduced difference spectra (Fig. 5B). The Fez+. Os complex for 20a,22R-dihydroxycholesterol was most noticeably different from the other sterols, having a clear shoulder (a band) at 580 nm and a charge transfer band at long wavelength (650 nm).
Spectral data for the various forms of the Fe". 0, complex are summarized in Table I. The lowest extinction for the Soret peak of the Fe'+ I O2 complex was for 20~~,22R-dihydroxycholesterol and the highest for cholesterol. Cholesterol also had the highest extinction for Amax minus A550 in the Fe2+.02 minus reduced difference spectrum while the substrate-depleted Fe" -O2 complex had the lowest (Table I). The latter might be due to the 10-15% loss of absorbance observed after completionoftheautooxidation ofthesubstrate-depleted Fe" . O2 complex, which may have occurred before spectra were recorded. The spectral characteristics of the oxyferro complexes did not appear to change with temperature over the range studied.
The Effect of Binding of Intermediates on the Stability of the Oxyferro Complex-The hydroxycholesteroi intermediates of the cholesterol side chain cleavage reaction markedly increased the stability of the Fe"-02 complex (Fig. 6, Table  11). The stability increased with the number of hydroxyl groups on the side chain of cholesterol. The activation energy for the autooxidation was relatively independent of the sterol bound to the cytochrome (Fig. 6) and values are slightly higher than reported for the autooxidation of the Fe2+.02 complex of P-450,,, (18-19 kcal/mol) (18). terol" terol lesterol ' Extinctions were calculated using an extinction of 103 mM" cm" for the reduced cytochrome at 411 nm (see "Experimental Procedures"). Wavelength with maximum absorbance in the oxyferro minus reduced difference spectrum.
"550 nm was chosen for calculation of the difference extinction coefficients since there is little variation in the absorption of the oxyferro complexes at this wavelength (see Fig. 5).
"Spectra were corrected for autooxidation to the time of 0 2 addition.
' Data are mean f deviation of duplicate determinations in 38% ethylene glycol at -17°C.  ethylene glycol were taken from the Arrhenius plots (Fig. 6).
' Mean -t deviation of duplicates.
Half-times for the decay of the Fez+. O2 complexes of cytochrome P-450,,, at -17 and 2 "C are summarized in Table 11. At 2 "C, it was possible to measure the half-times for the autooxidation of the Fe2+.02 complex for the cytochrome incorporated into fluid dioleoyl phosphatidylcholine vesicles, which more closely resemble the natural membrane environment of the cytochrome. In vesicles, increasing the number of hydroxyl groups on the side chain caused a comparable increase in the stability of the Fez+-O2 complex to that observed in 38% ethylene glycol. Half-times were, however, 20-50% lower than in the 38% ethylene glycol. For aqueous, cholesterol-bound cytochrome at 2 "C, a half-time of 1.55 f 0.15 min (mean f deviation of duplicates) was observed which is also lower than in 38% ethylene glycol but similar to the value in vesicles and illustrates the stabilizing influence of the ethylene glycol on the oxyferro complex.
Cardiolipin markedly promotes the binding of cholesterol to cytochrome P-450,,, and appears to bind to the cytochrome itself (12, 32, 33). It does not, however, affect the stability of the Fe".02 complex. The half-time for autooxidation of the Fez+. O2 complex of cholesterol-bound cytochrome in dioleoyl phosphatidylcholine vesicles containing 30% (w/w) cardiolipin, at 2 "C was 1.6 min, the same as in the absence of cardiolipin (Table 11).
Product Formation during Autooxidation-20a,22R-dihydroxycholesterol was used as substrate for cytochrome P-450,, to determine if any of the hydroxylation product (pregnenolone) was formed during autooxidation of the Fez+. 0 2 complex. The pregnenolone in triplicate extractions of the cytochrome with hexane was measured by a radioimmunoassay procedure previously described (24). The purified cytochrome contained 0.0039 +-0.0003 (f S.D.) mol of pregnenolone/mol of cytochrome. After autooxidation of the Fe".02 complex, there were 0.025 ? 0.001 mol of pregnenolone/mol of cytochrome, indicating that with 20a,22R-dihydroxycholesterol as substrate a small (2%) but measurable amount of turnover accompanies the autooxidation of the Fe2+-O:! complex.

DISCUSSION
The use of low temperatures and mixed solvent to stabilii the Fe2+. O2 complex of cytochrome P-450, has enabled us to study the interaction of substrate and intermediates with the complex. While we were limited to a minimum temperature of -17 "C, this was sufficient for analysis of the sterol-bound Fez+ .Or complexes. The Fe" O2 complex of substrate-depleted cytochrome decayed significantly at -17 "C during the time required to record its spectrum but computerized analysis enabled correction of the spectrum for autooxidation to the time of 0 2 addition.
The sterol-bound oxyferro complex of cytochrome P-450,, autooxidized to the oxidized enzyme by a f i s t order monophasic reaction. The Fez+. O2 complex of substrate-depleted cytochrome autooxidized by a first order biphasic process, with the initial phase corresponding to substrate-free enzyme and the slow phase due to remaining cholesterol complex, left from incomplete removal of cholesterol from the cytochrome. This is in contrast to substrate-free cytochrome P-4501, Fez+-O2 complex which autooxidizes in a biphasic manner in the absence of detectable substrate (22,23), but resembles the monophasic decay of the Fe" . 0 2 complex of P-450,,, (17,18).
If all the cholestero1 associated with purified cytochrome P-450,,, were bound to the active site, then only one catalytic cycle would be required for its complete conversion to pregnenolone which should occur rapidly. However, the ratio of cholesterol to purified cytochrome can be expected to be greater than 1 (11,31), indicating that some of the cholesterol must be bound by hydrophobic forces to secondary sites. Equilibration of cholesterol between the active and secondary sites, which might be expected to be slow based on the very low solubility of cholesterol, could therefore be rate limiting in the catalytic removal of choiesterol from the aqueous cytochrome. This would explain why overnight incubation of the cytochrome with the required electron transport components did not result in complete cholesterol removal.
The binding of cholesterol to cytochrome P-450,,, caused a 15-fold increase in the stability of the FeZ+.O2 complex at -17 "C. This is comparable to the 12-fold increase in half-time for the autooxidation of the Fe2+.02 complex of cytochrome P-450,,, at -20 "C in 50% ethylene glycol, when camphor is bound (18). The binding of the hydroxycholesterol intermediates to cytochrome P-450,, caused a further 3-17-fold increase in the stability of the Fe2'.02 complex in the order 20a,22R-dihydroxycholesterol > 22R-hydroxycholesterol > 20a-hydroxycholesterol > cholesterol. This order does not relate to the spin state of the oxidized cytochrome-sterol complex, as cholesterol and 20a,22R-dihydroxycholesterol are high spin inducers while 22R-dihydroxycholesterol and 20ahydroxycholesterol are low spin inducers (31). There is also a lack of correlation with the K d of the sterols which decrease in the order cholesterol > 20a-hydroxycholesterol > 20a,22Rdihydroxycholesterol > 22R-hydroxycholesterol(l2). The stability of the Fez'. O2 complex does, however, correlate with the number of hydroxyl groups on the cholesterol side chain. For the monohydroxycholesterols, greater stability is given by the hydroxyl group in the 22R position (4-5-fold over cholesterol), which is the major intermediate in the conversion of cholesterol to pregnenolone (2, 10) than in the 20a position (3-fold) where hydroxylation appears to be a minor pathway (9, 11). The stabilization appears specific for the hydroxyl group as the Fe2+<O2 complex of 22-ketocholesterol autooxidized at a rate similar to cholesterol (data not shown).
The Soret absorbance maximum of the oxyferro complex varied from 416 to 423 nm in the absolute spectrum and from 430 to 437 nm in the FeZ'.02 minus reduced difference spectrum depending on the sterol (Table I). It is interesting to note that the spectrum of the Fe2+ .02 complex for 20a,22Rdihydroxycholesterol, which has a change transfer band at 650 nm and a clear shoulder at 580 nm, is the similar to that of camphor-free or -bound P-450,., (17,18). In contrast, the spectra for the other sterols, particularly cholesterol, more closely resemble the Fe2+.02 complex of P-450,, (22,23).
Unlike cytochrome P-450,., and P-4501,, cytochrome p-450,,, catalyzes a triple hydroxylation with the product of one hydroxylation being the substrate for the next. We have previously shown that the hydroxycholesterol intermediates bind 100-300 times tighter to the oxidized cytochrome than does cholesterol (12,33). This thermodynamic stabilization of the enzyme-intermediate complexes thus prevents accumulation of free hydroxycholesterol intermediates during turnover. The present study shows that there is an increase in the stability of the Fe2+ .02 complex in the presence of the hydroxycholesterols over that observed for cholesterol. The 1electron-reduced oxygenated cytochrome-intermediate complexes are therefore kinetically stabilized, minimizing autooxidation, while awaiting the arrival of the second electron to complete the hydroxylation steps. The considerably tighter binding of hydroxycholesterol intermediates than cholesterol to the cytochrome and the increasing stability of the oxyferrosterol complexes with successive intermediates lead to a highly directed reaction sequence, with little opportunity for "leakage." Once the initial binding of cholesterol occurs, a binding facilitated by complex formation of cytochrome P-450.,, with adrenodoxin (27), complete hydroxylation becomes virtually inevitable.