Active complex between adrenodoxin reductase and adrenodoxin in the cytochrome P-450scc reduction reaction

In order to elucidate the mechanism of the electron transfer reaction of mitochondrial steroid hydroxylase, the reduction reaction of cytochrome P-450scc (P-450scc) catalyzed by covalently cross-linked complexes between adrenodoxin reductase (AR) and adrenodoxin (AD) was studied. The reduction rate with the covalent AR-AD complex was very slow (0.030 min-1, as the flavin turnover number) compared with the reduction catalyzed by AR and AD (4.6 min-1). When free AD was added to the reaction mixture containing the AR-AD complex, the rate increased about 30 times. The AD dimer [(AD)2], and a complex between AR and the AD dimer [AR-(AD)2] were then prepared. The Vmax for the P-450scc reduction activity of AR with (AD)2 was 50% of that of AR with AD. The Km value for the total concentration of AD in the P-450scc reduction reaction mixture containing AR and (AD)2 was found to be the same as that in the reaction mixture containing AR and AD. P-450scc reduction by AR-(AD)2 was about 5 times faster than that by AR-AD. The addition of free AD to the AR-(AD)2 complex enhanced the P-450scc reduction about 30 times. AR-AD and AR-(AD)2 were able to reduce external AD, cytochrome c, and acetylated cytochrome c.(ABSTRACT TRUNCATED AT 250 WORDS)     

A shunted system of electron transport from NAD(P)H to cholesterol-hydroxylating cytochrome P-450 in adrenal cortex mitochondria

The two main approaches presently used for cytochrome P-450scc modelling are as follows: i) the use of chemical compounds carrying activated oxygen species, e. g., peracids, organic hydroperoxides, iodosobenzene, etc., ii) the use of electrochemical reduction in the presence of redox-active compounds. In the present work, a new model system for simulation of steroidogenic electron transfer is proposed, which reduces cytochrome P-450 scc by NADPH in the absence of adrenodoxin reductase and adrenodoxin. Phenazine methosulfate is used as an electron carrier. More than 95% of cytochrome P-450scc is reduced in a model system. The reduction kinetics is characterized by a lag phase, thus indicating complex formation between cytochrome P-450scc and phenazine methosulfate or formation of intermediate reducing equivalents. NADH may also serve as an electron donor for cytochrome P-450scc. Phenazine methosulfate can reduce microsomal cytochrome P-450 LM2 and b5, but not cytochrome P-450 LM4. Superoxide dismutase does not affect the reduction, thus indicating that O9.- is not involved in the reduction process. The mechanism of hemoprotein reduction and the nature of intermediates which can be formed in the model system is proposed.     

The catalytic cycle of cytochrome P-450scc and intermediates in the conversion of cholesterol to pregnenolone

Cytochrome P-450scc as isolated is a cholesterol-depleted low-spin haemoprotein; addition of cholesterol results in formation of a high-spin complex. Cytochrome P-450scc--cholesterol is a one-electron acceptor on titration with NADPH. Cytochrome P-450scc--cholesterol can be anaerobically reduced to the ferrous state which, on oxygenation, forms an oxygenated cytochrome P-450scc--cholesterol complex. This oxygenated complex in the absence of adrenodoxin autoxidises to ferric cytochrome P-450scc--cholesterol without oxidation of cholesterol. The decay of the oxygenated complex is first-order, k = 9.3 X 10(-3) S-1 at 4 degrees C. The rate of autoxidation is influenced by pH, ionic strength and the chemical nature of bound sterol. The activation energy of autoxidation is 75 kJ mol-1. Addition of equimolar amounts of adrenodoxin to cytochrome P-450scc--cholesterol followed by stoichiometric reduction under anaerobic conditions and subsequent oxygenation, allows single catalytic turnover cycles of cytochrome P-450scc to be observed. This has led to detection of intermediates in the conversion of cholesterol to pregnenolone and a precursor/product sequence of cholesterol----22-hydroxycholesterol----20,22-dihydroxy-cholesterol ----pregnenolone has been established. Addition of oxidised adrenodoxin to oxygenated cytochrome P-450scc--cholesterol results in formation of 22-hydroxycholesterol.     

Inhibition of electron transfer from adrenodoxin to cytochrome P-450scc by chemical modification with pyridoxal 5'-phosphate: identification of adrenodoxin-binding site of cytochrome P-450scc

Covalent modification of cytochrome P-450scc (purified from bovine adrenocortical mitochondria) with pyridoxal 5'-phosphate (PLP) was found to cause inhibition of the electron-accepting ability of this enzyme from its physiological electron donor, adrenodoxin, without conversion to the "P-420" form. Reaction conditions leading to the modification level of 0.82 and 2.85 PLP-Lys residues per cytochrome P-450scc molecule resulted in 60% and 98% inhibition, respectively, of electron-transfer rate from adrenodoxin to cytochrome P-450scc (with beta-NADPH as an electron donor via NADPH-adrenodoxin reductase and with phenyl isocyanide as the exogenous heme ligand of the cytochrome). It was found that covalent PLP modification caused a drastic decrease of cholesterol side-chain cleavage activity when the cholesterol side-chain cleavage enzyme system was reconstituted with native (or PLP-modified) cytochrome P-450scc, adrenodoxin, and NADPH-adrenodoxin reductase. Approximately 60% of the original enzymatic activity of cytochrome P-450scc was protected against inactivation by covalent PLP modification when 20% mole excess adrenodoxin was included during incubation with PLP. Binding affinity of substrate (cholesterol) to cytochrome P-450scc was found to be increased slightly upon covalent modification with PLP by analyzing a substrate-induced spectral change. The interaction of adrenodoxin with cytochrome P-450scc in the absence of substrate (cholesterol) was analyzed by difference absorption spectroscopy with a four-cuvette assembly, and the apparent dissociation constant (Ks) for adrenodoxin binding was found to be increased from 0.38 microM (native) to 33 microM (covalently PLP modified).(ABSTRACT TRUNCATED AT 250 WORDS)     

Adrenal P-450scc catalyzes deoxycorticosterone 6 beta-hydroxylase reaction

Purified bovine P-450scc, the cholesterol side-chain cleaving P-450 in adrenal cortex mitochondria, was found to catalyze a deoxycorticosterone 6 beta-hydroxylase reaction. A turnover number (moles of product formed/min/mol of P-450) of 12 was found similar to that for cholesterol side chain cleavage activity. Conversion was dose-dependent in terms of P-450scc and no reaction took place when any one of the required electron donating components such as NADPH, NADPH-adrenodoxin reductase, or adrenodoxin was omitted. These results confirm and extend earlier observations that 21-hydroxypregnenolone is transformed into both deoxycorticosterone and 6 beta-hydroxydeoxycorticosterone by incubation of adrenal gland slices.     

Conformational change of the heme moiety of the ferrous cytochrome P-450scc-phenyl isocyanide complex upon binding of reduced adrenodoxin

Reduction of cytochrome P-450scc(SF) (SF, substrate free) purified from bovine adrenocortical mitochondria with sodium dithionite (Na2S2O4) or with beta-NADPH mediated by catalytic amounts of adrenodoxin and adrenodoxin reductase in the presence of phenyl isocyanide produced a ferrous cytochrome P-450scc(SF)-phenyl isocyanide complex with Soret absorbance maximum at 455 nm having a shoulder at 425 nm. On the other hand, when a preformed cytochrome P-450scc(SF)-adrenodoxin complex was reduced chemically or enzymatically under the same conditions, the absorbance spectrum showed drastic changes, i.e., an increase in intensity at 425 nm and a concomitant decrease in intensity at 455 nm. Similar spectral changes could be produced by addition of the same amount of reduced adrenodoxin afterward to the ferrous cytochrome P-450scc(SF)-phenyl isocyanide complex. Titration experiments with adrenodoxin showed that (1) a 1:1 stoichiometric saturation of the spectral change was obtained for both the absorbance increase at 425 nm and the absorbance decrease at 455 nm, (2) there was no spectral change in the presence of 0.35 M NaCl, and (3) there was no spectral change for cytochrome P-450scc(SF) whose Lys residue(s) essential to the interaction with adrenodoxin had been covalently modified with PLP. These results suggest that ternary complex formation of ferrous cytochrome P-450scc(SF)-phenyl isocyanide with reduced adrenodoxin caused a conformational change around the ferrous heme moiety. By analysis of temperature and pH dependencies of the spectral change of the ternary complex, it was suggested that this conformational change may reflect the essential step for electron transfer from reduced adrenodoxin to the ferrous-dioxygen complex of cytochrome P-450scc.     

Immunochemical characteristics of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria

Highly specific antibodies against hemeprotein were obtained by immunizing rabbits with a highly purified cholesterol-hydroxylating cytochrome P-450scc from adrenocortical mitochondria. The antibodies do not specifically interact with other components of the adrenocortical electron transport chain, e. g., adrenodoxin reductase and adrenodoxin. Using double immunodiffusion technique (Ouchterlony method), it was shown that the antibodies did not precipitate the microsomal cytochromes P-450 LM2 and LM4, cytochrome b5 and 11 beta-hydroxylating cytochrome P-450 from adrenocortical mitochondria. Antibodies against cytochrome P-450scc inhibited the cholesterol side chain cleavage activity of cytochrome P-450scc in a reconstituted system. Limited proteolysis with trypsin and immunoelectrophoresis in the presence of specific antibodies revealed that antigenic determinants are present of the heme-containing catalytic domain of cytochrome P-450scc (F1) as well as on the domain responsible for the interaction with the phospholipid membrane (F2).     

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.     

Stimulation by endozepine of the side-chain cleavage of cholesterol in a reconstituted enzyme system

Addition of endozepine in nanomolar concentrations to a system for side-chain cleavage reconstituted from highly purified P-450scc and electron carriers (adrenodoxin reductase and adrenodoxin) stimulates the conversion of cholesterol to pregnenolone (side-chain cleavage). This response is concentration and time-dependent and specific to the extent that a second steroidogenic P-450 located in the inner mitochondrial membrane (ie 11 beta-hydroxylase) was not stimulated by endozepine. Homogeneous endozepine prepared from bovine brain, the corresponding genetically engineered peptide and des(glu-ilu)-endozepine isolated from bovine adrenal cortex are all approximately equipotent in this system. Moreover, endozepine accelerates the rate of reduction of P-450scc by NADPH and the electron carriers. The results suggest that endozepine acts directly on P-450 and hence the rate of side-chain cleavage.     

Ferredoxin and cytochrome P-450scc concentrations in granulosa cells of porcine ovaries during follicular cell growth and luteinization

The concentrations of cytochrome P-450scc and ferredoxin, two of the three proteins which comprise the mitochondrial steroidogenic electron transport chain, were measured in granulosa and luteal cells from porcine ovaries by an immunoblot procedure. During the follicular phase of the ovarian cycle the concentration of cytochrome P-450scc increased 5-fold and ferredoxin increased 3-fold. When the large follicles developed into corpora lutea the cytochrome P-450scc concentration increased a further 7-fold while ferredoxin increased only 3-fold. These changes were coincident with an overall 4-fold increase in the concentration of ferredoxin reductase during follicular cell development and luteinization. Analysis of the data revealed that the concentration of ferredoxin, which shuttles electrons from ferredoxin reductase to cytochrome P-450scc, was always adequate to saturate both the reductase and cytochrome P-450scc. This came about from a co-ordinate increase in the concentration of cytochrome P-450scc and the concentration of ferredoxin minus ferredoxin reductase.     

Cross-linking studies of adrenocortical cytochrome P-450scc. Evidence for a covalent complex with adrenodoxin and localization of the adrenodoxin-binding domain

A cleavable cross-linking reagent, dimethyl-3,3'-dithiobispropionimidate, was used to study the molecular organization of adrenocortical cytochrome P-450scc. Extensive cross-linking was found to occur, resulting in the formation of heterologous oligomers up to octamer. The covalently cross-linked complex of adrenocortical cytochrome P-450scc with adrenodoxin has been obtained by using dimethyl-3,3'-dithiobispropionimidate. In the presence of NADPH and adrenodoxin reductase, electron transfer to cytochrome P-450scc occurs in the complex, and, in the presence of cholesterol, the latter effectively oxidizes to pregnenolone. By using covalently immobilized adrenodoxin and heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate, the adrenodoxin-binding site was shown to be located in the heme-containing, catalytic domain of cytochrome P-450scc. The data obtained indicate the existence of two different sites on the adrenodoxin molecule that are responsible for the interaction with adrenodoxin reductase and cytochrome P-450scc. This is consistent with the model mechanism of electron transfer in the organized complex.     

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.     

Chemical modification of steroid-hydroxylating monooxygenases with fluorescein isothiocyanate]

Chemical modifications of cytochrome P-450scc and cytochrome P-450(11) beta with fluorescein-, diiodofluorescein-, eosine- and rhodamine isothiocyanate have been carried out. At a low reagent/protein ratio and neutral pH, a selective chemical modification was known to take place which did not affect the spectral properties of cytochrome P-450scc. Covalent chromatography was found useful to discriminate between covalent modification of cytochrome P-450scc and non-specific binding of FITC with cytochrome P-450scc. Proteolytic modification of cytochrome P-450scc and structural analysis indicate that a lysine residue of the C-terminal sequence of cytochrome P-450scc is accessible to FITC. The residue was shown, by the analysis of the chymotryptic hydrolysate of the fragment F2, to be Lys338. Effect of modification with FITC on the interaction of cytochrome P-450scc with cholesterol or adrenodoxin, on the reduction kinetics and on the conversion of cholesterol to pregnenolone was also studied.     

Cytochrome P-450scc induces vesicle aggregation through a secondary interaction at the adrenodoxin binding sites (in competition with protein exchange

Addition of bovine adrenal cytochrome P-450scc to small unilamellar dioleoylphosphatidylcholine vesicles (DOPC-SUV) produces a complex sequence of interactions, indicating exceptional cytochrome mobility. First, cholesterol transfer from cytochrome to vesicles indicated rapid dissociation of P-450scc oligomers and integration of monomers into the membrane (delta A 390-420 nm; t1/2 = 2 s). After 10-15 s, P-450scc-induced aggregation of the vesicles starts, as indicated by increased turbidity (delta A 448 or 520 nm; complete in 6-8 min). Fluorescence quenching experiments indicate that this aggregation does not lead to measurable vesicle fusion during this period. Aggregation is prevented by mild heat denaturation of P-450scc, by addition of anti-P-450scc IgG, and also by 1:1 complex formation with the electron donor adrenodoxin (ADX). P-450scc, therefore, links two vesicles through two separate domains involved in, respectively, membrane integration (lipophilic) and ADX binding (charged). Although completely bound by DOPC-SUV, as evidenced by Sephadex elution, P-450scc has access within 1 min to cholesterol in secondary SUV. This is indicated by spectral changes (cholesterol complex formation) and by metabolism of secondary vesicle cholesterol. Since cholesterol equilibrates slowly between vesicles (t1/2 = 1-2 h), these changes arise from P-450scc transfer. This transfer was maximally slowed after a 5-min preincubation with primary vesicles, reflecting more extensive integration into the membrane than is necessary for the rapid initial cholesterol transfer to P-450scc. P-450scc transfer probably results from simultaneous interaction of P-450scc with two vesicles that may also initiate aggregation. Weaker integration into primary dimyristoylphosphatidylcholine vesicles facilitates exchange but prevents aggregation. Integration and aggregation are both enhanced by incorporation of 10% phosphatidylinositol into SUV, while exchange is slowed. This mobility of P-450scc is most probably a consequence of the absence of amino-terminal anchoring. P-450scc-induced association of inner mitochondrial membrane segments may contribute to the exceptionally vesiculated structure of adrenal and ovarian mitochondria that parallels increased P-450scc content.     

The use of a specific fluorescence probe to study the interaction of adrenodoxin with adrenodoxin reductase and cytochrome P-450scc

The single free cysteine at residue 95 of bovine adrenodoxin was labeled with the fluorescent reagent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS). The modification had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc, suggesting that the AEDANS group at Cys-95 was not located at the binding site for these molecules. Addition of adrenodoxin reductase, cytochrome P-450scc, or cytochrome c to AEDANS-adrenodoxin was found to quench the fluorescence of the AEDANS in a manner consistent with the formation of 1:1 binary complexes. F?rster energy transfer calculations indicated that the AEDANS label on adrenodoxin was 42 A from the heme group in cytochrome c, 36 A from the FAD group in adrenodoxin reductase, and 58 A from the heme group in cytochrome P-450scc in the respective binary complexes. These studies suggest that the FAD group in adrenodoxin reductase is located close to the binding domain for adrenodoxin but that the heme group in cytochrome P-450scc is deeply buried at least 26 A from the binding domain for adrenodoxin. Modification of all the lysines on adrenodoxin with maleic anhydride had no effect on the interaction with either adrenodoxin reductase or cytochrome P-450scc, suggesting that the lysines are not located at the binding site for either protein. Modification of all the arginine residues with p-hydroxyphenylglyoxal also had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc. These studies are consistent with the proposal that the binding sites on adrenodoxin for adrenodoxin reductase and cytochrome P-450scc overlap, and that adrenodoxin functions as a mobile electron carrier.     

Electron paramagnetic resonance studies of the purified cytochrome P-450scc and P-45011beta from bovine adrenocortical mitochondria.

Electron paramagnetic resonance studies have been carried out on two species of cytochrome P-450 (P-450scc and P-45011beta) purified from bovine adrenocortical mitochondria. The g values of the steroid-bound cytochromes in the high spin form were determined at 4.2 degrees K to be 8.07, 3.60 and 1.70 for P-450scc and 8.00, 3.65 and 1.71 for P-45011beta. The E/D values were estimated to be 0.103 for P-450scc and 0.099 for P-45011beta. Either high spin P-450 was converted into the low spin form by the treatment with an NADPH dependent electron donating system and subsequent gel filtration in order to remove the steroid. The g values of the low spin ferric cytochromes were 2.423, 2.247 and 1.914 for P-450scc and 2.430, 2.251 and 1.919 for P-45011beta at 77 degrees K. The values for magnitude of delta/gamma, magnitude of V/gamma and k were 5.69, 5.21 and 1.11 for P-450scc and 5.94, 5.38 and 1.16 for P-45011beta. These studies indicate that there are some differences in the ferric heme environment between P-450scc and P-45011beta.     

Biophysical and enzymological studies upon the interaction of trans-cinnamic acid with higher plant microsomal cytochromes P-450.

The interaction of trans-cinnamic acid with the cytochrome P-450 of microsomes derived from washed potato slices has been studied. The washing process increased the specific content of microsomal electron transport components and hence provided a useful material in which to study the interaction. Evidence is presented that the trans-cinnamic acid interacts with the cytochrome P-450, and that this interaction is analogous to "type 1" interactions of other cytochrome P-450 systems. This evidence includes the formation of a "type 1" substrate binding spectrum, an increased rate of reduction of cytochrome P-450 by NADPH in the presence of trans-cinnamic acid, an increased oxygen uptake and NADPH oxidation when trans-cinnamic acid is added to the microsomes in the presence of NADPH, and a close correlation between biophysical parameters of electron transport in the cytochrome P-450 system and enzymological parameters of the trans-cinnamic acid 4-hydroxulation reaction. The investigation has been extended to cytochrome P-450 systems of other tissues and it has been found that the trans-cinnamic acid 4-hydroxylation reaction cannot account for the presence of most of th cytochrome P-450 in several tissues. This suggests that other functions of higher plant cytochrome P-450 chains exist, and that the substrate specificityof the hemoprotein may vary in different plant tissues.     

Immunochemical study of cholesterol-hydroxylating cytochrome P-450. Topology of the peptide chain of the heme protein in the phospholipid membrane

The topology of adrenocortical cytochrome P-450scc in inner mitochondrial membrane was studied. To determine the polypeptide chain parts exposed to matrix or cytosol, two approaches were used, i.e. i) limited proteolysis of membrane-bound cytochrome P-450scc followed by the detection of the peptides formed by immunoblotting; ii) binding of monospecific antibodies against cytochrome P-450scc as well as fragments F1 and F2 representing N- and C-terminal sequences of the hemeprotein, to membrane structures (mitoplasts and submitochondrial particles). The data obtained confirm the transmembrane orientation of cytochrome P-450scc molecule, since antibodies against the hemeprotein as well as fragments F1 and F2 were found to be bound both on the matrix and cytosol surfaces of the inner mitochondrial membrane. It was shown that region 250-257 in cytochrome P-450scc connecting domains F1 and F2 is exposed to the matrix. A model of molecular organization of cytochrome P-450scc in inner mitochondrial membranes is proposed.     

Adrenal P-450scc modulates activity of P-45011 beta in liposomal and mitochondrial membranes. Implication of P-450scc in zone specificity of aldosterone biosynthesis in bovine adrenal.

Bovine adrenal P-45011 beta catalyzes the 11 beta- and 18-hydroxylation of corticosteroids as well as aldosterone synthesis. These activities of P-45011 beta were found to be modulated by another mitochondrial cytochrome P-450 species, P-450scc. The presence together of P-45011 beta and P-450scc in liposomal membranes was found to remarkably stimulate the 11 beta-hydroxylase activity of P-45011 beta and also stimulate the cholesterol desmolase activity of P-450scc. The stimulative effect of P-450scc on 11 beta-hydroxylase activity diminished by the addition of protein-free liposomes to proteoliposomes containing P-45011 beta and P-450scc, thus showing P-450scc to interact with P-45011 beta in the same membranes. Kinetic analysis of this effect indicated the formation of an equimolar complex between P-45011 beta and P-450scc on liposomal membranes. P-45011 beta in the complex had not only stimulated activity for 11 beta- and 18-hydroxylation of 11-deoxycorticosterone but also suppressed activity for production of 18-hydroxycorticosterone and aldosterone. When the inner mitochondrial membranes of zona fasciculata-reticularis from bovine adrenal were treated with anti-P-450scc IgG, aldosterone formation was stimulated to a greater extent than that of zona glomerulosa. This indicates the aldosterone synthesizing activity of P-45011 beta in the zona fasciculata-reticularis to be suppressed by interaction with P-450scc. The zone-specific aldosterone synthesis of P-45011 beta in bovine adrenal may possibly be induced by differences in interactions with P-450scc of mitochondrial membranes in each zone.