Cytochrome P-450 of bovine adrenal mitochondria. Ligand binding to two forms resolved by EPR spectroscopy.

The binding of cholest-5-ene-3beta,20alpha-diol (20alpha-hydroxycholesterol), 11-deoxycorticosterone, and aminoglutethimide to cytochrome P-450 in bovine adrenal mitochondria was measured by changes in optical spectra at room temperature and by EPR spectra at 14 K. The two methods provided nearly identical quantitation of these interactions with cytochrome P-450. Two distinct high spin forms of cytochrome P-450 were revealed by EPR spectra. The predominant high spin species (g = 8.2) was decreased by addition of 20alpha-hydroxycholesterol and elevated pH but was increased by addition of cholesterol. The minor high spin species (g = 8.1) was incrreased by addition of deoxycorticosterone but decreased by low concentrations of metyrapone. The two forms were evidently not in equilibrium and have been assigned to distinct forms of cytochrome P-450 involved in, respectively, cholesterol side chain cleavage (P-450scc) and steroid 11beta hydroxylation (P-450(11)beta). The high spin states are derived from complexes of these P-450 cytochromes with endogenous substrates, which are, respectively, cholesterol and deoxycorticoids. A high to low spin transition was observed when these complexes were turned over by initiating hydroxylation with malate. The contributions of cytochromes P-450(11)beta and P-450scc to the low spin spectrum were also resolved by similar means. At least 20% of P-450scc is in the low spin state while about 90% of P-450(11)beta is low spin in isolated beef adrenal mitochondria. Low spin complexes of cytochrome P-450scc with 20alpha-hydroxycholesterol and 3beta-hydroxypregn-5-ene-20-one (pregnenolone) gave distinct EPR spectra. Aminoglutethimide interacted with the total cytochrome P-450 content of the bovine adrenal mitochondria forming low spin complexes. Both optical and EPR data indicated binding to two forms of cytochrome P-450. These results suggest a detailed correlation between the spin state and absorbance changes seen at room temperature, illustrate that EPR allows the distinction of two principal forms of P-450, and suggest that there is no appreciable change in the spin state of either cytochrome between 14 K and 300 K.     

Role of Positively Charged Residues Lys267, Lys270, and Arg411 of Cytochrome P450scc (Cyp11A1) in Interaction with Adrenodoxin

Cytochrome P450scc and adrenodoxin are redox proteins of the electron transfer chain of the inner mitochondrial membrane steroid hydroxylases. In the present work site-directed mutagenesis of the charged residues of cytochrome P450scc and adrenodoxin, which might be involved in interaction, was used to study the nature of electrostatic contacts between the hemeprotein and the ferredoxin. The target residues for mutagenesis were selected based on the theoretical model of cytochrome P450scc-adrenodoxin complex and previously reported chemical modification studies of cytochrome P450scc. In the present work, to clarify the molecular mechanism of hemeprotein interaction with ferredoxin, we constructed cytochrome P450scc Lys267, Lys270, and Arg411 mutants and Glu47 mutant of adrenodoxin and analyzed their possible role in electrostatic interaction and the role of these residues in the functional activity of the proteins. Charge neutralization at positions Lys267 or Lys270 of cytochrome P450scc causes no significant effect on the physicochemical and functional properties of cytochrome P450scc. However, cytochrome P450scc mutant Arg411Gln was found to exhibit decreased binding affinity to adrenodoxin and lower activity in the cholesterol side chain cleavage reaction. Studies of the functional properties of Glu47Gln and Glu47Arg adrenodoxin mutants indicate that a negatively charged residue in the loop covering the Fe2S2 cluster, being important for maintenance of the correct architecture of these structural elements of ferredoxin, is not directly involved in electrostatic interaction with cytochrome P450scc. Moreover, our results indicate the presence of at least two different binding (contact) sites on the proximal surface of cytochrome P450scc with different electrostatic input to interaction with adrenodoxin. In the binary complex, the positively charged sites of the proximal surface of cytochrome P450scc well correspond to the two negatively charged sites of adrenodoxin: the "interaction" domain site and the "core" domain site.     

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

The effects of cholesterol and adrenodoxin binding on resonance Raman spectra of cytochrome P-450scc in both oxidized and CO-reduced states were examined. Upon cholesterol binding, oxidized cytochrome P-450scc showed a significant shift of spin equilibrium from low-spin to high-spin state. Addition of adrenodoxin caused a complete conversion of cholesterol-bound oxidized cytochrome P-450scc to a pure high-spin state that was considered to be in the hexacoordinated state judged by the v10 mode at 1620 cm-1 and v3 mode around 1485 cm-1. Cholesterol in substrate binding site may oppose a linear and perpendicular binding of carbon monoxide to the reduced heme iron, leading to the distorted Fe-C-O linkage. This is based on the following observations: (1) an increase of the Fe-CO stretching frequency to 483 from 477 cm-1 upon addition of cholesterol; (2) an enhanced photodissociability of bound carbon monoxide of CO complex of cytochrome P-450scc in the presence of cholesterol. As another aspect of the effect of cholesterol on the CO complex form of cytochrome P-450scc, the enhanced stability of the native form ("P-450" form) was observed. There was no additional effect of reduced adrenodoxin on the Raman spectra of the CO-reduced form of cytochrome P-450scc.     

Antagonistic effects of hydrostatic pressure and osmotic pressure on cytochrome P-450cam spin transition.

The combined effects of hydrostatic pressure and osmotic pressure, generated by polyols, on the spin equilibrium of fenchone-bound cytochrome P-450cam were investigated. Hydrostatic pressure indices a high spin to low spin transition, whereas polyols induce the reversed reaction. Of the four solutes used, glycerol, glucose, stachyose, and sucrose, only the last two would act on the spin transition by osmotic stress. The spin volume changes measured by both techniques are different, 29 and -350 ml/mol for hydrostatic pressure and osmotic pressure, respectively. It suggests that even if the two are perturbing water molecules, different properties are probed. From the volume change induced by osmotic stress, 19 water molecules are deduced that would be implicated in the spin transition of the fenchone-bound protein. This result suggests that water molecules other than the well defined ones located in the active site play a key role in modulating the spin equilibrium of cytochrome P-450cam.     

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.     

Placental cytochrome P450scc (CYP11A1): comparison of catalytic properties between conditions of limiting and saturating adrenodoxin reductase

The mitochondrial side-chain cleavage of cholesterol, catalysed by cytochrome P450scc, is rate-limiting in the synthesis of progesterone by the human placenta. Cytochrome P450scc activity is in turn limited by the concentration of adrenodoxin reductase (AR) in placental mitochondria. In order to better understand which components of the cholesterol side-chain cleavage system are important in the regulation of placental progesterone synthesis, we have examined their effects on P450scc activity with both saturating and limiting concentrations of AR. The present study reveals that decreasing the AR concentration causes a decrease in the K(m) of cytochrome P450scc for cholesterol, facilitating saturation of the enzyme with its substrate. Decreasing AR resulted in P450scc activity becoming less sensitive to changes in P450scc concentration. The adrenodoxin (Adx) concentration in mitochondria from term placentae is near-saturating for P450scc and under these conditions, we found that decreasing AR reduces the K(m) of P450scc for adrenodoxin. Increasing either the cholesterol or P450scc concentration increased the amount of AR required for P450scc to work at half its maximum velocity. A relatively small increase in AR can support considerably higher rates of side-chain cleavage activity when there is a coordinate increase in AR and P450scc concentrations. We conclude from this study that cholesterol is near-saturating for cytochrome P450scc activity in placental mitochondria due to the P450scc displaying a low K(m) for cholesterol resulting from the low and rate-limiting concentration of AR present. This study reveals that it is unlikely that cholesterol or adrenodoxin concentrations are important regulators of placental progesterone synthesis but AR or coordinate changes in AR and P450scc concentrations are likely to be important in its regulation.     

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.     

Probing the interaction of bovine cytochrome P450scc (CYP11A1) with adrenodoxin: evaluating site-directed mutations by molecular modeling

The present study was undertaken to evaluate the role of positively charged amino acid residues proposed to reside on the proximal surface of bovine cytochrome P450 cholesterol side chain cleavage (P450scc, CYP11A1) and to determine which residues may be involved in protein-protein interactions with the electron carrier adrenodoxin (Adx). In previous studies, nine different lysine residues were identified by chemical and immunological cross-linking experiments as potentially interacting with Adx, while in the present study, two arginine residues have been identified from sequence alignments. From these 11 residues, 13 different P450scc mutants were made of which only seven were able to be expressed and characterized. Each of the seven mutants were evaluated for their ability to bind Adx, to be reduced, and for their enzymatic activity. Among these, K403Q and K405Q showed a consistent decrease in Adx binding, the ability to be reduced by Adx, and enzymatic activity, with K405Q being affected to a much greater extent. More dramatic was the complete loss of Adx binding by R426Q, while still retaining its ability to be chemically reduced and bind carbon monoxide. Independently, a homology model of P450scc was constructed and docked with the structure of Adx. Four potential sites of interaction were identified: P450scc:K403 with Adx:D76, P450scc:K405 with Adx:D72; P450scc:R426 with Adx:E73, and P450scc:K267 with Adx:E47. Thus, the biochemical and molecular modeling studies together support the hypothesis that K267, K403, K405, and R426 participate in the electrostatic interaction of P450scc with Adx.     

Adrenodoxin-cytochrome P450scc interaction as revealed by EPR spectroscopy: comparison with the putidaredoxin-cytochrome P450cam system.

The cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc comprises three consecutive monooxygenase reactions (22R-hydroxylation, 20S-hydroxylation, and C(20)-C(22) bond scission) that produces pregnenolone. The electron equivalents necessary for the oxygen activation are supplied from a 2Fe-2S type ferredoxin, adrenodoxin. We found that 1:1 stoichiometric binding of oxidized adrenodoxin to oxidized cytochrome P450scc complexed with cholesterol or 25-hydroxycholesterol caused shifts of the high-spin EPR signals of the heme moiety at 5 K. Such shifts were not observed for the low-spin EPR signals. Ligation of CO or NO to the reduced heme of cytochrome P450scc complexed with reduced adrenodoxin and various steroid substrates did not cause any change in the axial EPR spectrum of the reduced iron-sulfur center at 77 K. These results are in remarkable contrast to those obtained for the cytochrome P450cam-d-camphor-putidaredoxin ternary complex, suggesting that the mode of cross talk between adrenodoxin and cytochrome P450scc is very different from that in the Pseudomonas system. The difference may be primarily due to the location of the charged amino acid residues of the ferredoxins important for the interaction with the partner cytochrome P450.     

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)     

Lipid regulation of bovine cytochrome P450(11) beta activity

Purified bovine adrenocortical cytochrome P450(11) beta has been reconstituted into phospholipid vesicles using a detergent dialysis procedure. Using this reconstituted system, we have examined the effect of changes in the fatty acyl substituents of the lipids on the catalytic activity of the enzyme. The studies reported here show that cytochrome P450(11) beta exhibits a completely different response to changes in the fatty acyl groups from that shown by cytochrome P450scc. Cytochrome P450(11) beta displays maximal activity in lipid vesicles composed of saturated lipids, such as dipalmitoyl and dimyristoyl phosphatidylcholines, with turnover numbers ranging from 35 to 60 min-1. Incremental increases of phospholipids such as diphytanoyl and dioleoyl phosphatidylcholines result in a progressive inhibition of 11 beta hydroxylase activity; most of this kinetic effect is attributable to a significant decrease in Vmax accompanied by modest changes in Km for the steroid substrate deoxycorticosterone. Diphosphatidyl glycerol (cardiolipin), which has been previously shown to activate cytochrome P450scc, is a potent inhibitor of the 11 beta hydroxylase activity of cytochrome P450(11) beta, with half maximum inhibition observed in vesicles containing 4-5 mol% diphosphatidyl glycerol. Kinetic analysis demonstrates that this inhibition by diphosphatidyl glycerol is reflected in both a decrease in Vmax and relatively large increases (up to sevenfold) in Km for the steroid substrate. These effects on the 11 beta hydroxylase activity may have important implications for the in vivo regulation of not only the 11 beta hydroxylase activity, but also the other catalytic activities of this enzyme, particularly 18- and 19-hydroxylase and oxidase activities.     

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.     

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.     

Oxidized adrenodoxin acts as a competitive inhibitor of cytochrome P450scc in mitochondria from the human placenta.

The conversion of cholesterol to pregnenolone by cytochrome P450scc is the rate-determining step in placental progesterone synthesis. The limiting component for placental cytochrome P450scc activity is the concentration of adrenodoxin reductase in the mitochondria, where it permits cytochrome P450scc to work at only 16% of maximum velocity. Adrenodoxin reductase serves to reduce adrenodoxin as part of the electron transfer from NADPH to cytochrome P450scc. We therefore measured the proportion of adrenodoxin in the reduced form in intact mitochondria from the human placenta during active pregnenolone synthesis, using EPR. We found that the adrenodoxin pool was only 30% reduced, indicating that the adrenodoxin reductase concentration was insufficient to maintain the adrenodoxin in the fully reduced state. As both oxidized and reduced adrenodoxin can bind to cytochrome P450scc we tested the ability of oxidized adrenodoxin to act as a competitive inhibitor of pregnenolone synthesis. This was done in a fully reconstituted system comprising 0.3% Tween 20 and purified proteins, and in a partially reconstituted system comprising submitochondrial particles, purified adrenodoxin and adrenodoxin reductase. We found that oxidized adrenodoxin is an effective competitive inhibitor of placental cytochrome P450scc with a Ki value half that of the Km for reduced adrenodoxin. We conclude that the limiting concentration of adrenodoxin reductase present in placental mitochondria has a two-fold effect on cytochrome P450scc activity. It limits the amount of reduced adrenodoxin that is available to donate electrons to cytochrome P450scc and the oxidized adrenodoxin that remains, competitively inhibits the cytochrome.     

Site-Directed Mutagenesis of Cytochrome P450scc. II. Effect of Replacement of the Arg425 and Arg426 Residues on the Structural and Functional Properties of the Cytochrome P450scc

Cytochrome P450-dependent monooxygenases, in spite of their wide distribution, can be simply divided into a few groups differing in the location of the electron transfer chain and their composition. The two main groups of cytochrome P450-dependent monooxygenases are the mitochondrial and microsomal enzymes. While in two-component microsomal cytochrome P450-dependent monooxygenases electrons are supplied to cytochrome P450 by a flavoprotein (NADPH-cytochrome P450 reductase), in three-component mitochondrial monooxygenases the electrons are supplied to cytochrome P450 by a low molecular weight protein (ferredoxin). The interaction of cytochrome P450 with NADPH-cytochrome P450 reductase and ferredoxin is the subject of intensive studies. Using chemical modification, chemical cross-linking, and sitedirected mutagenesis, we identified surface exposed positively charged residues of cytochrome P450scc which might be important for interaction with adrenodoxin. Theoretical analysis of the distribution of surface electrostatic potential in cytochrome P450 indicates that in contrast to microsomal monooxygenases, cytochromes P450 of mitochondrial type, and cholesterol side-chain cleavage cytochrome P450 (P450scc) in part, carry on the proximal surface an evidently positively charged site that is formed by residues Arg425 and Arg426. In the present work, to estimate the functional role of Arg425 and Arg426 of cytochrome P450scc, we used site-directed mutagenesis to replace these residues with glutamine. The results indicate that residues Arg425 and Arg426 are involved in the formation of a heme-binding center and electrostatic interaction of cytochrome P450scc with its physiological electron-transfer partner, adrenodoxin.     

Effects of phospholipid and detergent on the substrate specificity of adrenal cytochrome P-450scc. Substrate binding and kinetics of cholesterol side chain oxidation

Cytochrome P-450scc was isolated from mitochondria of bovine adrenal cortex by hydrophobic chromatography on octyl Sepharose followed by affinity chromatography on cholesterol-7-(thiomethyl)carboxy-3 beta-acetate-Sepharose. The partially purified eluate from the octyl Sepharose resin was free of adrenodoxin and adrenodoxin reductase and displayed biphasic binding characteristics for cholesterol, cholesterol sulfate, and cholesterol acetate (CA). Chromatography of the octyl Sepharose eluate on CA-Sepharose removed extraneous proteins and resolved the cytochrome P-450scc into two fractions, each of which displayed monophasic binding with all three substrates. These fractions behaved identically with respect to their ability to bind substrates, their kinetic properties, and their rate of migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dissociation constants of the cytochrome P-450scc.substrate complexes are 1.1, 2.6, and 1.3 microM for cholesterol, cholesterol sulfate, and cholesterol acetate, respectively. Addition of phospholipids isolated from adrenal cortex mitochondria or adrenodoxin had no effect on the equilibrium binding constants. Addition of Emulgen 913, however, decreased the binding affinities 10-20-fold. Emulgen 913 also inhibited the interaction of adrenodoxin with the cytochrome. An active side chain cleavage system was reconstituted with purified P-450 by addition of saturating amounts of adrenodoxin, adrenodoxin reductase, and NADPH-generating system. The apparent Km values for this reconstituted system of cholesterol, cholesterol sulfate, and cholesterol acetate are 1.8, 1.9, and 0.6 microM, respectively. Since the Km values of substrate oxidation are similar to the Kd values of the cytochrome P-450.substrate complexes, it seems likely that the binding of substrates, particularly when the side chain cleavage system is free of mitochondrial membranes, is not rate-limiting. Based on these results and electrophoretic data, it appears that one cytochrome P-450 present in adrenal mitochondria can oxidize cholesterol, its sulfate, and its acetate. This enzyme represented about 60% of the cytochrome P-450 present in the octyl Sepharose eluate. The factors responsible for the biphasic kinetics of oxidation by intact mitochondria and biphasic binding of sterol substrates by partially purified preparations of cytochrome P-450scc are still unknown.     

Site-specific mutations in human ferredoxin that affect binding to ferredoxin reductase and cytochrome P450scc

Ferredoxins found in animal mitochondria function in electron transfer from NADPH-dependent ferredoxin reductase (Fd-reductase) to cytochrome P450 enzymes. To identify residues involved in binding of human ferredoxin to its electron transfer partners, neutral amino acids were introduced in a highly conserved acidic region (positions 68-86) by site-directed mutagenesis of the cDNA. Mutant ferredoxins were produced in Escherichia coli, and separate assays were used to determine the effect of substitutions on the capacity of each mutant to bind to Fd-reductase and cytochrome P450scc and to participate in the cholesterol side chain cleavage reaction. Replacements at several positions (mutants D68A, E74Q, and D86A) did not significantly affect activity, suggesting that acidic residues at these positions are not required for binding or electron transfer interactions. In contrast, substitutions at positions 76 and 79 (D76N and D79A) caused dramatic decreases in activity and in the affinity of ferredoxin for both Fd-reductase and P450scc; this suggests that the binding sites on ferredoxin for its redox partners overlap. Other substitutions (mutants D72A, D72N, E73A, E73Q, and D79N), however, caused differential effects on binding to Fd-reductase and P450scc, suggesting that the interaction sites are not identical. We propose a model in which Fd-reductase and P450scc share a requirement for ferredoxin residues Asp-76 and Asp-79 but have other determinants that differ and play an important role in binding. This model is consistent with the hypothesis that ferredoxin functions as a mobile shuttle in steroidogenic electron transfer, and it is considered unlikely that a functional ternary complex is formed.     

Site-Directed Mutagenesis of Cytochrome b5 for Studies of Its Interaction with Cytochrome P450

We have shown earlier that microsomal cytochrome b ₅ can form a specific complex with mitochondrial cytochrome P450 (cytochrome P450scc). The formation of the complex between these two heme proteins was proved spectrophotometrically, by affinity chromatography on immobilized cytochrome b ₅, and by measuring the cholesterol side-chain cleavage activity of cytochrome P450scc in a reconstituted system in the presence of cytochrome b ₅. To further study the mechanism of interaction of these heme proteins and evaluate the role of negatively charged amino acid residues Glu42, Glu48, and Asp65 of cytochrome b ₅, which are located at the site responsible for interaction with electron transfer partners, we used sitedirected mutagenesis to replace residues Glu42 and Glu48 with lysine and residue Asp65 with alanine. The resulting mutant forms of cytochrome b ₅ were expressed in E. coli, and full-length and truncated forms (shortened from the C-terminal sequence due to cleavage of 40 amino acid residues) of these cytochrome b ₅ mutants were purified. Addition of the truncated forms of cytochrome b ₅ (which do not contain the hydrophobic C-terminal sequence responsible for interaction with the membrane) to the reconstituted system containing cytochrome P450scc caused practically no stimulation of catalytic activity, indicating an important role of the hydrophobic fragment of cytochrome b ₅ in its interaction with cytochrome P450scc. However, full-length cytochrome b ₅ and the full-length Glu48Lys and Asp65Ala mutant forms of cytochrome b ₅ stimulated the cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc by 100%, suggesting that residues Glu48 and Asp65 of cytochrome b ₅ are not directly involved in its interaction with cytochrome P450scc. The replacement of Glu42 for lysine, however, made the Glu42Lys mutant form of cytochrome b ₅ about 40% less effective in stimulation of the cholesterol side-chain cleavage activity of cytochrome P450scc, indicating that residue Glu42 of cytochrome b ₅ is involved in electrostatic interactions with cytochrome P450scc. Residues Glu42 and Glu48 of cytochrome b ₅ appear to participate in electrostatic interaction with microsomal type cytochrome P450.     

Specific and non-specific effects of potassium cations on substrate-protein interactions in cytochromes P450cam and P450lin

Substrate binding to cytochrome P450cam is generally considered to be a two-step process. The first step corresponds to the entrance of the substrate, camphor, into the heme pocket. The second step corresponds to a spin transition (low spin-->high spin) of the iron in the protein-substrate complex. This spin transition is related to the mobility of the substrate inside the active site [Biochim Biophys Acta 1338 (1997) 77]. Potassium cations (K(+)) have a specific effect on the spin equilibrium. This is generally attributed to the K(+) ion-induced conformational change of tyrosine 96, the hydroxyl group of which is hydrogen bonded to the keto group of camphor and results in optimum substrate orientation and reduced mobility of this substrate in the active site. In the present paper, we show that K(+) not only affects the substrate-Tyr 96 couple, but acts more globally since K(+) effects are also observed in the Tyr96Phe mutant as well as in complexes with camphor-analogues. Large compounds, that fit well in the heme pocket and bind with higher affinity than camphor, display high spin contents that are less dependent on the presence of K(+). In contrast, K(+) has a significant effect on the high spin content of substrate-cytochrome P450cam complexes with looser interactions. We conclude that large compounds with higher affinities than camphor have more van der Waals contacts with the active site residues. Their mobilities are then reduced and less dependent on the presence of K(+). In this study, we also explored, for comparison, the K(+) effect on the spin transition state of another member of the P450 superfamily, cytochrome P450lin. This effect is not as strong as those observed for cytochrome P450cam. Even though the spin equilibrium does not change dramatically in the presence of K(+) or Na(+), the value of the dissociation constant (K(d)) for linalool binding is significantly affected by ionic strength. Analysis of the thermodynamic parameters for the linalool binding strongly suggests that, similarly to our previous finding for cytochrome P450cam, electrostatic gates participate in the control of substrate access.     

Comparative study of topogenesis of cytochrome P450scc (CYP11A1) and its hybrids with adrenodoxin expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Hybrid proteins consisting of the mature form of cytochrome P450scc (mP) and adrenodoxin (Ad), attached to either the NH2- or COOH-terminus (Ad-mP and mP-Ad, respectively), were expressed in E. coli. Spectral and catalytic properties of P450scc were studied using the membrane fraction of E. coli cells. It has been shown that the Ad amino acid sequence attached to the termini of the P450scc-domain neither affects the insertion of a hybrid protein into the cytoplasmic membrane nor influences its heme binding ability. The results suggest that Ad attached to the NH2-terminus does not markedly affect the folding of the P450scc-domain, but cholesterol hydroxylase/lyase activity of the Ad-mP hybrid was found to be much lower than that of the native P450scc enzyme. The modification of the COOH-terminus does not alter the specific P450scc activity, but results in a dramatic increase in the amount of hybrid protein with incorrectly folded P450scc domain.