A covalently linked complex of cytochrome P-450 with adrenodoxin. Localization of the adrenodoxin binding site of cytochrome P-450

Cytochrome P-450scc and adrenodoxin were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The sample containing 94% of a cross-linked complex and 6% of free cytochrome P-450scc was obtained after purification on cholate-Sepharose. Cytochrome P-450scc in the cross-linked complex is not reduced in the presence of NADPH and adrenodoxin reductase, but completely preserves its high spin form in the presence of Tween-20 or pregnenolone. The use of radioactive labelled adrenodoxin, chemical cleavage of cytochrome P-450scc from the cross-linked complex by o-iodosobenzoic acid and HPLC for separation of peptides demonstrated that the cytochrome P-450scc complex with adrenodoxin was cross-linked through two amino acid sequences of cytochrome P-450scc, i.e., Leu 88-Trp108 and Leu368-Trp417.     

Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with adrenodoxin

Selective chemical modification of cytochrome P-450SCC has been carried out with lysine-modifying reagents. Modification of cytochrome P-450SCC with succinic anhydride was shown to result in loss of its ability to interact with intermediate electron transfer protein - adrenodoxin. To identify amino acid residues involved in charge-ion pairing with complementary carboxyl groups of adrenodoxin, cytochrome P-450SCC complex with adrenodoxin was modified with succinic anhydride. Adrenodoxin was then removed and cytochrome P-450 was additionally modified with isotopically labelled reagent. Subsequent chymotryptic hydrolysis of [14C]succinylated cytochrome P-450SCC and separation of digest obtained by combining various types of HPLC resulted in seven major radioactive peptides. The amino acid sequence of the peptides was determined by microsequencing. The major amino groups modified with radioactive succinic anhydride were found to be at Lys-73, -109, -110, -126, -145, -148 and -154 in the N-terminal sequence of cytochrome P-450SCC molecule and at Lys-267, -270, -338 and -342 in the C-terminal sequence. The role of electrostatic interactions in fixation of cytochrome P-450SCC complex with adrenodoxin is discussed.     

Cross-linking studies of steroidogenic electron transfer: covalent complex of adrenodoxin reductase with adrenodoxin

Bifunctional reagents 3,3'-dithiobis(succinimidyl propionate), 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide and N-succinimidyl 3-(2-pyridyldithio)propionate have been used in an attempt to study molecular organization and covalent cross-linking of adrenodoxin reductase with adrenodoxin, the components of steroidogenic electron transfer system in bovine adrenocortical mitochondria. There was no cross-linking of individual proteins by the bifunctional reagents used, except for adrenodoxin cross-linking with water-soluble carbodiimide. Substantial cross-linking of adrenodoxin reductase with adrenodoxin was observed when water-soluble carbodiimide was used as cross-linking reagent. However, the cross-linked complex failed to transfer electrons. Significant amounts of the functional cross-linked complex (up to 42%) were observed when the proteins were cross-linked with N-succinimidyl 3-(2-pyridyldithio)propionate. Using gel filtration, ion-exchange chromatography and affinity chromatography on adrenodoxin-Sepharose, the complex was obtained in a highly purified form. In the presence of cytochrome P-450scc or cytochrome c, the cross-linked complex of adrenodoxin reductase with adrenodoxin was active in electron transfer from NADPH to heme proteins. The data obtained indicate that there are distinct binding sites on the adrenodoxin molecule responsible for the adrenodoxin reductase and cytochrome P-450scc binding, which suggests that steroidogenic electron transfer may be realized in an organized complex.     

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.     

Purification and properties of cytochrome P-450 (SCC) from pig testis mitochondria

Cytochrome P-450 was purified from pig testis mitochondria to a specific content of 13.1 n mol/mg of protein. The purified preparation was found to contain a single species of P-450, on sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular weight of about 53000 +/- 2000. The cholesterol side chain-cleavage system could be reconstituted by mixing the purified cytochrome P-450, adrenodoxin reductase, adrenodoxin, cholesterol and NADPH. The rate of conversion of cholesterol to pregnenolone was 6.2 n mol/min/n mol of P-450 under the conditions employed. The absorption spectrum of the oxidized cytochrome P-450 had maxima at 416, 530 and 568 nm. The reduced CO-complex of the cytochrome P-450 exhibited an absorption maximum at 448 nm. The purified P-450 was subjected to microsequence analysis and its NH2-terminal amino acid sequence was found to show considerable homology with that of bovine adrenal P-450 (SCC).     

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.     

Protein rotation study of cytochrome P-450 in submitochondrial particles: effect of KCl and intermolecular interactions with redox partners

The rotational diffusion of cytochrome P-450 in submitochondrial particles (SMP) of bovine adrenocortical mitochondria was measured by detecting the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate the effect of KCl on intermolecular interactions involving cytochrome P-450 and to investigate the interactions of cytochrome P-450 with other redox partners. The rotational diffusion of cytochrome P-450 was significantly dependent on KCl concentration. When the KCl concentration was increased from 0 to 1,000 mM, the mobile population of cytochrome P-450 was increased from 33 to 82%. After removing the KCl, the mobile population of cytochrome P-450 returned to the original 33%. These results suggest that nonspecific protein aggregates are dissociated by the presence of KCl, possibly due to the change in electrostatic interactions, resulting in mobilization of cytochrome P-450. SMP were observed to be nearly free from adrenodoxin and adrenodoxin reductase. The addition of adrenodoxin to SMP increased the mobile population of cytochrome P-450 from 35 to 54%. Further addition of adrenodoxin reductase to SMP containing adrenodoxin immobilized cytochrome P-450 by 6%. The addition of only adrenodoxin reductase to SMP, however, did not immobilize cytochrome P-450. The present results are consistent with our previous observations [Ohta, Y., Mitani, F., Ishimura, Y., Yanagibashi, K., Kawamura, M., & Kawato, S. (1990) J. Biochem. 107, 97-104] that cholesterol-bearing P-450SCC forms a transient ternary association with adrenodoxin and adrenodoxin reductase.     

Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex

The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.     

Molecular organization of the reductase complex in adrenal cortex mitochondria. Study using bifunctional reagents

The water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, homobifunctional reagent 3,3'-dithiobis (succinimidyl propionate), and heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate have been used to cross-link adrenodoxin reductase and adrenodoxin, components of steroidogenic electron transfer system. Though maximal yield of the cross-linked complex was achieved with the water-soluble carbodiimide, this complex was inactive in the electron transfer from NADPH to cytochrome P-450. The functionally active complex was formed with N-succinimidyl 3-(2-pyridyldithio) propionate. The complex was purified to the apparent homogeneity and shown to be able to mediate the electron transfer. The data obtained indicate existence of different binding sites on adrenodoxin responsible for the adrenodoxin reductase and cytochrome P-450scc binding and do not contradict to the model of the steroidogenic electron transfer in an organized complex.     

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.     

Mechanism of electron transport in the cholesterol-hydroxylating system of adrenal cortex mitochondria: a triple complex of adrenodoxin reductase, adrenodoxin and cytochrome P-450

A cross-linked ternary adrenodoxin reductase-adrenodoxin-cytochrome P-450scc complex with an apparent molecular mass of 114 kD was obtained, using N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)-hexanoate. The composition of the cross-linked complex was determined by immunoblotting and radioactivity measurements, using N-ethyl [2.3-14C]maleimide-premodified adrenodoxin. The data obtained suggest that the ternary complex may exist in solution.     

Molecular properties of cytochrome P-45011 beta from adrenal cortex mitochondria.

Cytochrome P-45011 beta has been solubilized and partially purified from bovine adrenal cortex mitochondria using chromatography on Octyl-Sepharose CL-4B or DEAE-Sepharose CL-6B. The partially purified P-450 preparations were about 90% pure as judged by SDS-polyacrylamide gel electrophoresis. In the presence of purified preparations of adrenodoxin reductase and adrenodoxin, the partially purified P-450 preparations catalyzed NADPH-supported 11 beta-hydroxylation of unconjugated and sulphoconjugated deoxycorticosterone. In presence of Triton X-100 the partially purified cytochrome P-45011 beta had a Stoke's radius of 4.5 nm, a sedimentation coefficient of 3.1 S and a partial specific volume of about 0.85 cm3/g. These results indicate that the cytochrome P-45011 beta-Triton X-100 complex has a molecular weight of about 100 000 and that P-45011 beta bound about 1.1 g of Triton X-100 per g of protein. The P-45011 beta-Triton X-100 complex was catalytically active in hydroxylation reactions supported by NADPH or the hydroxylating agent ortho-nitroiodosobenzene, suggesting that the monomer of cytochrome P-45011 beta is an active form of the protein.     

Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.     

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.     

The active form of cytochrome P-45011beta from adrenal cortex mitochondria.

Cytochrome P-45011beta has been solubilized and partially purified from bovine adrenal cortex mitochondria by means of chromatography on Octyl-Sepharose CL-4B or DEAE-Sepharose CL-6B. The partially purified P-450 preparations were about 90% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but had a low specific content of P-450 (between 1 and 2 nmol of P-450 per mg of protein). In the presence of purified preparations of adrenodoxin reductase and adrenodoxin, the partially purified P-450 preparations catalyzed NADPH-supported 11beta-hydroxylation of unconjugated and sulfoconjugated deoxycorticosterone. In the reconstituted system the hydroxylation of deoxycorticosterone sulfate proceeded at a much higher rate than in intact mitochondria, indicating that in the former case interactions between the hydrophilic substrate and P-450 were facilitated. In the presence of Triton X-100 the partially purified cytochrome P-45011beta had a Stokes radius of 4.5 nm, a sedimentation coefficient of 3.1 S, and a partial specific volume of about 0.85 cm3/g. These results indicate that the cytochrome P-45011beta . Triton X-100 complex had a molecular weight of about 100,000 and that P-45011beta bound about 1.1 g of Triton X-100 per g of protein. The P-45011beta . Triton X-100 complex was catalytically active in hydroxylation reactions supported by NADPH or the hydroxylating agent ortho-nitroiodosobenzene, suggesting that the monomer of cytochrome P-45011beta is the active form of the protein.     

Purification and characterization of adrenal cortex mitochondrial cytochrome P-450 specific for cholesterol side chain cleavage activity.

Cytochrome P-450 was purified from bovine adrenal cortex mitochondria by affinity chromatography using an octylamine-substituted Sepharose column. The resulting optically clear preparation was stable at -20 degrees for months. The specific concentration of cytochrome P-450 in the preparation was about 5 nmol of heme per mg of protein. The preparations were free of adrenodoxin, adrenodoxin reductase, phospholipids, and other heme contaminations. Polyacrylamide gel electrophoresis of the purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed a single major band with a molecular weight of about 60,000. The optical absorption spectra of the preparation exhibited Soret maxima at 416, 416, and 448 nm for the Fe3+, Fe2+ and the C.Fe2+ complex, respectively. The EPR spectrum showed the characteristic features of the low spin form of ferric cytochrome P-450 with principal components 1.914, 2.241, and 2.415 of the g-tensor. The circular dichroism spectrum revealed two large negative ellipticities at 412 and 350 nm. Fluorescence spectra showed an excitation maximum at 285 nm and an emission maximum at 305 nm with a shoulder at 330 nm as the cytochrome P-450 molecule is excited at 285 nm, or an emission maximum at 335 nm when the cytochrome molecule is excited at 305 nm. After reconstitution with adrenodoxin and its reductase, this cytochrome P-450 was highly active for cholesterol desmolase with an NADPH-generating system as electron donor but was not active for steroid 11beta-hydroxylase.     

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.     

Study of the role of lysine residues of the cholesterol hydroxylating cytochrome P-450 by a method of chemical modification

Chemical modification of cytochrome P-450scc by lysine-specific reagents has been performed. Modification of the hemoprotein was shown to result in the loss of its ability to interact with adrenodoxin. With a view of identifying lysine residues involved in the interaction with adrenodoxin, cytochrome P-450scc was modified by succinic anhydride in the presence of adrenodoxin. After the removal of ferredoxin, the modification was performed with the use of a radioactively labeled reagent. Subsequent hydrolysis of the succinic hemoprotein by chymotrypsin and separation of the peptides obtained by high pressure liquid chromatography resulted in the isolation of seven chymotryptic peptides containing labeled lysine residues. These amino acid sequences were identified. The role of lysine residues of cytochrome P-450scc in complex formation with adrenodoxin is discussed.     

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)     

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

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