Spin label studies on the interactions of bovine adrenodoxin with NADPH-adrenodoxin reductase and with cytochrome P-450scc.

Adrenodoxin of bovine adrenocortical mitochondria was spin-labeled with two different spin-labeling reagents, N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)imidazole (I) and N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (II), without major loss of its activity for electron transport from NADPH to cytochrome c. The EPR spectrum of adrenodoxin spin-labeled with either of the reagents showed a pattern typical of a moderately immobilized spin label. When adrenodoxin was treated with (I), approximately two amino acid residues per molecule were spin-labeled, whereas a single residue was labeled by (II). While assition of NADPH to adrenodoxin spin-labeled with (I) did not diminish the EPR signal intensity, addition of the reductant to the labeled adrenodoxin in the presence of adrenodoxin reductase caused slow reduction of the spin label, the rate of which was dependent on the aerobicity. Addition of adrenodoxin reductase to adrenodoxin spin-labeled with (I) or (II) resulted in the appearance of a more immobilized component in the EPR spectrum. The ratio of the more immobilized component to the less immobilized component was saturated at a molar ratio of one to one. Addition of cytochrome P-450scc to adrenodoxin labeled with (I) had similar effects on the EPR spectrum.     

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)     

The formation of binary and ternary complexes of cytochrome P-450scc with adrenodoxin and adrenodoxin reductase.adrenodoxin complex. The implication in ACTH function.

Binary and ternary complexes of bovine adrenocortical mitochondrial cytochrome P-450scc with adrenodoxin and adrenodoxin reductase.adrenodoxin complex are formed in the presence of cholesterol and Emulgen 913. Both cholesterol and Emulgen 913 are required for the binding of cytochrome P-450scc with adrenodoxin. Since phospholipids are able to replace Emulgen 913 in this reaction, in vivo phospholipids of the mitochondrial inner membrane appear to play the function of the detergent. The dissociation constants of the cytochrome.adrenodoxin complex are 0.3 to 0.4 microM at 130 microM dimyristoylphosphatidylcholine and 0.9 microM at 120 microM Emulgen 913, whereas the dissociation constant for the ternary complex of cytochrome P-450scc with adrenodoxin reductase and adrenodoxin is 4.0 microM at 150 microM Emulgen 913. The stoichiometry of binary and ternary complexes reveals the 1:1 and 1:1:1 molar ratios, respectively, judging from chemical analyses after the fractionation of the complexes by gel filtration. Emulgen 913, Tween 20, ethylene glycol, myristoyllysophosphatidylcholine, dimyristoylphosphatidylcholine, and phosphatidylethanolamine show the enhanced activity of cholesterol side chain cleavage reaction with cytochrome P-450scc, adrenodoxin, adrenodoxin reductase, and NADPH. These results, in conjunction with earlier experiments, lead us to the proposal on the structure of the hydroxylase complex in the membrane and to the hypothesis on the regulation of the enzymatic activity by the availability of substrate cholesterol to the cytochrome. Hence, we propose a mobile P-450scc hypothesis for the response of the mitochondrion to adrenocorticotropic hormone stimuli.     

Modification of histidine 56 in adrenodoxin with diethyl pyrocarbonate inhibited the interaction with cytochrome P-450scc and adrenodoxin reductase

Three histidine residues of bovine adrenodoxin, His-10, His-56, and His-62, were modified with diethyl pyrocarbonate. The order of the modification among the three histidines were monitored by measuring the proton NMR spectra. The modified adrenodoxin exhibited reduced affinity for adrenodoxin reductase as determined in cytochrome c reductase activity. In the presence of cholesterol, the modified adrenodoxin induced a high spin form of cytochrome P-450scc on complex formation in the same manner as native adrenodoxin. The spectral titration showed that adrenodoxin modified with diethyl pyrocarbonate exhibited a 5-fold higher Kd value than that of native adrenodoxin. These effects of the modification of adrenodoxin on the affinities for the redox partners were not proportional to the number of modified histidines determined by the optical absorbance change at 240 nm. Modification of adrenodoxin up to 2 histidine residues did not affect the affinity for the redox partners, but further modification on the third one resulted in an increase of apparent Km in cytochrome c reductase activity by 2-fold and of Kd for cytochrome P-450scc by 5-fold. The 1H NMR spectra of the modified adrenodoxin unequivocally demonstrated that histidine residues at His-10 and His-62 reacted more readily with diethyl pyrocarbonate than His-56 did, indicating that modification of His-56 was responsible for the reduction of binding affinities of adrenodoxin for redox partners. These results are consistent with the proposal that the residue of His-56 in adrenodoxin has an essential role in the electron transfer mechanism where adrenodoxin functions as a mobile shuttle.     

Comparative structural-functional characterization of recombinant and natural adrenodoxin. Interaction with cytochrome P450scc.

The conditions for heterologous expression of recombinant bovine adrenodoxin in E. coli have been optimized, thus reaching expression levels up to 12-14 micromoles per liter of culture medium. A highly efficient method for purification of this recombinant ferredoxin from the E. coli cells has been developed. The structural-functional properties of the highly purified recombinant protein have been characterized and compared to those of natural adrenodoxin purified from bovine adrenocortical mitochondria. In contrast to natural adrenodoxin, which is characterized by microheterogeneity, the recombinant adrenodoxin is homogeneous as judged by N- and C-terminal amino acid sequencing, and its sequence corresponds to the full-length mature form of adrenodoxin containing 128 amino acid residues. The interactions of the natural and recombinant adrenodoxins with cytochrome P450scc have been studied and compared with respect to: the efficiency of their enzymatic reduction of cytochrome P450scc in a reconstituted system; the ability of the immobilized adrenodoxins to bind cytochrome P450scc; the ability of the adrenodoxins to induce a spectral shift of cytochrome P450scc and to effect the average polarity of exposed tyrosines in the low-spin cytochrome P450scc. The recombinant adrenodoxin is functionally active and in the reduced state as well as at low ionic strength it displays higher affinity to cytochrome P450scc as compared to the natural bovine adrenocortical adrenodoxin. The possible role of the C-terminal sequence of the adrenodoxin molecule in its interaction with cytochrome P450scc as well as the advantages of using the recombinant protein instead of the natural one are discussed.     

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.     

The concentration of adrenodoxin reductase limits cytochrome p450scc activity in the human placenta.

We have previously reported that cytochrome P450scc activity in the human placenta is limited by the supply of electrons to the P450scc [Tuckey, R. C., Woods, S. T. & Tajbakhsh, M. (1997) Eur. J. Biochem. 244, 835-839]. The aim of the present study was to determine whether it is adrenodoxin reductase, adrenodoxin or both which limits cytochrome P450scc activity and hence progesterone synthesis in the placenta. We found that the concentrations of adrenodoxin reductase and adrenodoxin in placental mitochondria were both considerably lower than the concentrations of these proteins in the bovine adrenal cortex. When P450scc activity assays were carried out at high mitochondrial protein concentrations, we found that the addition of exogenous adrenodoxin reductase to sonicated mitochondria rescued pregnenolone synthesis to a level above that for intact mitochondria, showing that adrenodoxin is near-saturating in vivo. In contrast, pregnenolone synthesis by sonicated mitochondria was almost zero even after the addition of human adrenodoxin. This shows that the concentration of endogenous adrenodoxin reductase was insufficient to support appreciable rates of pregnenolone synthesis, even when concentrated mitochondrial samples were used. Comparative studies with human and bovine adrenodoxin reductase have revealed that a twofold higher concentration of human adrenodoxin reductase is required for maximal P450scc activity in the presence of saturating human adrenodoxin. Thus, not only is the adrenodoxin concentration low in placental mitochondria, but the amount required for maximal P450scc activity is higher than that for the bovine reductase. Overall, the data indicate that the adrenodoxin reductase concentration limits the activity of P450scc in placental mitochondria and hence determines the rate of progesterone synthesis.     

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.     

The interaction of bovine adrenodoxin with CYP11A1 (cytochrome P450scc) and CYP11B1 (cytochrome P45011beta ). Acceleration of reduction and substrate conversion by site-directed mutagenesis of adrenodoxin.

The kinetics of protein-protein interaction and heme reduction between adrenodoxin wild type as well as eight mutants and the cytochromes P450 CYP11A1 and CYP11B1 was studied in detail. Rate constants for the formation of the reduced CYP11A1.CO and CYP11B1.CO complexes by wild type adrenodoxin, the adrenodoxin mutants Adx-(4-108), Adx-(4-114), T54S, T54A, and S112W, and the double mutants Y82F/S112W, Y82L/S112W, and Y82S/S112W (the last four mutants are Delta113-128) are presented. The rate constants observed differ by a factor of up to 10 among the respective adrenodoxin mutants for CYP11A1 but not for CYP11B1. According to their apparent rate constants for CYP11A1, the adrenodoxin mutants can be grouped into a slow (wild type, T54A, and T54S) and a fast group (all the other mutants). The adrenodoxin mutants forming the most stable complexes with CYP11A1 show the fastest rates of reduction and the highest rate constants for cholesterol to pregnenolone conversion. This strong correlation suggests that C-terminal truncation of adrenodoxin in combination with the introduction of a C-terminal tryptophan residue enables a modified protein-protein interaction rendering the system almost as effective as the bacterial putidaredoxin/CYP101 system. Such a variation of the adrenodoxin structure resulted in a mutant protein (S112W) showing a 100-fold increased efficiency in conversion of cholesterol to pregnenolone.     

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.     

Comparison of the multiple molecular forms of bovine adrenocortical P450scc with those of corpus luteum P450scc.

1. Bovine adrenocortical P450scc was resolved into several fractions by chromatography on AH-Sepharose 4B followed by gel filtration on Toyopearl HW55S. All fractions contained P450scc of the same molecular size and the P450scc could be resolved into 3-4 major and more than 10 minor isoelectric point forms by isoelectric focusing on polyacrylamide gel in the presence of Emulgen 913. 2. Both the AH-Sepharose chromatography profile and the isoelectric focusing pattern of the adrenocortical P450scc were more complex than those of the corpus luteum P450scc. The corpus luteum P450scc was practically devoid of the neutral to acidic isoelectric point forms. 3. Three to four P450scc subfractions with different isoelectric focusing pattern were obtained from a purified preparation of adrenocortical P450scc by ion-exchange chromatography on DEAE-Toyopearl 650S or DEAE-Sephadex A25. These P450scc subfractions showed essentially the same spectral properties, catalytic activity, molecular weight and N-terminal amino acid sequence. 4. The most acidic (the latest eluting) subfraction was composed mostly of the neutral to acidic isoelectric point forms. The sedimentation characteristics of this subfraction was also studied. 5. The structural basis of the multiple molecular forms was discussed.     

Mass spectrometry identification of cytochrome P450 2B4 interaction sites for NADPH: Cytochrome P450 reductase

The interaction sites for protein partners, cytochrome P450 2B4 (d-2B4) and NADPH: cytochrome P450 reductase (d-Fp), have been identified. These proteins form complexes during their functioning. Nonspecific covalent cross-linking of the d-2B4 complexes with d-Fp in the Emulgen 913 monomerized system was achieved by 4,4′- dithiobis-phenyl azide. Covalently cross-linked peptides of this complex were identified by ESI-MS/MS. Several binding sites have been identified for these proteins. Based on these sites a model for intermolecular interaction between these proteins has been proposed. This model includes 5 contact sites on d-2B4 for d-Fp (stabilized by the cross-linker); these include the following pairs of corresponding peptides of d-2B4 and d-Fp: 1) d-2B4_324–336 and d-Fp_570–585; 2) d-2B4_423–433 and d-Fp_102–109; 3) d-2B4_327–336 and d Fp_452–464; 4) d-2B4_192–197 and d-Fp_456–464; 5) d-2B4_134–139 and d-Fp_406–425. In the two last cases d-Fp peptides are located in the interdomain cleft and stabilize the protein-protein complex via the cross-linker and so the d 2B4/d-Fp complex formation by these sites may involve amino acid residues of the peptides d-Fp_456–464 and d-Fp_406–425, which surround the interdomain cleft.     

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.     

Phosphorylation of bovine adrenodoxin by protein kinase CK2 affects the interaction with its redox partner cytochrome P450scc (CYP11A1)

Adrenodoxin (Adx), a [2Fe-2S] vertebrate-type ferredoxin, transfers electrons from the NADPH-dependent flavoprotein Adx reductase (AdR) to mitochondrial cytochrome P450 enzymes of the CYP11A and CYP11B families, which catalyze key reactions in steroid hormone biosynthesis. Adx is a known phosphoprotein, but the kinases that phosphorylate Adx have remained mostly obscure. The aim of this study was to identify previously unknown Adx phosphorylating kinases and to acquire a deeper insight into the functional consequences of such a modification. Here, we show for the first time that bovine Adx is a substrate of protein kinase CK2, whereas bovine CYP11A1, CYP11B1, and AdR are not phosphorylated by this kinase. CK2 phosphorylation of mature Adx requires the presence of both the catalytic alpha-subunit and the regulatory beta-subunit of CK2 and takes place exclusively at residue Thr-71, which is located within the redox partner interaction domain of the protein. We created two Adx mutants, Adx-T71E (imitating a phosphorylation) and Adx-T71V (which cannot be phosphorylated at this site), respectively, and investigated how these mutations affected the interaction of Adx with its redox partners. These data were supplemented with detailed spectroscopic and functional assays using the phosphorylated protein. All Adx species behaved like wild type (Adx-WT) with respect to their redox potential, iron-sulfur cluster symmetry, and overall backbone structure. Substrate conversion assays catalyzed by CYP11A1 showed an increase in product formation when Adx-T71E or CK2-phosphorylated Adx were used as electron carrier instead of Adx-WT, whereas the activity toward CYP11B1 was not altered using these Adx species. Additionally, Adx-T71E represents the only full-length Adx mutant which leads to an increase in CYP11A1 product formation. Therefore, characterizing this full-length mutant helps to improve our knowledge on the functional effects of phosphorylations on complex redox systems.     

Deletions in the loop surrounding the iron-sulfur cluster of adrenodoxin severely affect the interactions with its native redox partners adrenodoxin reductase and cytochrome P450(scc) (CYP11A1)

The redox active iron-sulfur center of bovine adrenodoxin is coordinated by four cysteine residues in positions 46, 52, 55 and 92 and is covered by a loop containing the residues Glu-47, Gly-48, Thr-49, Leu-50 and Ala-51. In plant-type [2Fe-2S] ferredoxins, the corresponding loop consists of only four amino acids. The loop is positioned at the surface of the proteins and forms a boundary separating the [2Fe-2S] cluster from solvent. In order to analyze the biological function of the five amino acids of the loop in adrenodoxin (Adx) for this electron transfer protein each residue was deleted by site-directed mutagenesis. The resulting five recombinant Adx variants show dramatic differences among each other regarding their spectroscopic characteristics and functional properties. The redox potential is affected differently depending on the position of the conducted deletion. In contrast, all mutations in the protein loop influence the binding to the redox partners adrenodoxin reductase (AdR) and cytochrome P450(scc) (CYP11A1) indicating the importance of this loop for the physiological function of this iron--sulfur protein.     

Expression and regulation of adrenodoxin and P450scc mRNA in rodent tissues

The rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone. This reaction occurs in steroidogenic tissue in the inner mitochondrial membrane, and is mediated by the cholesterol side-chain cleavage enzyme. This enzyme system transfers electrons from NADPH to cholesterol through its three protein components: adrenodoxin reductase, adrenodoxin, and the terminal oxidase, P450scc. We have previously shown that P450scc mRNA is regulated by tropic hormones and cAMP by a cycloheximide-independent mechanism in mouse Leydig tumor MA-10 cells. We now show that the mRNA for adrenodoxin, another component of the cholesterol side-chain cleavage enzyme system, is regulated by tropic hormones and cAMP in MA-10 cells. We cloned rat adrenodoxin cDNA to analyze adrenodoxin mRNA in various rat tissues and in MA-10 cells by RNase protection assays. Adrenodoxin mRNA is found in virtually all rat tissues examined, although it is most abundant in adrenals, ovaries, and testes. MA-10 cells synthesize two species of adrenodoxin mRNA, one of 1.2 kb and the other of 0.8 kb. Both of these adrenodoxin mRNAs are increased approximately six-fold by 1 mM 8-Br-cAMP, five-fold by 10 microM forskolin, and three-fold by both 25 ng/ml hCG and by 100 ng/ml LH. Maximal adrenodoxin mRNA accumulation occurs by 4 h of hormonal stimulation. The cAMP-mediated increase in adrenodoxin mRNA accumulation is independent of protein synthesis, since treatment with cycloheximide or puromycin in the absence or presence of cAMP does not inhibit, and even increases, adrenodoxin mRNA accumulation.     

Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.     

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.     

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)