Cytochrome b5, cytochrome c, and cytochrome P-450 interactions with NADPH-cytochrome P-450 reductase in phospholipid vesicles

Upon incubation of detergent-solubilized NADPH-cytochrome P-450 reductase and either cytochrome b5 or cytochrome c in the presence of a water-soluble carbodiimide, a 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), covalently cross-linked complex was formed. The cross-linked derivative was a heterodimer consisting of one molecule each of flavoprotein and cytochrome, and it was purified to 90% or more homogeneity. The binary covalent complex between the flavoprotein and cytochrome b5 was exclusively observed following incubation of all three proteins including NADPH-cytochrome P-450 reductase, cytochrome b5, and cytochrome c in L-alpha-dimyristoylphosphatidylcholine vesicles, and no heterotrimer could be identified. The isolated reductase-cytochrome b5 complex was incapable of covalent binding with cytochrome c in the presence of EDC. No clear band for covalent complex formation between PB-1 and reductase was seen with the present EDC cross-linking technique. More than 90% of the cross-linked cytochrome c in the purified derivative was rapidly reduced upon addition of an NADPH-generating system, whereas approximately 80% of the cross-linked cytochrome b5 was rapidly reduced. These results showed that in the greater part of the complexes, the flavin-mediated pathway for reduction of cytochrome c or cytochrome b5 by pyridine nucleotide was intact. When reconstituted into phospholipid vesicles, the purified amphipathic derivative could hardly reduce exogenously added cytochrome c, cytochrome b5, or PB-1, indicating that the cross-linked cytochrome shields the single-electron-transferring interface of the flavoprotein. These results suggest that the covalent cross-linked derivative is a valid model of the noncovalent functional electron-transfer complex.     

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.     

The functional role of cytochrome b5 reincorporated into hepatic microsomal fractions

Incorporation of detergent-solubilized cytochrome b5 into phenobarbital-induced rabbit liver microsomal fractions decelerates hexobarbital-dependent reduction of ferric cytochrome P-450; this is accompanied by retardation of NADPH utilization and H2O2 formation in the assay media. Integration of manganese-substituted cytochrome b5 into the microsomal preparations fails to affect these parameters. Analysis of the cytochrome P-450 reduction kinetics in the presence of increasing amounts of cytochrome b5 reveals a gradual augmentation of the amplitude of slow-phase electron transfer at the expense of the relative contribution of the fast phase; finally, a slow, apparently monophasic reaction persists. This defect in enzymatic reduction is not due to detergent effects and also does not seem to reflect cytochrome b5-induced perturbation of anchoring of NADPH-cytochrome c(P-450) reductase to cytochrome P-450. Experiments with the highly purified cytochrome P-450 isozyme LM2, in which amino acid residue(s) close to the heme edge had undergone suicidal inactivation through covalent attachment of chloramphenicol metabolite(s) do not exclude the possibility that cytochrome b5 and reductase might compete for a common electron transmission site on the terminal acceptor. Hence, the inhibitory action of cytochrome b5 on the reduction of ferric cytochrome P-450 is tentatively attributed to partial substitution of the former pigment for reductase in direct transport of the first electron to the monooxygenase.     

Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme:isozyme interactions

The aim of the present study was to examine a recent proposal that inhibitory isozyme:isozyme interactions explain why membrane-bound isozymes of rat liver microsomal cytochrome P-450 exert only a fraction of the catalytic activity they express when purified and reconstituted with saturating amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. The different pathways of testosterone hydroxylation catalyzed by cytochromes P-450a (7 alpha-hydroxylation), P-450b (16 beta-hydroxylation), and P-450c (6 beta-hydroxylation) enabled possible inhibitory interactions between these isozymes to be investigated simultaneously with a single substrate. No loss of catalytic activity was observed when purified cytochromes P-450a, P-450b, or P-450c were reconstituted in binary or ternary mixtures under a variety of incubation conditions. When purified cytochromes P-450a, P-450b, and P-450c were reconstituted under conditions that mimicked a microsomal system (with respect to the absolute concentration of both the individual cytochrome P-450 isozyme and NADPH-cytochrome P-450 reductase), their catalytic activity was actually less (69-81%) than that of the microsomal isozymes. These results established that cytochromes P-450a, P-450b, and P-450c were not inhibited by each other, nor by any of the other isozymes in the liver microsomal preparation. Incorporation of purified NADPH-cytochrome P-450 reductase into liver microsomes from Aroclor 1254-induced rats stimulated the catalytic activity of cytochromes P-450a, P-450b, and P-450c. Similarly, purified cytochromes P-450a, P-450b, and P-450c expressed increased catalytic activity in a reconstituted system only when the ratio of NADPH-cytochrome P-450 reductase to cytochrome P-450 exceeded that normally found in liver microsomes. These results indicate that the inhibitory cytochrome P-450 isozyme:isozyme interactions described for warfarin hydroxylation were not observed when testosterone was the substrate. In addition to establishing that inhibitory interactions between different cytochrome P-450 isozymes is not a general phenomenon, the results of the present study support a simple mass action model for the interaction between membrane-bound or purified cytochrome P-450 and NADPH-cytochrome P-450 reductase during the hydroxylation of testosterone.     

Hepatic mitochondrial cytochrome P-450 system. Purification and characterization of two distinct forms of mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat liver

We have purified two distinct isoforms of mitochondrial cytochrome P-450 from beta-naphthoflavone (beta-NF)-induced rat liver to greater than 85% homogeneity and characterized their molecular and catalytic properties. One of these isoforms showing an apparent molecular mass of 52 kDa is termed P-450mt1 and the second isoform with 54-kDa molecular mass is termed P-450mt2. Cytochrome P-450mt2 comigrates with similarly induced microsomal P-450c (the major beta-NF-inducible form) on sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with polyclonal antibody monospecific for cytochrome P-450c. Cytochrome P-450mt2, however, represents a distinct molecular species since it failed to react with a monoclonal antibody to P-450c and produced V8 protease fingerprints different from P-450c. Cytochrome P-450mt1, on the other hand, did not show any immunochemical homology with P-450c or P-450mt2 as well as partially purified P-450 from control mitochondria. Electrophoretic comparisons and Western blot analysis show that both P-450mt1 and P-450mt2 are induced forms not present in detectable levels in control liver mitochondria. A distinctive property of mitochondrial P-450mt1 and P-450mt2 was that their catalytic activities could be reconstituted with both NADPH-cytochrome P-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, while P-450c showed exclusive requirement for NADPH-cytochrome P-450 reductase. Cytochromes P-450mt1 and P-450mt2 were able to metabolize xenobiotics like benzo(a)pyrene and dimethyl benzanthracene at rates only one-tenth with cytochrome P-450c. Furthermore, P-450mt1, P-450mt2, as well as partially purified P-450 from control liver, but not P-450c, showed varying activities for 25- and 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. These results provide evidence for the presence of at least two distinct forms of beta-NF-inducible cytochrome P-450 in rat hepatic mitochondria.     

Hepatic mitochondrial cytochrome P-450 system. Identification and characterization of a precursor form of mitochondrial cytochrome P-450 induced by 3-methylcholanthrene

Hepatic mitoplasts from 3-methylcholanthrene-treated rats contain cytochrome P-450 which can metabolize polycyclic aromatic hydrocarbons like benzo(a)pyrene. Mitochondrial cytochrome P-450 was partially purified and reconstituted in vitro using adrenodoxin and the adrenodoxin reductase electron transfer system and [3H]benzo(a)pyrene as the substrate. A polyclonal antibody to purified microsomal P-450c (a major 3-methylcholanthrene-inducible form) inhibited the activity of mitochondrial enzyme in a concentration-dependent manner and also reacted with a 54-kDa protein on the immunoblots. A monoclonal antibody having exclusive specificity for P-450c, on the other hand, did not inhibit the aryl hydrocarbon hydroxylase activity of the mitochondrial enzyme and showed no detectable cross-reaction with the 54-kDa mitochondrial protein. Similarly, two-dimensional analysis and immunodetection using the polyclonal antibody showed distinct molecular properties of the mitochondrial enzyme different from the similarly induced microsomal P-450c with respect to the isoelectric pH. In vitro translation of free polysomes from 3-methylcholanthrene-induced liver, transport of precursor proteins by isolated mitochondria in vitro, and immunoprecipitation with the polyclonal antibody showed the presence of a 57-kDa putative precursor which is transported and processed into mature 54-kDa species. These results present evidence for the true intramitochondrial location of the P-450c-antibody reactive isoform detected in 3-methylcholanthrene-induced rat liver mitochondria.     

The presence of essential carboxyl group for binding of cytochrome c in rat hepatic NADPH-cytochrome P-450 reductase by the reaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

NADPH-cytochrome P-450 reductase (EC 1.6.2.4) purified from rat hepatic microsomal fraction was inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a specific agent for modification of carboxyl groups in a protein. The inactivation exhibited pseudo-first order kinetics with a reaction order approximately one and a second-order-rate constant of 0.60 M-1 min-1 in a high ionic strength buffer and 0.08 M-1 min-1 in a low ionic strength buffer. By treatment of NADPH-cytochrome P-450 reductase with EDC, the pI value changed to 6.5 from 5.0 for the native enzyme, and the reductase activity for cytochrome c, proteinic substrate, was strongly inactivated. When an inorganic substrate, K3Fe(CN)6, was used for assay of the enzyme activity, however, no significant inactivation by EDC was observed. The rate of inactivation by EDC was markedly but not completely decreased by NADPH. Also, the inactivation was completely prevented by cytochrome c, but not by K3Fe(CN)6 or NADH. The sulfhydryl-blocked enzyme prepared by treatment with 5,5'-dithio-bis(2-nitrobenzoic acid), which had no activity, completely recovered its activity in the presence of dithiothreitol. When the sulfhydryl-blocked enzyme was modified by EDC, the enzyme in which the carboxyl group alone was modified was isolated, and its activity was 35% of the control after treatment with dithiothreitol. In addition, another carboxyl reagent, N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward reagent K), decreased cytochrome c reductase activity of NADPH-cytochrome P-450 reductase. These results suggest that the carboxyl group of NADPH-cytochrome P-450 reductase from rat liver is located at or near active-site and plays a role in binding of cytochrome c.     

NADPH-cytochrome P-450 reductase-cytochrome b5 interactions: crosslinking of the phospholipid vesicle-associated proteins by a water-soluble carbodiimide

Detergent-solubilized and purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome b5 were coreconstituted into phospholipid vesicles. When the proteoliposomes were incubated with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a new higher-molecular-weight band was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The band was purified by chromatography on DEAE-Sepharose CL-6B, 2'5'-ADP-Sepharose 4B, and Sephadex G-100. The heme absorption spectrum and fluorophotometric assay of flavin of the purified material demonstrate that this product is a 1:1 crosslinked complex containing one molecule each of the flavoprotein and cytochrome. Proteolysis of the crosslinked form indicates that the hydrophilic catalytic domains participate in the covalent attachment, and that the hydrophobic membrane-attachment peptide is necessary for the protein interaction. The purified crosslinked derivative showed no activities for reduction of either cytochrome c or ferricyanide. About half of the enzyme-associated flavin was reduced rapidly by NADPH, as was 20-30% of the crosslinked cytochrome, indicating that, in at least some of the complexes, the flavin-mediated pathway for reduction of cytochrome by pyridine nucleotide was intact. These data suggest that the output- rather than the input-electron transfer site(s) in the flavoprotein was (were) blocked by the covalently attached cytochrome.     

Studies on the mechanism of hepatic microsomal N-oxide formation. N-oxidation of NN-dimethylaniline by a reconstituted rabbit liver microsomal cytochrome P-448 enzyme system.

The N-oxidation of NN-dimethylaniline was studied by using a reconstituted rabbit liver microsomal enzyme system consisting of highly purified cytochrome P-448, NADPH-cytochrome c reductase and lipid factor. Both cytochrome P-448 and NADPH-cytochrome c reductase were required for optimum N-oxygenating activity; the catalytic capacity of the reductase fraction for supporting N-oxide formation varied with the isolation procedure applied. Addition of microsomal lipids to the assay media stimulated N-oxidation of the arylamine. N-Oxide formation appeared to be not generally controlled by electron transfer from cytochrome b5 to cytochrome P-448. The present work confirms that cytochrome P-448 can mediate about 44% of the rabbit liver microsomal N-oxidation of NN-dimethylaniline, thus reinforcing the existence of at least two distinct tertiary amine N-oxidases, i.e. haemoprotein and flavoprotein oxidase, in liver microsomal fractions.     

Hepatic mitochondrial cytochrome P-450 system. Distinctive features of cytochrome P-450 involved in the activation of aflatoxin B1 and benzo(a)pyrene

Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase activation of structurally different carcinogens such as aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), and dimethylnitrosamine. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) induce the level of mitochondrial cytochrome P-450 by 2.0- to 2.5-fold above the level of control mitoplasts. The enzyme activities for AFB1 (3-fold) and BaP (16-fold) metabolism were selectively induced by PB and 3-MC, respectively. Furthermore, the metabolism of AFB1 and BaP by intact mitochondria was supported by Krebs cycle substrates but not by NADPH. Both PB and 3-MC administration cause a shift in the CO difference spectrum of mitoplasts (control, 448 nm; PB, 451 nm; and 3-MC, 446 nm) suggesting that they induce two different forms of mitochondrial cytochromes P-450. Mitoplasts solubilized with cholate and fractionated with polyethylene glycol exhibit only marginal monooxygenase activities. The activity, however, was restored to preparations from both PB-induced and 3-MC-induced mitochondrial enzymes (AFB1 activation, ethylmorphine, and benzphetamine deamination and BaP metabolism) by addition of purified rat liver cytochrome P-450 reductase, and beef adrenodoxin and adrenodoxin reductase. The latter proteins failed to reconstitute activity to purified microsomal cytochromes P-450b and P-450c that were fully active with P-450 reductase. Monospecific rabbit antibodies against cytochrome P-450b and P-450c inhibited both P-450 reductase and adrenodoxin-supported activities to similar extents. Anti-P-450b and anti-P-450c provided Ouchterlony precipitin bands against PB- and 3-MC induced mitoplasts, respectively. We conclude that liver mitoplasts contain cytochrome P-450 that is closely similar to the corresponding microsomal cytochrome P-450 but can be distinguished by a capacity to interact with adrenodoxin. These inducible cytochromes P-450 are of mitochondrial origin since their levels in purified mitoplasts are over 10 times greater than can arise from the highest possible microsomal contamination.     

Reconstitution of cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata EHRH.).

Cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata) was solubilized from microsomal membranes with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulphonic acid). Cytochrome P-450 was separated from NADPH: cytochrome c (P-450) reductase by ion-exchange chromatography on DEAE-Sephacel. NADPH:cytochrome c (P-450) reductase was further purified by affinity chromatography on 2',5'-ADP-Sepharose 4B. This procedure resulted in a 248-fold purification of the enzyme; on SDS/polyacrylamide-gel electrophoresis after silver staining, only one band, corresponding to a molecular mass of 80 kDa, was present. The digitoxin 12 beta-hydroxylase activity could be reconstituted by incubating partially purified cytochrome P-450 and NADPH:cytochrome c (P-450) reductase together with naturally occurring microsomal lipids and flavin nucleotides. This procedure yielded about 10% of the original amount of digitoxin 12 beta-hydroxylase.     

Role of a hydrophobic polypeptide in the N-terminal region of NADPH-cytochrome P-450 reductase in complex formation with P-450LM.

Detergent-solubilized liver microsomal NADPH-cytochrome P-450 reductase is known to retain the ability to catalyze electron transfer to cytochrome P-450, whereas the trypsin-solubilized reductase does not. In the present study, treatment of the highly purified detergent-solubilized rabbit liver enzyme (m.w. 77,700) with trypsin was shown to yield a small peptide (m.w. 6,100) as well as the large peptide (m.w. 71,200) which retains the flavin prosthetic groups. The small peptide, which is hydrophobic in nature as shown by its amino acid composition and solubility properties, is apparently the moiety in the native reductase involved in binding to cytochrome P-450 and to the microsomal membrane. The C-terminal amino acid sequences of the native reductase and large fragment are identical [-Trp-(Leu, Val)-Asp-Ser-COOH], thereby indicating that the hydrophobic peptide is located in the N-terminal region of the enzyme.     

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.     

Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid

We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid.     

Purification of rat liver microsomal cytochrome P-450b without the use of nonionic detergent

Sodium cholate, Emulgen 911, and (3-[(-cholamidopropyl)-dimethyl- ammonio]-1-propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P-450, namely P-450a, P-450b, P-450c, and P-450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH- cytochrome P-450 reductase (reductase). The major phenobarbital-inducible form of rat liver microsomal cytochrome P-450, designated P-450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P-450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P-450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC-50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P-450b without the use of nonionic detergent. The protein is designated cytochrome P-450b^* to distinguish it from cytochrome P-450b purified with the use of Emulgen 911. NADPH-cytochrome P-450 reductase was also purified both with and without the use of nonionic detergent. The absolute spectra of cytochrome P-450b and P-450b^* were indistinguishable, as were the carbon monoxide (CO)- and metyrapone-difference spectra of the dithionite-reduced hemoproteins. When reconstituted with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine, cytochromes P-450b and P-450b^* catalyzed the N-demethylation of benzphetamine and aminopyrine, the 4-hydroxylation of aniline, the O-dealkylation of 7-ethoxycoumarin, the 3-hydroxylation of hexobarbital, and the 6-hydroxylation of zoxazolamine. Both hemo-proteins catalyzed the 16α- and 16β-hydroxylation of testosterone, as well as the 17-oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3-/9-hydroxylation. The rate of biotransformation catalyzed by cytochrome P-450b^* was up to 50% greater than the rate catalyzed by cytochrome P-450b when reconstituted with either reductase or reductase^*. The activity of cytochrome P-450b and P-450b^* increased up to 50% when reconstituted with reductase^* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P-450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P-450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911.     

Role of electrostatic interactions in the reaction of NADPH-cytochrome P-450 reductase with cytochromes P-450

Chemical modification of cytochrome P-450 reductase was used to determine the involvement of charged amino acids in the interaction between the reductase and two forms of cytochrome P-450. Acetylation of 11 lysine residues of the reductase with acetic anhydride yielded a 20-40% decrease in the apparent Km of the reductase for cytochrome P-450b or cytochrome P-450c using either 7-ethoxycoumarin or benzphetamine as substrates. A 20-45% decrease in the Vmax was observed except for cytochrome P-450b with 7-ethoxycoumarin as substrate, where there was a 27% increase. Modification of carboxyl groups on the reductase with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and methylamine, glycine methyl ester, or taurine as nucleophiles inhibited the interaction with the cytochromes P-450. We were able to modify 4.0, 7.9, and 5.9 carboxyl groups using methylamine, glycine methyl ester, or taurine, respectively. The apparent Km for cytochrome P-450c or cytochrome P-450b was increased 1.3- to 5.2-fold in a reconstituted monooxygenase assay with 7-ethoxycoumarin or benzphetamine as substrate. There were varied effects on the Vmax. There was no significant change in the conformation of the reductase upon chemical modification with either acetic anhydride or EDC. These results strongly suggest that electrostatic interactions as well as steric constraints play a role in the binding and electron transfer step(s) between the reductase and cytochrome P-450.     

Covalent cross-linking of the active sites of vesicle-bound cytochrome b5 and NADH-cytochrome b5 reductase

A water-soluble carbodiimide has been used to promote the formation of amide bonds between carboxyl residues on cytochrome b5 and lysyl residues on cytochrome b5 reductase. The visible and UV absorption spectrum of the purified cross-linked complex was identical with the sum of the spectra of the individual enzymes, and the average apparent molecular weight of the complex, determined by sodium dodecyl sulfate-gel electrophoresis, was within 12% of the sum of the apparent molecular weights of the two monomeric enzymes, indicating that the cross-linked derivative was a dimer containing one molecule each of cytochrome b5 and cytochrome b5 reductase. When reconstituted into phospholipid vesicles, the amphipathic derivative showed substantially reduced Vmax values with the soluble electron acceptors potassium ferricyanide, cytochrome b5 heme peptide and cytochrome c, and with the membrane-bound acceptors amphipathic cytochrome b5 and stearyl-CoA desaturase. The soluble catalytic fragment of the derivative, produced by limited digestion with subtilisin Carlsberg, showed similar decreases in Vmax values with the above soluble acceptors. In contrast, intradimer electron transfer in the soluble fragment, measured by stopped flow spectrophotometry at 2 degrees C was very efficient. Ninety per cent of the cytochrome b5 in the derivative was reduced with a first order rate constant of 51 s-1 upon the addition of NADH; the transfer of electrons from NADH to the reductase FAD prosthetic group, which is known to be the rate-limiting step in the reductase reaction mechanism, proceeded with an apparent rate constant of 57 s-1 under these conditions. These kinetic data show that the enzymes in the complex are cross-linked together at the surfaces involved in protein-protein contacts during electron transfer in an orientation similar to that assumed during electron transfer between the free proteins.     

Effect of monooxygenase reactions catalyzed by cytochrome P-450 on the microsomal membrane

Hydroxylation of dimethylaniline in rabbit liver microsomes is accompanied by inactivation of cytochrome P-450 and the formation of products inhibiting the catalytic activity of non-inactivated cytochrome P-450. Other enzymes and electron carriers of microsomal membrane (cytochrome b5, NADH-ferricyanide reductase, NADPH-cytochrome c and NADPH-cytochrome P-450 reductases) as well as glucose-6-phosphatase were not inactivated in the course of the monooxygenase reactions. Phospholipids and microsomal membrane proteins were also unaffected thereby. Consequently, the changes in the microsomal membrane during cytochrome P-450 dependent monooxygenase system functioning are confined to the inactivation of cytochrome P-450.     

The role of cytochrome b5 in adrenal microsomal steroidogenesis.

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17 alpha- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-450(17) alpha,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount of cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the first electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.     

Participation of the microsomal electron transport system involving cytochrome P-450 in ω-oxidation of fatty acids

The participation of the microsomal electron transport system involving cytochrome P-450 in ω-oxidation of fatty acids by a rat liver preparation was examined since ω-oxidation involves microsomal reactions requiring both NADPH and molecular oxygen.

ω-Oxidation of fatty acids was inhibited by CO and by the antibody against NADPH-cytochrome c reductase. The addition to the reaction mixture of drugs which interact with cytochrome P-450 inhibited ω-oxidation. It is concluded that the microsomal electron transport system involving cytochrome P-450 functions in ω-oxidation of fatty acids.