Kinetics of reduction of purified liver microsomal cytochrome P-450 in the reconstituted enzyme system studied by stopped flow spectrophotometry.

Stopped flow studies were undertaken to examine the kinetics of reduction of 5,6-benzoflavone-inducible P-450 LM4 by NADPH in the presence of NADPH-cytochrome P-450 reductase and phospholipid under anaerobic CO at 25 degrees C. The reaction exhibited biphasic kinetics irrespective of NADPH concentration or of the molar ratio of reductase to P-450 LM4. The apparent first order rate constants for the fast and slow phases were determined to be 0.9 to 1.0 and 0.25 s-1, respectively. With the reductase and P-450 LM4 present in equimolar amounts, the total amount of P-450 LM4 reduced increased linearly with NADPH concentration; the titration gave a stoichiometry of 2 mol of NADPH per mol of reductase-cytochrome complex. The NADPH concentration had no appreciable effect on the magnitude of the first order rate constants for the fast and slow phases. The kinetics obtained in the presence of benzphetamine were essentially indistinguishable from those seen in the absence of this substrate, while the amount of P-450 LM4 reduced in the fast phase, but not the rate constant for this phase, decreased when phospholipid was omitted from the reaction mixture. Nearly maximal rates of NADPH oxidation by P-450 LM2 OR LM4 were obtained with a molar ratio of reductase to P-450 LM of 1.0. Benzphetamine enhanced the oxidation of NADPH by P-450 LM2 but had no effect on the activity of P-450 LM4. Rates of NADPH oxidation in the presence of P-450 LM2 and LM4 decreased by 80 and 40%, respectively, when phospholipid was omitted from the reconstituted enzyme system. These studies provide evidence for the formation of a catalytically functional 1:1 complex between the reductase and P-450 LM4, and indicate that P-450 LM2 and LM4 differ in their dependence on phospholipid.     

Effects of substrates on the heat stability and on the reactivities of thiol groups of 3-phosphoglycerate kinase

The kinetics of the reduction of cytochrome P-450 LM2 mediated by NADPH-cytochrome P-450 reductase in reconstituted phospholipid vesicles was examined. An inefficient reduction of the hemoprotein in phosphatidylcholine vesicles was observed. However, by introducing negatively charged phospholipids into the membrane, the rate of reduction increased in a concomitant manner to the resulting net negative charge of the vesicles. In the presence of benzphetamine, the extent of cytochrome P-450 LM2 reduced 1 s after the addition of NADPH to the system was a linear function of the electrophoretic mobilities of the vesicles used. A similar relationship between the net negative charge of the vesicles, as measured electrophoretically, and the reduction rate was also attained in the absence of substrate. The enhanced reduction was mainly reflected in an altered phase distribution of the reduction; the extent of fast phase reduction in the absence or in the presence of added substrate was dependent upon the electrophoretic mobilities of the vesicles. A similar change in the distribution of the reduction phases was observed upon decreasing the phosphatidylcholine content of the vesicles; the fast phase reduction being more pronounced in membranes with higher relative amounts of the protein components. A decrease of the rate of O-demethylation of p-nitroanisole catalyzed by P-450 LM2 parallel to the extent of fast phase reduction was observed upon dilution of neutral phosphatidylcholine membranes with phospholipid. By contrast, no effect of lipid dilution was evident in negatively charged membranes. The results are consistent with the hypothesis that the extent of fast phase reduction is governed by the amount of complex formed between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membranes; negative membranes appear to favor the formation of such complexes, whereas similar complexes are less formed, or are not functional, in neutral membranes.     

Relationship between state of aggregation and catalytic activity for cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase

The detergent 1-O-n-octyl-beta-D-glucopyranoside (octylglucoside) was found to replace the phospholipid requirement in the demethylation of benzphetamine by cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase purified from phenobarbital-treated rabbit liver. At low enzyme concentration (0.1 microM) in the absence of glycerol and phosphate, the maximum rate of benzphetamine-specific NADPH oxidation was approximately 35% of that observed in the presence of dilauroylglyceryl-3-phosphoryl choline. At higher enzyme concentration (2.5 microM) and in the presence of 0.15 M phosphate, 20% glycerol, octylglucoside was as effective as phospholipid in stimulating the production of formaldehyde from benzphetamine. The detergent concentration required for maximal enzymatic activity was 2.5-4.0 g/liter, depending on the cytochrome preparation used. At higher octylglucoside concentrations (5-7 g/liter), activity decreased to zero, although neither enzyme appeared to be irreversibly denatured at these detergent concentrations. Sedimentation equilibrium experiments with P-450LM2 alone or in the presence of equimolar reductase showed that increasing octylglucoside levels promoted disaggregation of the cytochrome. Pentamers and hexamers predominated at detergent concentrations where maximal activity was observed, while higher levels of detergent where activity was absent produced cytochrome dimers and, ultimately, monomers. The reductase was monomeric at detergent levels between at least 3 and 7 g/liter. Moreover, both gel filtration and sedimentation equilibrium experiments demonstrated that a stable complex between P-450LM2 and its reductase was not formed at octylglucoside concentrations where high activity was evident. These results are consistent with a model of P-450/reductase interaction in which functional aggregates of three to six cytochrome polypeptides move laterally in the microsomal membrane and interact with the reductase by random collision.     

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. II. Kinetic parameters of reductase and monooxygenase reactions.

The kinetic parameters of NADPH-dependent cytochrome P450 LM2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM2 reduction was found in the system. Vmax values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas Km values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio at a constant total protein concentration allowed the Kd of the NADPH-cytochrome P450 reductase/cytochrome P450 LM2 complex to be estimated as 6.4 +/- 0.5 microM. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.     

Studies on the microsomal mixed function oxidase system: redox properties of detergent-solubilized NADPH-cytochrome P-450 reductase.

Hepatic microsomal NADPH-cytochrome P-450 reductase was solubilized from rabbit liver microsomes in the presence of detergents and purified to homogeneity by column chromatography. The purified reductase had a molecular weight of 78 000 and contained 1 mol each of FAD and FMN per mol of enzyme. On reduction with NADPH in the presence of molecular oxygen, an 02-stable semiquinone containing one flavin free radical per two flavins was formed, in agreement with previous work on purified trypsin-solubilized reductase. The reduction of oxidized enzyme by NADPH, and autoxidation of NADPH-reduced enzyme by air, proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The reductase reduced cytochrome P-450 (from phenobarbital-treated rabbits) and cytochrome P-448 (from 3-methylcholanthrene-treated rabbits). The rate of reduction of cytochrome P-450 increased in the presence of a substrate, benzphetamine, but that of cytochrome P-448 did not.     

Modification of cytochrome P-450 with fluorescein isothiocyanate

Fluorescein isothiocyanate (FITC) has been shown to be selectively attached to the N-terminus of cytochrome P-450 LM2. The N-demethylase activity of cytochrome P-450 LM2 reconstituted systems modified in this way was inhibited by 25%. As revealed by CD measurements the overall conformation as well as the immediate heme environment of cytochrome P-450 LM2 remained unchanged after attachment of the FITC molecule. The binding affinity of modified cytochrome P-450 LM2 toward benzphetamine and aniline and the cumene hydroperoxide- or H2O2-supported N-demethylation of benzphetamine are maintained. However, the introduction of the electron via NADPH-cytochrome P-450 reductase (EC 1.6.2.4) is impaired after modification of the alpha-amino group. The extent of reduced modified cytochrome P-450 LM2 in the cytochrome P-450 reductase-supported reduction reaction is diminished and the half-time of the reduction is increased. The diminished reducibility is ascribed to steric hindrance of groups directly involved in the interaction between cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase or to blocking of the charge-pair interactions between the alpha-amino group of P-450 LM2 and the respective negatively charged group of NADPH-cytochrome P-450 reductase. By energy-transfer measurements distances between the heme and the alpha-amino group of 2.65 and 3.97 nm for the oligomeric and the monomeric forms of P-450 LM2, respectively, have been determined.     

Cytochrome C (Fe2+) as a competitive inhibitor of NADPH-dependent reduction of cytochrome P450 LM2: locating protein-protein interaction sites in microsomal electron carriers.

The kinetics of NADPH-dependent reduction of cytochrome P450 LM2 in the soluble monomeric reconstituted system in the absence of any substrate is shown to be monophasic. We show that ferrous cytochrome c acts as a competitive inhibitor of the reduction. In the presence of 1 mM benzphetamine an additional extremely fast phase was observed. Under these conditions ferrous cytochrome c was found to be a competitive inhibitor of the slow phase of the reduction process, which accounted for 80% of the total reduction amplitude. Inhibition experiments yield a dissociation constant for the LM2-reductase complex of 3.0 +/- 1.5 microM. This constant was the same both in the presence and in the absence of benzphetamine. Based on these data we conclude that cytochromes P450 and c bind to the same center on the NADPH-cytochrome P450 reductase molecule. Comparative analysis of the amino acid sequences reveals a detectable similarity between cytochrome c and cytochrome P450 LM2 at positions 68-87 and 121-145, respectively. In addition, a substantial similarity was shown for sequence fragments 204-224 of NADPH-cytochrome P450 reductase and 40-60 of cytochrome b5. Based on these findings a hypothesis for the location of the centers of intermolecular interactions on the molecules of cytochrome P450 LM2 and NADPH-cytochrome P450 reductase is proposed.     

Differences in the spectral interactions between NADPH-cytochrome P-450 reductase and a series of cytochrome P-450 enzymes

The interaction between NADPH-cytochrome P-450 reductase and a series of cytochrome P-450 isozymes was investigated using UV-visible spectrophotometry. In the absence of substrate the interactions between the reductase and RLM3, RLM5, and RLM5a were tight, exhibiting sub-micromolar dissociation constants and resulted in type I spectra of varying magnitude from which the following increases in the proportion of high spin hemoprotein were calculated; RLM3 (7%), RLM5 (36%), RLM5a (6%), LM2 (29%), RLM2 (0%). Preincubation of LM2 with its type I substrate benzphetamine increased the affinity of the cytochrome for the reductase. Using initial estimates of the P-450 spin states in the absence of reductase in conjunction with the spectral binding data and equations relating these parameters to the microequilibria for the association of reductase with high or low spin P-450, RLM3, RLM5, RLM5a and LM2 were shown to bind significantly more tightly to high spin P-450. The relevance of this data to the understanding of spin state influence on P-450 reduction is discussed.     

Stoichiometry of microsomal oxidation reactions. Distribution of redox-equivalents in monooxygenase and oxidase reactions catalyzed by cytochrome P-450

The stoichiometry of NADPH oxidation in rabbit liver microsomes was studied. It was shown that in uncoupled reactions cytochrome P-450, besides O2- generation catalyzes direct two- and four-electron reduction of O2 to produce H2O2 and water, respectively. With an increase in pH and ionic strength, the amount of O2 reduced via an one-electron route increases at the expense of the two-electron reaction. In parallel, with a rise in pH the steady-state concentration of the oxy-complex of cytochrome P-450 increases, while the synergism of NADPH and NADH action in the H2O2 formation reaction is replaced by competition. The four-electron reduction is markedly accelerated and becomes the main pathway of O2 reduction in the presence of a pseudo-substrate--perfluorohexane. Treatment of rabbit with phenobarbital, which induces the cytochrome P-450 isozyme specific to benzphetamine results in a 2-fold increase in the degree of coupling of NADPH and benzphetamine oxidation. The experimental results suggest that the ratio of reactions of one- and two-electron reduction of O2 is controlled by the ratio of rates of one- and two-electron reduction of cytochrome P-450. In the presence of pseudo-substrates cytochrome P-450 acts predominantly as a four-electron oxidase; one of possible reasons for the uncoupling of microsomal monooxygenase reactions is the multiplicity of cytochrome P-450 isozymes.     

Purified liver microsomal cytochrome P-450. Electron-accepting properties and oxidation-reduction potential.

The reduction of highly purified cytochrome P-450 from rabbit liver microsomes under anaerobic conditions requires 2 electrons per molecule. Similar results were obtained with dithionite, NADPH in the presence of NADPH-cytochrome P-450 reductase, or a photochemical system as the electron donor, with CO or other ligands, with substrate or phosphatidylcholine present, after denaturation to form cytochrome P-420, or with cytochrome P-450 partially purified from rat or mouse liver microsomes. The reduced cytochrome P-450 donates 2 electrons to dichlorophenolindophenol or to cytochrome c. Reoxidation of reduced cytochrome P-450 by molecular oxygen restores a state where 2 electrons from dithionite are required for re-reduction. Although these unexpected findings indicate the presence of an electron acceptor in addition to the heme iron atom, significant amounts of non-heme iron, other metals or cofactors, or disulfide bonds were not found, and free radicals were not detected by electron paramagnetic resonance spectrometry. Resolution of the cytochrome with acetone and acid yielded the apoenzyme, which did not accept electrons, and ferriprotoporphyrin IX, which accepted a single electron. A reconstituted hemoprotein preparation with the spectral characteristics of cytochrome P-420 accepted as much as 0.7 extra electron equivalent per heme. The midpoint oxidation-reduction potential of purified cytochrome P-450 from rabbit liver microsomes at pH 7.0 is -330 mv, and with CO present this value is changed to about -150 mv. The oxidation-reduction potential is unaffected by the presence of phosphatidylcholine or benzphetamine, a typical substrate. Laurate, aminopyrine, and benzphetamine undergo hydroxylation in the presence of chemically reduced cytochrome P-450 and molecular oxygen. Neither NADPH nor the reductase is required for substrate hydroxylation under these conditions.     

Cytochrome P-450 LM2 reduction. Substrate effects on the rate of reductase-LM2 association

The effect of substrate on LM2 reduction was examined using a reconstituted system containing dilauroylphosphatidylcholine, NADPH-cytochrome P-450 reductase, and cytochrome P-450 LM2 in a 160:1.5:1 molar ratio. In general, most substrates increased the rate constants of both the first and second phases of reduction as well as the fraction of LM2 reduced in the first phase. The correlation between the high spin content of the cytochrome and each of these kinetic parameters was weaker than expected if spin state controlled LM2 reduction. Further, substrate was shown to exert a rapid effect on both the high spin content and stimulation of reduction indicating that the low spin to high spin shift cannot be responsible for the slow phase of reduction for this particular isoform. Cytochrome P-450 reduction was also examined in both phospholipid-containing and soluble systems where the LM2 and reductase were not present as a preformed complex. In these systems the reactions were substantially slower than with the standard reconstituted system. Addition of substrate enhanced the rate of reduction, indicating that the rate of association between LM2 and the reductase was increased by substrate addition. The strong correlation between the rate of LM2 reduction in a preformed complex and the logarithm of the rate of LM2 and reductase association implicates the rate of functional complex formation as the factor controlling the slow phase of reduction.     

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

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

Zwitterionic detergent mediated interaction of purified cytochrome P-450LM4 from 5,6-benzoflavone-treated rabbits with MADPH-cytochrome P-450 reductase

Hydroxylation of acetanilide catalyzed by purified cytochrome P-450LM4 and NADPH-cytochrome P-450 reductase was reconstituted with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The optimum rate of production of 4-hydroxyacetanilide was observed between 3 and 7 mM CHAPS and was about half that with 0.05 mM dilauroylglyceryl-3-phosphocholine (di-12-GPC). At higher detergent concentrations, hydroxylase activity decreased until at 15-20 mM CHAPS the system was inactive. The effect of CHAPS on the state of aggregation of P-450LM4 and on interaction between the cytochrome and P-450 reductase alone and under turnover conditions was investigated by ultracentrifugation. At 4 mM CHAPS, P-450LM4 was hexameric to heptameric (Mr 369,000). Neither reductase nor reductase plus acetanilide and NADPH altered the state of P-450LM4 aggregation, suggesting that a stable 1:1 P-450/reductase complex did not form under turnover conditions. Replacing CHAPS with 0.05 mM di-12-GPC resulted in formation of heterogeneous P-450 oligomers (Mr greater than 480,000). At CHAPS concentrations where substrate hydroxylation did not occur (15 and 22 mM), P-450LM4 was shown by sedimentation equilibrium measurements to be dimeric and monomeric, respectively. P-450 reductase was shown to reduce monomeric P-450LM4 in the presence of NADPH. Thus, the dependence of hydroxylase activity on [CHAPS] may be related to the state of aggregation of the cytochrome. An apparent correlation between P-450 aggregation state and NADPH-supported hydroxylation was also observed with phenobarbital-inducible P-450LM2 in the presence of detergents [Dean, W.L., & Gray, R.D. (1982) J. Biol. Chem. 257, 14679-14685; Wagner, S.L., Dean, W.L., & Gray, R.D. (1984) J. Biol. Chem. 259, 2390-2395].     

Evidence for a second microsomal trans-2-enoyl coenzyme A reductase in rat liver. NADPH-specific short chain reductase

Evidence for the existence of a previously unknown rat hepatic microsomal reductase, short chain trans-2-enoyl-CoA reductase (SC reductase) is presented. This reductase has a specific requirement for NADPH, is unable to utilize NADH, and catalyzes the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA to butyric acid and hexenoic acid at a rate of 5 and 65 nmol per min per mg of microsomal protein, respectively. Highly purified NADPH cytochrome P-450 reductase incorporated into liposomes prepared from dilauroyl phosphatidylcholine in the presence or absence of cytochrome P-450 possesses no SC reductase activity. These liposomal preparations did, however, catalyze mixed function oxidations of benzphetamine and testosterone. Rabbit antibody to rat liver NADPH cytochrome P-450 reductase had little to no effect on the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA, suggesting that the SC reductase accepts reducing equivalents directly from NADPH. When acetoacetyl-CoA was incubated with hepatic microsomes and either NADH or NADPH, no formation of butyrate was detected; however, when both cofactors were present, a rate of formation of 3 nmol of butyrate was determined per min per mg of microsomal protein. These results suggest the presence of a previously unknown short chain beta-ketoreductase which catalyzes the reduction of short chain beta-keto acids, only in the presence of NADH. Our results also indicate that the electrons from NADH to the beta-ketoreductase bypass cytochrome b5. The physiological significance is discussed in terms of lipogenesis and ketone body utilization by the liver.     

Induction of polysubstrate monooxygenase and aflatoxin production by phenobarbitone in Aspergillus parasiticus NRRL 3240

The presence of the components of polysubstrate monooxygenase (PSMO) activity, viz., cytochrome P-450 and NADPH cytochrome P-450 reductase has been established for the first time in the microsomes of $\text{Aspergillus parasiticus}$. The microsomes were able to metabolize benzphetamine. NADPH cytochrome P-450 reductase, benzphetamine metabolism and aflatoxin production was increased by the presence of phenobarbitone (PB, 2mg/ml) in the medium. These results demonstrate that induction of PSMO activity could be a prerequisite for increased production of aflatoxins, since hydroxylation of intermediates is an obligatory step in aflatoxin biosynthesis.     

Involvement of FMN and phenobarbital cytochrome P-450 in stimulating a one-electron reductive denitrosation of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea catalyzed by NADPH-cytochrome P-450 reductase

Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism.     

Butylated hydroxyanisole-stimulated NADPH oxidase activity in rat liver microsomal fractions

NADPH-dependent oxygen utilization by liver microsomal fractions was stimulated by the addition of increasing concentrations of butylated hydroxyanisole concomitant with the inhibition of benzphetamine N-demethylase activity. The apparent conversion of monooxygenase activity to an oxidase-like activity in the presence of the antioxidant was correlated with the partial recovery of the reducing equivalents from NADPH in the form of increased hydrogen peroxide production. The progress curve of liver microsomal NADPH oxidase activity in the presence of butylated hydroxyanisole displayed a lag phase indicative of the formation of a metabolite capable of uncoupling the monooxygenase activity. Ethyl acetate extracts of microsomal reaction mixtures obtained in the presence of butylated hydroxyanisole, oxygen, and NADPH stimulated the NADPH oxidase activity of either liver microsomes or purified NADPH-cytochrome c (P-450) reductase. Using high performance liquid chromatography, gas chromatography, and mass spectrometry techniques, two metabolites of butylated hydroxyanisole, namely t-butylhydroquinone and t-butylquinone, were identified. The quinone metabolite and/or its 1-electron reduction product interact with the flavoprotein reductase to directly link the enzyme to the reduction of oxygen which results in an inhibition of the catalytic activity of the cytochrome P-450-dependent monooxygenase.     

Cytochrome P-450 from mitochondria of bovine adrenal cortex: comparison of cholesterol side-chain cleavage P-450 with steroid 11beta-hydroxylation P-450 and immunochemical cross-reactivity between adrenal mitochondrial and liver microsomal cytochromes P-450.

Adrenocortical mitochondrial cytochrome P-450 specific to the cholesterol side-chain cleavage (desmolase) reaction differs from that for the 11beta-hydroxylation reaction of deoxycorticosterone. The former cytochrome appears to be more loosely bound to the inner membrane than the latter. Upon ageing at 0 degrees C or by aerobic treatment with ferrous ions, the desmolase P-450 was more stable than the 11beta-hydroxylase P-450. By utilizing artificial hydroxylating agents such as cumene hydroperoxide, H2O2, and sodium periodate, the hydroxylation reaction of deoxycorticosterone to corticosterone in the absence of NADPH was observed to a comparable extent with the reaction in the presence of adrenodoxin reductase, adrenodoxin and NADPH. However, the hydroxylation reaction of cholesterol to pregnenolone was not supported by these artificial agents. Immunochemical cross-reactivity of bovine adrenal desmolase P-450 with rabbit liver microsomal P-450LM4 was also investigated. We found a weak but significant cross-reactivity between the adrenal mitochondrial P-450 and liver microsomal P-450LM4, indicating to some extent a homology between adrenal and liver cytochromes P-450.     

Chemical characterization of protein-protein interactions between cytochrome P-450 and cytochrome b5

Native cytochrome b5 interacts with either RLM5 or LM2 to form tight equimolar complexes (Kd = 250 and 540 nM, respectively) in which the content of high spin cytochrome P-450 was substantially increased. Cytochrome b5 caused 3- and 7-fold increases in the binding affinities of RLM5 and LM2 for benzphetamine, respectively, and benzphetamine decreased the apparent Kd for cytochrome b5 binding. Upon formation of the ternary complex between cytochromes P-450, b5, and benzphetamine the percentage of cytochrome P-450 in the high spin state was increased from 28 to 74 (RLM5) and from 9 to 85 (LM2). Cytochrome b5 caused 13- and 7-fold increases in the rate of RLM5- and LM2-dependent p-nitroanisole demethylation, respectively. Amino-modified (ethyl acetimidate or acetic anhydride) cytochrome b5 produced results similar to those obtained above with native cytochrome b5. In contrast, modification of as few as 5 mol of carboxyl groups/mol of amidinated cytochrome b5 resulted in both a substantial loss of the spectrally observed interactions with either cytochrome P-450 LM2 or cytochrome P-450 RLM5, and in a loss of the cytochrome b5-mediated stimulation of p-nitroanisole demethylation catalyzed by either monooxygenase. In further studies, native and fully acetylated cytochromes b5 reoxidized carbonmonoxy ferrous LM2 at least 20 times faster than amidinated, carboxyl-modified cytochrome b5 derivatives. In contrast, amidination, or acetylation of amino groups, or amidination of amino groups plus methylamidination of the carboxyl groups did not appreciably slow the rate of reduction of the cytochrome b5 by NADPH-cytochrome P-450 reductase. Collectively, the results provide strong evidence for an essential role of cytochrome b5 carboxyl groups in functional interactions with RLM5 and LM2.     

Formation of reactive oxygen species during one-electron reduction of noradrenochrome catalyzed by NADPH-cytochrome P-450 reductase

One-electron reduction of noradrenochrome catalyzed by NADPH-cytochrome P-450 reductase resulted primarily in the formation of o-semiquinone and, probably, also o-hydroquinone. Under aerobic conditions these reduced form(s) autoxidize, accompanied by the formation of reactive oxygen species, as revealed by continuous NADPH oxidation and oxygen consumption.

The presence of manganese-pyrophosphate complex contributed to autoxidation of the o-semiquinone during the reduction of noradrenochrome catalyzed by NADPH-cytochrome P-450 reductase, since the addition of the metal chelator diethylenetriamine pentaacetic acid (DETAPAC) resulted in a 34% inhibition of NADPH oxidation.

Oxygen in the ground state was found to be predominantly involved in the autoxidation of o-semiquinone during the reduction of noradrenochrome catalyzed by NADPH-cytocbrome P-450 reductase, since the addition of superoxide dismutase (SOD) to the incubation mixture only inhibited NADPH oxidation 13% and 6% in the absence and presence of DETAPAC, respectively.

The addition of catalase to the incubation mixture resulted in a slight increase in NADPH oxidation, both in the absence and in the presence of diethylenetriamine pentaacetic acid. However, no effect of catalase and SOD together on NADPH oxidation was observed, either in the absence or presence of DETAPAC.