Reduction and catalytic properties of cytochrome P-450 LM2 in reconstituted system containing monomeric carriers

The membrane microsomal monooxygenase system can be reconstituted in solution from NADPH-specific flavoprotein and cytochrome P-450 which exist in the monomeric state in the presence of Emulgen 913 at molar ratio of the proteins and detergent of 1:1:300. Oxidized and dithionite-reduced monomers of cytochrome P-450 were much less thermostable than its initial aggregates, while thermal stability of NADPH-specific flavoprotein did not depend on its aggregation state. Binding spectra of cytochrome P-450 monomers with benzphetamine were atypical and had an absorbance minimum at 422 nm only. The addition of benzphetamine and/or flavoprotein to cytochrome P-450 monomers did not cause the spin equilibrium shift and the low-spin form content was higher than 85% in all cases. Investigation of the dependence of the initial rates of NADPH-dependent cytochrome P-450 reduction and benzphetamine oxidation on the stoichiometry of the flavoprotein and cytochrome P-450 at their constant total concentration showed that the molar ratio of 1:1 was required for maximal activity. Thus this system works in full accordance with the mass action law.     

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.     

Relation between the structure of benzphetamine analogues and their binding properties to cytochrome P-450 LM2

Twelve substrates of a homologous series of tertiary amines (type I substrates) have been reacted with cytochrome P-450 LM2 incorporated into unilamellar liposomes and in soluble form. The apparent spectral dissociation constants (Ks) of the substrate enzyme complexes and the induced high-spin shifts have been correlated with the electron density of distinct carbon atoms as monitored by 13C-NMR chemical shifts, the solubility of the amines and steric parameters of the substrate molecules. The results obtained led to the conclusion that two different intrinsic properties of the substrates can be discriminated in relation to the substrate-enzyme interaction. A diminished electron density at the nitrogen atom is accompanied by an increased binding affinity. The steric structure of the respective substrate determines its capability to shift the spin equilibrium to the high-spin state. Some characteristics of the active center of the enzyme are derived from the evidenced properties of the substrates.     

Effect of a zwitterionic detergent on the state of aggregation and catalytic activity of cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase

The zwitterionic detergent 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate (CHAPS) supports reconstituted cyclohexane hydroxylase activity of cytochrome P-450LM2 and NADPH-cytochrome reductase purified from phenobarbital-induced rabbit liver. Maximum activity (approximately 50% of that with phospholipid) was observed at 2 mM CHAPS. Inhibition took place at higher CHAPS, until at 20 mM CHAPS, no cyclohexane hydroxylase activity was observed. There was little denaturation of the two enzymes under these conditions. At 2 mM CHAPS, P-450LM2 was pentameric (Mr = 250,000) and reductase was dimeric (Mr = 139,500) by sedimentation equilibrium. P-450 was monomeric in 20 mM CHAPS. In addition, a stable complex between the two enzymes was not detected under conditions of maximum activity, even in the presence of saturating substrate. This confirms our previous conclusion that a stable complex between cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase is not a prerequisite for reconstituted xenobiotic hydroxylation (Dean, W. L., and Gray, R. D. (1982) J. Biol. Chem. 257, 14679-14685). Difference spectra of ferric P-450LM2 revealed that below 5 mM CHAPS, the high spin form of the cytochrome was slightly stabilized, while higher CHAPS levels stabilized the low spin form. Monomeric P-450LM2 formed with 20 mM CHAPS catalyzed the hydroxylation of toluene by cumene hydroperoxide. Thus, the reason that monomeric cytochrome P-450LM2 was inactive in NADPH-supported hydroxylation may either be because the bound detergent blocked productive interaction of the cytochrome with reductase or the monomer may be intrinsically incapable of interaction with reductase.     

ESR-spectroscopy of cytochrome P-450 LM2 in the soluble and membrane-bound state

Influence of microenvironment on the structure of spin-labeled cytochrome P-450 LM2 was studied. The distance between the spin-label which is covalently bound to cysteine-152 and heme ferrum in soluble protein is 17 A and 23 A in the membrane-bound one. The spin-label was exposed in water solution in both cases. It was shown that solubilized cytochrome P-450 LM2 in water solution forms the aggregates consisting of 4-6 monomers.     

Retinoic acid metabolism by a system reconstituted with cytochrome P-450

Feeding rats with a diet containing a hundred times the normal amount of vitamin A resulted, within 2 to 3 weeks, in an increase in total hepatic microsomal cytochrome P-450 content. This was associated, in isolated microsomes, with an enhanced conversion of all-trans-retinoic acid to polar metabolites, including a two- to threefold increased production of 4-hydroxy- and 4-oxo-retinoic acid, whether expressed per microsomal protein or per cytochrome P-450. Unlike effects of other inducers (e.g., phenobarbital or methylcholanthrene), activities of benzphetamine, aminopyrine, and ethylmorphine demethylases or benzopyrene hydroxylase were not increased. Furthermore, the CO-reduced difference spectral peak was shifted towards 449 nm. On sodium dodecyl sulfate-gel electrophoresis, one band was increased with electrophoretic mobility identical to that of cytochrome P-450f, a recently isolated new form which has a CO-reduced difference spectral peak at 448 nm. In a system reconstituted with NADPH-cytochrome P-450 reductase, NADPH, and phospholipid, purified cytochromes P-450f and b were discovered to promote conversion of retinoic acid to polar metabolites, including 4-hydroxy-retinoic acid.     

Phosphorylation of microsome-bound cytochrome P-450 LM2

The phosphorylation of a microsomal protein of rabbit liver by catalytic subunit of cyclic AMP-dependent protein kinase was shown, and the protein was identified as cytochrome P-450 LM2 on basis of comparative peptide-mapping. Acid hydrolysis of microsome-bound phosphorylated cytochrome P-450 revealed that phosphorylation occurred exclusively on serine residues. This serine residue was identified as the same residue phosphorylated in purified, soluble P-450, that is, serine in position 128.     

Cytochrome b5 as electron donor to rabbit liver cytochrome P-450LM2 in reconstituted phospholipid vesicles

When incorporated into phospholipid vesicles containing NADPH-cytochrome P-450 reductase and P-450_LM2, cytochrome b₅ enhanced the rate of NADPH-supported hydroxylation of 7-ethoxycoumarin or $\text{p}$-nitroanisole about 5-fold. Cytochrome b₅ did not affect the rate of NADPH-oxidation, nor the rate of NADPH-supported formation of the ferrous CO-complex of cytochrome P-450. However, the cytochrome b₅-mediated increase in product formation was found to be correlated with concomitant decreases in the production of H₂O₂ or $\text{O}{}_2{}^{\mathrm{?}}$ in the system, thus strongly indicating cytochrome b₅ being a more efficient donor of the second electron to cytochrome P-450 than is NADPH-cytochrome P-450 reductase.     

Active site model of cytochrome P-450 LM2

Based on (i) a detailed analysis of the physicochemical properties of selected benzphetamine derived substrates and (ii) the identification of Tyr-380 as active site residue trans to thiolate theoretical studies (computer aided molecular design) revealed a model of the substrate binding site of cytochrome P-450 LM2. The results indicate that substrates with a butterfly-like bulky conformation exhibit the highest intrinsic activity. Those substrates which preferably exist in an extended conformation are sterically hindered to intensively interact with the binding site which is demonstrated by computer graphics.     

The effect of NADPH concentration on the reduction of cytochrome P-450 LM2

Cytochrome P-450 LM2 reduction was measured at a series of NADPH concentrations in the absence of substrate and in the presence of 1 mM benzphetamine. In the absence of substrate reduction could be described as a biphasic process with 55% of the reaction occurring in the first phase (at 20 microM NADPH). When benzphetamine was present, the fraction of the reaction occurring in the first phase was increased to 91%. When examined either in the absence or presence of benzphetamine, the rate constant and fraction of LM2 reduced in the fast phase were decreased as the NADPH concentration was decreased. In each case the fraction of LM2 reduced in the second phase was not substantially altered over the NADPH concentrations examined. To explain the effect of NADPH concentration on the initial rate of LM2 reduction, the effect of NADPH on the reduction of NADPH-cytochrome P-450 reductase was examined. Due to the presence of two flavins within each reductase molecule, there would be nine possible oxidation-reduction states of the reductase which may be present at a given NADPH concentration. Based on the redox potentials for the flavin half-reactions and for NADPH oxidation, the relative concentrations of each of the reductase subspecies could be determined. Rate constants were assigned for the reduction of LM2 by the various reductase subspecies, and the theoretical initial rates of LM2 reduction at various NADPH concentrations were compared with values obtained experimentally. The experimental data are consistent with a model where, under the conditions of this assay, the fully reduced reductase is the form primarily responsible for the reduction of LM2.     

Multiple catalytic properties of the purified and reconstituted cytochrome P-450 (P-450sccII) system of pig testis microsomes

The catalytic properties of the testis microsomal P-450, termed P-450sccII, have been studied in a refined assay system which consists of P-450sccII (13 nmol of P-450 heme/mg of protein) and its reductase has been purified extensively from pig testis. The results indicated that P-450sccII was highly active in catalyzing hydroxylation of 11 beta-hydroxyprogesterone at the 17 alpha-position to give 21-deoxycortisol and cleavage of 17 alpha-hydroxyprogesterone at the 17-20 bond to give androstenedione with turnover numbers of 25 and 30 mol/min X mol of P-450, respectively. In contrast, many physiologically important corticosteroids we tested were found to be poor substrates for both the hydroxylase and lyase reactions. The possible reason for the importance of these substrate specificity of P-450sccII in production of both corticosteroids and androgens in the endocrine tissues is discussed. P-450sccII also catalyzed conversion of testosterone to androstenedione, but 18O experiments failed to show incorporation of atmospheric oxygen into the androstenedione formed. However, this does not preclude the possibility that the P-450-bound intermediate gem-diol stereoselectively dehydrates to give the nonlabeled ketosteroid. In addition to these steroid-oxidizing activities, P-450sccII revealed considerable specificities toward various xenobiotics, suggesting that P-450sccII and liver microsomal P-450 are basically similar as regards enzymatic functions and activities.     

Spin state transitions of liver microsomal cytochrome P-450.

Spin state transitions of membrane-bound cytochrome P-450 were investigated by difference spectrophotometry using the 'D'-charge transfer absorbance band at 645 nm as a measure of the amount of hemin iron present in the 5-coordinated state. The magnitude of the 'D'-absorbance band in the absence of exogenous substrates, e.g., the concentration of native high spin cytochrome P-450, was evaluated from the difference in absorbance at 645 nm between ferric cytochrome P-450 and the carbon monoxide derivative of the pigment in its ferrous state. The contribution of the native high spin species to the total cytochrome P-450 content of microsomes was calculated to be between 40% and 65% after induction with phenobarbital and polycyclic hydrocarbons, respectively. Up to 80% of the cytochrome P-450 was found to be present in the high spin state after the addition of exogenous substrates. Further, the steady state concentrations of high spin cytochrome P-450, observed in the presence of reduced pyridine nucleotides, suggest that the rate limiting step for microsomal mixed function oxidation reactions is variable and dependent on the substrate under investigation.     

Ring-andN-hydroxylation of 2-acetamidofluorene by rat liver reconstituted cytochrome P-450 enzyme system.

The N- and ring-hydroxylation of 2-acetamidofluorene were studied with a reconstituted cytochrome P-450 enzyme from microsomal fractions of liver from both control and 3-methylcholanthrene-pretreated rats. Proteinase treatment and Triton X-100 solubilization were two important steps for partial purification of the cytochrome P-450 fraction. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions were required for optimum N- and ring-hydroxylation activity. Hydroxylation activity was determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from pretreated animals was severalfold more active than the fraction from controls. Formation of N-hydroxylated metabolites with reconstituted systems from both control and pretreated animals was greater than that with their respective whole microsomal fractions.     

Mechanism of rate control of the NADPH-dependent reduction of cytochrome P-450 by lipids in reconstituted phospholipid vesicles

The NADPH-supported reduction of cytochrome P-450 LM2 (liver microsomal isozyme 2) in reconstituted phospholipid vesicles in general exhibits two-exponential kinetics. The physiologically relevant rapid partial reaction is favoured in amount with increasing reductase/P-450 ratio. A lipid specificity was observed in that negatively charged lipids favour that process, too. The rate constant increases concomitantly. The data are consistent with the formation of a reactive 1:1 complex the amount of which determines the rate constant. The dissociation constants amount to 0.048 microM for a microsomal lipid extract, 0.051 microM for a 3:1 (w/w) mixture of dioleoylglycerophosphoethanolamine and phosphatidylserine, and 0.47 microM for dioleoylglycerophosphocholine, respectively, in the respective reconstituted systems. At low reductase/P-450 ratio the amount of the rapidly reduced P-450 exceeds the equilibrium concentration of a 1:1 complex. Preformed 1:1 associates, therefore, cannot fit the derived mechanism. Instead, a cluster model based on P-450 association does correspond to the data.     

Spin equilibrium relaxation kinetics of cytochrome P450 LM2

The relaxation kinetics of the temperature-dependent spin equilibrium of cytochrome P450 LM2 has been resolved by means of a laser temperature-jump device. The Nd laser system in the Q-switched mode delivers 3 J/25 ns pulses which were Raman shifted to 1.89 μm by use of a high pressure H₂-gas cell. The LM2 equilibrium relaxation proceeds in the ns-range, the rate constants of the on- and off-reactions amount to about 10⁶ s^?1 in correspondence with respective processes of other hemoproteins and protein-free model compounds. Benzphetamine increases the on-rate constant (low-spin → high-spin) about 5 fold, the off-process is nearly unchanged.     

Reduction of hexavalent chromium in a reconstituted system of cytochrome P-450 and cytochrome b5

The reduction of hexavalent chromium (Cr(VI] by the monooxygenase components was studied. Both a reconstituted system of cytochrome P-450 (P-450) and cytochrome b5 (b5) with NADPH was capable of reducing Na2CrO4 (30 microM) provided anaerobic atmosphere. The rates were 1.29 nmol Cr.min-1 nmol P-450(-1) and 0.73 nmol Cr.min-1 nmol b5(-1). Using NADH instead of NADPH gave very low reducing activities, confirming the enzymic nature of the P-450 dependent Cr(VI) reductase reaction. Oxygen, 22% (air) and 0.1% gave 89% and 69% inhibition of Cr(VI) reducing activity, respectively. Carbon monoxide (100%) caused an inhibition of about 37% and 44% for P-450 and b5, respectively. Externally added flavin mononucleotide (FMN) (3 microM) or Fe-ADP (10 microM) to the complete system stimulated the enzymatic reaction about 2-fold and 3-fold, respectively.