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.     

Selective chemical modification of a functionally linked lysine in cytochrome P-450 LM2

Fluorescein isothiocyanate (FITC) has been selectively bound to the epsilon-amino group of lysine-382 in cytochrome P-450 LM2 (RH, reduced-flavoprotein: oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) at pH 8.15. Benzphetamine N-demethylase activity of the reconstituted FITC-modified cytochrome P-450 LM2 was inhibited by 25%. This inhibition has been shown to be due to an impaired electron transfer from the NADPH-cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) to the haemoprotein. The data indicate that cytochrome P-450 interacts with the flavoprotein via electrostatic interactions.     

Distance between lysine 384 and heme of cytochrome P-450 LM2 (P-450 IIB4) studied by fluorescence energy transfer measurements

The distance between FITC-modified lysine 384 of cytochrome P-450 LM2 and the active site, heme, was estimated by fluorescence energy transfer measurements. To avoid differential labelling of P-450 LM2 for protection of the alpha-amino group from FITC modification, deconvolution of measured fluorescence decay curves using a double exponential model was performed. A value of 2.7 nm was obtained for the distance FITC (lysine 384) - heme. This distance is too large to account for a direct electron tunneling from prosthetic group to prosthetic group at this interaction site between reductase and P-450 LM2.     

Purification and characterization of a new form of cytochrome P-450 (LM2b) from untreated male rabbit liver microsomes

A new form of cytochrome P-450 has been purified from untreated male rabbit liver microsomes. This form was designated P-450 LM2b on the basis of its electrophoretic mobility on SDS polyacrylamide gel, where it migrates as a polypeptide of apparent molecular weight of 50,250. This hemoprotein exhibits a maximum at 448.5 nm in the Soret band of the CO-Ferrous state spectrum. On the basis of its molecular, spectral, enzymologic and immunologic data, P-450 LM2b was shown to be distinct from the other P-450 forms, already characterized in rabbit liver microsomes. However P-450 LM2b and P-450 LM3b appear to be immunologically related proteins.     

Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450.

Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced animals, has a subunit molecular weight of 48,700. The best preparations contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone but is also present in phenobarbital-induced and untreated animals, was isolated from all three sources and found to have a subunit molecular weight of 55,300. The best preparations contain 17nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. Some of the purified preparations of the cytochromes, although electrophoretically homogeneous, contain apoenzyme due to heme loss during purification. The purified proteins contain no detectable NADPH-cytochrome P-450 reductase, cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of phospholipid (about 1 molecule per subunit). Amino acid analysis indicated that P-450LM2 and P-450LM4 are similar in composition, but the latter protein has about 60 additional residues. The COOH-terminal amino acid of P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that of P-450LM4 is lysine. NH2-terminal amino acid residues could not be detected. Carbohydrate analysis indicated that both cytochromes contain 1 residue of glucosamine and 2 of mannose per polypeptide subunit. The optical spectra of the oxidized and reduced cytochromes and carbon monoxide complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and 418 nm characteristic of a low spin hemeprotein, and P450LM4 from beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes has maxima at 645 and 394 nm, characteristic of the high spin state. The spectrum of -450lm4 becomes similar to that of P-450LM2 at high protein concentrations or upon the addition of detergent (Renex), whereas the spectrum of P-450LM2 is unaffected by the protein concentration or the presence of detergent. Electron paramagnetic resonance spectrometry of the purified cytochromes indicated that oxidized -450lm2 is in the low spin state, whereas P-450LM4 is largely, but not entirely, in the high spin state.     

Cytochrome P-450 in proteoliposomes: oligomeric structure of the LM2 isoform

Crosslinking of protein molecules with bifunctional reagents and subsequent electrophoresis of the modified proteins revealed the presence of cytochrome P-450 LM 2 oligomers in proteoliposome membranes obtained in different ways and differing in their phospholipid composition. Data from a comparative analysis of cytochrome P-450 oligomeric structures in solution and in membrane are suggestive of the hexameric organization of cytochrome P-450 LM 2 within proteoliposome membranes.     

High pressure induced inactivation of ferrous cytochrome P-450 LM2 (IIB4) CO complex: evidence for the presence of two conformers in the oligomer.

The effect of high pressure on the spectral properties of cytochrome P-450 LM2(Fe2+)-CO complex was studied. The application of high pressure was shown to induce the conversion of cytochrome P-450 to P-420. In the solution when P-450 was oligomeric only about 65% of the total converted to P-420. The remaining portion of cytochrome P-450 was stable at pressures up to 6 kbar. When P-450 was incorporated into membranes or when it was succinylated, the proportion of the pressure sensitive fraction was slightly higher (about 75%). Dissociation of P-450 oligomers into monomers was made by addition of 0.2% Triton N-101. Monomers were the most sensitive to pressure; they could be completely converted to P-420. These results have been interpreted as evidence for the existence of two different conformers of P-450 LM2, which differ in pressure stability. Splitting between these two states appears to be a result of the oligomeric organization of cytochrome P-450 in solution and in the membrane.     

Immunochemical characteristics of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria

Highly specific antibodies against hemeprotein were obtained by immunizing rabbits with a highly purified cholesterol-hydroxylating cytochrome P-450scc from adrenocortical mitochondria. The antibodies do not specifically interact with other components of the adrenocortical electron transport chain, e. g., adrenodoxin reductase and adrenodoxin. Using double immunodiffusion technique (Ouchterlony method), it was shown that the antibodies did not precipitate the microsomal cytochromes P-450 LM2 and LM4, cytochrome b5 and 11 beta-hydroxylating cytochrome P-450 from adrenocortical mitochondria. Antibodies against cytochrome P-450scc inhibited the cholesterol side chain cleavage activity of cytochrome P-450scc in a reconstituted system. Limited proteolysis with trypsin and immunoelectrophoresis in the presence of specific antibodies revealed that antigenic determinants are present of the heme-containing catalytic domain of cytochrome P-450scc (F1) as well as on the domain responsible for the interaction with the phospholipid membrane (F2).     

Secondary structure prediction of liver microsomal cytochrome P-450; proposed model of spatial arrangement in a membrane

The secondary structure of rabbit liver microsomal cytochrome P-450 LM2, rat liver microsomal cytochromes P-450b and P-450e (phenobarbital-inducible), and rat liver microsomal cytochromes P-450c, P-450d (3-methylcholanthrene-inducible) was predicted by a combination of methods (i) identifying the transmembrane parts of integral membrane proteins, and (ii) statistically predicting the secondary structure of globular proteins. The results are similar for all phenobarbital-inducible enzymes and make it possible to construct two structural models with seven or four transmembrane alpha-helices. The cytochromes of the second group obviously form a second structural family with four membrane-spanning alpha-helices. In both cases, a large ectodomain with several consecutive alpha-helices, which may provide the heme-binding pocket, is exposed out of the membrane.     

Association of cytochrome b5 and cytochrome P-450 reductase with cytochrome P-450 in the membrane of reconstituted vesicles

A protein-protein association of cytochrome P-450 LM2 with NADPH-cytochrome P-450 reductase, with cytochrome b5, and with both proteins was demonstrated in reconstituted phospholipid vesicles by magnetic circular dichroism difference spectra. A 23% decrease in the absolute intensity of the Soret band of the magnetic CD spectrum of cytochrome P-450 was observed when it was reconstituted with reductase. A difference spectrum corresponding to a 7% decrease in absolute intensity was obtained when cytochrome b5 was incorporated into vesicles that already contained cytochrome P-450 and cytochrome P-450 reductase compared to a decrease of 13% in absolute intensity when cytochrome b5 was incorporated into vesicles that contained only cytochrome P-450. The use of the magnetic circular dichroism confirmed that protein-protein associations that have been detected by absorption spectroscopy between purified and detergent-solubilized proteins also exist in membranes. High ionic strength was shown to interrupt direct electron flow from cytochrome P-450 reductase to cytochrome P-450 but not the electron flow from reductase through cytochrome b5 to cytochrome P-450. Upon incorporation of cytochrome b5 into cytochrome P-450- and cytochrome P-450 reductase-containing vesicles, an increase of benzphetamine N-demethylation activity was observed. The magnitude of this increase was numerically identical to the residual activity of the reconstituted vesicles measured in the presence of 0.3 M KCl. It is concluded that there is a requirement for at least one charge pairing for electron transfer from reductase to cytochrome P-450. These observations are combined in a proposed mechanism of coupled reversible association reactions in the membrane.     

The study of the active site of cytochrome P-450 LM2 using the chemical modification of tyrosine residues by tetranitromethane

Selective chemical modification of the hemoprotein by tetranitromethane was used in order to elucidate the functional role of tyrosine residues in the cytochrome P-450 LM2 molecule. It was shown that the degree of cytochrome P-450 LM2 modification can be determined, using the second derivative of the UV absorption spectra. Modification of one tyrosine residue resulted in the inactivation of cytochrome P-450 LM2. Nitration of the cytochrome was accompanied by changes in the spectral properties of the hemoprotein with the formation of spectra typical of hyperporphyrin structures, thus suggesting the involvement of tyrosine residues in the formation of the active center of cytochrome P-450 LM2.     

The identification of the heat-stable microsomal protein required for methoxyflurane metabolism as cytochrome b5

Methoxyflurane is an anesthetic whose metabolism by cytochrome P-450LM2 has been shown to be dependent upon a heat-stable microsomal protein (Canova-Davis, E., and Waskell, L. A. (1982) Biochem. Biophys. Res. Commun. 108, 1264-1270). Treatment of this protein with diethylpyrocarbonate, which modifies selected amino acids, caused a dose-dependent loss in its ability to effect the metabolism of methoxyflurane by purified cytochrome P-450LM2. This protein factor has been identified as cytochrome b5 by demonstrating that cytochrome b5 and the heat-stable factor coelute during cytochrome b5 purification. Neither ferriheme nor apocytochrome b5 was able to substitute for the activating factor, while cytochrome b5 reconstituted from apocytochrome b5 and heme exhibited an activity similar to that of native b5. Examination of the cytochrome b5 molecule by computer graphics suggested that diethylpyrocarbonate did not inactivate b5 by reacting with the anionic surface of the cytochrome b5 molecule. Maximal rates of methoxyflurane metabolism were obtained at a ratio of 1:1:1 of the three proteins, cytochrome P-450LM2:reductase:cytochrome b5. In summary, it has been demonstrated that the heat-stable protein, cytochrome b5, is obligatory for the metabolism of methoxyflurane by cytochrome P-450LM2. These data also suggest that cytochrome b5 may be acting as an electron donor to P-450LM2 in the O-demethylation of methoxyflurane.     

Fluorescent energy transfer measurements on fluorescein isothiocyanate modified cytochrome P-450 LM2

The distance between the heme iron and the N-terminus of cytochrome P-450 LM2 was determined by fluorescence energy transfer measurements. Fluorescein isothiocyanate which was covalently bound to the N-terminal methionine was used as donor chromophor. The Ro value between fluorescein isothiocyanate and the heme was calculated to be 3.98 nm. The distance between the nitrogen of the N-terminal methionine and the heme was estimated with 2.84 +/- 0.23 nm excluding most likely the N-terminal amino acid of cytochrome P-450 LM2 to participate in the electron transfer to the heme iron. A cytochrome P-450 LM2 membrane model is proposed.     

Influence of N,N-dimethylaniline on the association of phenobarbital-induced cytochrome P-450 and NADPH-cytochrome c(P-450) reductase in a reconstituted rabbit liver microsomal enzyme system

N,N-Dimethylaniline when added to reaction mixtures provokes deviation from Michaelis-Menten law of the interaction kinetics of NADPH-cytochrome c(P-450) reductase (NADPH:ferrihaemoprotein oxidoreductase, EC 1.6.2.4) with highly purified phenobarbital-induced rabbit liver microsomal cytochrome P-450 (P-450LM2). This phenomenon is not associated with the low-to-high spin transition in the iron-coordination sphere of the haemoprotein, as elicited by the arylamine. Substrate-triggered departure from linearity of the kinetics is abolished by inclusion into the assay media of p-chloromercuribenzoate, hinting at a vital role in the process of thiols. Similarly, the parabolic progress curve (nH = 1.7) is transformed to a straight line (nH = 1.01) when the N-terminal reductase-binding domain in the P-450LM2 molecule is selectively blocked through covalent attachment of fluorescein isothiocyanate (FITC); such a modification does not alter the affinity of the haemoprotein for the amine substrate. Steady-state fluorescence polarization measurements reveal that N,N-dimethylaniline perturbs the motional properties of the fluorophore-bearing reductase-binding region, suggesting the induction of a conformational change. Summarizing these results, the data possibly indicate N,N-dimethylaniline-induced cooperativity in the association of reductase with P-450LM2.     

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.     

Cytochrome P-450 isozyme LM3b from rabbit liver microsomes. Induction by triacetyloleandomycin purification and characterization

Oral administration of triacetyloleandomycin (TAO), 1 mmol/kg/day for 7 days to mature male New Zealand White rabbit results in a significant increase in the content of liver microsomal cytochrome P-450. This increase is accompanied by the occurrence on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the microsomes of a strong band in the zone of electrophoretic mobility associated with the LM3 isozymes and the stimulation of a number of monooxygenase activities of these microsomes including aminopyrine, chlorcyclizine, TAO, and erythromycin demethylation as well as 2-OH-estradiol and 6 beta OH-testosterone hydroxylation. Cytochrome P-450 LM3 (TAO) from these liver microsomes, purified to electrophoretic homogeneity, had Mr = 52,000 as determined by calibrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison with isozymes LM3a, LM3b, and LM3c isolated from control animals, by a number of criteria including spectral data, amino acid content, NH2-terminal sequence analysis, peptide mapping, immunological properties, and monooxygenase activities of reconstituted system, indicated that isozymes LM3 (TAO) and LM3b are very similar, if not identical, proteins. We conclude that TAO must be considered as a new type of inducer of microsomal cytochrome P-450 from rabbit liver.     

On the membrane topology of vertebrate cytochrome P-450 proteins

Hydropathy profiles of 34 aligned cytochrome P-450 sequences were compared to identify potential transmembrane segments. Eleven regions with the potential to cross a membrane in at least some P-450 sequences were detected. The known sidedness of several residues and peptides was used to eliminate some of these regions from consideration. Further arguments based on the location and orientation of the heme relative to the membrane excluded others. This process of elimination was continued until only two regions remained. These two segments, present in the first 66 amino acids of the P-450 NH2 termini, are proposed as the only transmembrane peptides of vertebrate microsomal P-450s. Mitochondrial P-450s may have a different membrane association. The three-dimensional structure of cytochrome P-450cam was examined for the location of conserved charged residues. These residues occurred mainly on the opposite surface from the substrate-binding site and along the edges of the flat triangular P-450cam. A model is proposed for vertebrate microsomal P-450s that is similar to P-450cam. The substrate-binding site faces the membrane, the heme is parallel to the membrane surface, and two NH2-terminal transmembrane segments anchor the protein to the bilayer.     

Membrane translocation and insertion of NH2-terminally anchored gamma-glutamyl transpeptidase require a signal recognition particle

The two subunits of the renal brush border enzyme, gamma-glutamyl transpeptidase (EC 2.3.2.2), are derived from a single-chain propeptide. The membrane-spanning domain consists of a hydrophobic sequence near its NH2-terminus and the protein is oriented with its NH2-terminus on the cytoplasmic side. The enzyme is synthesized without a cleavable signal sequence. Translocation and insertion of this enzyme have been shown to be dependent on the signal recognition particle and presumably require the same translocation machinery that other secretory and membrane proteins use for these processes.     

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.     

Catalytic properties of the liver microsomal hydroxylase system in reconstituted phospholipid vesicles.

Two types of cytochrome P-450, P-450LM2 and P-450LM3, have been purified from rabbit liver microsomes and incorporated into phospholipid vesicles by a cholate gel filtration technique together with purified preparations of NADPH-cytochrome P-450 reductase. The catalytic properties of the vesicles have been compared with a system reconstituted with small amounts of dilauroylphosphatidylcholine (DLPC). 6 beta-Hydroxylation of androstenedione proceeded at a rate 10 times higher in the vesicles compared to the DLPC-system. The kinetics for the reaction were the same in the vesicles as in intact microsomes i.e. sigmoidal substrate curves were obtained and Hill-coefficients of about 1.4 were calculated in these systems. In contrast, Michaelis-Menten kinetics were obtained for 6 beta-hydroxylation in the DLPC-system. The results could indicate cooperativity between different P-450 molecules in the intact membrane but not in the DLPC-system. P-450LM2-catalyzed 16-hydroxylation of androstenedione was in contrast to the situation with P-450LM3 inhibited in the vesicles as compared to the DLPC system. It is suggested that for evaluation of substrate specificity and other properties of different types of liver microsomal P-450, phospholipid vesicles may be a more relevant integration level than the DLPC-system.