Microsomal Cytochrome P-450 LM2 Research Articles

Conformational analysis of mitochondrial and microsomal cytochrome P-450 by resonance Raman spectroscopy.

Mitochondrial and microsomal cytochromes P-450SCC and P-450LM2 in the ferric substrate-free and substrate-bound states were studied by resonance Raman spectroscopy. In the spectra of cytochrome P-450SCC two conformational states (A and B) were detected, each of them constituting an equilibrium between a six-coordinated low-spin and a high-spin form. Both the conformational and the spin equilibria are pH- and temperature-dependent, which is in line with previously published results [Lange, R., Larroque, C., & Anzenbacher, P. (1992) Eur. J. Biochem. 207, 69-73)]. On the basis of well-resolved resonance Raman spectra, measured at different pH and temperatures, these equilibria were analyzed quantitatively. Both low-spin configurations of A and B exhibit different band patterns in the spin state marker band region, indicating differences in the active-site structures. While in the high-spin configuration of state A the heme iron remains weakly bound by a sixth ligand, the high-spin form of state B is five-coordinated. Binding of cholesterol to cytochrome P-450SCC causes a significant population of the high-spin forms, particularly of state A (62%). On the other hand, binding of 22R-hydroxycholesterol to the substrate-free enzyme leaves the overall spin equilibrium largely unchanged, i.e., six-coordinated low spin (76% A and 24% B). In both substrate-bound complexes, interactions between the substrate and the heme lead to small but distinct differences in the resonance Raman spectra of the low-spin form of state A. In contrast to cytochrome P-450SCC, the resonance Raman spectra of microsomal cytochrome P-450LM2 provide no indications for multiple conformers at 22 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein-protein interactions in microsomal cytochrome P-450 isozyme LM2 and their effect on substrate binding.

The effects of protein-protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P-450 LM2 have been analyzed by resonance Raman spectroscopy of the monomeric and oligomeric protein in solution. Also H2O2-dependent catalytic activities of the two states have been compared. The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P-450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P-450 LM2, benzphetamine binding to the oligomers of this isozyme additionally produces a shift of the spin-state equilibrium. This indicates that in the oligomer the substrate-binding pocket is converted by protein-protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five-coordinated high-spin configuration. In the monomer the substrate-binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P-450 LM2 produces essentially the same resonance Raman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein-protein interactions. H2O2-dependent N-demethylation of benzphetamine and aniline p-hydroxylation by cytochrome P-450 LM2 did not depend on its state of aggregation.

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-450 b and P-450 e (phenobarbital-inducible), and rat liver microsomal cytochromes P-450 c, P-450 d (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 α-helices. The cytochromes of the second group obviously form a second structural family with four membrane-spanning α-helices. In both cases, a large ectodomain with several consecutive α-helices, which may provide the heme-binding pocket, is exposed out of the membrane.

Heme release from rabbit liver microsomal cytochrome P-450 LM2.

Heme release from rabbit liver microsomal cytochrome P-450 LM2.

Effects of detergent on substrate binding and spin state of purified liver microsomal cytochrome P-450LM2 from phenobarbital-treated rabbits.

Spectral changes accompanying the binding of the nonionic detergent n-octyl beta-D-glucopyranoside (n-octyl glucoside) to cytochrome P-450LM2 purified from liver microsomes of phenobarbital-treated rabbits have been compared to changes in catalytic activity obtained in a reconstituted system consisting of various levels of detergent, P-450LM2, and NADPH-cytochrome P-450 reductase. In the absence of substrate and reductase, addition of n-octyl glucoside to 2-3 mM resulted in a difference spectrum (detergent-bound minus detergent-free cytochrome) characterized by a small maximum at 390 nm and a minimum at 410 nm, suggestive of a slight stabilization of the high-spin (S = 5/2) state of the cytochrome. As the detergent concentration was increased to 4-8 mM (corresponding to maximal activity and pentameric or hexameric P-450), a new peak appeared at 427 nm while the minimum remained at 410 nm. Between 10 and 30 mM n-octyl glucoside (conditions which produced catalytically inactive and monomeric P-450) the minimum in the difference spectrum shifted to 390 nm and the maximum to 425 nm, characteristic of a shift in spin equilibrium toward low-spin (S = 1/2) cytochrome. At low and high detergent concentrations, substrate [d-benzphetamine with n-octyl glucoside or cyclohexane with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)] was bound to P-450LM2 with formation of high-spin P-450, although the increase in high-spin cytochrome was less at high detergent levels than at low. The affinity of P-450 for substrate decreased by 2-3-fold at high detergent.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of rabbit liver cytochrome P-450 LM 2 and its effect on monooxygenase activity

The phosphorylation of rabbit liver microsomal cytochrome P-450 LM2 by catalytic subunit of cyclic AMP-dependent protein kinase (W. Pyerin et al. (1983) Carcinogenesis 4, 573) has now been studied in detail with purified soluble form of cytochrome P-450 as well as with the purified protein incorporated into model membranes. The apparent Km values for P-450 of the phosphorylation reaction in all experimental systems were in a range of 2-8 microM, while the Vmax values were dependent on the state of P-450. Upon phosphorylation, the reconstituted enzyme activities with benzphetamine (N-demethylation) and 7-ethoxycoumarin (O-deethylation) as substrates were reduced to 30-40% of control.

Complete amino acid sequence and predicted membrane topology of phenobarbital-induced cytochrome P-450 (isozyme 2) from rabbit liver microsomes.

The complete amino acid sequence of phenobarbital-induced isozyme 2 of rabbit liver microsomal cytochrome P-450 (P-450LM2) is presented. The polypeptide consists of 491 residues with a calculated Mr of 55,755. The rabbit isozyme is 77% identical to the corresponding rat cytochrome, P-450b, as deduced from cDNA, with 96% of the hydrophobic, 88% of the anionic, and 83% of the cationic positions conserved. The secondary structure of isozyme 2 was predicted and a model was developed for the membrane topology of this cytochrome. Of the two highly conserved cysteinyl peptides in P-450LM2, P-450b, and bacterial P-450cam, we favor, on the basis of our model, the one nearer the NH2 terminus (Cys-152 in P-450LM2) as the source of the thiolate ligand to the heme iron atom. The recently reported sequence of the apparently identical protein [Heinemann, F. S. & Ozols, J. (1983) J. Biol. Chem. 258, 4195-4201] has two fewer residues and differs in 14 other amino acid assignments.

The complete amino acid sequence of rabbit phenobarbital-induced liver microsomal cytochrome P-450.

The complete amino acid sequence of the major phenobarbital-induced cytochrome P-450 (P-450LM2) from rabbit liver microsomes has been determined. The protein contains 489 amino acid residues in a single polypeptide chain and has Mr = 55,464. The sequence was compared with the amino acid sequence of P-450CAM and the nucleotide sequence of cDNA obtained from phenobarbital-induced rat liver cytochrome P-450 mRNA. These comparisons suggest that, despite functional similarities, the structural homology between microbial and microsomal cytochromes P-450 is limited to a single 8-residue region, and, in contrast, the structure of inducible microsomal cytochrome P-450 isozymes is highly conserved among mammalian species. Furthermore, we propose that the thiolate heme ligand of cytochrome P-450 is contributed by a cysteinyl residue near the COOH terminus, position 434 in the rabbit P-450LM2 sequence, based on the homology in this region with P-450CAM. The NH2 terminus of the protein from residues 1-310 is characterized by 8 hydrophobic segments 18-23 residues long, each of which is terminated by a cluster of charged amino acid residues. Residues 320-443 comprise a hydrophilic region which contains the putative heme binding cysteinyl residue as well as segments of homology with a constitutive rabbit cytochrome P-450 isozyme. The sequence data suggest that cytochrome P-450LM2 contains multiple transmembranous segments as well as a hydrophilic cytoplasmic domain. The hydrophilic domain contains regions of homology with several other cytochromes P-450, and thus appears to have an essential role in the biological function of the protein.

Oxidation-reduction potential of soluble and membrane-bound rabbit liver microsomal cytochrome P-450 LM2.

The redox midpoint potentials of rabbit liver microsomal cytochromes P-450 and of soluble and membrane-bound rabbit liver microsomal cytochrome P-450 LM2 were determined using EPR-spectroscopy and absorption difference spectrometry with NADPH or dithionite as reductants. Using EPR, a redox midpoint potential of -0.36 V was obtained both for the low spin and the high spin components of microsomal cytochrome P-450. Spectrophotometrical determinations yielded very similar values: -0.37 V and -0.34 V for the low and high spin signals, respectively. Soluble cytochrome P-450 LM2 had a midpoint potential of -0.32 V. This redox potential was not significantly affected by incorporation of the protein into an artificial membrane structure or, furthermore, by the presence of cytochrome b5 the same membrane.

Cytochrome P-450 rotamers control mixed-function oxygenation in reconstituted membranes. Rotational diffusion studied by delayed fluorescence depolarization.

1. Rotational diffusion of purified rabbit-liver microsomal cytochrome P-450 LM2 in reconstituted lipid-vesicle membranes was investigated by measurement of time-dependent polarized emission of delayed fluorescence. 2. Cytochrome P-450 labelled with diiodofluorescein iodoacetamide exhibited strict uniaxial rotation about the normal to the membrane. 3. Benzphetamine retards rotation, while reduction of the cytochrome P-450 substrate-complex accelerates rotation. 4. A model is proposed in which cytochrome P-450 forms disc-shaped rotamers immersed in the bilayer membrane to a depth which is varied by substrate-induced and redox state-dependent conformational changes. The model describes a regulating mechanism of the electron transfer-controlling mixed-function oxygenation.

Direct evidence for electron transfer from ferrous cytochrome b5 to the oxyferrous intermediate of liver microsomal cytochrome P-450 LM2.

Interaction and electron transfer between highly purified microsomal cytochrome P-450 from phenobarbital-induced rabbits and cytochrome b5 from uninduced rabbits was investigated by difference and stopped-flow spectrophotometry. Formation of a 1:1 complex between ferric P-450 and b5, demonstrated by difference spectrophotometry, was observed only when both cytochromes were incorporated into micelles of phosphatidylcholine. The dissociation constant (Kd) of the complex was decreased from 2.3 microM to 0.4 microM in the presence of 1 mM benzphetamine. The apparent Kd of benzphetamine was reduced from 220 microM to 50 microM upon addition of b5. The influence of ferrous b5 on the autooxidation of the oxyferrous intermediate of P-450 in the presence and in the absence of substrate was investigated by stopped-flow spectrophotometry. Both cytochromes were reduced photochemically, so that experiments could be carried out in the absence of the corresponding reductases and reduced pyridine nucleotides. Kinetic analysis of the data showed that formation of a 1:1 complex between ferrous P-450 and b5 was a prerequisite for electron transfer between the cytochromes. Here again, incorporation of both cytochromes into micelles was absolutely required for this process. Kd was decreased from 7.5 microM to 2.2 microM in the presence of 1 mM benzphetamine. The rate of electron transfer from b5 to oxyferrous P-450, derived from the kinetics of reoxidation of either b5 or P-450, was increased from 2.5 s-1 to about 4 to 7 s-1 in the presence of 1 mM benzphetamine. These results provide the first quantitative data on the electron transfer between the b5 and P-450, whose rate constant is compatible with the observations made on the effect of b5 on hydroxylation reactions catalyzed by the P-450 enzyme system.

Membrane charge as effector of cytochrome P-450LM2 catalyzed reactions in reconstituted liposomes.

The phospholipid specificity of rabbit liver microsomal cytochrome P-450LM2 catalyzed hydroxylation reactions was examined in reconstituted phospholipid vesicles. An apparent linear relationship between the negative charge of the vesicles and the rate of P-450LM2-catalyzed O-dealkylation of p-nitroanisole or 7-ethoxycoumarin was obtained. The membrane charge-mediated increase in hydroxylation activities was found not to be due to (i) an altered lipid/water partition coefficient of the substrate, (ii) a change in the apparent Michaelis constant of P-450LM2 for the substrate, (iii) a different activation energy of the O-demethylation of p-nitroanisole, (iv) different spin states of P-450LM2 or (v) an altered secondary structure of this enzyme as monitored by circular dichroism. However, when the formation of the ferrous carbonyl complex of P-450LM2 was followed under aerobic or anaerobic conditions after the addition of NADPH to the vesicles, an increased negative charge of the membrane was accompanied by an increased reducibility of P-450LM2. A similar linear relationship between the reducibility of cytochrome b5 and the negative charge of the liposomes was also evident in membranes containing NADPH-cytochrome P-450 reductase and cytochrome b5. It is proposed that the interaction of the reductase with P-450LM2 is inefficient in neutral vesicles and thus rate determining for the overall hydroxylation activities.