Structural organization and localization of cytochromes P450 in the membrane

The secondary structure prediction of 19 microsomal cytochrome P-450s from two different families was made based on their amino acid sequences. It was shown that there is a structural similarity between the heme-binding sites of these enzymes and the bacterial P-450cam. An average predicted secondary structure of cytochrome P-450 proteins with 70% accuracy contains about 46% alpha-helices, 12% beta-strands, 9% beta-turns and 33% random coil. In the region of the 35-120 residues in microsomal P-450s two adjacent beta alpha beta-units (the Rossmann domain) were recognized, which may interact with the NADPH-cytochrome P-450 reductase. Using the procedure of identification of hydrophobic and membrane-associated alpha-helical segments of 23 cytochromes, only one N-terminal transmembrane anchor was predicted. Also the heme-binding site perhaps includes surface-bound helix. A model of vertebrate microsomal P-450s is proposed. That is an amphypathic membrane protein located on the cytoplasmic face of the endoplasmic reticulum, their active center lies out/on the bilayer border.     

Secondary structure and membrane topology of cytochrome P450s

The secondary structure prediction of 19 microsomal cytochrome P450s from two different families was made on the basis of their amino acid sequences. It was shown that there is structural similarity between the heme-binding sites in these enzymes and those in the bacterial P450cam. An average predicted secondary structure of cytochrome P450 proteins with 70% accuracy contains about 46% alpha-helices, 12% beta-sheets, 9% beta-turns, and 33% random coils. In the region of residues 35-120 in microsomal P450s two adjacent beta alpha beta-units (the Rossmann domain), were recognized and may be available to interact with the NADPH-cytochrome P450 reductase. Using the procedure for identification of hydrophobic and membrane-associated alpha-helical segments, only one N-terminal transmembrane anchor was predicted. Also the heme-binding site may include the surface-bound helix. A model for vertebrate microsomal P450s having an amphipathic membrane protein located on the cytoplasmic side of the endoplasmic reticulum membrane, with their active center lying outside or on the bilayer border, is proposed.     

Secondary structure prediction of 52 membrane-bound cytochromes P450 shows a strong structural similarity to P450cam

The secondary structure of 52 aligned cytochrome P450 sequences, all of which are membrane bound, is predicted and collectively compared with the crystal structure of the soluble cytochrome P450cam. Ten of 13 helical regions, 6 of 7 beta-pair regions, and beta-structure corresponding to a known beta-bulge near the active site of P450cam are predicted to exist in the membrane-bound P450s. Three turns associated with beta-structure in the soluble enzyme are also predicted for the membrane-bound forms. A strong structural similarity is evident between membrane P450s and the soluble P450cam. Consequently, a multitransmembrane structure involving much of P450 seems highly unlikely. A structure with two N-terminal transmembrane segments is compatible with these observations.     

Membrane topology of the mammalian P450 cytochromes.

The membrane topology of the mammalian P450 cytochromes has been studied intensively by computational approaches, proteolysis, chemical modification, genetic engineering, and immunochemistry. Initial results for the cytochromes of the endoplasmic reticulum appeared to indicate a polytopic, four to eight transmembrane anchor model with an active site buried in the membrane. However, recent findings show that the microsomal P450s are bound to the endoplasmic reticulum by only one or two transmembrane peptides located at the NH2-terminal end, and that the active site is part of a large cytoplasmic domain that may have one or two additional peripheral membrane contacts. The membrane-bound state is viewed as rather rigid, and the plane of the heme lies between perpendicular and parallel to the plane of the endoplasmic reticulum. The mitochondrial P450 cytochromes lack a hydrophobic NH2 terminus in the mature form, and thus differ from the microsomal isozymes in this significant way. However, although the exact topology of cytochrome P450 in the inner mitochondrial membrane remains to be elucidated, certain features are clearly comparable to those of microsomal P450. Therefore, the membrane topology of the P450 gene superfamily may follow a similar pattern.     

Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: a proposed molecular model for a cytochrome P-450cam electron-transfer complex

Cytochrome b5 has been genetically engineered to afford a fluorescent derivative capable of monitoring its association with cytochrome P-450cam from Pseudomonas putida [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. In the mutant cytochrome b5, threonine is replaced by a cysteine at position 65 (T65C) and has been labeled with the environmentally sensitive fluorophore acrylodan. In this paper, the physiological P-450cam reductant putidaredoxin, an Fe2S2.Cys4 iron-sulfur protein, is shown to competitively inhibit the cytochrome b5 association, suggesting that cytochrome b5 and putidaredoxin bind to a similar site on the cytochrome P-450cam surface. Since the crystal structures for both cytochrome b5 and cytochrome P-450cam have been solved to high resolution, the complex has been computer modeled, and a good fit was found on the proximal surface of nearest approach to the P-450cam heme prosthetic group. The proposed model includes electrostatic contacts between conserved cytochrome b5 carboxylates Glu-44, Glu-48, Asp-60, and the exposed heme propionate with cytochrome P-450cam basic residues Lys-344, Arg-72, Arg-112, and Arg-364, respectively. Putidaredoxin has similarly been shown to contain a carboxylate-based binding surface, and the current results suggest that if the model is correct, then it also interacts at the proposed site, probably utilizing similar P-450cam electrostatic contacts.     

Membrane topology of mammalian cytochromes P-450 from liver endoplasmic reticulum. Determination by trypsinolysis of phenobarbital-treated microsomes

We have studied the membrane topology of liver microsomal cytochromes P-450 derived from phenobarbital-treated rabbits via trypsinolysis of intact microsomes, recovery of solubilized peptide fragments by ultracentrifugation and liquid chromatography, primary structure determination by Edman microsequence analysis, and database searching to match isolated fragments with parent sequences. Relative to the primary structure of isozyme 2, the major phenobarbital-inducible form, fragments were isolated beginning at residues Glu86, Ile101, Arg126, Cys152, Leu198, Ser211, Asn237, Glu327, Asn385, Thr407, Phe408, Phe413, and Thr444. Such results show that this family of structurally related cytochromes is bound to the endoplasmic reticulum membrane by only one or two transmembrane segments, located at the NH2-terminal end of the polypeptide chain. The remainder of the protein, from residue approximately 50 to the COOH terminus must exist as a catalytic, heme-containing domain exposed on the cytosolic side of the membrane. Furthermore, our results indicate that the catalytic domain must be peripherally associated with the membrane surface. This would imply that substrates might have access to the active site of the cytochrome P-450 either by diffusion from the cytosol or from within the lipid bilayer.     

Molecular modeling of the 3-D structure of cytochrome P-450scc.

Sequence-alignment studies of the bovine mitochondrial cholesterol side-chain cleavage enzyme cytochrome P-450scc with the bacterial cytochrome P-450cam (camphor hydroxylating enzyme) have been undertaken. Our novel alignment of the sequences revealed 69 identical residues and many highly conserved regions. The results of the sequence alignment studies were used to model the 3-D structure of P-450scc based on the available crystal structure of P-450cam. The major insertions in the sequence are found mainly on four external-loop regions of the molecule, while the core structure of P-450cam is retained with subtle internal modifications. The most hydrophobic of these four external loops is proposed as a candidate for membrane attachment.     

Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site-specific antibodies

Fifteen peptides, ranging in length from 6 to 31 amino acids and corresponding in sequence to portions of the major phenobarbital-inducible form of rat liver cytochrome P-450 (P-450 PB-4), were previously synthesized chemically and used to prepare site-specific rabbit antibodies (Frey, A. B., D.J. Waxman, and G. Kreibich, 1985, J. Biol. Chem., 260:15253-15265). The antipeptide antibodies were affinity purified using Sepharose resins derivatized with the respective peptides and 14 preparations were obtained that in an ELISA assay showed affinities to immobilized P-450 judged to be adequate for binding studies on intact rat liver microsomes. The binding of these antibodies to rough microsomes from the livers of phenobarbital treated rats was assessed using 125I-labeled IgG and by immunoelectron microscopy employing protein A-gold as a marker. It was found that many of the antibodies bound to the cytoplasmic surface of the membrane but none bound to the luminal face of ruptured or inverted microsomal vesicles or to contaminating membranes of other organelles present in the preparations. These observations eliminate previously proposed models for the transmembrane disposition of P-450 that postulate the existence of multiple transmembrane domains and the exposure of several polar segments of the polypeptide on the luminal side of the membrane. The fact that an antibody raised to the first 31 residues of P-450 bound well to the purified P-450 but very poorly to rough microsomes, whereas an antibody to a peptide comprising residues 24-38 showed relatively strong binding to intact microsomes, is consistent with the proposal that the amino terminal segment of P-450 extending approximately to residue 20 is embedded in the phospholipid bilayer and the immediately following segment is exposed on the cytoplasmic surface of the membrane. All these results favor a model in which the cytochrome P-450 molecule is largely exposed on the cytoplasmic surface of the endoplasmic reticulum membrane to which it is anchored by its short amino terminal hydrophobic segment.     

The cytochrome P-450cam binding surface as defined by site-directed mutagenesis and electrostatic modeling

Cytochrome P-450cam cationic surface charges at Lys 344, Arg 72, and Lys 392 have been altered by site-directed mutagenesis techniques. The residues at Lys 344 and Arg 72 were previously suggested as salt bridge contacts in the cytochrome b5-cytochrome P-450cam association complex and implicated in the physiological putidaredoxin-cytochrome P-450cam complex [Stayton, P. S., Poulos, T. L., & Sligar, S. G. (1989) Biochemistry 28, 8201-8205]. Mutations to neutralize the basic charge at Arg 72 (R72Q) and to both neutralize and reverse the charge at Lys 344 (K344Q, K344E) resulted in alteration of NADH oxidation rates in the reconstituted physiological electron-transfer system, which is rate limited by putidaredoxin-cytochrome P-450cam electron transfer. The steady-state Vmax values were apparently unperturbed, suggesting that the observed rate differences were largely attributable to Km effects. The Km values observed for the K344Q (24 microM) and K344E (32 microM) mutants are in the direction expected for neutralization and reversal of a salt bridge charge interaction. A control mutation at a basic surface charge located away from the proposed site of interaction, Lys 392 (K392Q), resulted in overall activities quantitated by NADH oxidation rates that are similar to that of wild-type cytochrome P-450cam. Calculation of the cytochrome P-450cam electrostatic field revealed a patch of positive potential at the modeled cytochrome b5 interaction site lying directly above the nearest proximal approach to the buried heme prosthetic group. These results provide experimental and theoretical evidence for the modeled cytochrome P-450cam binding site implicated in both cytochrome b5 and putidaredoxin association.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation between the substrate-binding site and heme of cytochrome P-450 studied by excitation energy transfer

The conformation between the substrate-binding site and heme of cytochrome P-450 was studied by excitation energy transfer. Cytochrome P-450 was obtained from the hepatic microsomes of polychlorinated biphenyl-treated male rats, and ten polycyclic aromatic hydrocarbons were used as substrates. The energy transfer from the substrate to the heme of the enzyme was measured according to the F?rster equation. On the basis of the assumption that the substrates are bound at different positions in the plane of the same substrate-binding site, the position of the heme in relation to the substrate-binding site was determined in solution and in the presence of synthetic phospholipid. The results demonstrated that the distance between the substrate-binding site and the heme of cytochrome P-450 was greater when the enzyme was incorporated into micelles of phospholipid than when in solution, and that the conformational relationship of the substrate-binding site towards the heme was changed by an angle of 21 degrees on incorporation of the enzyme into phospholipid micelles.     

Apoprotein formation and heme reconstitution of cytochrome P-450cam

Apoprotein suitable for heme reconstitution has been prepared by an acid/butanone extraction of cytochrome P-450cam at pH 2.5. Absorption spectra of apo-P-450cam indicate less than 2% residual holoenzyme. Four tryptophan residues per molecule were estimated from the aromatic absorbance region of denatured apoprotein. Heme-reconstituted holoprotein was purified in 30% yield to a specific activity equivalent to the native enzyme. Absorption and EPR spectra of 57Fe- and 54Fe-heme-enriched P-450cam reveal complete restoration of the native active site.     

Localization of cytochrome c-binding domain on NADPH-cytochrome P-450 reductase

A covalent complex between purified rat liver microsomal NADPH-cytochrome P-450 reductase and horse cytochrome c was formed through cross-linking studies with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at low ionic strength. The purified cross-linked derivative shows that this product is a 1:1 complex containing one molecule each of the flavoprotein and cytochrome. The covalent complex had almost completely blocked the electron transfer from NADPH to exogenous cytochrome c or the rabbit liver microsomal cytochrome P-450 induced by phenobarbital, indicating that the cross-linked cytochrome c covers the electron-accepting site of the reductase. These results suggest that the covalently cross-linked derivative is a valid model of the noncovalent electron transfer complex. Although the exact number and site of the cross-linked location were not determinable, in cytochrome c the amide bond originates from Lys-13 and in reductase it might be at any one of six different side chain carboxyl groups in the two neighboring cluster acidic residues, Asp-207, -208, and -209, and Glu-213, Glu-214, and Asp-215. It is therefore proposed that the six clustered carboxyl groups on reductase are in an exposed location near the area where one heme edge comes close to the molecular surface.     

Studies on the molecular organization of cytochromes P-450 and b5 in the microsomal membrane.

1. The relative orientations of the heme groups of cytochromes P-450 and b5 in the microsomal membrane have been studied by the technique of electron paramagnetic resonance. The results show that the heme plane of cytochrome P-450 lies in the same plane as the membrane surface, whereas the cytochrome b5 heme plane has a random orientation. 2. No significant broadening or change in relaxation properties of the gz component of low spin cytochrome P-450 occurred when cytochrome b5 was reduced by redox poising. It is concluded that there is little or no paramagnetic coupling between the heme groups of the two species. 3. The results favor a model in which no tight complex between cytochromes P-450 and b5 is present, the species being independent and interacting only by random molecular collisions or via other intermediate species.     

Chemical modification and inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The alkylating agent 2-bromo-4'-nitroacetophenone (BrNAP) binds covalently to each of 10 isozymes of purified rat liver microsomal cytochrome P-450 (P-450a-P-450j) but substantially inhibits the catalytic activity of only cytochrome P-450c. Regardless of pH, incubation time, presence of detergents, or concentration of BrNAP, treatment of cytochrome P-450c with BrNAP resulted in no more than 90% inhibition of catalytic activity. Alkylation with BrNAP did not cause the release of heme from the holoenzyme or alter the spectral properties of cytochrome P-450c, data that exclude the putative heme-binding cysteine, Cys-460, as the major site of alkylation. Two residues in cytochrome P-450c reacted rapidly with BrNAP, for which reason maximal loss of catalytic activity was invariably associated with the incorporation of approximately 1.5 mol of BrNAP/mol of cytochrome P-450c. Two major radio-labeled peptides were isolated from a tryptic digest of [14C]BrNAP-treated cytochrome P-450c by reverse-phase high performance liquid chromatography. The amino acid sequence of each peptide was determined by microsequence analysis, but the identification of the residues alkylated by BrNAP was complicated by the tendency of the adducts to decompose when subjected to automated Edman degradation. However, results of competitive binding experiments with the sulfhydryl reagent 4,4'-dithiodipyridine identified Cys-292 as the major site of alkylation and Cys-160 as the minor site of alkylation by BrNAP in cytochrome P-450c.     

Crystal structure of substrate-free Pseudomonas putida cytochrome P-450

The crystal structure of Pseudomonas putida cytochrome P-450cam in the substrate-free form has been refined at 2.20-A resolution and compared to the substrate-bound form of the enzyme. In the absence of the substrate camphor, the P-450cam heme iron atom is hexacoordinate with the sulfur atom of Cys-357 providing one axial heme ligand and a water molecule or hydroxide ion providing the other axial ligand. A network of hydrogen-bonded solvent molecules occupies the substrate pocket in addition to the iron-linked aqua ligand. When a camphor molecule binds, the active site waters including the aqua ligand are displaced, resulting in a pentacoordinate high-spin heme iron atom. Analysis of the Fno camphor - F camphor difference Fourier and a quantitative comparison of the two refined structures reveal that no detectable conformational change results from camphor binding other than a small repositioning of a phenylalanine side chain that contacts the camphor molecule. However, large decreases in the mean temperature factors of three separate segments of the protein centered on Tyr-96, Thr-185, and Asp-251 result from camphor binding. This indicates that camphor binding decreases the flexibility in these three regions of the P-450cam molecule without altering the mean position of the atoms involved.     

Characterization of rabbit liver cytochrome P-450 (laurate omega-1 hydroxylase) synthesized in transformed yeast cells

Three cDNAs for chimeras between cytochrome P-450s (pHP3 and pHP2-1) were constructed and inserted between the alcohol dehydrogenase promoter and terminator regions of the yeast expression vector pAAH5 to form expression plasmids, pAH3P2, pAH3E2, and pAH3A2. pAH3P2 contained the entire coding sequence of cytochrome P-450 (pHP2-1) except for the 3rd, the 8th, the 36th, and the 42nd residues of the total of 490 amino acids. Nucleotide sequences of pAH3P2 were replaced with those of cytochrome P-450 (pHP3) in the region coding for the NH2-terminal 210 and 262 amino acid residues to yield pAH3E2 and pAH3A2, respectively. The three expression plasmids were introduced into Saccharomyces cerevisiae AH22 cells and cytochrome P-450 s (3P2, 3E2, and 3A2) were purified from the microsomal fractions of the transformed yeast cells. In the oxidized state either of the cytochromes exhibited a low- and high-spin mixed-type spectrum of cytochrome P-450. The reduced CO complex of the cytochromes showed a Soret absorption maximum at 450 nm. When laurate or caprate was added to ferric cytochrome P-450 s (3P2 and 3E2), the spectrum was converted to that of the typical high-spin type, indicating the binding of the fatty acids to the substrate site of the cytochromes. On the other hand, the addition of the fatty acids to ferric cytochrome P-450 (3A2) induced no spectral change. Only chemicals having a carboxyl group caused such spectral conversion of cytochrome P-450 (3P2) among dodecyl compounds examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the substrate-binding sites of liver microsomal cytochrome P-448.

The interaction of substrates of the microsomal mixed-function oxidases with cytochromes P-450 and P-448 was investigated by using liver microsomes from rats pretreated with phenobarbital or 3-methylcholanthrene, and with purified forms of the cytochromes isolated from rabbit liver. The two forms of the cytochrome have different substrate specificities; cytochrome P-450 has one type 1 substrate-binding site that can accommodate a large variety of substrates, but in contrast cytochrome P-448 may possess two type 1 substrate-binding sites, one of which is different to that of cytochrome P-450 in that it shows a specificity for substrates such as safrole and 9-hydroxy-ellipticine. These findings explain why the two forms of the cytochrome have different substrate specificities and play contrasting roles in the activation and deactivation of xenobiotics.     

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.     

The in vivo turnover of rat liver microsomal epoxide hydrolase and both the apoprotein and heme moieties of specific cytochrome P-450 isozymes

The in vivo turnover rates of liver microsomal epoxide hydrolase and both the heme and apoprotein moieties of cytochromes P-450a, P-450b + P-450e, and P-450c have been determined by following the decay in specific radioactivity from 2 to 96 h after simultaneous injections of NaH14CO3 and 3H-labeled delta-aminolevulinic acid to Aroclor 1254-treated rats. Total liver microsomal protein was characterized by an apparent biphasic exponential decay in specific radioactivity, with half-lives of 5-9 and 82 h for the fast- and slow-phase components, respectively. Most (approximately 90%) of the rapidly turning over microsomal protein fraction was immunologically distinct from membrane-associated serum protein, and thus appeared to represent integral membrane proteins. The existence of two distinct populations of cytochrome P-450a was suggested by the apparent biphasic turnover of both the heme and apoprotein moieties of the holoenzyme. The half-lives of the apoprotein were estimated to be 12 and 52 h for the fast- and slow-phase components, respectively, and 7 and 34 h for the heme moiety. The turnover of cytochromes P-450b + P-450e was identical to that of cytochrome P-450c, with half-lives of 37 and 28 h for the apoprotein and heme moieties, respectively. In all cases, the shorter half-lives of the heme component compared to the protein component were statistically significant. In contrast to the cytochrome P-450 isozymes, epoxide hydrolase (t1/2 = 132 h) turned over slower than the "average" microsomal protein (t1/2 = 82 h). The differential rates of degradation of these major integral membrane proteins during both the rapid and slow phases of total microsomal protein turnover argue against the concepts of unit membrane degradation and unidirectional membrane flow of liver endoplasmic reticulum.