Functional interaction of cytochrome P450 with its redox partners: a critical assessment and update of the topology of predicted contact regions.

The problem of donor-acceptor recognition has been the most important and intriguing one in the area of P450 research. The present review outlines the topological background of electron-transfer complex formation, showing that the progress in collaborative investigations, combining physical techniques with chemical-modification and immunolocalization studies as well as site-directed mutagenesis experiments, has increasingly enabled the substantiation of hypothetical work resulting from homology modelling of P450s. Circumstantial analysis reveals the contact regions for redox proteins to cluster on the proximal face of P450s, constituting parts of the highly conserved, heme-binding core fold. However, more variable structural components located in the periphery of the hemoprotein molecules also participate in donor docking. The cross-reactivity of electron carriers, purified from pro- and eukaryotic sources, with a diversity of P450 species points at a possible evolutionary conservation of common anchoring domains. While electrostatic mechanisms appear to dominate orientation toward each other of the redox partners to generate pre-collisional encounter complexes, hydrophobic forces are likely to foster electron transfer events by through-bonding or pi-stacking interactions. Moreover, electron-tunneling pathways seem to be operative as well. The availability of new P450 crystal structures together with improved validation strategies will undoubtedly permit the production of increasingly satisfactory three-dimensional donor-acceptor models serving to better understand the molecular principles governing functional association of the redox proteins.

Allosteric phenomena in cytochrome P450-catalyzed monooxygenations.

Allosteric regulation of monooxygenase activity is shown to occur with diverse cytochrome P450 isoforms and is characterized by kinetic patterns deviating from the Michaelis-Menten model. Homotropic and heterotropic phenomena are encountered in both substrate activation and productive coupling of the electron donors NADPH-cytochrome P450 reductase and cytochrome b5, and the lipid environment of the system also appears to play a role as an effector. Circumstantial analysis reveals the components of the electron transfer chain to be mutually beneficial in interactions with each other depending on the substrate used and type of cytochrome P450 operative. It is noteworthy that association of diatomic gaseous ligands may be amenable to allosteric regulation as well. Thus, dioxygen binding to cytochrome P450 displays nonhyperbolic kinetic profiles in the presence of certain substrates; the latter, together with redox proteins such as cytochrome b5, can exert efficient control of the abortive breakdown of the oxyferrous intermediates formed. Similarly, substrates may modulate the structural features of the access channel for solutes such as carbon monoxide in specific cytochrome P450 isozymes to either facilitate or impair ligand diffusion to the heme iron. The in vivo importance of allosteric regulation of enzyme activity is discussed in detail.

Interactions between redox partners in various cytochrome P450 systems: functional and structural aspects.

The various types of redox partner interactions employed in cytochrome P450 systems are described. The similarities and differences between the redox components in the major categories of P450 systems present in bacteria, mitochondria and microsomes are discussed in the light of the accumulated evidence from X-ray crystallographic and NMR spectroscopic determinations. Molecular modeling of the interactions between the redox components in various P450 mono-oxygenase systems is proposed on the basis of structural and mutagenesis information, together with experimental findings based on chemical modification of key residues likely to be associated with complementary binding sites on certain typical P450 isoforms and their respective redox partners.

Residue 285 in cytochrome P450 2B4 lacking the NH(2)-terminal hydrophobic sequence has a role in the functional association of NADPH-cytochrome P450 reductase.

Cytochrome P450 2B4 (CYP2B4) lacking the NH(2)-terminal signal anchor sequence (2-27) was used to study the impact of replacement of histidine with alanine at position 285 on electron transfer from NADPH-cytochrome P450 reductase (P450R). Absorption and circular dichroism spectra of the recombinant hemoproteins indicated that amino acid substitution neither grossly perturbed the geometry of the immediate heme vicinity nor the global polypeptide backbone folding. Fitting of the initial-velocity patterns of P450R-directed reduction of the ferric CYP2B4 (2-27) forms to the Michaelis-Menten kinetics revealed an approximately 3.5-fold increase in the apparent K(m) value for the electron donor of the H285A mutant, while its reductive capacity (V(max)) remained unchanged; this caused a strong drop in reductive efficiency of the engineered enzyme. Circumstantial analysis suggested that impaired association of the redox partners accounted for this phenomenon. Thus, deletion of the positive charge at position 285 of CYP2B4 (2-27) might have disrupted contacts with oppositely charged entities on the P450R surface. Measurements of the stoichiometry of aerobic NADPH consumption and H(2)O(2) production disclosed the oxyferrous H285A species to autoxidize more readily compared with the shortened wild type. This was assumed to arise from less efficient coupling of the system due to defective donation of the second electron by P450R. These results are consistent with the view that His-285 in the truncated CYP2B4 is of importance in the functional interaction with the flavoprotein reductase.

Identification of key residues in rabbit liver microsomal cytochrome P450 2B4: importance in interactions with NADPH-cytochrome P450 reductase.

A cytochrome P450 2B4 (CYP2B4) model was used to select key residues supposed to serve in interactions with NADPH-cytochrome P450 reductase (P450R). Eight amino acid residues located on the surface of the hemoprotein were chosen for mutagenesis experiments with CYP2B4(Delta2-27) lacking the NH(2)-terminal signal anchor sequence. The mutated proteins were expressed in Escherichia coli, purified, and characterized by EPR- and CD-spectral analysis. Replacement of histidine 226 with alanine caused a 3.8-fold fall in the affinity for P450R with undisturbed reductive capacity of the system. Similarly, the K225A, R232A, and R253A variants exhibited P450R-directed activity that was depressed to about half that of the control enzyme, suggesting that the deletion of positive charges on the surface of CYP2B4(Delta2-27) resulted in impaired electrostatic contacts with complementary amino acids on the P450R protein. While the Y235A mutant did not show appreciably perturbed reduction activity, the conservative substitution with alanine of the phenylalanine residues at positions 223 and 227 gave a 2.1- to 6. 1-fold increase in the K(m) values with unchanged V(max); this was attributed to the disruption of hydrophobic forces rather than to global structural rearrangement(s) of the engineered pigments. Measurement of the stoichiometry of aerobic NADPH consumption and H(2)O(2) formation revealed the oxyferrous forms of the F223A, H226A, and F227A mutants to autoxidize more readily owing to less efficient coupling of the systems. Noteworthy, the F244A enzyme did not exhibit significant reduction activity, suggesting a pivotal role of Phe-244 in the functional coupling of P450R. The residue was predicted to constitute part of an obligatory electron transfer conduit through pi-stacking with Phe-296 located close to the heme unit. All of the residues examined reside in the putative G helix of CYP2B4, so that this domain obviously defines part of the binding site for P450R.

The optical biosensor studies on the role of hydrophobic tails of NADPH-cytochrome P450 reductase and cytochromes P450 2B4 and b5 upon productive complex formation within a monomeric reconstituted system.

The optical biosensor study of interaction between microsomal proteins-NADPH-cytochrome P450 reductase, cytochrome P450 2B4, and cytochrome b5-was carried out in the monomeric reconstituted system in the absence of phospholipids. The formation of individual complexes was kinetically characterized and their association and dissociation rate constants were determined. The association rate constants for the complexes formed were found to be close to the diffusiion limit-(0.5-4) x 10(6) M-1 s-1-while their dissociation rate constants did not exceed 0.5 s-1. It was shown that the interprotein electron transfer can occur both through complex formation and due to random collision. The dominant role of hydrophobic membraneous protein fragments in formation of productive electron transfer complexes was demonstrated.

Some properties of mitochondrial adrenodoxin associated with its nonconventional electron donor function toward rabbit liver microsomal cytochrome P450 2B4.

Mitochondrial adrenodoxin (Adx) was found to cross-react with microsomal cytochrome P450 2B4 (CYP2B4) as the terminal electron acceptor. When compared with NADPH-cytochrome P450 reductase (P450R), the natural redox partner of CYP2B4, Adx was less efficient both in transferring the first electron and in coupling the system. The ferredoxin yielded an unusual reverse type I spectral change with low-spin CYP2B4, which underwent transformation to a typical type I optical perturbation upon deletion of the signal anchor sequence (Delta2-27) of the hemoprotein. Truncation of CYP2B4 slightly fostered electron transfer from Adx, but was deleterious to reduction of the engineered isozyme by P450R. Addition of manganese-substituted cytochrome b5, which failed to serve as an electron donor to CYP2B4, augmented the amount of hemoprotein existing in form of a low-spin complex with Adx and affected the ferredoxin-dependent reduction kinetics through causing a proportional rise in both Km and Vmax. Conservative replacement of Asp-76 with glutamate in the Adx molecule was associated with a drastic drop in reductive efficiency toward CYP2B4, while spectral binding of the mutant to the hemoprotein was marginally changed. The results support the concept of an evolutionary relationship between the various cytochrome P450 forms as regards the conservation of surface regions participating in contacts with heterologous donor proteins.

Influence of mutation of the amino-terminal signal anchor sequence of cytochrome P450 2B4 on the enzyme structure and electron transfer processes.

The role of the NH2-terminal hydrophobic patch of cytochrome P4502B4 (CYP2B4) in interactions with NADPH-cytochrome P450 reductase (P450R) and cytochrome b5 (b5) was assessed using a variant lacking the signal anchor sequence (Delta2-27). CD, second-derivative, and fluorescence emission spectra indicated that the structure of the deletion mutant slightly differed from that of the native CYP2B4. Fitting of the initial-velocity patterns for P450R- and b5-directed electron transfer to the ferric CYP2B4 forms to Michaelis-Menten kinetics revealed an approximately 2.3-fold decrease in the affinity of the two electron donors for the engineered enzyme, while the reductive efficiency remained unaffected. Circumstantial analysis suggested that impaired association of the redox proteins with P4502B4(Delta2-27) accounted for this phenomenon. Interestingly, spectral docking of P450R to the truncated pigment was not hampered, while the binding of b5 was blocked. The rates of substrate-triggered aerobic NADPH consumption in systems containing CYP2B4(Delta2-27) and P450R were 16 to 56% those obtained with the unchanged hemoprotein. Decelerated cofactor oxidation did not arise on defective substrate binding or perturbed utilization of the substrate-bound oxy complex. Experiments with b5 as the ultimate electron donor hinted at some damage to second-electron transfer to the truncated enzyme. The results are consistent with the proposal that the NH2-terminal hydrophobic region of CYP2B4 might be of importance in preservation of the catalytic competence of the enzyme.

Amino acid residue 250 has a functional role in the assembly of rabbit liver microsomal cytochrome P450 2B4.

Cytochrome P450 2B4 lacking amino acids 2-27, CYP2B4 (delta2-27), was mutated at position 250 and expressed in E. coli fused to glutathione S-transferase. Expression of the E250S variant (holo- plus apoenzyme) proceeded to an extent comparable with that of CYP2B4 (delta2-27), while the protein level of the E250P mutant averaged 42% that of the control pigment. Comparison of these data with the corresponding reduced CO difference spectra of the various CYP2B4 (delta2-27) forms revealed that, in the control and E250S preparations, about 90% and 44%, respectively, of the total amount of hemoprotein present existed in the form of holoenzyme, whereas the E250P derivative failed to produce a reduced carbonyl complex. Thus, replacement of the negatively charged E250 with an uncharged, polar serine residue substantially hampered assembly of CYP2B4 (delta2-27); introduction of an alpha-helix-disrupting proline completely blocked the formation of holoenzyme. These phenomena suggested that the negative charge of E250, residing in the putative G helix, underwent pairing with some positively charged group, possibly H285 located in the I helix. Deletion of the negative charge obviously perturbed the active-site geometry such as to affect both the incorporation and/or retention of the heme ligand and the spectral binding of substrates such as hexobarbital.

Primary aromatic amines: their N-oxidative bioactivation.

There exists a diversity of pathways in mammalian cells serving to activate primary aromatic amines. 1 N-Oxidative mixed-function turnover usually involves participation of the cytochrome P450 superfamily, while catalysis by the flavin-containing monooxygenases is restricted to a few amines capable of forming imine tautomers. Surprisingly, haemoglobin metabolizes cytotoxic and carcinogenic arylamines via a monooxygenase-like mechanism, but peroxygenase activity is also operative. 2 In extrahepatic tissues that exhibit only a low level of monooxygenases, peroxidative transformations, as are brought about by prostaglandin H synthase, myeloperoxidase or lactoperoxidase, predominate in amine activation. Non-mammalian peroxidases frequently used as model systems include horseradish peroxidase and chloroperoxidase. 3 Non-enzymatic, light-induced conversion of aromatic amines to free radical or N-oxy products proceeds either via direct photolysis of the nitrogenous compounds or through attack by lipid-derived reactive intermediates generated during irradiation. 4 The interplay of the various tissue-specific processes of arylamine activation serves to explain differences in susceptibility toward the biological actions of primary aromatic amines.

An optical biosensor study of the interaction parameters and role of hydrophobic tails of cytochrome P450 2B4, b5 and NADPH-flavoprotein in complex formation.

The real-time interactions of membrane proteins - cytochrome P450 2B4, NADPH cytochrome P450 reductase and cytochrome b5 - were studied by use of an optical biosensor system. The association and dissociation rate constants for the individual complexes were measured and the affinities of the redox partners for each other were estimated. The association rate constants of these complexes were found to be close to the diffusion limit and their dissociation rate constants were in the order of 1s-1. A dominant role of the interaction of the membraneous hydrophobic fragments in the formation of productive electron transferring complexes between the proteins was demonstrated.

Histidine residues in rabbit liver microsomal cytochrome P-450 2B4 control electron transfer from NADPH-cytochrome P-450 reductase and cytochrome b5.

Treatment of cytochrome P-450 2B4 (P-450 2B4) with diethylpyrocarbonate to introduce 10-11 equivalents of acylating agent per polypeptide chain resulted in the selective derivatization of histidine residues characterized by differential susceptibility toward the modifier. Second-derivative spectral analysis as well as fluorescence measurements disproved gross alterations in P-450 2B4 structure as a consequence of labelling. The modified haemoprotein retained its ability to bind hexobarbital and catalyse cumene hydroperoxide-sustained N-demethylation of the barbiturate. However, there was a steady attenuation of NAD(P)H-driven electron flux with increasing extent of P-450 2B4 carbethoxylation in reconstituted systems fortified with either NADPH-cytochrome P-450 reductase or NADH-cytochrome b5 reductase/cytochrome b5 as the redox partners, with 50% inhibition occurring when 6-7 histidines were blocked. Hampered P-450 2B4 reductase activities recovered to differing degrees upon treatment of the acylated mono-oxygenase with neutral hydroxylamine. Spectral data indicated that docking of the redox components to derivatized P-450 2B4 was not perturbed, so that disruption of the electron flows most likely resulted from some injury of the electron-transfer mechanisms.

Some aspects of the role of cytochrome P-450 isozymes in the N-oxidative transformation of secondary and tertiary amine compounds.

Indirect evidence of the participation of cytochrome P-450 (P-450) in the microsomal N-oxygenation of secondary and tertiary nitrogen functions is presented by studies employing diagnostic modifiers of the hemoprotein system as well as antibodies directed toward the diverse P-450 isoforms and NADPH-cytochrome P-450 reductase. Experiments with recombinant hemoproteins or P-450 isozymes directly purified from the tissues of various animal species support the results obtained by the inhibitor assays. Although the intermediacy of aminium radicals is thought to be restrictive to P-450-catalyzed N-oxygenation of secondary and tertiary amine groups bearing accessible hydrogens on the alpha-carbon, numerous exceptions to this rule are documented. It is proposed that aminium radicals partition between oxygen rebound and alpha-hydrogen abstraction to yield a finite level of N-oxygenated product in all P-450-mediated amine oxidations, the partition ratio depending on the amine structure and particular P-450 isozyme operative. In some instances, N-oxygenation appears to proceed by peroxidatic mechanisms. The relative contribution of P-450 to the N-oxygenation of secondary and tertiary amines in crude preparations or live animals, where competition with the flavin-containing monooxygenase (FMO) occurs, seems to be a function of the relative amounts and catalytic capacities of the two enzyme systems. Both parameters are species and tissue dependent. Accordingly, the extent to which P-450 contributes to total N-oxidative turnover of the amine substrates varies from minor to major.

Rabbit liver cytochrome P-450 2B5: high-level expression of the full-length protein in Escherichia coli, purification, and catalytic activity.

Rabbit liver cytochrome P-450 2B5 (P-450 2B5) was expressed in Escherichia coli using the D(+)-galactose-inducible expression vector pJL-2, containing the full-length cDNA encoding P-450 2B5. Stimulation by galactose of protein synthesis in the presence of the heme precursor 5-aminolevulinic acid peaked 72 h after addition to the inducer to yield 108 nmol membrane-bound P-450 2B5 per liter of culture medium. The recombinant enzyme was purified to near homogeneity by a two-column procedure involving chromatography on DE-52 cellulose and hydroxylapatite. The hemoprotein was isolated mainly in the low-spin iron configuration and exhibited a reduced CO-difference spectrum with a Soret band at 451 nm. Second-derivative spectral analysis in the middle-UV region revealed that type I binding of 4-nitroanisole to ferric P-450 2B5 abolished absorption bands ascribable to tyrosine residues within the polypeptide chain. Pseudo-first-order rates of NADPH-driven reduction of the pigment were lower when reconstituted with NADPH-cytochrome P-450 reductase than with the mitochondrial adrenodoxin/NADPH-adrenodoxin reductase redox couple. The enzyme was catalytically active toward 4-nitroanisole and androstenedione; metabolic rates were enhanced to different extents by the presence of cytochrome b5. The recombinant hemoprotein did not catalyze bioactivation of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, a potent pulmonary carcinogen. The methods described here should facilitate further studies on the biophysical basis of the complex interactions of P-450 2B5 with its redox partners.

Chemical modification of Tyr34 and Tyr129 in rabbit liver microsomal cytochrome b5 affects interaction with cytochrome P-450 2B4.

Rabbit liver microsomal cytochrome b5 was allowed to react with tetranitromethane. Up to three tyrosine residues in each cytochrome b5 molecule were found to be accessible to the nitrating agent. Co-modification of tryptophan and histidine residues could be disregarded. CD-spectral measurements disproved gross changes in cytochrome b5 structure as a consequence of derivatization. Introduction of 1.6 nitro groups/polypeptide chain resulted in a fivefold increase in binding affinity for cytochrome P-450 2B4 (P-450 2B4), whereas spectral interaction with cytochrome c remained unaffected. Furthermore, the capacity of nitrated cytochrome b5 to shift the spin equilibrium to the high-spin conformer of P-4502B4 was diminished by 44% compared with the control. This corresponded with the partial disruption of NADH-dependent electron flow to ferric P-450 2B4. Changes in the redox potential of cytochrome b5 could be discounted as being responsible for this effect. The overall oxidative turnover of 4-nitroanisole did not respond to cytochrome b5 modification. MS analysis and sequencing of peptide fragments produced by tryptic digestion of modified cytochrome b5 permitted the detection of three nitrated tyrosine residues located at positions 11, 34 and 129. Derivatization of cytochrome b5 in the presence of a protective amount of P-450 2B4 provided evidence of the involvement of Tyr34 and Tyr129 in complexation of the two hemoproteins. It is proposed that Tyr129 might control docking of cytochrome b5 to P-450 2B4, whereas Tyr34 could be of functional importance in electron transfer.

Regulatory mechanisms in the activation of nitrogenous compounds by mammalian cytochrome P-450 isozymes.

Metabolic activation of nitrogenous compounds by the cytochrome P-450 system is a highly complex process. Inherent substrate factors, such as basicity, electronic state, lipophilicity, and conformation control binding of the diverse classes of amines to cytochrome P-450. Accommodation of these compounds in the enzyme cavity and proper orientation of the molecules are governed by intrinsic properties of the peptide structure of cytochrome P-450, which may be subject to modification by the action of effectors. On the membrane level, phospholipid might have some impact on substrate binding. On the other hand, bound amine substrate is beneficial to the productive interaction of the electron transport chains with the terminal acceptor, improving economy of the system. Certain amines appear to regulate O2 association with cytochrome P-450 and stabilize the various oxy species formed. Considering the selective prerequisites for oxidative attack by cytochrome P-450 at vulnerable nitrogen centers, many cytotoxic amines belonging to the category of relatively rigid, planar molecules undergo N-oxidative activation by the cytochrome P-450IA subfamily, while more bulky amines with flexible conformation are N-oxygenated preferentially by phenobarbital-inducible cytochromes P-450. Small differences in protein structure between the various cytochrome P-450 subforms might serve to stabilize aminium radicals to permit oxygen rebound. Collectively, the selective regulatory mechanisms operative in the bioactivation of nitrogen-containing compounds appear to be determined largely by the type of substrate used and the isozyme involved in catalysis. With respect to the latter, the interplay of the multiple cytochromes P-450 in the various organs of animal species thus serves to rationalize the differences in the particular selectivities for amine substrates. These are responsible for the mode and/or extent to which activation of nitrogenous compounds, including promutagens and procarcinogens, occurs, and this may explain the tissue-specific response to the tumorigenic action of these agents.

Metabolic N-oxide formation by rabbit-liver microsomal cytochrome P-4502B4: involvement of superoxide in the NADPH-dependent N-oxygenation of N,N-dimethylaniline.

NADPH-sustained N-oxygenation of N,N-dimethylaniline (DMA) was investigated with the aid of a reconstituted membranous cytochrome P-4502B4 system. The N-oxidative process did not appear to be supported by hydroxyl radicals or products arising from lipid peroxidation. However, superoxide dismutase was a very potent scavenger of N-oxide formation, while catalase was ineffective. Superoxide by itself did not bring about N-oxygenation of DMA. Therefore, O2- was presumed to serve as a source of the actual proximate oxidant. The reconstituted hemoprotein system catalyzed N-oxygenation of DMA when excess H2O2 substituted for NADPH/O2. This 'peroxygenase' process was entirely dependent on the presence of native enzyme and was not inhibited by CO or metyrapone. By contrast, cyanide severely blocked metabolic transformation. Among some other hemeproteins tested, only horseradish peroxidase was efficient in producing appreciable amounts of N-oxide in the presence of H2O2. Peroxidatic DMA N-oxygenation in intact liver microsomes fortified with cumene hydroperoxide was 2-fold stimulated by pretreatment of rabbits with phenobarbital, whereas administration of 3-methylcholanthrene or ethanol decreased turnover. Studies with uninduced hepatic microsomes, in which the activity of the flavin-containing monooxygenase had been partially suppressed by thermal treatment, revealed pronounced susceptibility of the NADPH-dependent N-oxide formation to the inhibitory action of both superoxide dismutase and antibody to NADPH-cytochrome P-450 reductase. These findings were interpreted to mean that at least 23% of the total amount of N-oxide produced in these preparations resulted from superoxide-dependent conversion of DMA by the P-450 system.

Inactivation of phenobarbital-inducible rabbit-liver microsomal cytochrome P-450 by allylisopropylacetamide: impact on electron transfer.

Application of a single dose of allylisopropylacetamide (AIA) to phenobarbital-pretreated rabbits resulted in partial destruction of the heme moiety of liver microsomal cytochrome P-450. A minor fraction of chromophore loss was accounted for by heme-derived product(s) covalently attached to microsomal proteins. Interestingly, cytochrome P-450 appeared to have undergone significant drug-mediated alkylation of the apohemoprotein. The modified species was purified to apparent homogeneity and shown to arise from AIA-induced blockage of about 2 histidines in the cytochrome P-450LM2 molecule located close to the heme edge. AIA administration to the animals caused inhibition of hexobarbital-promoted electron flow from NADPH-cytochrome P-450 reductase to phenobarbital-inducible ferricytochrome P-450 both in microsomal particles and reconstituted systems. The impaired interaction between the proteins was shown not to originate from decreased capacity to bind each other but more likely to be due to some defect in a step subsequent to complex formation. In contrast, treatment with the porphyrogenic agent did not affect microsomal electron transmission from cytochrome b5 to the ferric monooxygenase. However, when the intermediate carrier was to donate reducing equivalents to the ferrous oxycytochrome in the presence of benzphetamine, there was a pronounced deceleration of the electron flux observable. These findings were interpreted to mean that there exist multiple reductase- and cytochrome-b5-binding domains in phenobarbital-inducible cytochrome P-450, some of which seem to be common to the two redox proteins. This sheds interesting light on the molecular organization of the catalytic electron transfer complexes.

Chemical modification of lysine residues in cytochrome P450LM2 (P450IIB4): influence on heme liganding of arylamines.

Treatment of cytochrome P450LM2 with fluorescein isothiocyanate to introduce up to two equivalents of fluorophore per polypeptide chain resulted in the selective derivatization of lysine residues. CD-spectral measurements revealed the overall conformation as well as the immediate heme environment of the hemoprotein to remain unaffected by attachment of the label. Modification caused decreased affinity of p-phenylenediamine and other 4-substituted anilines for the heme site, whereas there was a rise in the extent of substrate interaction. Experiments with pigment containing acetylated lysines gave analogous results, suggesting that the observed phenomenon was due to charge neutralization. There was linear correlation between the Hammett sigma P values and both the optical dissociation constants for arylamine binding to intact enzyme and the dipole moments of the anilines, indicating that basicity along with electronic factors controlled heme liganding; lipophilicity appeared to be of minor importance. Introduction of fluorescein isothiocyanate into the oxygenase was found to influence the bond-making process through modulating basicity of the nitrogenous compounds, but perturbation of optimal spacial orientation of the amine nitrogen toward the heme iron also might have been operative. The lysines studied seem to represent metabolically inactive elements of the substrate channel located on the cytosolic surface of the aggregates, as evidenced by steady-state fluorescence measurements. A hydrophilic segment in the cytochrome P450LM2 molecule that would accommodate the critical residues is discussed.

Evidence of the existence of structurally distinct hepatic and pulmonary forms of microsomal flavin-containing monooxygenase in the rabbit.

The flavin-containing monooxygenase has been purified from rabbit liver and lung microsomes. SDS-PAGE analysis shows that both enzyme forms migrate as a single band with an apparent Mr of 59000. The NH2-terminus of both forms is blocked. The liver oxidase contains a lower percentage of glutamine/glutamate and a greater amount of phenylalanine than does the lung flavoprotein. Polyclonal antibodies to a 14-amino-acid peptide obtained after CNBr cleavage of the liver oxidase cross-react with the microsomal and purified liver enzyme, but do not recognize the lung oxidase. HPLC profiles of tryptic digests of the liver and lung enzymes exhibit different patterns. Sequence alignment of selected peptides from the liver and lung oxidases reveals aberrant residues within homologous segments. These findings are interpreted to mean that both enzymes represent distinct gene products.