Use of spin-labelled substrate analogs for a comparative study of microsomal cytochrome P-450 and P-448

The previously described, iodine-labeled alkylating stable nitroxyl radicals located at different distances between the N-O. group and the iodine atom were used for a comparative study of the structure of microsomal cytochromes P-450 and P-448 active centers. The radicals were shown to change the optical spectra of Fe3+ located in the active site of the enzyme that are similar to those induced by cytochrome P-450 substrates. Some differences in the type of the radicals binding to control, phenobarbital- and 3-methylcholanthrene-induced microsomes were revealed. The alkylating radical substrate analogs covalently bound to microsomal cytochrome P-450 in the vicinity of the active center, resulting in the inhibition of oxidation of type I and II substrates (e. g., aniline and naphthalene). The value of the spectral binding constant (Ks) for naphthalene in the presence of the radical covalently bound to the cytochrome P-450 active center showed a tendency to increase. Using the ESR technique, the interaction between Fe3+ and the radical localized in the active site of cytochrome P-450 was demonstrated. The contribution of Fe3+ to the relaxation of the radicals covalently bound to cytochrome P-450 was evaluated from the values of the spin label ESR spectra saturation curves at 77K. The distances between the N-O. group of these radicals and Fe3+ in the enzyme active center for the three types of microsomes were determined. The data obtained point to structural peculiarities of the active center of cytochrome P-450, depending on the microsomal type.     

Spin-labeling studies of rat liver NADPH-cytochrome p450 reductase: conformation and function relationship.

ESR spin-labeling studies designed to yield information regarding the relationship between function and conformation of rat liver NADPH-cytochrome P450 reductase (EC 1.6.4.2) were carried out. The purified enzyme was spin labeled by a nitroxide derivative of p-chloromercuribenzoate. Two conditions for spin labeling were employed: (i) the presence of NADP+, yielding an active site-protected spin-labeled reductase, and (ii) the absence of NADP+, yielding completely spin-labeled reductase. Reductase in which the active site was protected by binding NADP+ and then spin-labeled retains most of its enzymatic activity; on the other hand, completely spin-labeled reductase is devoid of any enzymatic activity. Completely spin-labeled reductase yields a two-component resolved ESR spectrum that reflects two classes of spin-labeled binding sites, a strongly immobilized (S) and a weakly immobilized (W) site. The ratio of W/S provides a valuable parameter for studying the relationship between function and conformation. Structural perturbants, such as urea, KCl, and pH, were employed to determine their effects on the activity of the enzyme and their relationship to changes in the conformational state of the reductase. It was further observed that the enzymatically active spin-labeled derivative generated superoxide radical in the presence of NADPH and cytochrome c, which in turn reduced completely the attached spin-label.     

Adaptations for the oxidation of polycyclic aromatic hydrocarbons exhibited by the structure of human P450 1A2

Microsomal cytochrome P450 family 1 enzymes play prominent roles in xenobiotic detoxication and procarcinogen activation. P450 1A2 is the principal cytochrome P450 family 1 enzyme expressed in human liver and participates extensively in drug oxidations. This enzyme is also of great importance in the bioactivation of mutagens, including the N-hydroxylation of arylamines. P450-catalyzed reactions involve a wide range of substrates, and this versatility is reflected in a structural diversity evident in the active sites of available P450 structures. Here, we present the structure of human P450 1A2 in complex with the inhibitor alpha-naphthoflavone, determined to a resolution of 1.95 A. alpha-Naphthoflavone is bound in the active site above the distal surface of the heme prosthetic group. The structure reveals a compact, closed active site cavity that is highly adapted for the positioning and oxidation of relatively large, planar substrates. This unique topology is clearly distinct from known active site architectures of P450 family 2 and 3 enzymes and demonstrates how P450 family 1 enzymes have evolved to catalyze efficiently polycyclic aromatic hydrocarbon oxidation. This report provides the first structure of a microsomal P450 from family 1 and offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members.     

Interactions of microsomal cytochrome P-450 with spin-labeled substrate analogs

The iodine-containing stable iminoxyl radicals with various distances between the N-O-group and the iodine atom are proposed to be used to study the structure of the active center of the microsomal cytochrome P-450. The radicals used induce changes in the optical spectra of the Fe3+ ion located in the active center of the enzyme, as in the case of type 1 substrates and inhibit essentially the microsomal oxidation of cytochrome P-450 substrates of type 1 and 2. This inhibition is neither due to suppression of the NADPH-cytochrome c reductase activity nor to cytochrome P-450 conversion to cytochrome P-420. Cytochrome P-450 substrates (aminopyrine) protect the enzyme against the radical-induced inactivation. The iodine-containing radicals are covalently bound to cytochrome P-450 in the vicinity of active center. The values of dissociation constants for the reversible enzyme-radical constants and the rate constants for the monomolecular transformation in the complex, k, were determined. The EPR method was used to detect the coupling between Fe3+ and the radical located in the active center of cytochrome P-450. The saturation curves of radical SPR spectra at 77 degrees K were employed to determine the contribution of Fe3+ to the relaxation time, T1, of the radicals covalently bound to cytochrome P-450 and to estimate the distances between the Fe3+ ion and the N-O-group of these radicals in the enzyme active center.     

Crystal structure of inhibitor-bound P450BM-3 reveals open conformation of substrate access channel

P450BM-3 is an extensively studied P450 cytochrome that is naturally fused to a cytochrome P450 reductase domain. Crystal structures of the heme domain of this enzyme have previously generated many insights into features of P450 structure, substrate binding specificity, and conformational changes that occur on substrate binding. Although many P450s are inhibited by imidazole, this compound does not effectively inhibit P450BM-3. Omega-imidazolyl fatty acids have previously been found to be weak inhibitors of the enzyme and show some unusual cooperativity with the substrate lauric acid. We set out to improve the properties of these inhibitors by attaching the omega-imidazolyl fatty acid to the nitrogen of an amino acid group, a tactic that we used previously to increase the potency of substrates. The resulting inhibitors were significantly more potent than their parent compounds lacking the amino acid group. A crystal structure of one of the new inhibitors bound to the heme domain of P450BM-3 reveals that the mode of interaction of the amino acid group with the enzyme is different from that previously observed for acyl amino acid substrates. Further, required movements of residues in the active site to accommodate the imidazole group provide an explanation for the low affinity of imidazole itself. Finally, the previously observed cooperativity with lauric acid is explained by a surprisingly open substrate-access channel lined with hydrophobic residues that could potentially accommodate lauric acid in addition to the inhibitor itself.     

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

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

Resonance Raman studies of cytochrome P450 2B4 in its interactions with substrates and redox partners

Resonance Raman studies of P450 2B4 are reported for the substrate-free form and when bound to the substrates, benzphetamine (BZ) or butylated hydroxytoluene (BHT), the latter representing a substrate capable of inducing an especially effective conversion to the high-spin state. In addition to studies of the ferric resting state, spectra are acquired for the ferrous CO ligated form. Importantly, for the first time, the RR technique is effectively applied to interrogate the changes in active site structure induced by binding of cytochrome P450 reductase (CPR) and Mn(III) cytochrome b 5 (Mn cyt b 5); the manganese derivative of cyt b 5 was employed to avoid spectroscopic interferences. The results, consistent with early work on mammalian P450s, demonstrate that substrate structure has minimal effects on heme structure or the FeCO fragment of the ferrous CO derivatives. Similarly, the data indicate that the protein is flexible and that substrate binding does not exert significant strain on the heme peripheral groups, in contrast to P450 cam, where substantial effects on heme peripheral groups are seen. However, significant differences are observed in the RR spectra of P450 2B4 when bound with the different redox partners, indicating that the heme structure is clearly sensitive to perturbations near the proximal heme binding site. The most substantial changes are displacements of the peripheral vinyl groups toward planarity with the heme macrocycle by cyt b 5 but away from planarity by CPR. These changes can have an impact on heme reduction potential. Most interestingly, these RR results support an earlier observation that the combination of benzphetamine and cyt b 5 binding produce a synergy leading to unique active site structural changes when both are bound.     

A molecular model for the enzyme cytochrome P450(17 alpha), a major target for the chemotherapy of prostatic cancer

The enzyme cytochrome P450(17 alpha) catalyses two key steps in the biosynthesis of the androgens from pregnanes: the 17 alpha hydroxylation step and the subsequent 17-20 lyase reaction. Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P450(17 alpha) based on that of P450cam, the only cytochrome P450 enzyme for which the crystal structure is known. The model suggests the possibility of two modes of binding of steroid substrates at the active site, perhaps reflecting the dual functionality of the enzyme.     

Inhibitory analogs of ubiquinol act anti-cooperatively on the Yeast cytochrome bc1 complex. Evidence for an alternating, half-of-the-sites mechanism of ubiquinol oxidation.

The cytochrome bc(1) complex is a dimeric enzyme that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is re-reduced at a second center, referred to as center N. To understand better the mechanism of ubiquinol oxidation, we have examined the interaction of several inhibitory analogs of ubiquinol with the yeast cytochrome bc(1) complex. Stigmatellin and methoxyacrylate stilbene, two inhibitors that block ubiquinol oxidation at center P, inhibit the yeast enzyme with a stoichiometry of 0.5 per bc(1) complex, indicating that one molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme. This stoichiometry was obtained when the inhibitors were titrated in cytochrome c reductase assays and in reactions of quinol with enzyme in which the inhibitors block pre-steady state reduction of cytochrome b. As an independent measure of inhibitor binding, we titrated the red shift in the optical spectrum of ferrocytochrome b with methoxyacrylate stilbene and thus confirmed the results of the inhibition of activity titrations. The titration curves also indicate that the binding is anti-cooperative, in that a second molecule of inhibitor binds with much lower affinity to a dimer in which an inhibitor molecule is already bound. Because these inhibitors bind to the ubiquinol oxidation site in the bc(1) complex, we propose that the yeast cytochrome bc(1) complex oxidizes ubiquinol by an alternating, half-of-the-sites mechanism.     

Spatial arrangement of coenzyme and substrates bound to L-3-hydroxyacyl-CoA dehydrogenase as studied by spin-labeled analogues of NAD+ and CoA.

The synthesis of nitroxide spin-labeled derivatives of S-acetoacetyl-CoA, S-acetoacetylpantetheine, and S-acetoacetylcysteamine is described. These compounds are active substrates of L-3-hydroxyacyl-CoA dehydrogenase [(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.35] exhibiting vmax values from 20% to 70% of S-acetoacetyl-CoA itself. S-Acetoacetylpantetheine and S-acetoacetylcysteamine form binary complexes with the enzyme and exhibit ESR spectra typical for immobilized nitroxides. In the case of spin-labeled pantetheine, the radical is more mobile. When spin-labeled substrates are bound simultaneously to each active site of this dimeric enzyme, spin-spin interactions differentiate between two alternate orientations of the substrate [Birktoft, J.J., Holden, H.M., Hamlin, R., Xuong, N.H., & Banaszak, L.J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8262-8266]. The fatty acid moiety is thought to be located in a cleft between two domains whereas a large part of the CoA moiety probably extends into the solution. NAD+, spin-labeled at N6 of the adenine ring, is an active coenzyme of L-3-hydroxyacyl-CoA dehydrogenase (60% vmax). Complexes with the enzyme exhibit ESR spectra typical of highly immobilized nitroxides. Binding of coenzyme NAD+ causes conformational changes of the binary enzyme/substrate complex as revealed by changes in the ESR spectrum of spin-labeled S-acetoacetylpantetheine.     

Interaction of cytochrome P-450 with phospholipids in dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol mixtures

ESR and microcalorimetry methods were employed to investigate the thermotropic properties and structure of proteoliposomes that incorporate cytochrome P450 and DMPC-DMPG binary mixtures depending on cytochrome P450 content and phospholipid composition. The microcalorimetry data demonstrated that the incorporation of cytochrome P450 into the phospholipid mixture resulted in bilayer thermal stabilization. The maximum shift of the temperature and proteoliposome transition enthalpy were achieved at the protein/lipid molar ratio of 1:1000 in almost equimolar phospholipid mixture. Using fatty acids that were spin-labeled at different positions (C5, C12, C16), it has been shown that the incorporation of cytochrome P450 into lipid mixtures containing 0-100% DMPG decreases C12 and C16 mobility and increases the C5 order parameter at transition phase (30 degrees C) and liquid crystal phase (37 degrees C) of bilayer. The maximum alteration amplitude of the probes used was not characteristic for the separate DMPC and DMPG but rather for the mixture at the molar ratio close to equimolar value. It is proposed that cytochrome P450 incorporation into the binary mixture initiated the formation of the bilayer crystal-like phase.     

Localization of the active center of nitroxide radical reduction in rat liver microsomes: its relation to cytochrome P-450 and membrane fluidity

The properties and localization of the active center of NADPH-dependent nitroxide radical reduction in rat liver microsomes were investigated with the following five spin-probes as substrates; tetramethylpiperidinol-N-oxyl (TEMPOL) and four spin-labeled stearic acid derivatives with a nitroxide radical at the 5th, 7th, 12th, or 16th position of the hydrocarbon chain (abbreviated as 5SLS, 7SLS, 12SLS, and 16SLS, respectively). The ESR signals of these spin-probes in microsomes decreased on the addition of NADPH, and the decay was inhibited by pretreatment with SKF-525A. Experiments with various microsomal preparations induced by phenobarbital (PB), polychlorinated biphenyls (PCB), or 3-methylcholanthrene (3-MC) revealed that the reduction rate was correlated to the concentration of cytochrome P-450 but not to that of NADPH reductase. Thus, the nitroxide radicals of the SLSs and TEMPOL seem to be reduced by the combined action of NADPH-cytochrome P-450 reductase and cytochrome P-450. The decay showed a lag time, but no distinct correlation was observed between the lag time and the spin-probe species. On the other hand, the initial velocity of the nitroxide reduction depended strongly on the spin-probe species. Among the five spin-probes, 7SLS was reduced most quickly, followed by 5SLS, 12SLS, TEMPOL, and 16SLS in that order. The reduction rate varied from 0.18/min for 7SLS to 0.08/min for 16SLS. There was a linear relation between the cytochrome P-450 content and the reduction rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen peroxide-mediated inactivation of microsomal cytochrome P450 during monooxygenase reactions

Cytochrome P450 can undergo inactivation following monooxygenase reactions in liver microsomes of untreated, phenobarbital and 3-methylcholanthrene-treated rats and rabbits. The acceleration of cytochrome P450 loss in the presence of catalase inhibitors (sodium azide, hydroxylamine) indicates that hydrogen peroxide is involved in hemoprotein degradation. It was revealed that cytochrome P450 is inactivated mainly by H₂O₂ formed through peroxy complex breakdown, whereas H₂O₂ formed via the dismutation of superoxide anions produces a slight inactivating effect. The hydrogen peroxide added outside or formed by a glucose-glucose oxidase system has less of an inactivating effect than H₂O₂ produced within the cytochrome P450 active center. Self-inactivation of cytochrome P450 during oxygenase reactions is highly specific. Other components of the monooxygenase system, such as cytochrome b₅, NADH- and NADPH-specific flavorproteins, undergo no inactivation. The alterations in phospholipid content and in the rate of lipid peroxidation were not observed as well. The inactivation of cytochrome P450 by H₂O₂ is the result of heme loss or destruction without cytochrome P420 formation. Such. a mechanism operates with different substrates and cytochrome P450 species catalyzing the partially coupled monooxygenase reactions.     

Modeling and synthesis of novel tight-binding inhibitors of cytochrome P450 2C9

Cytochrome P450 2C9 (2C9) is one of the three major drug metabolizing cytochrome P450 enzymes in human liver. Although the crystal structure of 2C9 has been solved, the important physicochemical properties of substrate-enzyme interactions remain difficult to be determined. This is due in part to the conformational flexibility of mammalian P450 enzymes. Therefore, probing the active-site with high-affinity substrates is important in further understanding substrate-enzyme interactions. Three-dimensional quantitative structure-activity relationships (3D-QSAR) and docking experiments have been shown to be useful tools in correlating biological activity with structure. In particular we have previously reported that the very tight-binding inhibitor benzbromarone can provide important information about the active-site of 2C9. In this study we report the binding affinities and potential substrate-enzyme interactions of 4H-chromen-4-one analogs, which are structurally similar to benzbromarone. The chromenone structures are synthetically accessible inhibitors and give inhibition constants as low as 4.2 nM, comparable with the very tightest-binding inhibitors of 2C9. Adding these compounds to our previous 2C9 libraries for CoMFA models reinforces the important electrostatic and hydrophobic features of substrate binding. These compounds have also been docked in the 2C9 crystal structure and the results indicate that Arg 108 plays significant roles in the binding of chromenone substrates.     

X-ray structure of human aromatase reveals an androgen-specific active site

Aromatase is a unique cytochrome P450 that catalyzes the removal of the 19-methyl group and aromatization of the A-ring of androgens for the synthesis of estrogens. All human estrogens are synthesized via this enzymatic aromatization pathway. Aromatase inhibitors thus constitute a frontline therapy for estrogen-dependent breast cancer. Despite decades of intense investigation, this enzyme of the endoplasmic reticulum membrane has eluded all structure determination efforts. We have determined the crystal structure of the highly active aromatase purified from human placenta, in complex with its natural substrate androstenedione. The structure shows the binding mode of androstenedione in the catalytically active oxidized high-spin ferric state of the enzyme. Hydrogen bond-forming interactions and tight packing hydrophobic side chains that complement the puckering of the steroid backbone provide the molecular basis for the exclusive androgenic specificity of aromatase. Locations of catalytic residues and water molecules shed new light on the mechanism of the aromatization step. The structure also suggests a membrane integration model indicative of the passage of steroids through the lipid bilayer.     

Affinity modification of membrane-bound microsomal cytochrome P-450 with lipophilic analogs of substrates

The following lipophilic spin-labeled cytochrome P-450 analogs were synthesized: 2-octyl-4-(3-iodine-2-oxopropylidene)-2,3,5,5-tetramethylimidaz olidine-1-oxyl (RIII), 2-nonyl-4-(3-iodine-2-oxopropylidene)-2,3,5,5-tetramethylimidaz olidine-1-oxyl (RIV), 2-hepta-decyl-4-(3-iodine-2-oxopropylidene)-2,3,5,5-tetramethyl imidazolidine-1- oxyl (RV). The distribution coefficients, k, in water--lipid and water--octanol systems as well as the theoretical estimates of k for these and previously synthesized analogs, i.e., 4-(3-iodine-2-oxo-propylidenyl)-2,2,3,5,5-pentamethylimidazolid ine-1-oxyl (RI) and 2-hexyl-4-(3-iodine-2-oxopropylidene)-2,3,5,5-tetramethylimidaz olidine- 1-oxyl (RII) were determined. It was shown that RIII and RIV bind as type I substrates to cytochrome P-450 from rat microsomes induced with phenobarbital or 3-methylcholanthrene as well as to those from control rats. Radicals RIII and RIV inhibit the oxidation of aniline, aminopyrine and benzphetamine. RIII-RV strongly inhibit the O-deethylation of 7-etoxyresorufin. The inhibitory activity of the radicals increases in the following order: RV less than RIV less than or equal to RI less than or equal to RIII less than RII. The experimental results suggest that the inhibitory properties are nonmonotonuesly related to the lipophility. The high lipophility of RIII and its strong inhibitory properties permit to render the latter to the class of inhibitors which can be transported by liposome membrane vehicles to the liver, inhibit the in vivo activity of the microsomal system and thus prolong the effects of drugs oxidized by cytochrome P-450.     

Role of protein and substrate dynamics in catalysis by Pseudomonas putida cytochrome P450cam

The role of protein structural flexibility and substrate dynamics in catalysis by cytochrome P450 enzymes is an area of current interest. We have addressed these in cytochrome P450(cam) (P450(cam)) and its Y96A mutant with camphor and its related compounds using fluorescence spectroscopy. Previously [Prasad et al. (2000) FEBS Lett. 477, 157-160], we provided experimental support to dynamic fluctuations in P450(cam), and substrate access into the active site region via the channel next to the flexible F-G helix-loop-helix segment. In the investigation described here, we show that the dynamic fluctuations in the enzyme are substrate dependent as reflected by tryptophan fluorescence quenching experiments. The orientation of tryptophan relative to heme (kappa(2)) for W42 obtained from time-resolved tryptophan fluorescence measurements show variation with type of substrate bound to P450(cam) suggesting regions distant from heme-binding site are affected by physicochemical and steric characteristics/protein-substrate interactions of P450(cam) active site. We monitored substrate dynamics in the active site region of P450(cam) by time-resolved substrate anisotropy measurements. The anisotropy decay of substrates bound to P450(cam) indicate that mobility of substrates is modulated by physicochemical and steric characteristics/protein-substrate interactions of local active site structure, and provides an understanding of factors controlling observed hydroxylated products for substrate bound P450(cam) complexes. The present study shows that P450(cam) local and peripheral structural flexibility and heterogeneity along with substrate mobility play an important role in regulating substrate binding orientation during catalysis and accommodating diverse range of substrates within P450(cam) heme pocket.     

Formation of benzoquinol moiety in cornoside by salidroside mono-oxygenase,a cytochrome P450 enzyme,from <Emphasis Type="Italic">Abeliophyllum distichum </Emphasis>cell suspension cultures

A microsomal fraction prepared from Abeliophyllum distichumNakai (Oleaceae) cell suspension cultures oxidized salidroside, a glucoside of 4-hydroxyphenylethyl alcohol, to cornoside possessing a unique benzoquinol ring. The enzyme named salidroside mono-oxygenase required NADPH as the only cofactor, and molecular oxygen. The reaction was strongly inhibited by CO as well as several cytochrome P450 inhibitors, such as cytochrome c and miconazole, indicating the involvement of a cytochrome P450 enzyme. Salidroside mono-oxygenase accepted salidroside as the only substrate, but did not oxidize 4-hydroxyphenylethyl alcohol, the salidroside aglucone, and 4-hydroxybenzoic acid. The optimum pH of the reaction was 7.5, and apparent K(m) values for salidroside and NADPH were 44 micro M and 33 micro M, respectively. The benzoquinol ring formation mechanism is discussed in comparison to the mechanism for ipso substitution of 4-hydroxybenzoate by active oxygen species followed by elimination leading to hydroquinone.     

Study of the binding of substrates and paramagnetic Mn2+ ions with phosphoglycerate kinase by saturating ESR spectra of a spin-labelled protein]

A single SH-group of phosphoglycerate kinase from yeast was modified by mercury-containing spin label. The saturation curves of ESR spectra of the spin-labeled enzyme were studied. The paramagnetic ions of Mn2+ bound to the centre of ion nonspecific binding or active centre in the complex with ATP can influence the saturation of the spin-labeled enzyme. The saturation curves of the ESR signal of the spin-labeled enzyme in the presence of paramagnetic complex of CrATP were studied. It has been demonstrated that the second nonspecific centre of ATP binding is located at the active site of the enzyme (3-phosphoglycerate binding centre).