Ferric P-450 Research Articles

Engineering cytochrome c peroxidase into cytochrome P450: a proximal effect on heme-thiolate ligation.

In an effort to investigate factors required to stabilize heme-thiolate ligation, key structural components necessary to convert cytochrome c peroxidase (CcP) into a thiolate-ligated cytochrome P450-like enzyme have been evaluated and the H175C/D235L CcP double mutant has been engineered. The UV-visible absorption, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectra for the double mutant at pH 8.0 are reported herein. The close similarity between the spectra of ferric substrate-bound cytochrome P450cam and those of the exogenous ligand-free ferric state of the double mutant with all three techniques support the conclusion that the latter has a pentacoordinate, high-spin heme with thiolate ligation. Previous efforts to prepare a thiolate-ligated mutant of CcP with the H175C single mutant led to Cys oxidation to cysteic acid [Choudhury et al. (1994) J. Biol. Chem. 267, 25656-25659]. Therefore it is concluded that changing the proximal Asp235 residue to Leu is critical in forming a stable heme-thiolate ligation in the resting state of the enzyme. To further probe the versatility of the CcP double mutant as a ferric P450 model, hexacoordinate low-spin complexes have also been prepared. Addition of the neutral ligand imidazole or of the anionic ligand cyanide results in formation of hexacoordinate adducts that retain thiolate ligation as determined by spectral comparison to the analogous derivatives of ferric P450cam. The stability of these complexes and their similarity to the analogous forms of P450cam illustrates the potential of the H175C/D235L CcP double mutant as a model for ferric P450 enzymes. This study marks the first time a stable cyanoferric complex of a model P450 has been made and demonstrates the importance of the environment around the primary coordination ligands in stabilizing metal-ligand ligation.

Oxidation Kinetics of Ethanol by Human Cytochrome P450 2E1: RATE-LIMITING PRODUCT RELEASE ACCOUNTS FOR EFFECTS OF ISOTOPIC HYDROGEN SUBSTITUTION AND CYTOCHROME b5 ON STEADY-STATE KINETICS

A number of cytochrome P450 (P450) 2E1 substrates are known to show kinetic deuterium isotope effects of approximately 5 on Km (DK = DKm/HKm), but not on kcat, in rat liver microsomes (e.g. N-nitrosodimethylamine, ethanol, and CH2Cl2). We observed DKm values of 3-5 for recombinant human P450 2E1-catalyzed ethanol oxidation. Replacing NADPH and O2 with the oxygen surrogate cumene hydroperoxide yielded similar results. Ferric P450 2E1 reduction was fast (k >1000 min-1) even in the absence of substrate. These results indicate that the basis for the increase in Km is in the latter portion of the catalytic cycle. The intrinsic isotope effect (Dk) for ethanol oxidation was determined (competitively) to be 3.8, indicating that C-H bond cleavage is isotopically sensitive. Pre-steady-state studies showed a burst of product formation (k = 410 min-1), with the burst amplitude corresponding to the P450 concentration. Deuteration of ethanol resulted in an isotope effect of 3.2 on the rate of the burst. We conclude that product release is rate-limiting in the oxidation of ethanol to acetaldehyde by P450 2E1. The steady-state kinetics can be described by a paradigm in which the kcat approximates the rate of product release, and Km is an expression in which the denominator is dominated by the rate of C-H bond breaking.

Absorption spectral studies on heme ligand interactions of P-450 nor

Heme-external ligand interactions of P-450 nor were examined spectrophotometrically and compared with those of other P-450s. Most nitrogenous ligands induced type II spectral changes on binding to ferric P-450 nor, as did other P-450s. In contrast with other P-450s, 2-methylpyridine and 1-butanol induced type I changes in the spectrum of P-450 nor. No spectral interaction of ferrous P-450 nor with these ligands was observed. The absorption spectra of the alkyl isocyanide complexes of ferrous P-450 nor exhibited the Soret peak at 427 nm with a slight shoulder at around 455 nm at neutral pH, and this shoulder was intensified as the pH was increased, suggesting that the isocyanide complexes of P-450 nor existed in two states (the 430 and 455 nm states) which were in pH-dependent equilibrium in a similar manner to microsomal P-450s. However, the equilibrium was shifted mostly to the 430 nm state in the complexes of P-450 nor. The findings suggest that P-450 nor, especially its ferrous form, has some distinct features from P-450 cam and microsomal P-450s in the distal heme environment.

Lack of Electron Transfer from Cytochrome b5 in Stimulation of Catalytic Activities of Cytochrome P450 3A4: CHARACTERIZATION OF A RECONSTITUTED CYTOCHROME P450 3A4/NADPH-CYTOCHROME P450 REDUCTASE SYSTEM AND STUDIES WITH APO-CYTOCHROME b5

Many catalytic activities of cytochrome P450 (P450) 3A4, the major human liver P450 enzyme, require cytochrome b5 (b5) for optimal rates. The stimulatory effect of b5 on P450 reactions has generally been thought to be due to transfer of electrons from ferrous b5 to the ferrous P450-O2-substrate complex. We found that apo-b5, devoid of heme, could substitute for b5 in stimulating two prototypic activities, testosterone 6beta hydroxylation and nifedipine oxidation. The stimulatory effect was not seen with albumin, hemoglobin, catalase, or cytochrome c. Apo-b5 could not substitute for b5 in a testosterone 6beta hydroxylation system composed of NADH-b5 reductase and P450 3A4. Rates of electron transfer from NADPH-P450 reductase to ferric P450 3A4 were too slow (<2 min-1) to support testosterone 6beta hydroxylation ( approximately 14 min-1) unless b5 or apo-b5 was present, when rates of approximately 700 min-1 were measured. The oxidation-reduction potential (Em,7) of the ferric/ferrous couple of P450 3A4 was unchanged ( approximately -310 mV) under different conditions in which the kinetics of reduction were altered by the addition of substrate and/or apo-b5. Rapid reduction of P450 3A4 required substrate and a preformed complex of P450 3A4, NADPH-P450 reductase, and b5; the rates of binding of the proteins to each other were 2-3 orders of magnitude less than reduction rates. We conclude that b5 can facilitate some P450 3A4-catalyzed oxidations by complexing with P450 3A4 and enhancing its reduction by NADPH-P450 reductase, without directly transferring electrons to P450.

Roles of Cytochrome b5in the Oxidation of Testosterone and Nifedipine by Recombinant Cytochrome P450 3A4 and by Human Liver Microsomes

NADH-dependent testosterone 6β-hydroxylation and nifedipine oxidation activities could be reconstituted in systems containing cytochrome b5(b5), NADH–b5reductase, and bacterial recombinant cytochrome P450 (P450) 3A4 with a synthetic phospholipid mixture, cholate, MgCl2, and reduced glutathione. Replacement of NADH–b5reductase with NADPH–P450 reductase produced an eightfold increase in testosterone 6β-hydroxylation activity. Further stimulation could be obtained when NADPH was used as an electron donor instead of NADH. Removal of b5from the NADH- and NADPH-supported systems caused a 90% loss of testosterone 6β-hydroxylation activities in the presence of NADPH–P450 reductase but resulted in complete loss of the activities in the absence of NADPH–P450 reductase. These results suggested that about 10% of the activities was due to electron flow from NADPH–P450 reductase to P450 3A4 in the absence of b5. In the presence of testosterone and MgCl2, P450 3A4 was reduced by b5and NADH–b5reductase, although the rate of P450 3A4 reduction was much slower than that by NADPH–P450 reductase. Anti-human b5immunoglobulin G (IgG) (purified using rabbit b5affinity chromatography) inhibited testosterone 6β-hydroxylation activity catalyzed by human liver microsomes more strongly in NADH- than in NADPH-supported reactions. However, anti-rat NADPH–P450 reductase IgG inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. Addition of NADH enhanced NADPH-supported testosterone and nifedipine oxidations in human liver microsomes. MgCl2stimulated rates of reduction of b5by NADPH–P450 reductase, but not by NADH–b5reductase, in reconstituted systems. These results suggest that b5is an essential component in P450 3A4-catalyzed testosterone hydroxylation and nifedipine oxidation in human liver microsomes. Our previous observation that rates of reduction of ferric P450 3A4 by NADPH–P450 reductase are accelerated by complexation with substrates and b5is supported in this study.

Role of Cytochrome P450 in the Metabolism and Toxicity of Hydroperoxides in Isolated Rat Hepatocytes

The contributions of cytochromes P450 (P450) to the metabolism and toxicity of hydroperoxides in freshly isolated rat hepatocytes were investigated utilizing 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclo-hexadienone (BHTOOH). This hydroperoxide was rapidly degraded in cell suspensions, and the products were identical to those determined previously with subcellular preparations of ferric P450. With 250 μMBHTOOH, the ratio of glutathione peroxidase-mediated:P450-mediated metabolism was estimated to be about 3:1. Surprisingly, BHTOOH was found to be a more potent cytotoxin than cumyl hydroperoxide (CuOOH), despite the fact that it caused substantially less lipid peroxidation than the latter. P450 inhibition enhanced the toxicity of BHTOOH, but lowered the toxicity of CuOOH. These data demonstrate that intracellular ferric P450 can compete with glutathione peroxidase to reduce hydroperoxides by 1- and 2-electron processes. If the alkoxy radical from homolytic cleavage of the O–O bond can undergo facile intramolecular reactions to nontoxic products, as with BHTOOH, the role of P450 is detoxification. On the other hand, if the alkoxy radical preferentially attacks membrane lipids, as with CuOOH, P450 contributes to lipid peroxidation and toxicity. It was determined that the levels of glutathione, protein thiols, and ATP decreased in parallel with BHTOOH-induced cell death, but no conclusions are possible concerning mechanisms underlying the relatively potent toxicity of BHTOOH. Toxicity may be related to the high lipophilicity of this hydroperoxide which, presumably, facilitates its passage into cells and distribution to various intracellular sites. BHTOOH appears to be an excellent model compound for investigating mechanisms of hydroperoxide-medi ated cytotoxicity which do not involve lipid peroxidation.

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 of 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 b 5. 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.

High-pressure-induced transitions in microsomal cytochrome P450 2B4 in solution: Evidence for conformational inhomogeneity in the oligomers

Pressure-induced changes in ferric P450 2B4 (LM2) were studied as a function of benzphetamine concentration (0.05/2 mM) and state of aggregation of the hemoprotein in solution. Application of factor analysis to the spectral changes in the Soret region allowed us to resolve two particular pressure-induced processes in 2B4 oligomers. The first process was identified as the conversion of the low-spin P450 into the P420 state. At 25°C it was followed by decay (bleaching) about 50% of the newly formed P420. The second process was a pressure-induced high- to low-spin shift. Both transitions were reversible, except the hemoprotein bleaching. The applitude of the P450 → P420 transition accounted for 67 ± 5% of the total hemoprotein content. Furthermore, the fraction of the hemoprotein exposed to spin equilibrium was not affected by the P450 → P420 conversion and was estimated to be only about 31 ± 5% of the total hemoprotein content. After the dissociation of the oligomers by 0.2% Triton N-101, the inhomogeneity vanished: 95% of the monomers were involved in the P450 → P420 transition (Δ V° = −86 ml/mol) followed by intense bleaching of the hemoprotein. This agrees with our earlier observations on the reduced carbonyl complex of P450 2B4 and suggests some conformational difference between subunits in P450 LM2 oligomers. The parameters of the P450 → P420 conversion (Δ V° = −32 ml/mol, P 1 2 = 1560 bar) show no dependency on the substrate concentration. Analysis of the pressure-induced spin shift versus benzphetamine concentration shows this transition to be caused mainly by changes in the spin equilibrium of both substrate-bound (Δ V° = −49 ml/mol) and substrate-free ( ΔV° = −21 ml/mol) hemoprotein, whereas the substrate binding step itself has a very weak pressure dependency ( ΔV° = −8 ml/mol).

Interactions of Peroxyquinols with Cytochromes P450 2B1, 3A1, and 3A5 - Influence of the Apoprotein on Heterolytic Versus Homolytic O-O Bond-Cleavage

In studies of the mechanisms involved in oxygen activation by cytochromes P450 (P450s), organic hydroperoxides are frequently used to model the putative peroxyferrous complex formed immediately prior to oxygen-oxygen bond cleavage during the reaction cycle of this monooxygenase. Heterolysis of the O-O bond by ferric P450 generates the catalytically competent oxo-ferryl intermediate analogous to peroxidase Compound I, and homolysis produces the Compound II analog capable only of one-electron oxidations. As P450s have been shown to catalyze both modes of O-O bond scission, the present investigation was focused on the influence of the apoprotein on the relative contributions of these competing processes. Liver microsomes from rats treated with the P450-inducing agents phenobarbital, β-naphthoflavone, and dexamethasone, as well as purified P450s 2B1, 3A1, and 3A5 were incubated with 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2, 5-cyclohexadienone (BHTOOH). Ratios of heterolysis to homolysis were determined by analyzing the products derived from this hydroperoxide. The data demonstrate that BHTOOH is cleaved with a ratio of approximately 1.0 with all of the liver microsomal or purified P450s investigated, except with liver microsomes from dexamethasone-treated rats or with P450s 3A1 and 3A5. In these cases, heterolysis predominated over homolysis by factors of 2.5 to 4.0. On the other hand, microsomes rich in P450 2B1 catalyzed predominantly heterolysis with analogs of BHTOOH containing smaller alkyl substituents. The data are consistent with a requirement for solvent access to the peroxyferrous complex and general acid catalysis of heterolytic O-O bond cleavage by water.

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.

Expression of Modified Human Cytochrome P450 1A1 in Escherichia coli: Effects of 5′ Substitution, Stabilization, Purification, Spectral Characterization, and Catalytic Properties

Human cytochrome P450 (P450) 1A1 is primarily an extrahepatic enzyme and is important because of its roles in the activation of polycyclic hydrocarbons and other xenobiotic chemicals. Purification of active enzyme from human tissues has not been successful. We report the expression and purification of the recombinant enzyme from Escherichia coli. A full-length cDNA of human cytochrome P450 1A1 and several modified constructs were engineered into a pCW vector and used to transform E. coli cells. Little expression was observed with the native sequence and several modified constructs, but successful expression (20-25 nmol membrane-bound P450 1A1 per liter of culture) was achieved with a construct in which the Ala codon GCT was placed in the second position and the 5′-terminal codons were maximized for AT content and minimized for the potential of secondary structure formation of the mRNA transcript. α-Naphthoflavone was found to protect against denaturation by detergents during solubilization and was added to buffers used for purification. The recombinant P450 1A1 was purified to electrophoretic homogeneity after two ion-exchange chromatography steps in ∼50% yield. N-Terminal amino acid sequence analysis verified the expected first 21 residues, with the exception of the terminal Met. The isolated human ferric P450 1A1 was predominantly in the high spin state, in contrast to the orthologous rat and rabbit enzymes. Recombinant P450 1A1 catalyzed 7-ethoxyresorufin O-deethylation and benzo[ a]pyrene 3-hydroxylation with K m values of 0.58 and 15 μM and V max values of 8.3 and 2.5 nmol min −1 (nmol P450 1A1) −1, respectively. The successful expression and purification of human P450 1A1 should increase the availability of this enzyme and the generation of antibodies for further biochemical and other biological studies.

Comparison of heme environment at the putative distal region of P-450 s utilizing their external and internal nitrogenous ligand bound forms

Thr-303 to Lys-mutated P-450 2E1 1 1 Individual forms of P-450 are designated according to the nomenclature recommended by Nebert et al. [1]. , as well as Thr-301 to Lys-mutated P-450 2C2, had absorption spectra characteristic of a nitrogenous ligand-bound form of P-450, such as the pyridine complex of P-450 2E1; (i) in the ferric state, the red-shifted Soret band, compared with the typical low-spin type spectmm of P-450, and the more intense β band than the α band and (ii) in the ferrous state, two Soret peaks at around 447 and 422 nm, the relative intensities of which depended on pH, indicating the existence of two interconvertible states. The equilibrium between the two states of the mutated P-450 2E1 appeared to be shifted toward the 422 nm state, compared with the mutated P-450 2C2. The corresponding mutant of P-450 2C14 had similar spectral properties to those of both mutated P-450 s except that the shorter of the two Soret bands of its ferrous form was relatively broad and appeared at 418 mn. These findings suggest that the ϵ-amino-nitrogen of the Lys of the mutated P-450 s is located in the appropriate position to occupy the sixth coordination position with the heme iron and spatial differences exist in the essentially conserved structure of the distal heme domain among the three ferrous Lys-mutated P-450 s.

Expression of Modified Human Cytochrome P450 2E1 in Escherichia coli, Purification, and Spectral and Catalytic Properties

Human cytochrome P450 (P450) 2E1 is of interest because of its role in the oxidation of numerous drugs and carcinogens. The purification of the protein from human liver is difficult, and we report the development of a system for relatively high-level expression in Escherichia coli. A cDNA was prepared from liver cDNA by polymerase chain reaction methods and several variants with modified 5′-termini were constructed. Analysis of seven of these indicated that the highest levels of expression were found when the first 21 codons of the native sequence were deleted and the Trp immediately following the resulting N-terminal Met was changed to Ala (GCT). Levels of 40-nmol membrane-bound P450 2E1 (liter culture) −1 mere routinely recovered. The recombinant P450 2E1 was purified to electrophoretic homogeneity from the bacterial membranes in two ion-exchange steps in >80% yield. Ferric P450 2E1 was isolated in a mixed spin state. The enzyme was active in chlorzoxazone 6-hydroxylation; the addition of human liver cytochrome b 5 lowered the K m for the substrate and increased V max. N-Terminal amino acid sequence analysis yielded the expected first 21 residues. The expression system should facilitate the availability of human P450 2E1 and antibodies for studies of the enzyme.

Spectral Intermediate in the Reaction of Ferrous Cytochrome P450 cam with Superoxide Anion

The addition of KO 2 to ferrous cytochrome P450 cam in the presence of d-camphor under anaerobic condition resulted in the formation of exo- and endo-5-hydroxycamphor. The product formation was inhibited by the addition of superoxide dismutase, not catalase. The ferrous form of P450 cam reacts with O − 2 to form an intermediate species of the enzyme, of which spectrum is closely similar to that of the oxygenated form of P450 cam. Subsequently, the intermediate was converted to native ferric P450 cam. In contrast, ferrous form of horseradish peroxidase reacted with O − 2 to form Compound I of the enzyme without detection of intermediates.

Essential Role of His163 of Cytochrome P450 1A2 in Catalytic Functions Associated with Cytochrome b 5

The effect of cytochrome b 5 on the activity of cytochrome P450 1A2 (P450 1A2) and three site-directed mutants designed to alter surface charges has been investigated. Cytochrome b 5 increased the rate of oxidation of methanol and/or 7-ethoxycoumarin 2- to 10-fold in both wild type and mutant P450. Cytochrome b 5 also increased the coupling of electron transfer to substrate hydroxylation as opposed to the uncoupling reaction which leads to peroxide production. The effect of cytochrome b 5 on both wild type and the Lys99Glu and Lys401Glu mutants are similar. In contrast, turnover numbers in the His163Glu mutant did not change in the presence of cytochrome b 5. The reduction potential of the His163Glu mutant decreased approximately −40 mV while the Lys99Glu and Lys401Glu mutants exhibited little change. The rate of photoreduction decreased from 1.1 × 10 −1 min −1 to 8.3 × 10 −3 min −1 in the His163Glu mutant while the rate of electron transfer from ferrous P450 to ferric cytochrome b 5 increased from 0.02 min −1 to > 5 min −1. Overall, the present study suggests that His163 is important to keep an appropriate redox potential of P450 1A2 for optimum electron transfer to occur from cytochrome b 5. Based on the P450 101 crystal structure, His163 is not expected to directly contact the heme. Therefore, the resulting change in redox properties in the His163Glu mutant is probably not the result of a direct electrostatic change in the heme environment.

Photoaffinity labeling of cytochrome P450 2B4: capture of active site heme ligands by a photocarbene.

Spiro[adamantane-2,2'-diazirine], which produces adamantyl carbene upon photolysis, binds tightly to P450 2B4 (KS = 3.2 microM), giving a normal substrate binding difference spectrum. Irradiation of 2-[3H]adamantane diazirine at 365 nm in the presence of native, ferric P450 2B4 resulted in first-order photolysis (t1/2 = 1.8 min). The main product was 2-[3H]adamantanol, with about 6% of the radioactivity covalently bound to P450 2B4. With the ferrous carbonyl form of P450 2B4, 2-adamantanol production decreased and protein labeling increased to 12%. When ferric cyanide 2B4 was used, 2-adamantanecarbonitrile was formed in addition to 2-adamantanol. The nitrile appears to have resulted from capture of the iron-bound cyanide ligand by the carbene. The use of multiple cycles of photolysis increased the percentage of protein labeling to 76%. Photolabeling was inhibited by known 2B4 substrates and inhibitors. Also, N-demethylation of benzphetamine and generation of a substrate binding difference spectrum by benzphetamine were both inhibited stoichiometrically with the fraction of radiolabeled protein. The labeled protein was permanently converted to the high-spin state, as indicated by the characteristic change in the absorbance spectrum, demonstrating irreversible occupation of the substrate binding site by the adamantyl residue. Mild acid hydrolysis of radiolabeled 2B4 at the five Asp-Pro bonds generated a 2-kDa peptide which carried 78% of the radioactivity. These results are interpreted as the result of the active site carbene reacting by three competing pathways: capture of the heme sixth ligand to yield either 2-adamantanol or 2-adamantanecarbonitrile, capture of an unbound active site water molecule to yield adamantanol, and covalent attachment to a protein residue. Thus, the P450 2B4 active site appears to contain at least one unbound water molecule in addition to the heme aquo sixth ligand, even when substrate is present.

Cytochrome P-450 55A1 (P-450dNIR) acts as nitric oxide reductase employing NADH as the direct electron donor.

Cytochrome P-450dNIR (P-450dNIR), involved in the fungal denitrification by Fusarium oxysporum, was purified to homogeneity. The cytochrome P-450 (P-450) exhibited a potent nitric oxide (NO) reductase activity to form nitrous oxide (N2O) employing NADH but not NADPH as the sole effective electron donor. The apparent maximum turnover rate against NO was estimated as high as 31,500 min-1. The stoichiometry of the reaction between NO:NADH:N2O was 2:1:1. The reaction required neither an artificial electron-carrying mediator nor other proteinaceous components. An anaerobic incubation of ferric P-450dNIR.NO complex, but not of free ferric P-450dNIR, resulted in rapid reduction of the P-450, indicating that P-450dNIR.NO complex was reduced directly by NADH. Spectral changes during catalytic turnover indicated that decomposition of the ferrous P-450.NO complex might be rate-limiting. The reaction was not inhibited by carbon monoxide at all, suggesting that the free ferrous P-450 is not formed during turnover. On the basis of these results a possible reaction mechanism was considered. The present results demonstrated not only the unique reaction catalyzed by P-450, but also the first P-450-dependent reaction where the electron transport from NAD(P)H to P-450 is not supported by other components. Now that the physiological function of P-450 55A1 (P-450dNIR) has been found, we propose to alter the trivial name to P-450nor.

Inhibition by a novel azole antifungal agent with a geranyl group on lanosterol 14α-demethylase of yeast

AFK-108 (1-[2-(2,4-dichlorophenyl)-2-((2 E)-3,7-dimethylocta-2,6-dienyloxy)ethyl]-1 H-imidazole) is a new imidazole derivative characterized by a geranyl substituent showing strong antifungal activity. Azole antifungal agents are known to be potent inhibitors of lanosterol 14α-demethylase (P450 14DM) of fungi. The role of the geranyl group of AFK-108 on interaction of AFK-108 with the target was studied by using Saccharomyces cerevisiae P450 14DM as the model enzyme. AFK-108 and some of its derivatives bound to oxidized P450 14DM with one-to-one stoichiometry and inhibited the demethylase activity. AFK-108 derivatives having the longer farnesyl or the shorter prenyl group showed lower affinity than AFK-108 for the enzyme. AFK-108 caused 100% inhibition at the equivalent concentration to P450 14DM in the reaction mixture (0.07 μM), while the farnesyl derivative inhibited the activity by 60% at the same concentration. AFK-108 interfered with the binding of CO to the ferrous P450 14DM. However, the interfering effect of the prenyl derivative was lower than that of AFK-108. Another AFK-108 derivative having the saturated 3,7-dimethyloctyl group was also a weaker inhibitor than AFK-108. These experiments suggest that the geranyl group of AFK-108 interacts with the substrate binding site of P450 14DM that recognises the side chain of the substrate. AFK-108 is the first example of an azole derivative interacting with the side chain recognising region of the substrate binding site of P450 14DM.

Nitrogenous Ligation at the Sixth Coordination Position of the Thr-301 to Lys-Mutated P450IIC21 Heme Iron2

Threonine-301 of P450IIC2 was replaced by lysine via site-directed mutagenesis. The Lys-mutated P450 exhibited absorption spectra that were characteristic of the nitrogenous-ligand-bound form of P450. In the oxidized form, the Soret band was red-shifted as compared with the typical ferric low-spin form of P450 and the beta band was more intense than the alpha band. In the reduced form, two Soret peaks were observable at 447 and 423 nm and their relative heights were dependent on pH, indicating the existence of two interconvertible states of ferrous Lys-mutated P450 which are in equilibrium. In addition, the interaction of external ligands with the P450 heme iron was profoundly inhibited both in the oxidized and reduced forms. These findings suggest that epsilon-amino nitrogen of Lys-301, which was introduced by amino acid substitution, occupies the 6th coordination position with the heme iron of the Lys-mutated P450, because, owing to conformation of the P450 protein, the epsilon-amino group may be located at just the right position for coordination as the internal 6th ligand.

Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae

A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation. Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine. Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes). The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family.