Fast photochemical reactions of cytochrome P450 at subzero temperatures.

Several reactions of the cytochrome P450 multi-step cycle have been studied by fast light activation combined with subzero temperatures. A flash device was adapted to an Aminco-Chance DW 2 spectrophotometer equipped for subzero temperature thermostatisation. The first electron can be introduced into the cycle by non specific reducing agents such as reduced flavin mononucleotide (FMNH2) or methylviologen radical (MV.). This first reduction remains a fast process even at subzero temperatures. The oxy-compound Fe2+-O2 can thus be formed either directly from Fe2+ or via the photodissociation of the carboxy-ferro adduct. Fe2+-O2 is stable at subzero temperatures towards spontaneous autoxidation as well as further reduction by FMNH2 or MW.. In addition, the recombination of CO after flash photodissociation of Fe2+-CO was used to study in more details the specific behaviors of the purified microsomal cytochrome.     

Absorption spectra of highly purified liver microsomal cytochrome P-450 in non-equilibrium conformational states at low temperatures

Absorption spectra of highly purified liver microsomal cytochrome P-450 in non-equilibrium states were obtained at 77 K by reduction with trapped electrons, formed by gamma-irradiation of the water-glycerol matrix. In contrast to the equilibrium form of ferrous cytochrome P-450 with the heme iron in the high-spin state the non-equilibrium ferrous state has a low-spin heme iron. The absorption spectrum of the non-equilibrium ferrous cytochrome P-450 is characterized by two bands at 564 (-band) and 530 nm (-band). When the temperature is increased to about 278 K this non-equilibrium form of the reduced enzyme is relaxed to the corresponding equilibrium form with a single absorption band at 548 nm in the visible region characteristic for a high-spin heme iron.     

Spectral perturbation of human microsomal cytochrome P-450 by flavonoid binding

Aromatase is the cytochrome P-450 complex responsible for oestrogen biosynthesis in vivo. Inhibitors of this enzyme complex might therefore serve to modulate oestrogen-dependent processes by interfering with the production of oestrogens. Thus, these agents may be useful in reproductive processes and in treating oestrogen-dependent disease states such as breast and endometrial cancer. We have demonstrated that inhibitors such as the naturally occurring flavonoids having 5,7-dihydroxy substituents can bind to human placental cytochrome P-450 with affinity comparable to their ability to inhibit aromatization of androstenedione and testosterone to oestradiol and oestrone, respectively. It appears that the mechanism of this inhibition requires the flavonoid to bind to the active site of the cytochrome P-450 without prior generation of metabolic intermediate products. Our data also suggest that the presently known differences in potency of inhibition of cytochrome P-450-mediated aromatization of steroids by different hydroxylated derivatives of 5,7-dihydroxyflavones may arise from their different binding affinity to the enzyme, particularly those compounds hydroxylated in the C3 position in ring C of the flavonoid nucleus.     

Spectral and catalytic properties of cytochrome P-450 from a diazinon-resistant housefly strain

Microsomes from the diazinon-resistant Rutgers strain of housefly contain amounts of cytochrome P-450 that are larger than those reported for rat liver, but the specific activity expressed as nmole of cytochrome P-450 per mg protein is much lower. The hemoprotein shows that spectral changes type I, II and IV are essentially in the low-spin form as judged by the n-octylamine and ethyl isocyanide difference spectra, and is unstable at pH below 6.5 and above 8.0. Cytochrome P-420 is also produced with time when CO-difference spectra are recorded. This is accelerated at pH above 8.0. The presence of contaminating amounts of cytochrome P-420, due to denaturation during spectral analysis or to the method used to isolate the microsomes, makes questionable the practice of characterizing the hemoprotein on the basis of the 455 nm peak in the ethyl isocyanide spectra, since a 434 nm peak is produced with concomitant decrease of the 455 nm peak. Microsomes hydroxylate naphthalene, aminopyrine and aniline, but the activity when expressed as nmole of product per nmole of cytochrome P-450 is the same or lower than that reported for other resistant housefly strains.     

Substrate binding to solubilized cytochrome P-450 from rabbits at different temperatures.

The binding affinities of selected type I- and type II-substrates to partially purified cytochrome p-450 from rabbit liver microsomes were studied and found to differ from those of rats. The temperature dependence of the apparent binding constants qualitatively exhibited the same characteristics compared with that of rats. For type I-substrates endothermic and for type II-substrates exothermic reaction characteristics were observed. Taking into account the partition coefficients of the substrates so far investigated it is obvious that type I substrates with increasing hydrophobicity are bound more strongly while type II-substrates show a more complicated behvaiour. This may due to the fact that other types of binding are included besides the hydrophobic interactions.     

Comparative study of the reaction kinetics of cytochrome P-450 reduction by NADPH-cytochrome P-450 reductase and dithionite

The reactions of NADPH- or dithionite-dependent reduction of cytochrome P-450 were studied using a stopped flow technique. It was found that the kinetic curves for both reactions may be fitted by a sum of the two exponents. The arrhenius plots for the fast phase rate constants are linear for both reactions. On the contrary, the breaks on the corresponding plots for the slow phase rate constants are observed at 22 and 33 degrees C for cytochrome P-450 reduction by dithionite and at 31 degrees C for NADPH-dependent reduction of cytochrome P-450. The coincidence of the values of the rate constants and activation energy (56 +/- 5 kJ/mol) for the fast phase of NADPH-dependent reduction of cytochrome P-450 with values of catalytic constants and activation energy for demethylation of tertiary amines suggests that the first electron transfer process from NADPH-cytochrome P-450 reductase to cytochrome P-450 may be the rate-limiting step. A diverse character of the kinetic parameters for the two cytochrome P-450 reduction reactions is indicative of different nature of biphasity of these processes.     

Electron paramagnetic resonance detectable states of cytochrome P-450cam

Cytochrome P-450cam is a low-spin Fe3+hemoprotein (g = 2.45, 2.26, and 1.91) which is made 60% high spin (g = 7.85, 3.97, and 1.78) at 12 K by the addition of 1 mol of substrate per mol of enzyme. Low-temperature EPR spectra show that the low-spin fraction of substrate-bound P-450cam contains two magnetic species. The majority species has an unusual EPR spectrum (g = 2.42, 2.24, and 1.97) which connot be simulated by using the range of crystal field parameters known for other heme proteins. The minority species has the same g values as substrate-free enzyme. Both low-spin species show Curie law temperature dependence below 50 K and have similar saturation behavior. Above 50 K the g = 2.42, 2.24, and 1.97 species rapidly loses signal intensity. The distribution of low-spin species is pH dependent (apparent pKa = 6.2) with the g = 2.42, 2.24, and 1.97 magnetic species favored at high pH. The substrate binding stoichiometry and the equilibria observed in the low-spin fraction suggest that there are not multiple protein forms of cytochrome P-450cam. Putidaredoxin and other effector molecules which specifically catalyze hydroxylation convert either the high-spin or the g = 2.42, 2.24, and 1.97 low-spin species to another new magnetic species (g = 2.47, 2.26, and 1.91). This species is only seen in the presence of substrate, and its stability reflects the catalytic potency of the effector molecule. The EPR and UV-visible spectra of cytochrome P-420 depend upon the manner in which the P-420 is generated. Incubation with acetone or reaction with N-ethylmaleimide or diethyl pyrocarbonate generates P-420 with different spectral characteristics. Through identification of active-site amino acids by chemical modification and comparison with porphyrin model complexes, the range of ligands likely to participate in each of the EPR detectable species is assigned. Mechanisms of interconversion of these species and their bearing on catalysis are discussed.     

Kinetics of elementary steps in the cytochrome P-450 reaction sequence. III. NADPH reduction of cytochrome P-450LM at different integrational levels.

The aerobic NADPH reduction of cytochrome P-450LM has been investigated on microsomes, as well as on the solubilized enzyme system in the associated, disintegrated, and reconstituted state, respectively. P-450 exhibits biphasic reduction kinetics of about 70/30% phase distribution and rate constants differing 10-fold. The partial reactions are due to organizational asymmetries, the cytochrome being either incorporated into P-450/reductase associates (cluster) or localized outside (randomly distributed, homoassociated, weakly cluster-associated). Triton N-101 disintegrates the different associate structures, consequently followed by the disappearance of the rapid reaction phase. The enzyme system can be reconstituted; at microsomal stoichiometry the respective standard parameters are approached, depending on the composition and structural organization of the phospholipid. The reorganization without any membrane matrix is obviously thermodynamically determined.     

Studies on cytochrome P-450-dependent lipid hydroperoxide reduction

A reconstituted mixed-function oxidase system containing cytochrome P-450, cytochrome P-450 reductase, phosphatidylcholine, and NADPH catalyzed the reduction of 13-hydroperoxy-9,11-octadecadienoic acid to 13-hydroxy-9,ll-octadecadienoic acid. Activity was stimulated by the addition of type I substrates, while carbon monoxide and oxygen inhibited the reaction. Perfluoro-n-hexane stimulated the reduction of lipid hydroperoxide to lipid alcohol in the reconstituted system but not by cytochrome P-450 alone. Incubation of cytochrome P-450 with only lipid hydroperoxide resulted in destruction of the hemoprotein. Addition of substrates such as aminopyrine decreased cytochrome P-450 destruction. Addition of reducing equivalents from a reconstituted electron transport system also decreased cytochrome P-450 destruction.     

Proton activities for three states of cytochrome P-450cam.

Changes in proton concentration during the binding of dioxygen, carbon monoxide, and for the exchange of dioxygen by carbon monoxide, at ferrous-cytochrome P-450cam were measured by direct titration. Insufficient proton release was observed to support protonation-deprotonation of an axial cysteinyl sulfur donor as a mechanism for generation of hyper spectra in only the carbonylated ferrous state. Measurement of the $\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ value for CO binding as a function of pH (the carbon monoxide Bohr effect) confirms the direct titration data.     

Spectral properties of a novel cytochrome P-450 of a Saccharomyces cerevisiae mutant SG1. A cytochrome P-450 species having a nitrogenous ligand trans to thiolate

An altered cytochrome P-450 (SG1 P-450) was partially purified from Saccharomyces cerevisiae mutant SG1 which is defective in lanosterol 14 alpha-demethylation. Oxidized SG1 P-450 showed a Soret peak at 422 nm and the alpha peak was lower than the beta peak. This spectrum was considerably different from those of known low-spin P-450s, indicating a unique ligand structure of SG1 P-450. The absorption spectrum of ferric SG1 P-450 was superimposable on that of the imidazole complex of ferric P-450, suggesting the presence of a nitrogenous ligand such as histidine of the apoprotein at the 6th coordination position. SG1 P-450 was immunochemically indistinguishable from cytochrome P-450 of S. cerevisiae catalyzing lanosterol 14 alpha-demethylation (P-45014DM) but had no lanosterol 14 alpha-demethylase activity.     

Induction of cytochrome P-450 in rat liver by the antibiotic troleandomycin: partial purificaton and properties of cytochrome P-450-troleandomycin metabolite complexes

Liver cytochrome P-450 from rats treated intraperitoneally with troleandomycin (TAO) were solubilized and partially purified using DE 52 anion exchange chromatography. The major TAO-induced cytochrome P-450 form appears in fraction A which is not bound on the DE 52 column. It is different from the major form induced in rats by phenobarbital or 3-methylcholanthrene in terms of absolute visible spectroscopy, gel electrophoresis (M 45000) and reactions with antibodies. This TAO-induced form mainly exists $\text{in vivo}$ as an iron-TAO metabolite complex and exhibits a characteristic Soret peak at 456 nm. Reconstitution experiments using this partially purified form, after dissociation of its iron-metabolite bond by ferricyanide treatment, underline its particular ability to demethylate TAO itself. TAO also leads to an important induction of other cytochromes P-450 that are present in fraction B (retained on DE 52 column) like the major phenobarbital-induced form, but are immunologically distinct from it.     

Possible higher valence states of cytochrome P-450 during oxidative reactions

The addition of the organic hydroperoxide, cumene hydroperoxide, to liver microsomes results in the appearance of a transient spectral change associated with cytochrome P-450. In addition, unique electron paramagnetic resonance signals are observed with liver microsomal cytochrome P-450 comparable to signals obtained when peroxides interact with metmyoglobin. It is suggested that higher valence states of cytochrome P-450 may function during the activation of oxygen for the hydroxylation of a variety of xenobiotics.     

NADPH reduction of cytochrome P-450 at different integrational levels of the enzyme system.

The complex monooxygenatic enzyme exhibits different functional behaviour at different integrational levels, thus indicating distinct organizational states. The aerobic NADPH reduction of microsomes, solubilized and reconstituted systems follows a biphasic kinetics, the two phases are attributed to associated state (cluster) and random cytochrome P-450 reduction. States of different cytochrome P-450/reductase ratio (associates) could not be differentiated in rate. Detergents (Triton N-101, cholate) are capable of disintegrating the system, at last only monophasic slow reduction is observed. The hydroxylation activity follows the respective reduction behaviour. Sedimentation analysis proves the distinct structural states. Reconstitution of the system can be achieved by means of detergent dilution as well as by combining the constituents. The activity of the reconstituted system depends on the composition of the phospholipids as well as on its organizational state. The reassociation of the solubilized enzyme system at nearly microsomal components stoichiometry (Triton N-101 dilution) proves to be thermodynamically governed leading to self-organization of the system without matrix prerequisite. Individual step rate constants of the reduction reaction and other system parameters are accessible by means of a model treatment of the disintegrated system. Further application to mixed kinetics systems is in progress.     

On the mechanism of action of cytochrome P-450. Oxidation and reduction of the ferrous dioxygen complex of liver microsomal cytochrome P-450 by cytochrome b5

The effects of cytochrome b5 on the decay of the ferrous dioxygen complexes of P-450LM2 and P-450LM4 from rabbit liver microsomes were studied by stopped-flow spectrophotometry. The P-450 (FeIIO2) complexes accept an electron from reduced cytochrome b5 and, in a reaction not previously described, donate an electron to oxidized cytochrome b5 to give ferric P-450. A comparison with the electron-transferring properties of ferrous P-450 under anaerobic conditions allowed determination of the limiting steps of the two reactions involving the oxygenated complex. The rate of decay of the dioxygen complex was increased in all cases with b5 present; however, with oxidized b5 a large increase in the rate was observed with P-450 isozyme 4 but not with isozyme 2, whereas the opposite situation was found when reduced b5 was used. The reactions between b5 and ferrous dioxygen P-450 were not at thermodynamic equilibrium under the conditions employed. From the results obtained, a model is proposed in which the ferrous dioxygen complex decomposes rapidly into another species differing from ferric P-450 in its spectral properties and from the starting complex in its electron-transferring properties. A scheme is presented to indicate how competition among spontaneous decay, cytochrome b5 oxidation, and cytochrome b5 reduction by the ferrous O2 complex may influence substrate hydroxylation.