Stimulatory effect of FMN and methyl viologen on cytochrome P-450 dependent reduction of tertiary amine N-oxide.

FMN or methyl viologen stimulated anaerobic reduction of tertiary amine N-oxides by liver microsomes and this stimulatory effect was completely inhibited by carbon monoxide. Spectral study indicated that FMN or methyl viologen is reduced by NADPH-cytochrome c reductase and reduced FMN or methyl viologen is reoxidized by cytochrome P-450 in the presence of tertiary amine N-oxides. In the presence of FMN, xanthine oxidase-hypoxanthine system rapidly reduced tiaramide N-oxides through the reduction of cytochrome P-450: the maximum reduction rate of tiaramide N-oxide was about 100 nmoles/mg protein/min.     

Alterations in microsomal electron transport, oxidative N-demethylation and azo-dye cleavage in carbon tetrachloride and dimethylnitrosamine-induced liver injury

The effect of administration of carbon tetrachloride and dimethylnitrosamine in vivo on hepatic microsomal function related to drug metabolism was measured. It was found that the capacity of isolated microsomes to demethylate dimethylaniline was diminished during the first hour after carbon tetrachloride poisoning and during the second hour after dimethylnitrosamine poisoning. Thereafter the microsomes from carbon tetrachloride-poisoned livers showed a continuous decline in activity so that at 24hr. there was little residual capacity to undertake demethylation. Microsomes from dimethylnitrosamine-poisoned animals were not different from controls at 24hr. During the first 3hr. there was a transient rise in the accumulation of the N-oxide intermediate in carbon tetrachloride-poisoned livers, with a subsequent fall to below control values. In dimethylnitrosamine poisoning there was a parallel decrease in N-oxide accumulation with decreased demethylation. In the latter part of the first 24hr. the ratio of N-oxide accumulation to demethylation was increased in both instances. At 2hr. after poisoning with either compound there was no evidence of altered NADPH(2)-dependent neotetrazolium reduction or lipid peroxidation. NADPH(2)-dependent azo-dye cleavage was decreased. There was no difference in microsomal cytochrome b(5) content, but there was a decrease in the amount of cytochrome P-450. This latter change was correlated with the decreased capacity for NADPH(2)-dependent oxidative demethylation. It is suggested that dimethylnitrosamine is associated with a defect in microsomal NADPH(2)-dependent electron transport at the level of cytochrome P-450. In addition to affecting cytochrome P-450, carbon tetrachloride is associated with a second severe block involving the release of formaldehyde from the N-oxide intermediate.     

Mechanisms of hydroxyl radical formation and ethanol oxidation by ethanol-inducible and other forms of rabbit liver microsomal cytochromes P-450

The hydroxyl radical-mediated oxidation of 5,5-dimethyl-1-pyrroline N-oxide, benzene, ketomethiolbutyric acid, deoxyribose, and ethanol, as well as superoxide anion and hydrogen peroxide formation was quantitated in reconstituted membrane vesicle systems containing purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochromes P-450 LM2, P-450 LMeb , or P-450 LM4, and in vesicle systems devoid of cytochrome P-450. The presence of cytochrome P-450 in the membranes resulted in 4-8-fold higher rates of O-2, H2O2, and hydroxyl radical production, indicating that the oxycytochrome P-450 complex constitutes the major source for superoxide anions liberated in the system, giving as a consequence hydrogen peroxide and also, subsequently, hydroxyl radicals formed in an iron-catalyzed Haber-Weiss reaction. Depletion of contaminating iron in the incubation systems resulted in small or negligible rates of cytochrome P-450-dependent ethanol oxidation. However, small amounts (1 microM) of chelated iron (e.g. Fe3+-EDTA) enhanced ethanol oxidation specifically when membranes containing the ethanol and benzene-inducible form of cytochrome P-450 (cytochrome P-450 LMeb ) were used. Introduction of the Fe-EDTA complex into P-450 LMeb -containing incubation systems caused a decrease in hydrogen peroxide formation and a concomitant 6-fold increase in acetaldehyde production; consequently, the rate of NADPH consumption was not affected. In iron-depleted systems containing cytochrome P-450 LM2 or cytochrome P-450 LMeb , an appropriate stoichiometry was attained between the NADPH consumed and the sum of hydrogen peroxide and acetaldehyde produced. Horseradish peroxidase and scavengers of hydroxyl radicals inhibited the cytochrome P-450 LMeb -dependent ethanol oxidation both in the presence and in the absence of Fe-EDTA. The results are not consistent with a specific mechanism for cytochrome P-450-dependent ethanol oxidation and indicate that hydroxyl radicals, formed in an iron-catalyzed Haber-Weiss reaction and in a Fenton reaction, constitute the active oxygen species. Cytochrome P-450-dependent ethanol oxidation under in vivo conditions would, according to this concept, require the presence of non-heme iron and endogenous iron chelators.     

Evidence for the involvement of cytochrome P-450 in reduction of benzo(a)pyrene 4,5-oxide by rat liver microsomes.

Benzo(a)pyrene 4,5-oxide is reduced to benzo(a)pyrene by microsomes in the presence of NADPH. Carbon monoxide and oxygen inhibit this reduction. The liver has highest activity which is almost lackng in new-born rats. Phenobarbital as well as 3-methylcholanthrene pretreatment increases the epoxide reduction. Additions of FMN or methylviologen stimulate the epoxide reduction; dimethylaniline N-oxide and cumene hydroperoxide are inhibitory. These results indicate that benzo(a)pyrene 4,5-oxide is reduced by the reduced form of cytochrome P-450.     

The ratio of two isozyme groups in microsomal cytochrome P-450 under exogenous influence of carbon tetrachloride and cyclophosphamide

A method for measuring the content of two groups of microsomal cytochrome P-450 isozymes--cytochromes P-450W and P-450L--with the active sites directed into the water phase and membrane lipids, respectively, has been developed. The method is based on the ability of the xanthine oxidase-menadione complex to reduce microsomal cytochromes b5 and P-450 under anaerobic conditions by transferring electrons to hemoproteins with the active sites directed into the water phase. Cytochrome b5 is completely reduced (to the dithionite level) and cytochrome P-450 is reduced partially (only a group of cytochromes P-450W). The amount of cytochromes P-450L is estimated using the difference between the total content of cytochrome P-450 reduced by sodium dithionite and the content of cytochromes P-450W. The possibility of controlling the ratio of these two isozyme groups in cytochrome P-450 in vivo in membranes of the endoplasmic reticulum by pretreatment of animals with a variety of chemicals has been demonstrated. The ratio of cytochromes P-450W and P-450L has been shown to decrease two-fold 18 days after three injections of phenobarbital into mice. Carbon tetrachloride and cyclophosphamide also decrease this ratio in vivo.     

Metyrapone - reduced cytochrome P-450 complex: A specific method for the determination of the phenobarbital inducible form of rat hepatic microsomal cytochrome P-450

Using homogeneous cytochrome P-450, we have shown that the well-known metyrapone-dithionite reduced cytochrome P-450 complex is specific for the cytochrome P-450b induced by phenobarbital. A linear relationship was observed between the absorbance of metyrapone-reduced cytochrome P-450 complex and the one of CO-reduced cytochrome P-450 complex, the usual method for the determination of cytochrome P-450. A method has been proposed for the specific determination of the cytochrome P-450b.     

Cytochrome P-450cam and putidaredoxin interaction during electron transfer.

Cytochrome P-450cam, the bacterial hemeprotein which catalyzes the 5-exo-hydroxylation of d-camphor, requires two electrons to activate molecular oxygen for this monooxygenase reaction. These two electrons are transferred to cytochrome P-450cam in two one-electron steps by the physiological reductant, putidaredoxin. The present study of the kinetics of reduction of cytochrome P-450cam by reduced putidaredoxin has shown that the reaction obeys first order kinetics with a rate constant of 33 s-1 at 25 degrees C with respect to: 1) the appearance of the carbon monoxide complex of Fe(II) cytochrome P-450cam; 2) the disappearance of the 645 nm absorbance band of high-spin Fe(III) cytochrome P-450cam; and 3) the disappearance of the g = 1.94 EPR signal of reduced putidaredoxin. This data was interpreted as indicative of the rapid formation of a bimolecular complex between reduced putidaredoxin Fe(III) cytochrome P-450cam. The existence of the complex was first shown indirectly by kinetic analysis and secondly directly by electron paramagnetic resonance spectroscopic analysis of samples which were freeze-quenched approximately 16 ms after mixing. The direct evidence for complex formation was the loss of the EPR signal of Fe(III) cytochrome P-450cam upon formation of the complex while the EPR signal of reduced putidaredoxin decays with the same kinetics as the appearance of Fe(II) cytochrome P-450. The mechanism of the loss of the EPR signal of cytochrome P-450 upon formation of the complex is not apparent at this time but may involve a conformational change of cytochrome P-450cam following complex formation.     

Interaction of ligands with cytochrome P-450. On the 442 nm spectral species generated during the oxidative metabolism of pyridine.

When added to aerobic rabbit liver microsomal fractions fortified with an NADPH-generating system, pyridine initially produces a type II difference spectrum such as is observed with other aromatic amines. There is a time-dependent conversion of this perturbation into a new spectral species characterized by an absorbance maximum at 442 nm and a minor peak at 389 nm. Experiments with inhibitors of the cytochrome P-450-dependent electron-transport chain suggest that these species originate from binding to the haemoprotein of metabolic intermediate(s) derived from the amine substrate. Analysis of the incubation mixtures by t.l.c., high-pressure liquid chromatography, u.v.- and mass-spectrometry reveals the presence of a single metabolite arising from cytochrome P-450-catalysed oxidation of the heteroaromatic tertiary amine, which was identified as pyridine N-oxide, obviously accounting for adduct formation. This view is supported by comparative studies on the spectral changes generated by exogenous amine oxide with NADPH-reduced cytochrome P-450. Moreover, dithiothreitol, a potent N-oxidase inhibitor, strongly suppresses development of the 442 nm and 389 nm complexes. The ability of forming low-spin adducts with ferrous cytochrome P-450 absorbing around 440 nm appears to be an inherent property of different types of N-oxides. Considering the dipole character of the N+-O- function, a co-ordinate iron-oxygen bond is proposed to be formed in these complexes.     

Cytochrome P-450 isozymes from the marine teleost Stenotomus chrysops: their roles in steroid hydroxylation and the influence of cytochrome b5

Two new cytochrome P-450 forms were purified from liver microsomes of the marine fish Stenotomus chrysops (scup). Cytochrome P-450A (Mr = 52.5K) had a CO-ligated, reduced difference spectrum lambda max at 447.5 nm, and reconstituted modest benzo[a]pyrene hydroxylase activity (0.16 nmol/min/nmol P-450) and ethoxycoumarin O-deethylase activity (0.42 nmol/min/nmol P-450). Cytochrome P-450A reconstituted under optimal conditions catalyzed hydroxylation of testosterone almost exclusively at the 6 beta position (0.8 nmol/min/nmol P-450) and also catalyzed 2-hydroxylation of estradiol. Cytochrome P-450A is active toward steroid substrates and we propose that it is a major contributor to microsomal testosterone 6 beta-hydroxylase activity. Cytochrome P-450A had a requirement for conspecific (scup) NADPH-cytochrome P-450 reductase and all reconstituted activities examined were stimulated by the addition of purified scup cytochrome b5. Cytochrome P-450B (Mr = 45.9K) had a CO-ligated, reduced difference spectrum lambda max at 449.5 nm and displayed low rates of reconstituted catalytic activities. However, cytochrome P-450B oxidized testosterone at several different sites including the 15 alpha position (0.07 nmol/min/nmol P-450). Both cytochromes P-450A and P-450B were distinct from the major benzo[a]pyrene hydroxylating form, cytochrome P-450E, by the criteria of spectroscopic properties, substrate profiles, minimum molecular weights on NaDodSO4-polyacrylamide gels, peptide mapping and lack of cross-reaction with antibody raised against cytochrome P-450E. Cytochrome P-450E shares epitopes with rat cytochrome P-450c indicating it is the equivalent enzyme, but possible homology between scup cytochromes P-450A or P-450B and known P-450 isozymes in other vertebrate groups is uncertain, although functional analogs exist.     

Studies on the mechanism of hepatic microsomal N-oxide formation. The role of cytochrome P-450 and mixed-function amine oxidase in the N-oxidation of NN-dimethylaniline.

Evidence is established for the existence of alternative metabolic routes of N-oxidation of NN-dimethylaniline in rabbit liver microsomal fraction. One pathway involves the participation of two types of cytochrome P-450 with different sensitivities towards heat. Both types may represent distinct haemoprotein species or two physical forms of a single pigment. The other pathway is represented by the mixed-function amine oxidase. The enzyme lacks NADPH dehydrogenase activity and is insensitive to treatment with 2-bromo-4'-nitroacetophenone and steapsin: it catalyses N-oxidation of imipramine, trimethylamine and NN-dimethylaniline in molar proportions considerably different from those of the cytochrome P-450-supported reactions. Cytochrome P-450 is estimated to account for the formation of at least 50-60% of the total NN-dimethylaniline N-oxide formed in the intact rabbit liver microsomal fraction, the remainder arising from the action of the mixed-function amine oxidase.     

The reoxidation of cytochrome P-450 by paraquat inhibits aldosterone biosynthesis from 18-hydroxycorticosterone

Paraquat is an artificial electron carrier that captures electrons from reduced cytochrome P-450 instead of the natural acceptors, thus decreasing the concentration of reduced mitochondrial cytochrome P-450. In the present study, paraquat inhibited the biosynthesis of aldosterone from 18-hydroxycorticosterone by mitochondria from duck adult adrenal gland, under aerobic conditions. Since paraquat did not induce any change in the absorption spectrum of highly purified cytochrome P-450 11 beta, the possibility of a displacement of steroid by the drug is ruled out. Moreover, paraquat did not affect oxidative phosphorylating chain nor did it alter by itself the chemical structure of 18-hydroxycorticosterone. In our conditions, the inhibitory role of paraquat seems restricted to a capture of electrons from reduced cytochrome P-450. Under the same conditions metopirone and spironolactone, known to bind cytochrome P-450 11 beta at the steroid binding site, also inhibited the reaction. Altogether these results show that for aldosterone synthesis from 18-hydroxycorticosterone to take place, the steroid binding site on cytochrome P-450 must be accessible to 18-hydroxycorticosterone and that the cytochrome P-450 must be the direct donor of reducing equivalents. Hence, cytochrome P-450 appears as the final linking point between 18-hydroxycorticosterone and the reducing equivalents provided by NADPH.     

Purified liver microsomal cytochrome P-450. Electron-accepting properties and oxidation-reduction potential.

The reduction of highly purified cytochrome P-450 from rabbit liver microsomes under anaerobic conditions requires 2 electrons per molecule. Similar results were obtained with dithionite, NADPH in the presence of NADPH-cytochrome P-450 reductase, or a photochemical system as the electron donor, with CO or other ligands, with substrate or phosphatidylcholine present, after denaturation to form cytochrome P-420, or with cytochrome P-450 partially purified from rat or mouse liver microsomes. The reduced cytochrome P-450 donates 2 electrons to dichlorophenolindophenol or to cytochrome c. Reoxidation of reduced cytochrome P-450 by molecular oxygen restores a state where 2 electrons from dithionite are required for re-reduction. Although these unexpected findings indicate the presence of an electron acceptor in addition to the heme iron atom, significant amounts of non-heme iron, other metals or cofactors, or disulfide bonds were not found, and free radicals were not detected by electron paramagnetic resonance spectrometry. Resolution of the cytochrome with acetone and acid yielded the apoenzyme, which did not accept electrons, and ferriprotoporphyrin IX, which accepted a single electron. A reconstituted hemoprotein preparation with the spectral characteristics of cytochrome P-420 accepted as much as 0.7 extra electron equivalent per heme. The midpoint oxidation-reduction potential of purified cytochrome P-450 from rabbit liver microsomes at pH 7.0 is -330 mv, and with CO present this value is changed to about -150 mv. The oxidation-reduction potential is unaffected by the presence of phosphatidylcholine or benzphetamine, a typical substrate. Laurate, aminopyrine, and benzphetamine undergo hydroxylation in the presence of chemically reduced cytochrome P-450 and molecular oxygen. Neither NADPH nor the reductase is required for substrate hydroxylation under these conditions.     

Cytochrome P-450 revealed: the effect of the respiratory cytochromes on the spectrum of bacterial cytochrome P-450

Soluble extracts of Bacillus megaterium ATCC 14581 prepared by centrifuging a sonicated cell suspension at 40,000 xg for 30 min apparently contained no cytochrome P-450 unless the culture had been grown in the presence of an inducer: a reduced+CO minus reduced spectrum was used to measure cytochrome P-450 concentration. When the 40,000 xg supernatants from the uninduced cultures were recentrifuged at 105,000 xg the respiratory cytochromes, including one like cytochrome a1, were sedimented, and cytochrome P-450 was observed to be 100 nM or 30 +/- 9 p mol cytochrome P-450/mg protein (n=9). Measurements of cytochrome P-450 in cultures induced with phenobarbital were always higher after ultracentrifugation. There was soluble cytochrome o in all extracts. When cytochrome a1 was present a deep trough at 441 nm developed in the reduced +CO minus reduced difference spectrum of the 40,000 xg supernatant of both the uninduced and the induced cultures. The 40,000 xg supernatant obtained after lysing protoplasts of B. megaterium did not contain cytochrome a1 and always gave a good measure of cytochrome P-450.     

Absorbance spectral change of the cytochrome P-450S21-phenylisocyanide complex upon binding of reduced NADPH-cytochrome-P-450 reductase.

Reduction of cytochrome P-450S21 (SF) (SF, substrate-free; purified from bovine adrenocortical microsomes) with sodium dithionite (Na2S2O4) in the presence of phenylisocyanide produced a ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex with Soret absorbance maxima at 429 and 456 nm. On the other hand, when a preformed ferric cytochrome P-450S21 (SF)-NADPH-cytochrome-P-450 reductase (Fp2) complex was reduced chemically or enzymatically under the same conditions, the absorbance spectrum of the ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex changed drastically, as characterized by an increase in absorbance intensity at 429 nm and a decrease at 456 nm. Similar spectral changes were observed by addition of reduced Fp2 to the preformed ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex. Experiments to reduce a ferric cytochrome P-450S21 (SF)-phenylisocyanide complex with sodium dithionite in the presence of various amounts of Fp2 showed that; (1), the spectral change reached maxima for both absorption increase at 429 nm and decrease at 456 nm when cytochrome P-450S21 and Fp2 were previously mixed at the cytochrome P-450S21:Fp2 ratio of 1:5; (2), the spectral change was suppressed in 300 mM potassium phosphate buffer (pH 7.4). These results suggest that the absorbance spectral change is due to a conformational change around the heme moiety induced by association with reduced Fp2.     

Regulation of arachidonic acid metabolism by cytochrome P-450 in rabbit kidney.

Renal microsomal cytochrome P-450-dependent arachidonic acid metabolism was correlated with the level of cytochrome P-450 in the rabbit kidney. Cobalt, an inducer of haem oxygenase, reduced cytochrome P-450 in both the cortex and medulla in association with a 2-fold decrease in aryl-hydrocarbon hydroxylase, an index of cytochrome P-450 activity, and a similar decrease in the formation of cytochrome P-450-dependent arachidonic acid metabolites by renal microsomes (microsomal fractions). Formation of the latter was absolutely dependent on NADPH addition and was prevented by SKF-525A, an inhibitor of cytochrome P-450-dependent enzymes. Arachidonate metabolites of cortical microsomes were identified by g.c.-m.s. as 20- and 19-hydroxyeicosatetraenoic acid, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid. The profile of arachidonic acid metabolites was the same for the medullary microsomes. Induction of cytochrome P-450 by 3-methylcholanthrene and beta-naphthoflavone increased cytochrome P-450 content and aryl-hydrocarbon hydroxylase activity by 2-fold in the cortex and medulla, and this correlated with a 2-fold increase in arachidonic acid metabolites via the cytochrome P-450 pathway. These changes can also be demonstrated in cells isolated from the medullary segment of the thick ascending limb of the loop of Henle, which previously have been shown to metabolize arachidonic acid specifically via the cytochrome P-450-dependent pathway. The specific activity for the formation of arachidonic acid metabolites by this pathway is higher in the kidney than in the liver, the highest activity being in the outer medulla, namely 7.9 microgram as against 2.5 micrograms of arachidonic acid transformed/30 min per nmol of cytochrome P-450 for microsomes obtained from outer medulla and liver respectively. These findings are consistent with high levels of cytochrome P-450 isoenzyme(s), specific for arachidonic acid metabolism, primarily localized in the outer medulla.     

Purification of a Single Major Form of Microsomal Cytochrome P-450 from Tulip Bulbs (Tulipa gesneriana L.)

Microsomal cytochrome P-450 from tulip bulbs (Tulipa gesneriana L., Balalaika) was purified to an almost electrophoretically homogeneous preparation. The specific content of cytochrome P-450 in the final preparation was 6.68 nmol/mg protein, which was 30-fold enriched from that of the solubilized fractions of microsomes. The molecular weight of purified cytochrome P-450 by SDS-gel electrophoresis is 52,500. The Oxidized form of the purified cytochrome P-450 had absorption peaks at 392, 552, and 645 nm and the absolute reduced CO spectrum peaked at 448 nm. Judged spectrally, the purified cytochrome P-450 is in high spin in the oxidized state. Antiserum against this cytochrome P-450 previously has shown to be highly specific for its antigen but showed a single precipitin line with solubilized microsomal proteins from tulip bulbs of several other cultivars. The physiological role of this cytochrome P-450, however, is unknown in these dormant tulip bulbs.     

Cytochrome P-450 from lodderomyces elongisporus: Its purification and some properties of the highly purified protein

An effective method, based on the chromatography on ω-aminooctyl Sepharose 4B, for the purification of the alkane-induced cytochrome P-450 is described. The purified cytochrome P-450 was homogeneous in SDS/polyacrylamide gel electrophoresis. In the oxidized state it showed a low spin type absorption spectrum. The reduced CO-complex is characterized by a Soret peak at 447 nm. The alkane hydroxylating enzyme system could be reconstituted combining purified cytochrome P-450 with partially purified NADPH-cytochrome P-450 reductase from the yeast microsomal fraction.     

Gene structure of a major form of phenobarbital-inducible cytochrome P-450 in rat liver

The gene structure of cytochrome P-450b, a major form of phenobarbital-inducible cytochrome P-450 in rat livers was elucidated by sequence analysis of the cloned genomic DNAs and was compared with the previously determined gene structures of cytochrome P-450e, a minor form of phenobarbital-inducible cytochrome P-450 and two forms of 3-methylcholanthrene-inducible cytochrome P-450 (P-450c and -d). The gene for cytochrome P-450b is 23 kilobase pairs (kb) long and is separated into 9 exons by 8 intervening sequences. This gene structure is very similar to that of cytochrome P-450e except for the first intron, the first intron being much longer in cytochrome P-450b gene (approximately 12 kb) than in cytochrome P-450e gene (3.2 kb), but differs greatly from the gene structures of two 3-methylcholanthrene-inducible cytochrome P-450s as pointed out previously (Sogawa, K., Gotoh, O., Kawajiri, K. & Fujii-Kuriyama, Y. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5066-5070). The nucleotide sequences in all 9 exons and their flanking regions in introns show very close homology between the two phenobarbital-inducible cytochrome P-450 genes. Forty base substitutions are found in approximately 1900 nucleotides of all exonic sequences, and 15 of them result in 14 amino acid replacements. These base substitutions occur in relatively limited regions of the gene sequences. Most of them are found in exons 6, 7, 8, and 9, most frequently in exon 7 as described previously (Mizukami, Y., Sogawa, K., Suwa, Y., Muramatsu, M. & Fujii-Kuriyama, Y. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3958-3962). The close sequence homology between the two phenobarbital-inducible cytochrome P-450 genes is also found to extend to the promoter region with one notable exception. The simple repeated sequences of (CA)n which is present at -254 position in cytochrome P-450e gene is also observed at the equivalent position in cytochrome P-450b gene, but the repetitiveness is greatly reduced in cytochrome P-450b gene ((CA)5 for P-450b versus (CA)19 for P-450e), and this may somehow be related to the difference in the level of cytochrome P-450b and P-450e in the inductive phase of phenobarbital administration.     

Electrochemical investigations on the oxygen activation by cytochrome P-450.

The application of cytochrome P-450 in substrate conversion is complicated both due to the limited stability and the cofactor regeneration problems. To overcome the disadvantages of NADPH consumption the transfer of the reduction equivalents from an electrode into the cytochrome P-450-system was studied: 1. NADPH was cathodically reduced at a mercury pool electrode. By immobilization of NADP on dialdehyde Sephadex the reductive recycling was possible. 2. Different forms of reduced oxygen were produced by the cathode: a) The reaction of O2- with deoxycorticosterone yields a carboxylic acid derivative. In contrast the cytochrome P-450 catalyzed NADPH-dependent reaction with the same substrate gives corticosterone, O2- represents only an intermediate in the activation of oxygen and is not the "activated oxygen" species. b) Molecular oxygen was reduced to HO2- and H2O2, respectively. The interaction of adsorbed cytochrome P-450 on the electrode surface with the reduced oxygen species in the absence of NADPH was studied. The electrochemically generated peroxide seems to be more active than added H2O2. 3. In a model of electro-enzyme-reactor several substrates were hydroxylated by microsomal cytochrome P-450 with cathodically reduced oxygen which substitutes NADPH.     

Inactivation of sodium dithionite reduced cytochromes P-450 of different origins

The inactivation of five dithionite reduced soluble cytochrome P-450 isoforms has been studied. The inactivation of microsomal rabbit liver isoform LM2 and bacterial linalool cytochrome P-450 is followed by its conversion into cytochrome P-420. Microsomal rabbit liver isoform LM4, bacterial camphor and p-cymene cytochromes P-450 were not inactivated under these conditions. The inactivation of linalool cytochrome P-450 and LM2 isoform is a first order reaction; the rate constants for linalool cytochrome P-450 and LM2 are 0.3 and 0.1 min-1, respectively. In the case of linalool cytochrome P-450 its carboxycomplex (Fe2+-CO) is inactivated, while in the case of LM2 the inactivation affects its oxycomplex (Fe2+-O2). The amino acid residues of linalool cytochrome P-450 are probably modified due to a direct electron transfer in its carboxycomplex. The amino acid residues of LM2 isoform are modified, presumably due to oxidation by oxygen active species which are released during the oxycomplex decay.