Monooxygenase activities of dioxygenases. Benzphetamine demethylation and aniline hydroxylation reactions catalyzed by indoleamine 2,3-dioxygenase

Benzphetamine demethylase and aniline hydroxylase activities were determined with various hemoproteins including indoleamine 2,3-dioxygenase in a cytochrome P-450-like reconstituted system containing NADPH, NADPH-cytochrome P-450 reductase, and O2. The highest specific activities, almost comparable to those of liver microsomal cytochrome P-450, were detected with indoleamine 2,3-dioxygenase from the rabbit intestine. The indoleamine 2,3-dioxygenase-catalyzed benzphetamine demethylation reaction was inhibited by catalase but not by superoxide dismutase. Exogenous H2O2 or organic hydroperoxides was able to replace the reducing system and O2. The stoichiometry of H2O2 added to the product formed was essentially unity. These results indicate that the dioxygenase catalyzes the demethylation reaction by the so-called "peroxygenation" mechanism using H2O2 generated in the reconstituted system. On the other hand, the dioxygenase-catalyzed aniline hydroxylation reaction was not only completely inhibited by catalase but also suppressed by superoxide dismutase by about 60%. Although the O2- and H2O2-generating system (e.g. hypoxanthine-xanthine oxidase) was also active as the reducing system, neither exogenous H2O2 nor the generation of O2- in the presence of catalase supported the hydroxylation reaction, indicating that both H2O2 and O2- were essential for the hydroxylation reaction. However, typical scavengers for hydroxyl radical and singlet oxygen were not inhibitory. These results suggest that a unique, as yet unidentified active oxygen species generated by H2O2 and O2- participates in the dioxygenase-mediated aniline hydroxylation reaction.     

Substrate reduction and oxygen activation during microsomal metabolism of quinones

2-Dimethylamino-3-chloro-1,4-naphthaquinone (DCNQ) was used to study oxygen and substrate activation in microsomal system. DCNQ was shown to be bound to microsomal cytochrome P-450 as a type I substrate; its N-demethylation was catalyzed by cytochrome P-450. Cytochrome P-450 and NADPH-cytochrome P-450 reductase are capable of DCNQ reduction to semi- and hydroquinones. The OH-radical formed in the presence of DCNQ, NADPH and reductase was detected, using a spin trap (5,5-dimethylpyrroline-N-oxide). The OH-radical formation was shown to be stimulated by the Fe-EDTA complex. Using the OH-radical scavengers (mannitol, N-butanol, alpha-naphthol) and the catalase inhibitor sodium azide, it was shown that the OH-radical participates in microsomal oxidation of DCNQ and aminopyrine. It was assumed that in the course of microsomal oxidation the reduced DCNQ is responsible for: i) stimulation of molecular oxygen reduction to H2O2; ii) reduction of Fe ions (Fe3+----Fe2+) which cause the decomposition of H2O2 in the Fenton reaction resulting in the formation of a strong oxidizing agent--a hydroxyl radical.     

Stoichiometry of microsomal oxidation reactions. Distribution of redox-equivalents in monooxygenase and oxidase reactions catalyzed by cytochrome P-450

The stoichiometry of NADPH oxidation in rabbit liver microsomes was studied. It was shown that in uncoupled reactions cytochrome P-450, besides O2- generation catalyzes direct two- and four-electron reduction of O2 to produce H2O2 and water, respectively. With an increase in pH and ionic strength, the amount of O2 reduced via an one-electron route increases at the expense of the two-electron reaction. In parallel, with a rise in pH the steady-state concentration of the oxy-complex of cytochrome P-450 increases, while the synergism of NADPH and NADH action in the H2O2 formation reaction is replaced by competition. The four-electron reduction is markedly accelerated and becomes the main pathway of O2 reduction in the presence of a pseudo-substrate--perfluorohexane. Treatment of rabbit with phenobarbital, which induces the cytochrome P-450 isozyme specific to benzphetamine results in a 2-fold increase in the degree of coupling of NADPH and benzphetamine oxidation. The experimental results suggest that the ratio of reactions of one- and two-electron reduction of O2 is controlled by the ratio of rates of one- and two-electron reduction of cytochrome P-450. In the presence of pseudo-substrates cytochrome P-450 acts predominantly as a four-electron oxidase; one of possible reasons for the uncoupling of microsomal monooxygenase reactions is the multiplicity of cytochrome P-450 isozymes.     

On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450. Products of oxygen reduction

This laboratory has recently reported that, in a reconstituted enzyme system containing alcohol-induced isozyme 3a of liver microsomal cytochrome P-450, the sum of acetaldehyde generated by the monooxygenation of ethanol and of hydrogen peroxide produced by the NADPH oxidase activity is inadequate to account for the O2 and NADPH consumed. Studies on the stoichiometry have revealed the occurrence of an additional reaction involving an overall 4-electron transfer to molecular oxygen which is presumed to yield water: O2 + 2 NADPH + 2H+----2 H2O + 2 NADP+. The occurrence of a peroxidase reaction in which free H2O2 is reduced to water by NADPH was ruled out. When the 4-electron oxidase activity is taken into account, measurements of NADPH oxidation and O2 consumption are in accord with the amounts of products formed in the presence of various P-450 isozymes, either in the absence or presence of typical substrates, including those which undergo hydroxylation, N- or O-demethylation, or oxidation of hydroxymethyl to aldehyde groups. Of the substrates examined, some had no effect on the oxidase reaction yielding hydrogen peroxide or the 4-electron oxidase reaction, some were inhibitory, and some were stimulatory, but the same substrate did not necessarily have the same effect on the two reactions.     

Fatty acid hydroxylation in rat kidney cortex microsomes

Rat kidney microsomes have been found to catalyze the hydroxylation of medium-chained fatty acids to the omega- and (omego-1)-hydroxy derivatives. This reaction, which requires NADPH and molecular oxygen, is a function of monooxygenase system present in the kidney microsomes, containing NADPH-cytochrome c reductase and cytochrome P-450K. NADH is about half as effective as an electron donor as NADPH and there is an additive effect in the presence of both nucleotides. Cytochrome P-450K absorbs light maximally at 452-3 nm, when it is reduced and bound to carbon monoxide. The extinction coefficient of this complex is 91 mM(-1) cm(-1). Electrons from NADPH are transferred to cytochrome P-450K via the NADPH-cytochrome c reductase. The reduction rate of cytochrome P-450K is stimulated by added fatty acids and the reduction kinetics reveal the presence of endogenous substrates bound to cytochrome P-450K. Both cytochrome P-450K concentration and fatty acid hydroxylation activity in kidney microsomes are increased by starvation. On the other hand, phenobarbital treatment of the rats has no effect on either the hemoprotein or the overall hydroxylation reaction and 3,4-benzpyrene administration induces a new species of cytochrome P-450K not involved in fatty acid hydroxylation. Cytochrome P-450K shows, in contrast to liver P-450, high substrate specificity. The only substances forming enzyme-substrate complexes with cytochrome P-450K are the medium-chained fatty acids and certain derivatives of these acids. The chemical requirements for substrate binding include a carbon chain of medium length and at the end of the chain a carbonyl group and a free electron pair on a neighbouring atom. The distance between the binding site for the carbonyl group and the active oxygen is suggested to be in the order of 16 A. This distance fixes the ratio of omega- and (omega-1)-hydroxylated products formed from a certain fatty acid by the single species of cytochrome P-450K involved. The membrane microenvironment seems also to be of importance for the substrate specificity of cytochrome P-450K, since removal of the cytochrome from the membrane lowers its binding specificity to some extent. A comparison between the liver and kidney cytochrome P-450 systems suggests that the kidney cytochrome P-450K system is specialized for fatty acid hydroxylation.     

Biophysical and enzymological studies upon the interaction of trans-cinnamic acid with higher plant microsomal cytochromes P-450.

The interaction of trans-cinnamic acid with the cytochrome P-450 of microsomes derived from washed potato slices has been studied. The washing process increased the specific content of microsomal electron transport components and hence provided a useful material in which to study the interaction. Evidence is presented that the trans-cinnamic acid interacts with the cytochrome P-450, and that this interaction is analogous to "type 1" interactions of other cytochrome P-450 systems. This evidence includes the formation of a "type 1" substrate binding spectrum, an increased rate of reduction of cytochrome P-450 by NADPH in the presence of trans-cinnamic acid, an increased oxygen uptake and NADPH oxidation when trans-cinnamic acid is added to the microsomes in the presence of NADPH, and a close correlation between biophysical parameters of electron transport in the cytochrome P-450 system and enzymological parameters of the trans-cinnamic acid 4-hydroxulation reaction. The investigation has been extended to cytochrome P-450 systems of other tissues and it has been found that the trans-cinnamic acid 4-hydroxylation reaction cannot account for the presence of most of th cytochrome P-450 in several tissues. This suggests that other functions of higher plant cytochrome P-450 chains exist, and that the substrate specificityof the hemoprotein may vary in different plant tissues.     

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.     

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.     

Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect

In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, [1,2,6,7,16,17-3H]progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting [1,7,16-3H]progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of 3H2O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of [1,7,16-3H]progesterone and [4-14C]progesterone was employed.     

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.     

Mechanisms of hydroxylation by cytochrome P-450: Exchange of iron-oxygen intermediates with water

Highly-purified rat liver microsomal cytochrome P-450 converted cyclohexane to cyclohexanol in the presence of iodosobenzene. Oxygen from ¹⁸O-iodosobenzene was not incorporated into cyclohexanol but oxygen from H₂¹⁸O was readily incorporated. Cytochrome P-450 catalyzed the facile exchange of oxygen between iodosobenzene and water but neither cytochrome P-420 nor the apoenzyme did. Under these conditions cytochrome P-450 readily incorporated oxygen from ¹⁸O₂ into cyclohexanol in the presence of NADPH-cytochrome P-450 reductase and NADPH. The results are interpreted in a mechanism in which cytochrome P-450 forms a common hydroxylating species in the presence of iodosobenzene or O₂ plus NADPH.     

Enzymic studies on adrenocortical deoxycorticosterone 11beta-hydroxylase system.

Radiometric methods for the assay of deoxycorticosterone 11beta-hydroxylase and for the determination of NADP on a microscale were developed. The determination of NADP was based on the quantitative conversion of 6-phospho[1-14C]gluconate to 14CO2 by the action of 6-phosphogluconate dehydrogenase. Using these methods NADPH oxidase activity of the adrenodoxin reductase-adrenodoxin system as well as kinetic properties of deoxycorticosterone 11beta-hydroxylase (cytochrome P-450) were investigated. The NADPH oxidase activity observed in the presence of adrenodoxin reductase, adrenodoxin, and O2, but in the absence of cytochrome P-450 and deoxycorticosterone, were functions of O2 and adrenodoxin concentrations and represented the autooxidation of reduced adrenodoxin which resulted in the production of H2O2. Due to the rapid autooxidizability of reduced adrenodoxin, only a small fraction of electrons conveyed from NADPH to adrenodoxin by way of adrenodoxin reductase was utilized for the deoxycorticosterone 11beta-hydroxylase reaction under the conditions employed.     

Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung

The ethanol-inducible form of cytochrome P-450 (P-450IIE1) has previously been shown to exhibit an unusually high rate of oxidase activity with the subsequent formation of reactive oxygen species, e.g., hydrogen peroxide, and to be the main contributor of microsomal oxidase activity in liver microsomes from acetone-treated rats [Ekstr?m & Ingelman-Sundberg (1989) Biochem. Pharmacol. (in press)]. The results here presented indicate that oxygen exposure of rats causes an about 4-fold induction of P-450IIE1 in rat liver and lung microsomes. The induction in liver was not accompanied by any measurable increase in the P-450IIE1 mRNA levels, but the enhanced amount of P-450IIE1 accounted for 60% of the net 50% increase in the level of hepatic P-450 as determined spectrophotometrically. The induction of P-450IIE1 was maximal after 60 h of O2 exposure, and concomitant increases in the rates of liver microsomal CCl4-dependent lipid peroxidation, O2 consumption, NADPH oxidation, O2- formation, H2O2 production, and NADPH-dependent microsomal lipid peroxidation were seen. Liver microsomes from oxygen-treated rats had very similar properties to those of microsomes isolated from acetone-treated rats with respect to the P-450IIE1 content and catalytic properties, but different from those of thyroxine-treated animals. Treatment of rats with the P-450IIE1 inducer acetone in combination with oxygen exposure caused a potentiation of the NADPH-dependent liver and lung microsomal lipid peroxidation and decreased the survival time of the rats. The results reached indicate a role for cytochrome P-450 and, in particular, for cytochrome P-450IIE1 in oxygen-mediated tissue toxicity.     

Cytochrome P-450 mediated interaction between hydroperoxide and molecular oxygen. A proposed method for P-450 kinetic studies

Rat liver cytochrome P-450 mediates a novel reaction between equimolar quantities of dissolved oxygen and organic hydroperoxides. The reaction shares some of the properties of both NADPH-O2 dependent hydroxylation and NADPH-O2 independent peroxidase reactions, but does not require either NADPH, phosphatidylcholine, or any substrates other than hydroperoxide and oxygen. It proceeds at a rate approximately 100 times faster than other well known P-450 hydroxylation reactions. Monitoring the rate of O2 consumption in this novel reaction may be a simple and rapid means for studying the kinetics of cytochrome P-450.     

Cytochrome P-450 from mitochondria of bovine adrenal cortex: comparison of cholesterol side-chain cleavage P-450 with steroid 11beta-hydroxylation P-450 and immunochemical cross-reactivity between adrenal mitochondrial and liver microsomal cytochromes P-450.

Adrenocortical mitochondrial cytochrome P-450 specific to the cholesterol side-chain cleavage (desmolase) reaction differs from that for the 11beta-hydroxylation reaction of deoxycorticosterone. The former cytochrome appears to be more loosely bound to the inner membrane than the latter. Upon ageing at 0 degrees C or by aerobic treatment with ferrous ions, the desmolase P-450 was more stable than the 11beta-hydroxylase P-450. By utilizing artificial hydroxylating agents such as cumene hydroperoxide, H2O2, and sodium periodate, the hydroxylation reaction of deoxycorticosterone to corticosterone in the absence of NADPH was observed to a comparable extent with the reaction in the presence of adrenodoxin reductase, adrenodoxin and NADPH. However, the hydroxylation reaction of cholesterol to pregnenolone was not supported by these artificial agents. Immunochemical cross-reactivity of bovine adrenal desmolase P-450 with rabbit liver microsomal P-450LM4 was also investigated. We found a weak but significant cross-reactivity between the adrenal mitochondrial P-450 and liver microsomal P-450LM4, indicating to some extent a homology between adrenal and liver cytochromes P-450.     

A shunted system of electron transport from NAD(P)H to cholesterol-hydroxylating cytochrome P-450 in adrenal cortex mitochondria

The two main approaches presently used for cytochrome P-450scc modelling are as follows: i) the use of chemical compounds carrying activated oxygen species, e. g., peracids, organic hydroperoxides, iodosobenzene, etc., ii) the use of electrochemical reduction in the presence of redox-active compounds. In the present work, a new model system for simulation of steroidogenic electron transfer is proposed, which reduces cytochrome P-450 scc by NADPH in the absence of adrenodoxin reductase and adrenodoxin. Phenazine methosulfate is used as an electron carrier. More than 95% of cytochrome P-450scc is reduced in a model system. The reduction kinetics is characterized by a lag phase, thus indicating complex formation between cytochrome P-450scc and phenazine methosulfate or formation of intermediate reducing equivalents. NADH may also serve as an electron donor for cytochrome P-450scc. Phenazine methosulfate can reduce microsomal cytochrome P-450 LM2 and b5, but not cytochrome P-450 LM4. Superoxide dismutase does not affect the reduction, thus indicating that O9.- is not involved in the reduction process. The mechanism of hemoprotein reduction and the nature of intermediates which can be formed in the model system is proposed.     

The nature of the rate-limiting step in aniline hydroxylation involving cytochrome p-450 rat liver microsomes.

The kinetics of aniline hydroxylation was studied with: (1) rat liver microsomes involving NADPH and O2 (system 1), (2) hepatic microsomes and tert-butylhydroperoxide (system 2) and (3) microsomes and cumyl hydroperoxide (system 3) at 15--37 degrees C. The reactions were characterized by the values of the aniline oxidation rate constants, k2 = V/E0, where E0 is the initial concentration of cytochrome P-450: K 1/2 = 1.60 - 10(8) EXP (-13 400/RT) sec-1, k 2/2 = 1.66 - 10(9) exp (-14 500/RT) sec-1, k 3/2 = 6.83 - 10(9) exp (-15 300/RT) sec-1. The values of delta H0 and delta S0, were calculated and compared for the three systems. The evidence suggests that oxygen insertion into the substrate molecule is the rate-limiting step in the reaction of aniline oxidation for the mentioned systems. The nature of aniline binding to cytochrome P-450 and that of the hydroxylating agent have been discussed.     

Inactivation of glutamine synthetase by a purified rabbit liver microsomal cytochrome P-450 system

Several mixed-function oxidation systems catalyze inactivation of Escherichia coli glutamine synthetase and other key metabolic enzymes. In the presence of NADPH and molecular oxygen, highly purified preparations of cytochrome P-450 reductase and cytochrome P-450 (isozyme 2) from rabbit liver microsomes catalyze enzyme inactivation. The inactivation reaction is stimulated by Fe(III) or Cu(II) and is inhibited by catalase, Mn(II), Zn(II), histidine, and the metal chelators o-phenanthroline and EDTA. The inactivation of glutamine synthetase is highly specific and involves the oxidative modification of a histidine in each glutamine synthetase subunit and the generation of a carbonyl derivative of the protein which forms a stable hydrazone when treated with 2,4-dinitrophenylhydrazine. We have proposed that the mixed-function oxidation system (the cytochrome P-450 system) produces Fe(II) and H2O2 which react at the metal binding site on the glutamine synthetase to generate an activated oxygen species which oxidizes a nearby susceptible histidine. This thesis is supported by the fact that (a) Mn(II) and Zn(II) inhibit inactivation and also interfere with the reduction of Fe(III) to Fe(II) by the P-450 system; (b) Fe(II) and H2O2 (anaerobically), in the absence of a P-450 system, catalyze glutamine synthetase inactivation; (c) inactivation is inhibited by catalase; and (d) hexobarbital, which stimulates the rate of H2O2 production by the P-450 system, stimulates the rate of glutamine synthetase inactivation. Moreover, inactivation of glutamine synthetase by the P-450 system does not require complex formation because inactivation occurs when the P-450 components and the glutamine synthetase are separated by a semipermeable membrane. Also, if endogenous catalase is inhibited by azide, rabbit liver microsomes catalyze the inactivation of glutamine synthetase.     

Subterminal hydroxylation of fatty acids by a cytochrome P-450-dependent enzyme system from a fungus, Fusarium oxysporum

The cell-free extract of a cytochrome P-450-producing fungus, Fusarium oxysporum, was found to catalyze the hydroxylation of fatty acids. Three product isomers were formed from a single fatty acid. The products from lauric acid were identified by mass-spectrometry as 9-, 10-, and 11-hydroxydodecanoic acids, and those from palmitic acid as 13-, 14-, and 15-hydroxyhexadecanoic acids. The ratio of the isomers formed was 50 : 36 : 14 in the case of laurate hydroxylation, and 37 : 47 : 16 in the case of palmitate. The reaction was dependent on both NADPH (or NADH) and molecular oxygen,and was strongly inhibited by carbon monoxide, menadione, or the antibody to purified Fusarium P-450. Further, lauric acid induced a type I spectral change in purified Fusarium P-450. Further, lauric acid induced a type I spectral change in purified Fusarium P-450 with an apparent Kd of 0.3 mM. The hydroxylase activity together with cytochrome P-450 could be detected in both the soluble and microsome fractions, and the activity was almost proportional to the amount of cytochrome P-450 reducible with NADPH. It can be concluded from these results that Fusarium P-450 reducible with NADPH. It can be concluded from these results that Fusarium P-450 is involved in the (omega-1)-, (omega-2)-, and (omega-3)-hydroxylation of fatty acids catalyzed by the cell-free extract of the fungus.     

Role of cytosolic superoxide dismutase as a stimulator in anthranilamide hydroxylation by a microsomal monooxygenase system in rat liver

We have isolated a protein factor from rat liver which stimulates anthranilamide hydroxylation by the microsomes in the presence of NADPH and oxygen and showed this factor to contain Cu and Zn and to have superoxide dismutase activity [Biochim. Biophys. Acta 365, 148-157 (1974)]. In the present study, this protein factor was confirmed to be a superoxide dismutase (SOD) by comparison of the recovery of SOD activity with that of anthranilamide hydroxylation-stimulating activity at each step of its purification, by inhibition of SOD activity with NaCN and hydrogen peroxide (H2O2), and by recovery of the SOD activity of the protein factor after reconstitution with Cu2+ and/or Zn2+. At a given SOD activity level, there was no difference among the rat liver SOD, Cu,Zn-SOD from bovine erythrocytes, and Mn-SOD from Serratia marcescens in their ability to stimulate anthranilamide hydroxylation not only by rat liver microsomes, but also by the reconstituted cytochrome P-450-containing monooxygenase system. Rat liver SOD stimulated anthranilamide hydroxylation by the reconstituted system in proportion to its amount below a protein concentration of 1 microgram/ml. In anthranilamide hydroxylation by the reconstituted system without SOD, only a slight hydroxylase activity was found at the initial stage of the reaction and a marked increase in the amounts of NADPH oxidized and H2O2 formed was observed after a lag time. In the presence of rat liver SOD, however, the hydroxylase activity was markedly and continuously increased almost proportionally to reaction time with a concomitant decrease in the amounts of NADPH oxidized and H2O2 formed. In addition, a trace of 3-OH anthranilamide, one of the products, not only stimulated NADPH-dependent H2O2 formation in the reconstituted system, but also inhibited the apparent reduction of cytochrome P-450 by NADPH in the reconstituted system. These effects of 3-OH anthranilamide were diminished by rat liver SOD. When a trace of 3-OH anthranilamide were added to a system composed of NADPH-cytochrome c (P-450) reductase and NADPH, H2O2 formation and NADPH oxidation were markedly stimulated. However, on addition of 3-OH anthranilamide to the system containing rat liver SOD, no stimulation on either H2O2 formation or NADPH oxidation was found.(ABSTRACT TRUNCATED AT 400 WORDS)