Redox regulation of yeast flavin-containing monooxygenase.

The flavin-dependent monooxygenase from yeast (yFMO) oxidizes biological thiols such as cysteine, cysteamine, and glutathione. The enzyme makes a major contribution to the pools of oxidized thiols that, together with reduced glutathione from glutathione reductase, create the optimum cellular redox environment. We show that the activity of yFMO, as a soluble enzyme or in association with the ER membrane of microsomal fractions, is correlated with the redox potential. The enzyme is active under conditions normally found in the cytoplasm, but is inhibited as GSSG accumulates to give a redox potential similar to that found in the lumen of the ER. Site-directed mutations show that Cys 353 and Cys 339 participate in the redox regulation. Cys 353 is the principal residue in the redox-sensitive switch. We hypothesize that it may initiate formation of a mixed disulfide that is partially inhibitory to yFMO. The mixed disulfide may exchange with Cys 339 to form an intramolecular disulfide bond that is fully inhibitory.

Size limits of thiocarbamides accepted as substrates by human flavin-containing monooxygenase 1.

Microsomes isolated from Spodoptera frugiperda (Sf)9 cells infected with human flavin-containing monooxygenase (FMO)1 recombinant baculovirus catalyzed the NADPH- and O2-dependent oxidation of methimazole, thiourea, and phenylthiourea. However, there was no detectable activity with 1,3-diphenylthiourea or larger thiocarbamides. Microsomes from control Sf9 cells were devoid of methimazole or thiourea S-oxygenase activity. Trimethylamine up to 1.0 mM had no detectable effect on the oxidation of 10 microM methimazole (Km = 5 microM) but 1.0 mM N,N-dimethylaniline or chlorpromazine inhibited the oxidation of 1.0 mM methimazole 50 and 70%, respectively. Although products were not isolated, the pronounced inhibition of methimazole S-oxygenation suggests that these amines are alternate substrates for human FMO1. Because 1,3-diphenylthiourea is apparently completely excluded from the catalytic site, tricyclic amine drugs are probably approaching the upper size limits of xenobiotics accepted by human FMO1. The substrate specificity of this isoform in humans appears considerably more restricted than that of pig or guinea pig FMO1. Differences in the size of nucleophiles accepted must be considered in attempting to extrapolate the extensive structure-activity studies available for pig FMO1 to this FMO isoform in humans.

Lysine 219 participates in NADPH specificity in a flavin-containing monooxygenase from Saccharomyces cerevisiae.

The flavin-containing monooxygenase from Saccharomyces cerevisiae (yFMO) uses NADPH and O(2) to oxidize thiol containing substrates such as GSH and thereby generates the oxidizing potential for the ER. The enzyme uses NADPH 12 times more efficiently than NADH. Amino acid sequence analysis suggests that Lys 219 and/or Lys 227 may act as counterions to the 2' phosphate of NADPH and to help determine the preference for pyridine nucleotides. Site directed mutations show that Lys 219 makes the greater contribution to cosubstrate recognition. Conversion of Lys 219 to Ala reduces NADPH dependent activity 90-fold, but has no effect on NADH-dependent activity. Conversion of Lys 227 to Ala reduces NADPH-dependent activity fivefold and NADH-dependent activity threefold. Dissociation constants for NADP(+) to oxidized yFMO were measured spectroscopically. K(d) is 12 microM for the wild-type enzyme and 243 microM for the K219A mutant, consistent with the role of Lys 219 in pyridine nucleotide binding.

Yeast flavin-containing monooxygenase generates oxidizing equivalents that control protein folding in the endoplasmic reticulum.

The flavin-containing monooxygenase from yeast (yFMO) catalyzes the O2- and NADPH-dependent oxidations of biological thiols, including oxidation of glutathione to glutathione disulfide (GSSG). Glutathione and GSSG form the principle redox buffering system in the cell, with the endoplasmic reticulum (ER) being more oxidizing than the cytoplasm. Proper folding of disulfide-bonded proteins in the ER depends on an optimum redox buffer ratio. Here we show that yFMO is localized to the cytoplasmic side of the ER membrane. We used a gene knockout strain and expression vectors to show that yFMO has a major effect on the generation of GSSG transported into the ER. The enzyme is required for the proper folding, in the ER, of test proteins with disulfide bonds, whereas those without disulfide bonds are properly folded independently of yFMO in the ER or in the cytoplasm.

N-Glycosylation of pig flavin-containing monooxygenase form 1: determination of the site of protein modification by mass spectrometry.

By using a combination of biochemical methods (i.e., endoglycosidase H digestion and immunoblot and plant lectin binding studies), it was verified that pig flavin-containing monooxygenase (FMO1) was N-glycosylated. By using mass spectrometry approaches [i.e., peptide mapping, gas chromatography/mass spectrometry, microbore HPLC/electrospray ionization mass spectrometry (LC/ESI/MS), chemical ionization gas chromatography/mass spectrometry (CI/GC/MS), and matrix-assisted laser desorption mass spectrometry (MALDI/MS)], we were able to confirm that pig FMO1 was N-glycosylated and we were able to identify the site of N-glycosylation. Pig FMO1 contains two putative consensus sites of N-glycosylation. The results showed that pig FMO1 amino acid Asn120 was selectively N-glycosylated. Highly purified pig FMO1 avidly bound concanavalin A and reacted positively for carbohydrates by the periodic acid/Schiff's base method of analysis. In addition, treatment of pig FMO1 with endo-N-acetylglucosaminidase converted the enzyme to another species with a molecular mass approximately 5000 Da lower than that of the parent protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot experiments. Peptide mapping of pig FMO1 showed that the protein used in the study was not contaminated with another glycoprotein. MALDI/MS experiments showed that pig FMO1 was present with the expected molecular mass but that higher-molecular mass forms consistent with the presence of N-linked high-mannose oligosaccharide structures were also covalently attached to the enzyme. The presence of N-acetylglucosamine isolated from acid hydrolysates of the N-linked high-mannose oligosaccharide of pig FMO1 was confirmed by high-pH anion exchange HPLC studies and verified by CI/GC/MS studies of derivatized monosaccharide fractions. Further analysis of pig FMO1 proteolytic peptides by LC/ESI/MS showed that the only residue that was N-glycosylated in pig FMO1 was Asn120. Knowledge of the structural aspects of FMO may be useful in understanding the membrane association properties of the enzyme.

Cost-effectiveness of clozapine therapy for severe psychosis.

The cost-effectiveness of using the atypical antipsychotic medication clozapine for severe psychosis was examined in a rural public-sector community mental health setting in Virginia. Based on a sample of 20 patients, use of clozapine resulted in estimated cost savings of between $3,000 and $9,000 per patient per year, including the costs of dropouts from treatment. Savings were mainly due to a decline in hospitalization from 47.7+/-59.8 days per patient in the year before clozapine treatment to 4.6+/-11.3 days in the year after. Although this study had methodological limitations, the results suggest that clozapine may be cost-effective in this setting.

Molecular cloning and kinetic characterization of a flavin-containing monooxygenase from Saccharomyces cerevisiae.

An open reading frame from yeast coding for a homologue of flavin containing monooxygenase (FMO) has been cloned into several Escherichia coli expression vectors. A His10 peptide attached to the amino terminus produced a high yield of soluble protein when coexpressed with GroEL and GroES. The protein was purified on an affinity column and characterized. The protein binds one mole per mole of flavin but the binding is relatively weak and 50 microM exogenous FAD is used to maintain full occupancy. The yeast enzyme, like mammalian enzymes, exhibits NADPH oxidase activity. The enzyme does not catalyze the oxidation of amines, but thiols, including glutathione, cysteine, and cysteamine, show substrate activity. The Km values for these are 7.0, 9.9, and 1.3 mM, respectively; kcat values are 94, 246, and 94 per min, respectively. The enzyme apparently does not accept xenobiotic compounds but may be involved in maintaining cellular reducing potential, probably through its action on cysteamine. This activity may represent the initial role of the FMO family of enzymes, giving rise to the multigene family of drug metabolizing enzymes seen in modern mammals.

Inhibition of purified and membrane-bound flavin-containing monooxygenase 1 by (N,N-dimethylamino)stilbene carboxylates.

(E)-[2-(4-(Dimethylamino)phenyl)vinyl]benzenes bearing a nitrile or carboxyl group in the 2', 3', or 4' position were synthesized and tested for substrate activity with purified pig liver flavin-containing monooxygenase (FMO1). Although the nitrile derivatives were too insoluble to saturate the catalytic site at pH 7.4, they appeared to be substrates with K(m)'s somewhat above their maximum solubility (approximately equal to 0.1 mM) in the assay medium. Of the three carboxylic acid analogs, (E)-4-[2-(4(dimethylamino)phenyl)vinyl]benzoic acid had no detectable water solubility at pH 7.4, and measurements were restricted to (E)-3-[2-(4-(dimethylamino)phenyl)vinyl]benzoic acid (DS3CO) and (E)-2-[2-(4-(dimethylamino)phenyl)vinyl]benzoic acid (DS2CO). While DS3CO and DS2CO were substrates, they also inhibited FMO1 turnover. DS3CO was the more effective inhibitor, and at 2 mM it inhibited FMO1 and microsomal-catalyzed oxidation of methimazole (N-methyl-2-mercaptoimidazole) by 80-90%. Kinetic studies indicated that the aminostilbene carboxylates were noncompetitive with both the xenobiotic substrate, methimazole, and NADPH. However, inhibition constants calculated from double reciprocal plots of velocity vs NADPH were K(i)(comp) 130 and 150 microM for DS3CO and DS2CO, respectively, whereas the uncompetitive Ki's were 10-15 times higher, which suggests that inhibition of NADPH binding may be primarily responsible for inhibition of FMO1 by the aminostilbene carboxylates. This model is also consistent with inhibition of cyclohexanone monooxygenase, a bacterial analog of FMO. DS3CO and DS2CO were again noncompetitive with methimazole but primarily competitive with NADPH. The aminostilbene carboxylates had no detectable effects on activity of pig or rat liver NADPH-cytochrome P450 reductase, which suggests that they are not nonspecific flavoprotein antagonists.

Oxidation of aldehydes catalyzed by pig liver flavin-containing monooxygenase.

Flavin-containing monooxygenase-1 (FMO1) purified to homogeneity from pig liver microsomes catalyzes NADPH- and oxygen-dependent oxidation of salicylaldehyde to pyrocatechol and formate. These products, formed in equimolar amounts, were the only ones detected that suggests that FMO1 catalyzes the oxidation of salicylaldehyde by Baeyer-Villiger chemistry. In addition to salicylaldehyde, 2-hydroxy-1-naphthaldehyde, 5-chlorosalicylaldehyde, 5-nitrosalicylaldehyde, ferrocene carboxaldehyde, 2-pyridine carboxaldehyde, and acetylacetone also stimulated NADPH-dependent oxygen uptake in the presence of FMO1. On the other hand, benzaldehyde, 2-methoxybenzaldehyde, 4-pyridine carboxaldehyde and 3- or 4-hydroxybenzaldehyde, and none of the alkylaldehydes tested had detectable substrate activity. Pig liver FMO1 apparently only catalyzes C-oxidation of reactive aldehydes with a hydrogen ion acceptor function adjacent to the carbonyl.

Multisubstrate flavin-containing monooxygenases: applications of mechanism to specificity.

Kinetic studies on mechanism of the flavin-containing monooxygenase (FMO1) from pig liver microsomes are described in detail with special emphasis on the interpretation of constants derived from the rate equation. The evidence reviewed indicates that oxidation of xenobiotic substrates by the 4a-hydroperoxyflavin form of the enzyme is a second order reaction not saturable by substrate. Under steady-state conditions decomposition of the hydroxyflavin (an intermediate form of the enzyme that does not require enzyme-substrate or enzyme-product equilibrium complexes) is rate limiting. The lack of detectable equilibrium binding is also consistent with rate constants defining Km deduced from steady-state measurements. A model consistent with all evidence currently available indicates that at saturating concentrations of xenobiotic substrates that catalytic site on the enzyme is unoccupied most of the time. This property may explain why non-substrate analogs of xenobiotic substrates do not inhibit FMO activity. Rate constants for the oxidation of xenobiotics by the enzyme-bound and synthetic 4a-hydroperoxyflavin indicate that while enzyme protein accelerates the reaction with xenobiotics bearing nitrogen, it has only marginal effects on the oxidation of substrates bearing sulfur. Differences in the nucleophilicity of compounds bearing these heteroatoms may be primarily responsible but other, as yet undefined, factors may also contribute. In addition, analysis of rate constants affected by protonated lipophilic amines indicates that these allosteric effectors apparently modify enzyme structure so as to affect two or more rate constants and, depending on the nature and concentration of the xenobiotic substrate, protonated amines can either stimulate or inhibit catalytic activity.

The oxidation of ebselen metabolites to thiol oxidants catalyzed by liver microsomes and perfused rat liver.

The oxidation of 2-(methylseleno)benzanilide and 2-selenylbenzanilide, metabolites of the antioxidant drug ebselen, was examined in reactions catalyzed by rat, pig, and guinea pig liver microsomes and in perfused rat liver. Microsomes from all three species catalyzed NADPH- and oxygen-dependent oxidation of the selenide and the selenol to thiol-reactive metabolites. The oxidation product of the selenide was similar in properties to the chemically synthesized selenoxide [2-(methylseleninyl)benzanilide]. The selenoxide oxidized GSH and thiocholine at rate constants of 1.2 x 10(2) and 7.2 x 10(2) M-1 s-1, respectively at pH 7.4, 37 degrees C. n-Octylamine stimulated the oxidation of the ring-opened metabolites of ebselen catalyzed by pig and guinea pig liver microsomes but it had essentially no effect on these activities in rat liver microsomes. The selenoxidase activity of microsomes from all three species was partially (30-50%) sensitive to N-benzylimidazole. The effects of n-octylamine and the imidazole suggest that the oxidation of the selenide was catalyzed primarily by enzymes with the properties of flavin-containing and P450-dependent monooxygenases, but the nature of enzymes responsible for a small fraction of the N-benzylimidazole-sensitive activity was not fully resolved. The 2-(methylseleno)benzanilide oxidase activity of pig liver microsomes sensitive to N-benzylimidazole was only partially sensitive to antisera to pig liver NADPH-cytochrome P450 reductase. While neither 2-(methylseleno)benzanilide nor ebselen affected bile flow, the biliary efflux of GSSG was stimulated about fourfold in rat liver perfused with either of these selenium compounds. The increased GSSG efflux produced by 5 microM ebselen or its methyl metabolite was abolished by N-benzylimidazole.

Liver microsome and flavin-containing monooxygenase catalyzed oxidation of organic selenium compounds.

Eight commercially available selenides, a selenol, and selenourea were examined for substrate activity with purified pig liver flavin-containing monooxygenase (FMO). While none of the aromatic heterocyclic selenides tested showed detectable activity, all dialkyl- and alkylaryl-selenides free from ionic groups stimulated NADPH- and FMO-dependent oxygen uptake. With limiting substrate the molar ratio of oxygen reduced to selenide added was 1:1. Analysis of products from N-[2-(methylseleno)ethyl]benzamide demonstrated that the selenide was quantitatively oxidized to selenoxide. Further oxidation of either the FMO-generated or synthetic selenoxides was not detected. The dialkyl- and alkylaryl-selenoxides are potent thiol oxidants and reaction rates for the oxidation of thiols by methylphenylselenoxide and N-[2-(methylseleninyl)ethyl]benzamide followed second order kinetics with rate constants from 3-4 x 10(3) M-1 s-1 at pH 7.4, 37 degrees C. The rapid oxidation of thiols by these selenoxides demonstrates that the oxidation of selenides to selenoxides catalyzed by crude tissue preparations can be measured by following selenide-dependent oxidation of thiocholine by the procedure described earlier for the oxidation of thiourea (WXA Guo and D. M. Ziegler, 1991, Anal. Biochem. 198, 143-148). Activity measurements by this procedure indicated that the oxidation of dialkyl selenides by rat, guinea pig, or pig liver microsomes was catalyzed primarily by a monooxygenase with the properties of FMO. However, from 20 to 40% of the microsome-catalyzed oxidation of selenides bearing aryl substituents was sensitive to N-benzylimidazole, suggesting that P450-dependent monooxygenases also contribute, at least in part, to the oxidation of these xenobiotics.

The mammalian flavin-containing monooxygenases: molecular characterization and regulation of expression.

The flavin-containing monooxygenase (FMO) has been characterized in several mammalian species, including human. The FMO forms a stable NADP(H)- and oxygen-dependent 4 alpha-hydroperoxy flavin enzyme intermediate in the absence of an oxygenatable substrate. As such, substrate specificity appears to be controlled by access to this stabilized intermediate, resulting in this enzyme's ability to metabolize a wide variety of xenobiotics. These include tertiary and secondary alkyl- and arylamines, many hydrazines, thiocarbamides, thioamides, sulfides, disulfides, thiols, and other soft nucleophiles. Although some of these compounds are oxidized to less active derivatives, several examples of metabolic activation to potentially toxic intermediates also exist. Mercapto-pyrimidines and thiocarbamides, for example, appear to be activated predominantly by FMO. Thus, this enzyme system may play an important role in the early steps of chemical toxicity. Often, the contribution of FMO to the metabolism of a given compound can be assessed by its unique stereoselectivity relative to other oxygenases. For example, the cytochromes P450 oxidize (S)-nicotine to a mixture of cis- and trans-N-1'-oxides. In contrast, (S)-nicotine is oxidized by human FMO3 exclusively to the trans-N-1'-oxide. With the purification and cloning of FMO from multiple tissues and species it became apparent that more than one FMO exists. Further, there are considerable tissue- and species-specific differences in FMO expression that likely contribute to observed differences in detoxication competency and toxicant susceptibility.

Flavoenzymes inhibited by indomethacin.

The effect of indomethacin on the activity of five different flavoenzymes, three dehydrogenases and six hydrosases, was determined. Indomethacin at concentration 1.0 mM inhibited the activity, in decreasing order of sensitivity, of the following flavoenzymes: D-amino acid oxidase (pig kidney), flavin-containing monooxygenases (pig liver microsomal), cyclohexanone monooxygenase (Acinetobacter), NADPH-quinone reductase (pig liver), and glutathione reductase (yeast), but it had no effect on the activity of glucose oxidase (Aspergillus) or liver microsomal NADPH-cytochrome P-450 reductase. Indomethacin was competitive with D-alanine for the D-amino acid oxidase (Ki=30 microM) and with NADPH for all other flavoenzymes sensitive to this compound (Kis 170-500 microM). While indomethacin also inhibited two of the three NAD(P)+-dependent dehydrogenases tested, the Kis were relatively high (<1, 500 microM), and of the six different hydrolases tested only one, liver microsomal esterase, was inhibited by indomethacin (Ki=600 microM). Indomethacin also inhibited aminopyrine demethylation catalyzed by the liver microsomal P-450 monooxygenase (Ki=1,000 microM). Although the exact mechanism for the inhibition of functionally different flavoenzymes sensitive to indomethacin is not known, the inhibition is probably not due to the detergent properties of this drug.

Oxidation of desmethylpromethazine catalyzed by pig liver flavin-containing monooxygenase. Number and nature of metabolites.

Homogenous preparations of pig liver flavin-containing monooxygenase catalyze the oxidation of desmethylpromethazine to six distinct metabolites. The products, identified with the aid of chemically synthesized reference compounds, arise by N-oxygenation of the side-chain nitrogen and by S-oxygenation of the phenothiazine ring sulfur. Although kinetic constants (KM) differ somewhat for the R and S isomers, both are initially oxidized to either the desmethylpromethazine sulfoxide or a secondary hydroxylamine. All other metabolites detected are formed by further oxidation of the latter product. Reaction rates determined with intermediates indicate that the secondary hydroxylamine is oxidized to an oxime sulfoxide via the following intermediates: nitrone-->N-hydroxydidesmethylpromethazine-->oxime-->oxime sulfoxide. All of the steps, except for the hydrolysis of the nitrone, are enzymic, and the data presented demonstrate unambiguously for the first time that the oxidative steps in the metabolism of a secondary amine to the oxime are enzymic. In addition, sulfoxidation of desmethylpromethazine is the first demonstration that flavin-containing monooxygenase can catalyze sulfoxidation of a phenothiazine drug bearing a basic side-chain nitrogen.

Oxidation of aldose reductase inhibitors ALO-4114 and ALO-3152 catalyzed by liver microsomes.

Rat and Cynomolgus monkey liver microsomes catalyze the oxidation of 2.7-difluoro-4.5-dimethoxyspiro (9H-fluorene-9,4'-imidazolidine)-2',5'-dione (ALO-4114) to its monomethoxymetabolite (ALO-4417). Formation of this product by O-demethylation of ALO-4114 is catalyzed by NADPH and oxygen-dependent microsomal enzymes with the properties of P-450 monooxygenases. The reaction is blocked by inhibitors selective for these enzymes and activity increases about 2-fold in rats pretreated with phenobarbital or methylcholanthrene. The increase in the O-demethylation of ALO-4114 was, however, considerably less than the increase in benzphetamine N-demethylation or nitrophenetole O-deethylation activities in liver microsomes from rats pretreated with either phenobarbital or methylcholanthrene. Rats pretreated with 20 or 40 mg/kg of ALO-4114 for 3-4 days failed to change significantly the rate of ALO-4114 O-demethylase activity of liver microsomes. O-Demethylation of the achiral ALO-4114 yields the chiral ALO-4417. The enantiomers separated on a Daicel Chiracel AS column by HPLC indicated that O-demethylation of ALO-4114 by microsomes from untreated rats was only slightly stereoselective. However, rats pretreated with methylcholanthrene not only enhanced activity, but also increased the formation of one enantiomer. Further oxidative metabolism of the enantiomers was slow and barely detectable in vitro. Studies conducted with Cynomolgus monkey liver microsomes from one male and one female per experimental group were generally consistent with those from the rat, but some differences were noted. Whether the differences are real or only reflect individual variations caused by the small sample size is not known at present.

Use of thiocarbamides as selective substrate probes for isoforms of flavin-containing monooxygenases.

The oxidation of thiourea, phenylthiourea, 1,3-diphenylthiourea, 1,3-bis-(3,4-dichlorophenyl)-2-thiourea and 1,1-dibenzyl-3-phenyl-2-thiourea was measured in reactions catalyzed by purified pig liver flavin-containing monooxygenase (FMO-1) and by microsomal fractions isolated from pig, guinea pig, chicken, rat and rabbit tissues. The reactions, followed by measuring substrate-dependent thiocholine oxidation [Guo and Ziegler, Anal Biochem 198: 143-148, 1991], were carried out in the presence of 2 mM 1-benzylimidazole to minimize potential interference from reactions other than those catalyzed by isoforms of the flavin-containing monooxygenase (FMO). While at saturating substrate concentrations the Vmax for purified FMO-1 catalyzed oxidation of all five thiocarbamides was essentially constant, velocities for the microsomal catalyzed reactions varied not only with tissue and species but also with the van der Waals' surface area of the thiocarbamide. Rat liver, rat kidney and rabbit liver microsomes failed to catalyze detectable oxidation of thiocarbamides larger than 1,3-diphenylthiourea and lung microsomes from a female rabbit only accepted substrates smaller than 1,3-diphenylthiourea. On the other hand, liver microsomes from chickens, pigs and guinea pigs catalyzed the oxidation of larger thiocarbamides, but the rates decreased with increasing substrate size and chicken liver microsomes showed no detectable activity with the largest thiocarbamide tested. To define more precisely the parameters affecting thiocarbamide substrate specificity of microsomal preparations, activities present in detergent extracts of guinea pig liver microsomes were separated into three distinct fractions. The substrate specificities of these partially purified fractions were different and consistent with the difference observed with microsomal catalyzed reactions. This strongly suggests that thiocarbamides that differ in size may be useful probes for measuring the number of activities of FMO isoforms in crude tissue preparations.

NADPH-dependent oxidation of reduced ebselen, 2-selenylbenzanilide, and of 2-(methylseleno)benzanilide catalyzed by pig liver flavin-containing monooxygenase.

The selenazole ring-opened metabolites of ebselen, 2-selenylbenzanilide and 2-(methylseleno)benzanilide, are substrates for flavin-containing monooxygenase from pig liver. The Km values were 25 and 3 microM, respectively, measured at 37 degrees C, pH 7.4, in the presence of 1 mM GSH. The Vmax values were 390 mU/mg of protein, similar to those obtained with methimazole or other substrates for FMO1. Although ebselen also appears to be a substrate in the absence of GSH, it progressively inactivates the enzyme, apparently by binding covalently to essential enzyme thiols. The oxidation products of the selenol and methylseleno derivatives are rapidly reduced by GSH, regenerating the parent substrates. Rapid reduction of the selenide oxide by GSH was unexpected and suggests that, unlike S-oxidation of sulfides, Se-oxidation of selenides may be a route for bioactivation. The data show that in the presence of FMO1 micromolar amounts of either of these ring-opened metabolites establish a futile cycle catalyzing the oxidation of GSH to GSSG by NADPH and oxygen.