Reduction of S -nitrosoglutathione by human alcohol dehydrogenase 3 is an irreversible reaction as analysed by electrospray mass spectrometry Jesper J. Hedberg*, William J. Griffiths, Stina J. F. Nilsson † and Jan-Olov Ho¨o¨g From the Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Human alcohol dehydrogenase 3/glutathione-dependent formaldehyde dehydrogenase was shown to rapidly and irreversibly catalyse the reductive breakdown of S-nitroso- glutathione. The steady-state kinetics of S-nitrosogluta- thione reduction was studied for the wild-type and two mutated forms of human alcohol dehydrogenase 3, muta- tions that have previously been shown to affect the oxidative efficiency for the substrate S-hydroxymethylglutathione. Wild-type enzyme readily reduces S-nitrosoglutathione with a k cat /K m approximately twice the k cat /K m for S-hydroxy- methylglutathione oxidation, resulting in the highest cata- lytic efficiency yet identified for a human alcohol dehydrogenase. In a similar manner as for S-hydroxy- methylglutathione oxidation, the catalytic efficiency of S-nitrosoglutathione reduction was significantly decreased by replacement of Arg115 by Ser or Lys, supporting similar substrate binding. NADH was by far a better coenzyme than NADPH, something that previously has been suggested to prevent reductive reactions catalysed by alcohol dehydro- genases through the low cytolsolic NADH/NAD + ratio. However, the major products of S-nitrosoglutathione reduction were identified by electrospray tandem mass spectrometry as glutathione sulfinamide and oxidized glutathione neither of which, in their purified form, served as substrate or inhibitor for the enzyme. Hence, the reaction products are not substrates for alcohol dehydrogenase 3 and the overall reaction is therefore irreversible. We propose that alcohol dehydrogenase 3 catalysed S-nitrosoglutathione reduction is of physiological relevance in the metabolism of NO in humans. Keywords: alcohol dehydrogenase; glutathione-dependent formaldehyde dehydrogenase; mass spectrometry; nitric oxide; S-nitrosoglutathione. The biological action of nitric oxide (NO) includes vaso- dilation, inhibition of platelet aggregation and neurotrans- mission [1]. In addition to the endogenous production, NO is also a common air pollutant, a component of cigarette smoke and is generated during metabolism of several pharmaceutical drugs [2,3]. It has been suggested that many intracellular processes of NO involve nitrosylation of thiols [4]. S-nitrosothiols have been proposed to affect ventilation [5], alter protein function [4], act as bioactivators of nitrites and nitrates [6] or serve as a ÔpoolÕ of NO [7]. Recently, attention has been drawn to the S-nitrosothiol of glutathi- one (GSH), i.e. S-nitrosoglutathione (GSNO) where GSH may alternatively act as a scavenger for NO to withstand nitrosative stress [8] or act as a modulator of the action of NO [9]. GSNO has been proposed to be produced under physiologically relevant conditions [10–12] and indeed, it has been detected in various biological systems including rat cerebellum and primary glial cell cultures [8,9] as well as in human airways [13]. As GSNO potentially has significant roles in various biological processes, its fate and breakdown is of consider- able interest. In addition to its complex chemical fate [14,15], the enzymatic reduction of GSNO by rat alcohol dehy- drogenase 3 (ADH3), also known as GSH-dependent formaldehyde dehydrogenase, has been investigated [16]. More recently studies on ADH3–/– mice and yeast have demonstrated that ADH3 is important for GSNO metabo- lism and may regulate intracellular S-nitrosothiol levels in these model systems [17]. Furthermore, plant ADH3 also possesses GSNO reductase activity indicating the activity to be general [18]. It is noteworthy with respect to ADH activities, that reductive reactions are generally not believed to be of physiological relevance due to the low NADH/ NAD + ratio in the cytosol [19,20]. Naturally this notion is also applicable to the metabolism of GSNO by ADH3. ADH3 belongs to the medium chain alcohol dehydro- genase (ADH) system which, according to current nomen- clature, is divided into five classes in man ADH1–ADH5 [21] in which the ADH proteins and genes have been designated the same number. All ADHs display oxidative/ reductive enzymatic activities for a variety of alcohols/ aldehydes of both endogenous and exogenous origin [22,23]. The preferred coenzymes are NAD + and NADH for Correspondence to J O. Ho ¨ o ¨ g, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden. Fax: +468 338453, Tel.: +468 728 7740, E-mail: jan-olov.hoog@mbb.ki.se Abbreviations:ADH,alcoholdehydrogenase;ADH3,alcoholdehy- drogenase 3/GSH-dependent formaldehyde dehydrogenase; GSNO, S-nitrosoglutathione; HMGSH, S-hydroxymethylglutathione; NO, nitric oxide. *Present address: Amersham Biosciences, SE-751 84 Uppsala, Sweden. Present address: Department of Medical Biochemistry and Micro- biology, Uppsala University, SE-751 23 Uppsala, Sweden. (Received 4 November 2002, revised 17 January 2003, accepted 27 January 2003) Eur. J. Biochem. 270, 1249–1256 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03486.x oxidation and reduction of substrates, respectively. Most likely, one physiological substrate for human ADH3 is the spontaneously formed complex of formaldehyde and GSH, S-hydroxymethylglutathione (HMGSH). On the basis that the enzyme is considered to be the prime guardian against formaldehyde [24–26], shows extensive evolutionary con- servation [27] and is expressed in all tissues examined [28,29], ADH3 is regarded as essential for basic cell metabolism possibly also including retinoic acid production during growth [30]. GSNO is a recent addition to the ADH3 substrate repertoire and here we investigate the enzymatic activity of the human form of ADH3 for this compound. Recombi- nant expression and purification of wild-type and mutated forms of the enzyme enabled kinetic characterization and investigation of substrate–enzyme interactions. Electrospray tandem MS (MS/MS) was used to identify the products of the enzymatic reduction of GSNO, which were previously in doubt. Finally, we conclude that the glutathione sulfinamide formed is not a substrate for ADH3, thus ADH3 catalysed GSNO reduction is irreversible. Materials and methods Enzyme purification and chemicals Generation of expression vectors for recombinant expres- sion of the wild-type and mutant forms of ADH3 have previously been described [31]. The various forms of ADH3 were expressed in Escherichia coli and purified to homo- geneity in a three-step procedure essentially as described [31]. Protein concentrations were determined colourimetri- cally [32] and the purity was analysed by SDS/PAGE. NAD + (crystalline lithium salt) and NADH (disodium salt) were purchased from Boehringer Mannheim. GSH, GSSG, glutathione sulfonic acid, methylglutathione, NADP + , NADPH and GSNO were from Sigma. Formaldehyde solutions in acetonitrile were made from newly opened glass ampoules, 20% solutions (Ladd Research Industries). Kinetic analysis Enzymatic activity was monitored by following the absorb- ance change at 340 nm with a Hitachi U-3000 spectro- photometer. For HMGSH/NAD + oxidation/reduction a molar absorptivity of 6220 M )1 Æcm )1 wasused.ForGSNO/ NADH reduction/oxidation the additive molar absortivity of NADH and GSNO was used, i.e. 7060 M )1 Æcm )1 [16]. HMGSH concentrations were calculated according to [33] with a K eq of 1.77 m M for the adduct formation. Deter- mination of kinetic constants for NADH and NADPH were performed with a GSNO concentration of 0.5 m M . Determination of kinetic constants for NAD + and NADP + , were performed with GSH and formaldehyde concentrations of 1 m M each. Kinetic constants for HMGSH and GSNO were determined with 2.4 m M NAD + and 0.1 m M NADH, respectively. All kinetic determinations were performed in 0.1 M potassium phos- phate pH 7.5. To fit lines to data points a weighted nonlinear regression analysis program was used ( FIG P for Windows; Biosoft). k cat values are based on a molecular mass of 40 kDa for all proteins. Standard errors for K m and k cat values were <10% except for GSNO reduction by ADH3–Arg115Ser and GSNO reduction using NADPH as coenzyme. Under these conditions the enzymes could not be saturated and kinetic constant determinations should be regarded with caution. For mass spectrometric analyses, reduction of GSNO was performed in 10 m M ammonium bicarbonate pH 7.5, as phosphate ions severely impair mass spectrometric analysis. Reaction velocities were not signifi- cantly different in this buffer as compared to that used for kinetic analyses. MS Electrospray mass and tandem mass spectra were recorded on an AutoSpec-OATOFFPD high-resolution magnetic sector-orthogonal acceleration time-of-flight (OATOF) tan- dem mass spectrometer (Micromass). Both positive- and negative-ion nano-electrospray spectra were recorded by spraying sample mixtures from metal coated borosilicated capillaries. Tandem mass spectra were recorded by selecting the desired precursor ion with the double focussing sectors of the instrument (MS1) focussing them into an inter- mediate collision cell containing Xe and orthogonally accelerating undissociated precursors and fragment ions into the time-of-flight (TOF) analyser (MS2). Product isolation In an effort to isolate the major product, i.e. glutathione sulfinamide, a reaction mixture initially containing 1 m M GSNO, 2 m M NADH and 100 lg ADH3 in 10 m M ammonium bicarbonate pH 7.5 was incubated at 37 °C for 2 h to reach equilibrium. The resulting reaction products were resolved on a strong anion exchange column Table 1. Steady state kinetic constants for HMGSH/GSNO oxidation/reduction at pH 7.5, catalysed by wild-type and mutant forms of ADH3. K m -andk cat -values were determined from initial velocity experiments at 25 °C with substrate concentrations varied over a 10-fold range. Values are given as means (± SE) of three independent experiments. NAD + and NADH concentrations were fixed at 2.4 m M and 0.1 m M , respectively. Enzyme HMGSH GSNO K m (l M ) k cat (min )1 ) k cat /K m (min )1 Æm M )1 ) K m (l M ) k cat (min )1 ) k cat /K m (min )1 Æm M )1 ) ADH3wt 2 ± 0.6 115 ± 5 58 000 27 ± 8 2400 ± 400 90 000 ADH3-Arg115Ser 270 ± 70 135 ± 10 500 700 ± 250 a 2750 ± 800 a 3900 a ADH3-Arg115Lys 150 ± 50 150 ± 5 1000 140 ± 30 3400 ± 400 24 000 a Values were determined under nonsaturating conditions. 1250 J. J. Hedberg et al. (Eur. J. Biochem. 270) Ó FEBS 2003 (Resource Q, 1 mL; Amersham Biosciences) with a linear gradient from 10 m M to 450 m M ammonium bicarbonate pH 7.5 with 20% acetonitrile, flow rate 2 mLÆmin )1 ,using an A ¨ KTA HPLC system (Amersham Biosciences). The elution profile was monitored at 214 nm. The identity of the isolated products were confirmed by MS as described above. The concentration of the collected glutathione sulfinamide was determined spectrophotometrically at 214 nm using standardized GSH solutions as references. Results Steady-state kinetics Steady-state kinetic constants for HMGSH, GSNO, NAD + and NADH were determined for wild-type and mutated forms of ADH3 (Tables 1 and 2). In addition, kinetic constants for NADP + and NADPH were deter- mined for the wild-type enzyme (Table 2). Human ADH3 was found to readily reduce GSNO with NADH as coenzyme yielding a k cat /K m of 90 000 min )1 Æm M )1 .Reduc- tive capacity for GSNO was drastically reduced by the substitution of Arg115 for Ser or Lys. The lowered catalytic efficiencies were due to 25- and 5-fold increases in K m values for the Arg115Ser and Arg115Lys mutants, respectively. NAD + and NADH were by far the best coenzymes for HMGSH oxidation and GSNO reduction, with K m values in the micromolar range. The catalytic capacity for HMGSH oxidation and GSNO reduction using NADP + and NADPH as coenzymes were two to three orders of magnitudes lower than with NAD + and NADH as coenzymes. For the two mutants, kinetic constants for NAD + and NADH were unchanged as compared to the wild-type enzyme. K m values for HMGSH and GSNO were not significantly changed when using NADP + and NADPH (data not shown). GSSG, GSH or methylgluta- thione did not inhibit ADH3 significantly up to 4 m M (data not shown). Electrospray MS/MS and isolation of the products To determine the structure of the product generated by ADH3 catalysed reduction of GSNO, nano-electrospray mass spectra of the reaction mixture with and without the addition of enzyme were recorded (Fig. 1). In addition, the same experiment was performed with and without GSH. This allowed the source of contaminant ions to be identified (data not shown). After an incubation period of 1 h following addition of enzyme, the peaks corresponding to [GSNO]H + (m/z 337) and [NADH]H + (m/z 710) were found to decrease in intensity, and new peaks at m/z 339 and 708 to appear. In the reaction mixtures containing GSH, the peak at m/z 708 corresponded to NAD + and the peak at m/z 613 corresponded to protonated GSSG. As GSH levels in the initial reaction mixture were increased the peak corresponding to protonated GSSG increased in intensity. When a 15-fold excess of GSH was applied, the peak at m/z 339 and the peak corresponding to protonated GSSG were approximately equal in intensity. In addition, a correspond- ing reaction mixture without enzyme was analysed yielding similar results with respect to GSSG formation. The identity of the compound responsible for the peak at m/z 339 was Table 2. Steady state kinetic constants for the coenzymes NAD + , NADH, NADP + and NADPH at pH 7.5, catalyzed by wild-type and mutant forms of ADH3. K m and k cat values were determined from initial velocity experiments at 25 °C with substrate concentrations varied over a 10-fold range. Values are given as means (± SE) of three independent experiments. HMGSH and GSNO concentrations were fixed at 0.3 m M (i.e. 1 m M GSH and 1 m M formaldehyde [33]) and 0.5 m M , respectively. ND, not determined. Enzyme NAD + NADH NADP + NADPH K m (l M ) k cat (min )1 ) k cat /K m (min )1 Æm M )1 ) K m (l M ) k cat (min )1 ) k cat /K m (min )1 Æm M )1 ) K m (l M ) k cat (min )1 ) k cat /K m (min )1 Æm M )1 ) K m (l M ) k cat (min )1 ) k cat /K m (min )1 Æm M )1 ) ADH3wt 7 ± 1.5 90 ± 5 13 000 8 ± 2.5 2700 ± 400 340 000 3600 ± 550 110 ± 16 30 >5000 a ND 600 a ADH3-Arg115Ser 11 ± 2.8 80 ± 7 7300 10 ± 6 1800 ± 800 180 000 ND ND ND ADH3-Arg115Lys 11 ± 3 110 ± 20 10 000 8 ± 2 1900 ± 400 240 000 ND ND ND a Values were determined under nonsaturating conditions. Ó FEBS 2003 GSNO reduction by human alcohol dehydrogenase 3 (Eur. J. Biochem. 270) 1251 determined by MS/MS. The MS/MS spectra of the peaks at m/z 337, [GSNO]H + ,andm/z 339 are displayed in Fig. 2. The spectra show many similarities and are characteristic of glutathione conjugates (Fig. 2 [34]). However, the spectra differ in that the ion at m/z 307, specific for the glutathione conjugation moiety is absent in MS/MS spectrum of the ion at m/z 339. In its place a new fragment at m/z 322 is observed. Fig. 2. Mass fragmentation analyses of substrate and products obtained from the enzymatic reaction. Tandem mass spectrometric fragmentation spectra of [GSNO]H + m/z 337 (upper), and protonated major product m/z 339 (lower). The schematic structures are depicted to the right with some fragmentation breaks. For a more detailed description see Yang et al. [34]. Fig. 1. Nano-electrospray mass spectra of reaction mixtures. The upper spectrum shows components without the addition of enzyme (background) and the lower spectrum shows components after the addition of ADH3. Peaks corresponding to NAD + , [NADH]H + ,[GS i ]H + , [GSNO]H + , [GSSG]H + and the major product are indicated. The peaks at m/z 359 and 361 correspond to Na + adducts of GSNO and the Ômajor productÕ, those at m/z 666/688 and 664/686 are associated with NADH and NAD + , respectively, while those at m/z 279, 280 and 316 are small contaminants. Peaks at m/z from 524 to 587 detected in the mass spectra with enzyme are also present in the spectra without enzyme although at much lower intensities. 1252 J. J. Hedberg et al. (Eur. J. Biochem. 270) Ó FEBS 2003 In an effort to understand the fragmentation spectrum of the ion at m/z 339, reference spectra were recorded of protonated glutathione sulfinic acid (generated through acidification of the reaction mixture after incubation as described [16]), glutathione sulfonic acid and HMGSH (Fig. 3). From the spectra of these glutathione conjugates it is evident that oxidation of the glutathione sulfur effects fragmentation in such a way that the ion at m/z 307 is no longer observed in the MS/MS spectrum. This, along with a comparison with reference spectra of other glutathione conjugates recorded on this instrument [34], demonstrates that the ion at m/z 339 has the structure of a glutathione sulfinamide (Scheme 1). The glutathione sulfinamide was purified using an anion exchange column (Fig. 4A) and the identity and purity was confirmed by MS (Fig. 4B). In addition to the peak corresponding to glutathione sulfinamide additional peaks corresponding to NAD + , NADH, glutathione sulfinic acid and GSSG were resolved. Notably, glutathione sulfinic acid, GSSG, GSNO, NAD + or NADH (m/z 340, 613, 337, 708 and 710, respectively) were not found to be present in the glutathione sulfinamide fraction, i.e. peak 3 in Fig. 4A. The glutathione sulfinamide was not a substrate for ADH3 using NAD + as coenzyme nor did it inhibit octanol or HMGSH oxidation by ADH3 to any extent at concentrations up to 100 l M (data not shown). During the HPLC purification, glutathione sulfinic acid was detected (Fig. 4). However, this compound was never observed during the initial reaction analysis as described above. Probably this compound is spontaneously formed during the exceptionally long incu- bation used to reach equilibrium. The formation of this glutathione sulfinic acid can be enhanced by addition of acid as described [16]. Discussion Human ADH3 readily catalyses the reduction of GSNO. The two glutathione conjugates HMGSH and GSNO are by far the best substrates identified for ADH3. Further, the catalytic efficiency for GSNO reduction is almost twofold higher than for HMGSH oxidation with the result that GSNO is the best substrate for the human ADH3 yet identified. Notably, GSNO concentrations have been reported to reach micromolar levels under certain circum- stances [8,13]. These observations, together with the fact that ADH3 is expressed in all tissues, lend support to the notion that ADH3 may serve as a GSNO metabolizer in humans. It has been suggested however, that due to the ÔnormallyÕ low NADH/NAD + ratio in the cytoplasm [19], reductive reactions exerted by ADHs are not favourable [20]. Analysis of the major product by electrospray tandem MS demonstrated that in addition to GSSG, the other Fig. 3. Tandem mass spectrometric fragmentation spectra of protonated glutathione sulfinic acid m/z 340 (upper) and protonated HMGSH m/z 338 (lower). The schematic structures are depicted to the right with some fragmentation breaks. For a more detailed description see Yang et al. [34]. Scheme 1. Transformation of GSNO to glutathione sulfinamide by ADH3. Ó FEBS 2003 GSNO reduction by human alcohol dehydrogenase 3 (Eur. J. Biochem. 270) 1253 major product is glutathione sulfinamide (Scheme 1). This finding is in line with the previously suggested but not confirmed structure of Jensen and coworkers [16]. The isolated glutathione sulfinamide did not serve as substrate or inhibitor for ADH3. Other sulfinamides have been shown to be formed spontaneously by rearrangement of the corres- ponding semimercaptale [35], a mechanism where the S¼O oxygen originates from the solvent. It is most likely that the isolated glutathione sulfinamide is also formed via a semimercaptale intermediate (Scheme 1). Hence, the over- all reduction of GSNO catalysed by ADH3 is clearly an irreversible reaction. With these observations taken into account, the reaction may therefore be forced in the reductive direction thereby ÔoverridingÕ the influence of a low cytoplasmic NADH/NAD + ratio and lending support to the physiological relevance. There are circumstances under which the NADH/NAD + ratio is significantly changed. For instance, during ethanol intake increased levels of enzyme coenzyme complexes in rat hepatocytes have been observed [36] and ethanol consump- tion has also been shown to increase the output of lactate from liver resulting in an increased NADH/NAD + ratio in kidney [37]. It is possible that such a change may also occur in other tissues, i.e. where GSNO is produced, and thereby influence its metabolism. Naturally, such a change in redox potential would presumably also influence HMGSH meta- bolism. Similar reasoning has included other ADHs and their metabolism of various endogenous substrates, e.g. certain serotonin metabolites, during ethanol intake [23]. The observation of GSSG as a product is in line with findings of previous investigators. However, for the bacter- ial ADH3, Liu et al. do not report any detection of glutathione sulfinamide but propose that the intermediate semimercaptale reacts with NADH to form glutathione amine, which is subsequently oxidized to GSSG by an additional GSH [17]. We do not detect any glutathione amine for the human ADH3-catalysed reaction. Moreover, Jensen et al. proposes that for the mammalian enzyme, GSSG is only a minor product [16]. We find that when excess GSH is added, approximately equal amounts of GSSG and glutathione sulfinamide are formed, and so both products are probably physiologically relevant. Of note is that GSSG is one of the major products spontaneously formed from GSH and GSNO [14] and so it is conceivable that the GSSG formed is not generated from the enzyme catalysed reaction but rather is formed spontaneously during the incubation. This was also confirmed in a control experiment without enzyme. Fig. 4. Isolation and analysis of reaction products. (A) After incubating the ADH3- catalysed GSNO reduction for 2 h at 37 °C (see Materials and methods), the components were resolved on a strong anion exchange column (Resource Q) using 10 m M ammo- nium bicarbonate pH 7.5, 20% acetonitrile as the initial solvent and 1 M ammonium bicar- bonate pH 7.5, 20% acetonitrile as the elution solvent (buffer B). The elution positions (1) NAD + (2) NADH (3) glutathione sulfina- mide (4) glutathione sulfinic acid and (5) GSSG are indicated by arrows. (B) MS of the fraction corresponding to peak 3 in (A) dem- onstrating the identity and purity of the glutathione sulfinamide. Notably, no con- taminants of GSSG, GSNO, NAD + or NADH (m/z 613, 337, 708 and 710, respect- ively) were detected. 1254 J. J. Hedberg et al. (Eur. J. Biochem. 270) Ó FEBS 2003 The effect of substituting Arg115 by Ser or Lys in ADH3 has previously been investigated [31]. For HMGSH, an exact positioning in the active site is essential for efficient catalysis. The substrate binding is accomplished in part by a charge interaction between Arg115 and the glycine carb- oxylate group in the GSH moiety. However, both mutants studied stress the requirement for exact positioning of interacting groups in the substrate and the polypeptide chain. Like the results for HMGSH, GSNO reduction was impaired by the replacement of Arg115 by both Ser and Lys due to increased K m values. These findings show that GSNO and HMGSH bind in similar, if not identical, manners in the ADH3 active site cleft. Moreover, HMGSH, GSNO, GSH and methylglutathione show extensive struc- tural similarities. Still, GSH or methylglutathione did not inhibit the enzyme, which further illustrates the exactness with which the HMGSH or GSNO molecule interacts with the substrate pocket. Addition of millimolar concentrations of NADPH to cytosol fractions of rat liver has been reported to increase the rate of GSNO disappearance [38]. NADPH could indeed be utilized as coenzyme, but with three orders of magnitude lower efficiency, primarily due to higher K m as compared to NADH (Table 2). NADPH concentrations have been estimated to be in the range of 50–100 l M in the hepatocyte cytosol and even lower in other tissues, including heart and brain [39], indicating that NADPH is unlikely to be utilized in vivo by ADH3 as coenzyme for GSNO reduction. In conclusion, human ADH3 shows high catalytic capacity for GSNO reduction with NADH as coenzyme. Mutational analysis demonstrates that GSNO binds in a similar manner to HMGSH to ADH3. As the major products formed are GSSG and glutathione sulfinamide, neither of which function as substrate or inhibitor for the enzyme, it is now clear that the overall reaction is irreversible. We propose that human ADH3 may serve as a GSNO metabolizer in vivo. Acknowledgements We thank B. Agerberth and M. Tollin for the use of equipment and valuable help with HPLC. This work was supported by the Alcohol Research Council of the Swedish Alcohol Retailing Monopoly, the Swedish Match and Karolinska Institutet. References 1. Moncada, S. (1994) Nitric oxide. J. Hypertens. Suppl. 12, S35–S39. 2. IARC (1986) Tobacco Smoking. International Agency for Research on Cancer, Lyon. 3. Graedel, T. E. (1988) Ambient levels of anthropogenic emissions and their atmospheric transformation products. In Air Pollution, the Automobile and Public Health (Watson, A.Y., Bates, R.R. & Kennedy, D., eds), pp. 133–160. Health Effects Institute, Wash- ington DC. 4. Stamler, J.S. (1994) Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 78, 931–936. 5. Lipton, A.J., Johnson, M.A., Macdonald, T., Lieberman, M.W., Gozal, D. & Gaston, B. (2001) S-Nitrosothiols signal the venti- latory response to hypoxia. Nature 413, 171–174. 6. Ignarro, L.J., Lippton, H., Edwards, J.C., Baricos, W.H., Hyman, A.L., Kadowitz, P.J. & Gruetter, C.A. (1981) Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiols as active intermediates. J. Pharmacol. Exp. Ther. 218, 739–749. 7. Stamler, J.S., Jaraki, O., Osborne, J., Simon, D.I., Keaney, J., Vita, J., Singel, D., Valeri, C.R. & Loscalzo, J. (1992) Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc. Natl Acad. Sci. USA 89, 7674–7677. 8. Chatterjee, S., Noack, H., Possel, H. & Wolf, G. (2000) Induction of nitric oxide synthesis lowers intracellular glutathione in microglia of primary glial cultures. Glia 29, 98–101. 9. Kluge,I.,Gutteck-Amsler,U.,Zollinger,M.&Do,K.Q.(1997) S-nitrosoglutathione in rat cerebellum: identification and quanti- fication by liquid chromatography-mass spectrometry. J. Neuro- chem. 69, 2599–2607. 10. Kharitonov, V.G., Sundquist, A.R. & Sharma, V.S. (1995) Kinetics of nitrosation of thiols by nitric oxide in the presence of oxygen. J. Biol. Chem. 270, 28158–28164. 11. Gow, A.J., Buerk, D.G. & Ischiropoulos, H. (1997) A novel reaction mechanism for the formation of S-nitrosothiol in vivo. J. Biol. Chem. 272, 2841–2845. 12. Tsikas, D., Sandmann, J., Denker, K. & Fro ¨ lich, J.C. (2000) Is S-nitrosoglutathione formed in nitric oxide synthase incubates? FEBS Lett. 483, 83–85. 13. Gaston, B., Reilly, J., Drazen, J.M., Fackler, J., Ramdev, P., Arnelle, D., Mullins, M.E., Sugarbaker, D.J., Chee, C., Singel, D.J., Loscalzo, J. & Stamler, J.S. (1993) Endogenous nitrogen oxides and bronchodilator S-nitrosothiols in human airways. Proc. Natl Acad. Sci. USA 90, 10957–10961. 14. Singh, S.P., Wishnok, J.S., Keshive, M., Deen, W.M. & Tan- nenbaum, S.R. (1996) The chemistry of the S-nitrosoglutathione/ glutathione system. Proc.NatlAcad.Sci.USA93, 14428–14433. 15. Wong, P.S., Hyun, J., Fukuto, J.M., Shirota, F.N., DeMaster, E.G., Shoeman, D.W. & Nagasawa, H.T. (1998) Reaction between S-nitrosothiols and thiols: generation of nitroxyl (HNO) and subsequent chemistry. Biochemistry 37, 5362– 5371. 16. Jensen, D.E., Belka, G.K. & Du Bois, G.C. (1998) S-nitroso- glutathione is a substrate for rat alcohol dehydrogenase class III isoenzyme. Biochem. J. 331, 659–668. 17. Liu,L.,Hausladen,A.,Zeng,M.,Que,L.,Heitman,J.&Stamler, J.S. (2001) A metabolic enzyme for S-nitosothiol conserved from bacteria to humans. Nature 410, 490–494. 18. Sakamoto, A., Ueda, M. & Morikawa, H. (2002) Arabidopsis glutathione-dependent formaldehyde dehydrogenase is an S-nitrosoglutathione reductase. FEBS Lett. 515, 20–24. 19. Bu ¨ cher,T.(1970)The State of the DPN System in Liver (Sund, H., eds), pp. 439. Springer-Verlag, Berlin. 20. Svensson, S., Some, M., Lundsjo ¨ , A., Helander, A., Cronholm, T. &Ho ¨ o ¨ g, J O. (1999) Activities of human alcohol dehydrogenases in the metabolic pathways of ethanol and serotonin. Eur. J. Bio- chem. 262, 324–329. 21. Duester, G., Farre ´ s, J., Felder, M.R., Holmes, R.S., Ho ¨ o ¨ g, J O., Pare ´ s,X.,Plapp,B.V.,Yin,S.J.&Jo ¨ rnvall, H. (1999) Recommended nomenclature for the vertebrate alcohol dehydro- genase gene family. Biochem. Pharmacol. 58, 389–395. 22. Edenberg, H.J. & Bosron, W.F. (1997) Alcohol dehydrogenases. In Comprehensive Toxicology (Guengerich, F. P., eds) pp. 119– 131. Pergamon Press, Inc., New York. 23. Ho ¨ o ¨ g, J O., Hedberg, J.J., Stro ¨ mberg, P. & Svensson, S. (2001) Mammalian alcohol dehydrogenase – functional and structural implications. J. Biomed. Sci. 8, 71–76. 24. Uotila, L. & Koivusalo, M. (1974) Formaldehyde dehydrogenase from human liver. Purification, properties, and evidence for the formation of glutathione thiol esters by the enzyme. J. Biol. Chem. 249, 7653–7663. Ó FEBS 2003 GSNO reduction by human alcohol dehydrogenase 3 (Eur. J. Biochem. 270) 1255 25. Deltour, L., Foglio, M.H. & Duester, G. (1999) Metabolic defi- ciencies in alcohol dehydrogenase Adh1, Adh3, and Adh4 null mutant mice – overlapping roles of Adh1 and Adh4 in ethanol clearance and metabolism of retinol to retinoic acid. J. Biol. Chem. 274, 16796–16801. 26. Hedberg, J.J., Ho ¨ o ¨ g, J O., Nilsson, J.A., Xi, Z., Elfwing, A ˚ .& Grafstro ¨ m, R.C. (2000) Expression of alcohol dehydrogenase 3 in tissue and cultured cells from human oral mucosa. Am. J. Pathol. 157, 1745–1755. 27. Danielsson, O., Shafqat, J., Estonius, M. & Jo ¨ rnvall, H. (1994) Alcohol dehydrogenase class III contrasted to class I. Charac- terization of the cyclostome enzyme, the existence of multiple forms as for the human enzyme, and distant cross-species hybridization. Eur. J. Biochem. 225, 1081–1088. 28.Estonius,M.,Svensson,S.&Ho ¨ o ¨ g, J O. (1996) Alcohol dehydrogenase in human tissues: localisation of transcripts coding for five classes of the enzyme. FEBS Lett. 397, 338–342. 29. Uotila, L. & Koivusalo, M. (1997) Expression of formaldehyde dehydrogenase and S-formylglutathione hydrolase activities in different rat tissues. Adv. Exp. Med. Biol. 414, 365–371. 30. Molotkov, A., Fan, X., Deltour, L., Foglio, M.H., Martras, S., Farre ´ s, J., Pare ´ s, X. & Duester, G. (2002) Stimulation of retinoic acid production and growth by ubiquitously expressed alcohol dehydrogenase Adh3. Proc. Natl Acad. Sci. USA 8, 5337–5342. 31. Hedberg, J.J., Stro ¨ mberg, P. & Ho ¨ o ¨ g, J O. (1998) An attempt to transform class characteristics within the alcohol dehydrogenase family. FEBS Lett. 436, 67–70. 32. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. 33. Sanghani, P.C., Stone, C.L., Ray, B.D., Pindel, E.V., Hurley, T.D. & Bosron, W.F. (2000) Kinetic mechanism of human glutathione- dependent formaldehyde dehydrogenase. Biochemistry 39, 10720– 10729. 34. Yang, Y., Rafter, J., Gustafsson, J A ˚ ., Sjo ¨ vall, J. & Griffiths, W.J. (1997) Differentiation of isomeric mercapturic acid pathway metabolites of benzo[a]pyrene. Eur. Mass. Spec. 3, 396–399. 35. Kazanis, S. & McClelland, R.A. (1992) Electrophilic intermediate in the reaction of glutathione and nitrosarenes. J. Am. Chem. Soc. 114, 3052–3059. 36. Cronholm, T. (1987) Effect of ethanol on the redox state of the coenzyme bound to alcohol dehydrogenase studied in isolated hepatocytes. Biochem. J. 248, 567–572. 37. Cherrik, G.R. & Leevy, C.M. (1965) The effect of ethanol meta- bolism on levels of oxidized and reduced nicotinamide-adenine dinucleotide in liver, kidney and heart. Biochem. Biophys. Acta 107, 29–37. 38. Jensen, D.E. & Belka, G.K. (1997) Enzymic denitrosation of 1,3- dimethyl-2-cyano-1-nitrosoguanidine in rat liver cytosol and the fate of the immediate product S-nitrosoglutathione. Biochem. Pharmacol. 53, 1279–1295. 39. Sies, H. (1982) Nicotinamide Nucleotide Compartmentation. In Metabolic Compartmentation (Sies, H., ed.), pp. 205–231. Academic Press Ltd, London. 1256 J. J. Hedberg et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . Reduction of S -nitrosoglutathione by human alcohol dehydrogenase 3 is an irreversible reaction as analysed by electrospray mass spectrometry Jesper J. Hedberg*,. conditions. Ó FEBS 20 03 GSNO reduction by human alcohol dehydrogenase 3 (Eur. J. Biochem. 270) 1251 determined by MS/MS. The MS/MS spectra of the peaks at m/z 33 7, [GSNO]H + ,andm/z 33 9 are displayed in. see Yang et al. [34 ]. Scheme 1. Transformation of GSNO to glutathione sulfinamide by ADH3. Ó FEBS 20 03 GSNO reduction by human alcohol dehydrogenase 3 (Eur. J. Biochem. 270) 12 53 major product is