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Pii: s0891-5849(01)00477-4

Free Radical Biology & Medicine, Vol. 30, No. 7, pp. 803– 808, 2001 Copyright 2001 Elsevier Science Inc.
PII S0891-5849(01)00477-4
Fast Track Paper
NITROXYL OXIDIZES NADPH IN A SUPEROXIDE DISMUTASE NDREAS REIF,* LUIGI ZECCA,*† PETER RIEDERER,* MARTIN FEELISCH,‡ and HARALD H. H. W. SCHMIDT *Clinical Neurochemistry, Department of Psychiatry and Psychotherapy, Julius-Maximilians-University, Wu¨rzburg, Germany; †Institute of Advanced Biomedical Technologies, National Research Council, Segrate, Italy; ‡Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA; and §Rudolf-Buchheim-Institute for Pharmacology, University of Gießen, Gießen, Germany (Received 14 July 2000; Revised 28 November 2000; Accepted 16 January 2001) Abstract—Nitric oxide synthases (NOS) convert L-arginine and N␻-hydroxy-L-arginine to nitric oxide (•NO) and/or
nitroxyl (NOϪ) in a NADPH-dependent fashion. Subsequently, •NO/superoxide (OϪ-derived peroxynitrite (ONOOϪ)
consumes one additional mol NADPH. The related stoichiometry of NOϪ and NADPH is unclear. We here describe thatNOϪ also oxidizes NADPH in a concentration-dependent manner. In the presence of superoxide dismutase (SOD),which also converts NOϪ to •NO, nitrite accumulation was almost doubled and no oxidation of NADPH was observed.
Nitrate yield from NOϪ was low, arguing against intermediate ONOOϪ formation. Thus, biologically formed NOϪ mayfunction as an effective pro-oxidant unless scavenged by SOD and affect the apparent NADPH stoichiometry of theNOS reaction.
Keywords—NO synthase, Nitroxyl, NADPH, Superoxide dismutase, Nitrite, Nitrate, Peroxynitrite, Free radicals
pal products which may be converted into •NO have beensuggested, including nitroxyl (NOϪ) [11,12]. As NOϪ Nitrogen monoxide (nitric oxide, •NO) is a physiologi- can be converted to •NO by stoichiometric amounts of cally and pathophysiologically important messenger SOD [12,13], principal NOϪ formation would explain molecule synthesized by a family of NO synthases why •NO can only be detected in the presence of SOD.
[NOS; L-arginine, NADPH:oxygen oxidoreductases (ni- Alternatively, it was suggested that NOS produces su- tric oxide forming); EC]. These multifunc- peroxide [14,15], which rapidly reacts with •NO to form tional enzymes require the presence of NADPH, FAD, oxoperoxonitrate(1Ϫ) (peroxynitrite, ONOOϪ) [16]. Un- FMN, heme, and tetrahydrobiopterin (H Bip) [1,2], a der these conditions, SOD would be needed to scavenge structural Zn, as well as the substrates L-arginine and superoxide in order to enable the detection of •NO.
molecular oxygen [3–7]. NADPH donates electrons for However, such a role of SOD would not explain why the the reduction of FAD and FMN [8], which function as absolute amounts of enzyme (5 kU mlϪ1) required to prosthetic groups shuttling the electrons to the heme detect NO from purified NOS are far in excess of the where molecular oxygen is activated [9].
amounts required to catalyze the dismutation of super- Numerous studies were carried out in order to mea- sure directly •NO formation from NOS either electro- To resolve this issue, it is important to determine the chemically or by gas phase chemiluminescence [10 –12].
NADPH stoichiometry of the principal NOS reaction.
However, when purified neuronal NOS (NOS-I) was Literature values differ between 1, 1.5, and 2 mol used, the presence of supracatalytic amounts of SOD was NADPH per mol L-citrulline [17–19], depending on the required to detect •NO [10,12]. Thus, alternative princi- presence of cofactors in the incubation media. We re-cently reported an NADPH/citrulline-stoichiometry of Address correspondence to: Dr. Andreas Reif, Department of Psy- 1.6, which was significantly lowered to 0.8 by SOD [12].
chiatry and Psychotherapy, Julius-Maximilians-University, Fu¨chslein- A NOS-derived N-oxide may interfere with the observed str. 15, D-97080 Wu¨rzburg, Germany; Tel: ϩ49 (931) 201-7672; Fax: ϩ49 (931) 201-7755; E-Mail:
NADPH stoichiometry, as ONOOϪ was shown to con- sume one mol NADPH [12]. Moreover, Marletta and lowing modifications. Aliquots of the Angeli’s salt stock coworkers recently examined the reactions of H Bip-free solution (100, 200, or 400 ␮M) were added to 0.5 M NOS [20] utilizing N␻-hydroxy-L-arginine, the proposed TEA buffer (100 ␮l, pH 7.0). The samples were then intermediate of the NOS-reaction [17], as a substrate.
incubated for 5 min at 37°C in the absence or presence of Under these conditions, NOϪ was formed with a stoichi- 100 ␮M NADPH, with or without 5 kU mlϪ1 SOD.
ometry of 1.5:1 NADPH/NOϪ, whereas the theoretical Thereafter, samples were frozen in liquid nitrogen and value for this reaction should be 1:1. Again, this suggests stored at Ϫ80°C. For analysis, a 50 ␮l aliquot of the that an N-oxide interferes with the NOS/NADPH stoi- reaction mixture was directly injected into the HPLC chiometry. It is known that NOϪ is able to oxidize system without further sample processing. The HPLC biological molecules (e.g., thiols and DNA), especially system used was a Series 10 solvent delivery pump in the presence of oxygen [21]. We examined therefore (Perkin Elmer, Norwalk, CT, USA) equipped with an the possible nonenzymatic redox reaction of NADPH injection valve (Type 7125, Rheodyne, Cotati, CA, with NOϪ and the effects of SOD, which converts NOϪ USA) and connected to an UV detector operated at 230 to •NO. Furthermore, the amounts of the •NO decompo- nm. A C-18 reverse phase chromatography column (5 sition products nitrite and nitrate were determined.
␮m particle size, 250 mm, 4.6 mm I.D.; CPS Analitica,Milano, Italy) was connected to a guard column (C-18, 5 EXPERIMENTAL PROCEDURES
␮m, 10 mm). The mobile phase contained 5 mM tetrabu-tylammonium hydrogensulfate and 10 mM Na HPO and was adjusted to pH 6.4 with 0.2 M phosphoric acid.
NADPH was obtained from Applichem (Darmstadt, The system was operated at a flow rate of 1.5 ml minϪ1.
Germany); Cu,Zn-SOD, from Roche Molecular Bio- Calibration curves were prepared with standard solutions chemicals (Penzberg, Germany). Angeli’s salt (AS) was of potassium nitrite and nitrate. Chromatograms were synthesized as published [21] and kindly provided by Dr.
recorded and processed with the use of an A C-R4A David Wink (National Cancer Institute, Bethesda, MD, integrator (Shimadzu Corporation, Kyoto, Japan).
USA). All other materials were of the highest purityavailable and obtained from either Sigma Chemicals RESULTS AND DISCUSSION
(Deisenhofen, Germany) or Merck AG (Darmstadt, Ger-many). Water was deionized to 18 M⍀ cm (Milli-Q; To investigate whether NOϪ, derived from AS, is able Millipore, Eschborn, Germany) and de-oxygenated with to oxidize NADPH, different concentrations of AS were incubated together with 100 ␮M NADPH and the reac-tion was followed at 340 nm (A tions of AS and in the absence of SOD, 1 mol NADPHwas oxidized by about 4 moles AS (Fig. 1, open circles).
Angeli’s salt (AS) was dissolved freshly in 10 mM At concentrations above 100 ␮M AS, the relationship NaOH; NADPH, in 0.5 M triethanolamine (TEA) buffer between AS concentration and NADPH consumption (pH 7.0). Aliquots of the Angeli’s salt stock solution became nonlinear and reached a plateau (Fig. 1, inset), were added to 94 well microtiter plates containing 100␮ most likely due to competing reactions of NOϪ, such as M NADPH in 0.5 M TEA buffer (100 ␮l). To controls, dimerization yielding N O; however, the latter concen- equal amounts of NaOH were added. The mixtures were trations are about 10 times higher than product concen- then incubated under protection from light in the absence trations typically achieved in NOS activity assays.
or presence of 5 kU mlϪ1 SOD for 5 min at 37°C.
The stoichiometry of this reaction with respect to Thereafter, the extinction was measured at 340 nm (A NOϪ cannot be determined exactly, as the yield of NOϪ with a SpectraMax 340 microplate reader (Molecular from AS varies— depending on the presence of oxy- gen— between 83 and 48% [24]. When the slight decom- measure of NADPH oxidation, as this assay is commonly position (12%) of the AS preparation used in this study used to determine the redox state of NADPH, although (see below) is taken into account, between 42 and 73 concurring reactions resulting in a similar loss in absor- mol% NOϪ could have been formed per mol AS under bance cannot be ruled out entirely. The results shown are the investigated conditions. Thus, an initial reaction stoi- representative of at least three individual experiments, chiometry of 2 mol NOϪ per mol NADPH appears to be When the nonenzymatic redox reaction of NADPH Determination of nitrite and nitrate with NOϪ is taken into account, the recently published The NO-related metabolites, nitrite and nitrate, were 1:1.5 NADPH/NOϪ stoichiometry for H Bip-free NOS determined as previously described [22,23] with the fol- with N␻-hydroxy-L-arginine as substrate [20] may be Fig. 1. NADPH oxidation by AS-derived NOϪ. NADPH (100 ␮M) was incubated for 5 min at 37°C in the presence of differentconcentrations of the NOϪ-donor Angeli’s salt. Thereafter, the amount of oxidized NADPH was determined as described inExperimental Procedures. Open circles, reaction without SOD; closed circles, with addition of 5 kU mlϪ1 SOD. The dashed lineindicates an initial 1:2 stoichiometry of the NADPH oxidation with respect to NOϪ based on the considerations given in the text. Inset,NADPH oxidation at higher NOϪ concentrations. Data represent means Ϯ SEM (n ϭ 3–10 individual experiments, each performedin triplicate). An asterisk indicates statistical significance between the conditions with and without SOD (p Ͻ .01).
explained as follows: 1 mol NADPH would be consumed H O formation or other uncoupled NOS reactions, al- enzymatically; in addition, 0.5 mol would react directly though they regarded this as unlikely under the condi- with NOϪ to a yet-to-be-identified product (Fig. 2). The tions applied. Our results now provide an alternative authors of the aforementioned study suggested additional explanation that does not imply such uncoupled NOS reac-tions. Recently, Stuehr’s laboratory found that NOS pro-duces different N-oxides depending on the presence ofH Bip [25], resulting of course in varying NADPH sto- ichiometries. Thus the NADPH stoichiometry of the NOSreaction still remains a matter of debate and varies depend-ing on the availability of cofactors. The present studymight therefore be helpful in the interpretation of differ-ent experimentally observed NADPH/citrulline ratios.
Furthermore, we examined the effects of SOD, which can convert NOϪ to •NO [12,26], on the oxidation ofNADPH by NOϪ. In this reaction system, SOD does notserve as a superoxide dismutating enzyme (under ourexperimental conditions, a source for superoxide canhardly be envisaged) but as a reactant in the reactionCu(II)SOD ϩ NOϪ N Cu(I)SOD ϩ •NO [13,26]. Asexpected, the addition of SOD (5 kU mlϪ1) markedlyprevented the oxidation of NADPH (Fig. 1, closed cir-cles). This is consistent with the rapid oxidation of NOϪ Fig. 2. NOS-derived NOϪ. Different N-oxides generated by H Bip-free by SOD to •NO, which by itself does not consume NOS [20] from N␻-hydroxy-L-arginine and the corresponding NADPHstoichiometry.
Table 1. The Yield of Nitrite and Nitrate from the Reaction of reactive nitrogen intermediate. That this reactive nitro- Angeli’s Salt with NADPH and the Influence of SOD gen species is ONOOϪ, generated by the reaction of NOϪ with oxygen, is unlikely. ONOOϪ decomposes to both nitrite and nitrate at a pH of 7.5 with a ratio of approximately 1:2 [27]. The oxidation of NADPH by ONOOϪ should result in the formation of nitrite, in analogy to the oxidation of NADH by ONOOϪ [28].
Both the decomposition as well as the oxidation reaction Angeli’s salt (100 ␮M) was incubated with or without 100 ␮M NADPH in the absence or presence of 5 kU SOD mlϪ1 as described in would thus have resulted in an increased level of either Experimental Procedures. Thereafter, both NO metabolites were mea- nitrite or nitrate, none of which was observed. The oxi- sured by HPLC analysis. The inclusion of NADPH to the reaction dation of NADPH by NOϪ therefore appears to be di- mixture significantly decreased the amount of nitrite, whereas thepresence of SOD markedly increased nitrite levels. Nitrate levels were rectly NOϪ-mediated. Candidate products for the metab- almost one magnitude lower. Similar results have been obtained with olites of NOϪ include the reduced form of the NOϪ AS at concentrations of 200 and 400 ␮M. The results represent the dimerization product, hyponitrous acid, as well as hy- means Ϯ SEM of n independent experiments. Student’s t-test wasperformed and p Ͻ .05 was considered to be statistically significant; *p droxylamine (NH OH), a known product of both •NO Ͻ .05 compared to control (AS, 100 ␮M).
and NOϪ reduction [29 –31], or— by reaction with oxy-gen—a recently suggested derivative of ONOOϪ [32]. In To examine possible products of the NADPH/NOϪ the present study, we did not carry out anaerobic exper- reaction, we determined the amounts of nitrite and nitrate iments, as we focus on the relevance of our findings with formed (Table 1). In the absence of NADPH, 100 ␮M respect to the aerobic NO synthase reaction. Neverthe- AS (at a pH of 7.0) generated 88.3 Ϯ 9.4 ␮M nitrite, less, reinvestigating the reaction described here in the while the yield of nitrate was as low as 5.3 Ϯ 0.6 ␮M.
absence of oxygen would be of interest.
One mol of AS theoretically generates one mol of nitrite, As nitrite levels were decreased when NADPH was besides NOϪ. The yield of nitrite was, however, only oxidized by NOϪ, the NADPH/NOϪ reaction product 0.88 mol per mol of AS, suggesting that approximately might further react with nitrite. A possible reaction of 12% of the AS was decomposed. Similar values were hydroxylamine with nitrite was therefore considered. Ni- obtained at AS concentrations of 200 and 400 ␮M (data trite (100 ␮M) was incubated with an equimolar amount of hydroxylamine (100 ␮M) for 5 min; thereafter, nitrite In the presence of NADPH, the amount of nitrite levels were determined by HPLC analysis as described generated during AS decomposition was significantly above. The presence of hydroxylamine however had no lowered to an average of 66 Ϯ 5 ␮M, whereas that of influence on nitrite levels (99 Ϯ 6% of control); there- nitrate remained almost unchanged (see Table 1). The fore, the formation of hydroxylamine from NOϪ and possibility exists that NOϪ reacts with NADPH via a NADPH seems unlikely, although it cannot be ruled out Fig. 3. AS-derived NOϪ. AS generates equimolar amounts of nitrite and NOϪ. The latter can dimerize yielding hyponitrous acid, whichdecomposes to nitrous oxide and water, or it can be converted to •NO by SOD. •NO can react with superoxide yielding peroxynitrite,which decomposes to nitrate and nitrite in a ratio of approximately 1:2. In the absence of superoxide, •NO decomposes predominantlyto nitrite. If NADPH is present, NOϪ will oxidize this cofactor. The metabolite of NOϪhas yet to be established; it appears to reactfurther with nitrite to a yet-to-be-identified reactive nitrogen species (confer also to [32]).
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