Journal of NeurochemistryLippincott—Raven Publishers, Philadelphia© 1998 International Society for Neurochemistry

Interaction of a-Phenyl-N-tert-Butyl Nitrone and AlternativeElectron Acceptors with Complex I Indicates a SubstrateReduction Site Upstream from the Rotenone Binding Site

Kenneth Hensley, Quentin N. Pye, Michael L. Maidt, Charles A. Stewart, Kent A. Robinson,Fatima Jaffrey, and Robert A. Floyd

Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation,Oklahoma City, Oklahoma, U.S.A.

Abstract: Mitochondrial complexes I, II, and III were stud-ied in isolated brain mitochondrial preparations with thegoal of determining their relative abilities to reduce 02 tohydrogen peroxide (H202) or to reduce the alternativeelectron acceptors nitroblue tetrazolium (NBT) and di-phenyliodonium (DPI). Complex I and II stimulationcaused H202 formation and reduced NBT and DPI asindicated by dichlorodihydrofluorescein oxidation, nitro-formazan precipitation, and DPI-mediated enzyme inacti-vation. The 02 consumption rate was more rapid undercomplex II (succinate) stimulation than under complex I(NADH) stimulation. In contrast, H202 generation andNBT and DPI reduction kinetics were favored by NADHaddition but were virtually unobservable during succi-nate-linked respiration. NADH oxidation was stronglysuppressed by rotenone, but NADH-coupled H202 fluxwas accelerated by rotenone. a-Phenyl-N-tert-butyl ni-trone (PBN), a compound documented to inhibit oxida-tive stress in models of stroke, sepsis, and parkinsonism,partially inhibited complex I-stimulated H202 flux and NBTreduction and also protected complex I from DPI-medi-ated inactivation while trapping the phenyl radical prod-uct of DPI reduction. The results suggest that complex Imay be the principal source of brain mitochondrial H20~synthesis, possessing an “electron leak” site upstreamfrom the rotenone binding site (i.e., on the NADH side ofthe enzyme). The inhibition of H202 production by PBNsuggests a novel explanation for the broad-spectrum an-tioxidant and antiinflammatory activity of this nitrone spintrap. Key Words: Mitochondrial complexes—Hydrogenperoxide—a-Phenyl-N-tert-butyl nitrone—Electron ac-ceptors.J. Neurochem. 71, 2549—2557 (1998).

Mitochondrial complex I (NADH-CoQ reductase,NADH dehydrogenase) and complex II (succinate-C0Q reductase, succinate dehydrogenase) are struc-turally complex metallofiavoenzymes that catalyzeNADH- and succinate-mediated reduction of ubiqui-none, respectively. Flavin dehydrogenases such ascomplex I and II are distinguished from flavin oxidases

by differences in the propensity with which their re-duced flavohydroquinone prosthetic groups react withdioxygen. Flavin oxidases preferentially reduce 02,yielding the superoxide radical anion (02~) and thedismutation product hydrogen peroxide (H202) andregenerating fiavoquinone (Harris and Massey, 1997;Hiran et al., 1997). In contrast, flavin dehydrogenasesdo not readily autooxidize in such a manner, presum-ably because of structural features that preclude associ-ation of oxygen with the fiavohydroquinone. 02 andH202 production by isolated mitochondria is nonethe-less a commonly reported phenomenon, constituting1—2% of the total oxygen consumption by these organ-elles (Boveris and Chance, 1973; Boveris, 1984). Theusual explanation for mitochondrial reactive oxygenspecies (ROS) leakage is redox cycling between ubi-quinone and ubihydroquinone with concomitant 02 re-duction near the ubiquinone—complex III junction(Loschen et al., 1974; Boveris et al., 1976; Cadenaset al., 1977; Boveris, 1984; Turrens et al., 1985; Liu,1997; Turrens, 1997). A significant fraction of mito-chondrial ROS production, however, seems to occurat the level of complex I (Cadenas et al., 1977;Turrensand Boveris, 1980). The exact biochemistry of mito-chondrial ROS production is unclear.

In the current study, isolated brain mitochondriawere stimulated with NADH (the complex I respira-

Received May 19, 1998; revised manuscript received June 30,1998; accepted July 2, 1998.

Address correspondence and reprint requests to Dr. K. Hensleyat Free Radical Biology and Aging Research Program, OklahomaMedical Research Foundation, Oklahoma City, OK 73104, U.S.A.

Abbreviations used: DMPO, 5,5-dimethyl- 1-pyrroline N-oxide;DMSO, dimethyl sulfoxide; DPI, diphenyliodonium; EPR, electronparamagnetic resonance; H2DCFDA, dichlorodihydrofluorescein di-acetate; HRP, horseradish peroxidase; KHP, buffer containingHEPES, potassium phosphate, and potassium chloride; MPP~, I-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1 ,2,5,6-tet-rahydropyridine; NBT, nitroblue tetrazolium; PBN, ce-phenyl-N-tert-butyl nitrone; ROS, reactive oxygen species; SOD, superoxide dis-mutase.

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2550 K. HENSLEY ET AL.

tory substrate) or succinate (which stimulates respira-tion through complex II + III). These two respiratorypathways were compared with respect to their abilityto reduce alternative electron acceptors (i.e., reduciblesubstrates other than ubiquinone). The alternativeelectron acceptors chosen in this studywere 02 (reduc-ible to H202), nitroblue tetrazolium (NBT), whichis reducible to a blue formazan (Slater et al., 1963;Belyakovich, 1983; Bitton and Koopman, 1986), anddiphenyliodonium (DPI), which is reduced by fiavo-hydroquinones to form a phenyl radical that recom-bines with the fiavosemiquinone to inactivate the en-zyme (O’Donnell et al., 1994). Despite the findingthat succinate-stimulated 02 consumption was three tofive times more rapid than NADH-stimulated respira-tion, it was observed that succinate stimulation causedvery little observable H202 generation and was an inef-ficient means to reduce the alternative electron ac-ceptors NBT and DPI. Rotenone strongly inhibitedNADH oxidation and 02 consumption while accelerat-ing H202 generation and only mildly affecting NBTreduction kinetics. 02, NBT, and DPI reduction bycomplex I were all partially inhibited by a-phenyl-N-tert-butyl nitrone (PBN), a compound documented topossess broad-spectrum but unexplained antioxidantand antiinfiammatory properties in vivo. The findingspresented indicate that complex I is a major and under-estimated source of H202 in brain mitochondria andsuggest the presence of an “electron leak” site withincomplex I but distinct from the rotenone binding andthe ubiquinone reduction sites. Furthermore, the datasuggest that interaction of PBN with mitochondrialfiavin dehydrogenases effects subtle alterations in elec-tron transit through these enzymes, which may contrib-ute to its action as a biological antioxidant, antiin-flammatory, and neuroprotective agent.

MATERIALS AND METHODS

Mitochondrial preparationsAdult Sprague—Dawley rats were obtained from Charles

River Laboratories (Wilmington, MA, U.S.A.) and main-tained in the Oklahoma Medical Research Foundation Labo-ratory Animal Care facility until used. Brain mitochondrialpreparation followed a variation on the protocol of Mecocciet al. (1993). Cerebral cortices (.-~1.5g each) were pooledfrom two rats and minced thoroughly with dissecting scis-sors, then rinsed once in ~-~20ml of ice-cold isolation me-dium [0.3 M sucrose, 25 mM tris(hydroxymethyl)amino-methane, 2 mM EDTA, pH 7.31. Tissue was then suspendedin 12 ml of ice-cold isolation medium, homogenized at 4°Cwith 10 strokes of amotor-driven glass-walled Dounce-typehomogenizer equipped with a Teflon pestle (0.25-mm clear-ance), and then diluted to 26 ml. Tissue thus homogenizedwas centrifuged at 10—15°Cand 2,100 g for 3 mm in a fixed-angle rotor. Supernatant was decanted and centrifuged at12,500 g for 10 mm. The pellet from the second centrifuga-tion was gently dispersed into 20 ml of isolation mediumand recentrifuged at 12,500 g for 10 mm. The final pelletwas resuspended in 12 ml of isolation medium and rotatedfor 10—20 mm at 60 rpm to gently disperse particulates.

Protein content was determined by the method of Lowry etal. (1951) and adjusted to 10 mg/ml. This final mitochon-drial preparation was maintained on ice until use.

Determination of 02 consumptionState 4 oxygen utilization by mitochondrial preparations

was followed using a stirred, Clark-type oxygen electrodemaintained at 30°C(Barjaet al., 1994). The analyte solutioncontained mitochondria (0.5 mg/ml), KHP (20 mMHEPES, 5 mMpotassium phosphate, 80mM KC1, pH 7.3),and 1 mM substrate (succinate or NADH). The electrodesystem was calibrated by addition of sodium hydrosulfite toquantitatively remove 02, assumed to be present initially at200 1iM.

Kinetic assays of dehydrogenase activitiesAll kinetic assays were performed in a microplate format

using a Molecular Devices (Sunnyvale, CA, U.S.A.) Ther-moMax instrument maintained at 30°Cincubation tempera-ture. Assay mixtures consisted of mitochondria (1.5 mgIml of protein in final reaction mixture), substrates (finalconcentration of 0.4 mM NADH or succinate and 0.5 mMNBT unless otherwise stated), KHP, and indicated concen-trations of nitrone spin trap. Total reaction volume was 0.2ml/well, and reactions were initiated by addition of mito-chondria to prepared substrate solutions. Nitroblue formazanstandards were purchased (Sigma Chemical, St. Louis, MO,U.S.A.) or prepared by treatment of NBT with potassiumsuperoxide. Standard curves for nitro blue formazan wereprepared by addition of the formazan [dissolved in dimethylsulfoxide (DMSO) I to mitochondrial preparations. NADHoxidation (i.e., NADH dehydrogenase activity) was fol-lowedat 340 nm. NADH- or succinate-coupled NBT reduc-tion (i.e., NADH-NBT reductase activity or succinate-NBTreductase activity) was followed at 660 nm.

Measurement of hydrogen peroxideHydrogen peroxide produced by stimulated mitochondria

(state 4 conditions) was measured fluorogenically usinghorseradish peroxidase (HRP) -mediated, H2O2-dependentchemistries similar to those previously described (Boveriset al., 1976; Nowak, 1990; Yakes and Van Houten, 1997).Dichlorodihydrofluorescein diacetate (H2DCFDA; 2 gil, ini-tially dissolved to 10 mM in ethanol) was mixed with 30~l of mitochondria (initially at 10 mg/ml), 10 ~tl of HRP(initially at 2 mg/ml), and 138 ~.elof KHP. Reactions wereinitiated by addition of 20 ~il of concentrated substrate(NADH, succinate, or buffer). H202 production was mea-sured as picomoles H2DCFDA oxidized per milligram ofprotein per minute using a Molecular Devices Fmax instru-ment operating at 37°C with an excitation wavelength of485 nm and an emission wavelength of 538 nm. Authenticdichlorofluorescein was used as astandard. In specific exper-iments, rotenone was added as a solution in DMSO (finalDMSO concentration 1%), and care was taken to includeequivalent quantities of DMSO in samples that did not re-ceive rotenone. Specific experiments were conducted to con-firm that mitochondrial stimulants and inhibitors used in thisstudy did not affect HRP activity or HRP-catalyzed, H2O2-dependent H2DCFDA oxidation.

Electron paramagnetic resonance (EPR)spectroscopy

EPR samples were prepared in 0.2 ml of reaction volumeand immediately placed in a 0.2-mi quartz EPR flat cell.EPR spectra were acquired at ambient temperature (22—

.1. Neuroehem., Vol. 71, No. 6, 1998

NITRONE EFFECTS ON MITOCHONDRIA 2551

FIG. 1. Oxygen consumption by brain mitochondria stimulatedwith succinate or NADH at indicated time points (5 mm afterdilution of mitochondria into buffer). Full scale on the verticalaxis is —.100 p~M02.

24°C)on a Bruker 300 EPR instrument operating at 20-mWmicrowave power with a resonance frequency of 9.79 GHzand a modulation amplitude of 0.9 G.

StatisticsData were assessed by ANOVA and Student’s t tests. A

value of p < 0.05 was considered statistically significant.

RESULTS

Measurement of 02 consumption using an oxygenelectrode (Fig. 1) confirmed that brain mitochondrialpreparations used in this study were competent to con-sume 02 upon stimulation with respiratory substrates(succinate, NADH, malate plus glutamate, or ADP).NADH stimulation of intact mitochondria is possiblebecause NADH can be effectively transferred acrossthe inner membrane through the action of the malate/oxaloacetate/aspartate shuttle and similar shuttle sys-tems thatpartially co-purify with mitochondria (Ceder-baum et al., 1973). Direct stimulation with NADHallows concurrent monitoring of substrate oxidation(monitored optically at 340 nm, the absorbance maxi-mum for NADH) and other respiratory parameterssuch as oxygen utilization or peroxide leakage. Asshown in Fig. 1, succinate-stimulated 02 consumptionrates exceeded NADH-stimulated rates by a factor ofthree- to fivefold (maximal rate ‘-~ 15 nmol of 02/mgof protein/mm). These respiratory rates are muchlower than those commonly reported for liver or heartmitochondrial preparations (Radi et al., 1994; Hensleyet al., 1 997a) but consistent with previous data regard-ingbrain mitochondria (Radi et al, 1994; Hensley et al.,1997b). Substrate-stimulated respiration was almostcompletely inhibited by classic inhibitors (rotenone,antimycin A, and KCN) and was accelerated by theclassic uncoupler dinitrophenol (data not shown). Thefinding that rotenone virtually stopped NADH oxida-tion (discussed below) and 02 consumption indicatedthat almost all NADH oxidation occurring in the mito-chondrial preparation was complex I dependent.

FIG. 2. Hydrogen peroxide generation by NADH-stimulated mi-tochondria as measured fluorogenically using the H2DCFDAIHRP system. Reaction mixtures were stimulated with the indi-cated concentration of NADH or succinate. Error bars indicateSEM of 12 samples.

Electronleakage from mitochondrial flavin dehydro-genases would be expected to yield 02 - or the dismu-tation product H202. In this study, stimulation of unin-hibited mitochondria with NADH produced a quanti-fiable H202 flux that was linear with respect to NADHconcentration up to 1 mM (Fig. 2). Comparison ofNADH oxidation rate with H202 synthesis rate indi-cated 1—2% of NADH oxidation resulted in hydrogenperoxideleakage, in agreement withprevious estimates(Boveris and Chance, 1973). Additionally, removalof mitochondria by centrifugation prior to H202 assayabolished HRP-catalyzed DCF oxidation in the super-natant fraction (not shown), indicating minimal in-volvement of soluble dehydrogenase enzymes inNADH-coupled H202 generation. Despite the propen-sity with which mitochondrial preparations utilize suc-cinate as an electron donor (Fig. 1), succinate wasfound to be a very poor substrate for H202 synthesisin uninhibited mitochondria (Fig. 2). Succinate stimu-lation caused substantial H202 flux only in mitochon-dna that had been treated with the complex III inhibitorantimycin A (Fig. 3). In antimycin-treated mitochon-

FIG. 3. H202 flux from the complex Il/complex Ill respiratorypathway in the absence and presence ofthe complex Ill inhibitorantimycin A (ant; 25 jiM) and PBN (0.5 mM). Error bars indicateSEM of 12 samples. suc, succinate.

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2552 K HENSLEY ET AL.

dna, succinate-stimulated H202 generation approachedthat seen in uninhibited NADH-stimulated mitochon-dna (Fig. 3). These findings substantiate previouswork indicating a source of mitochondrial H202 pro-duction within the ubiquinone/complex III junction(Bovenis and Chance, 1973; Cadenas et al., 1977; Tur-rens et al., 1985); however, this source may be signifi-cant only in compromised organelles.

Addition of rotenone caused a concentration-depen-dent inhibition of NADH oxidation while strongly ac-celerating NADH-linked H202 synthesis (Fig. 4).Antimycin produced a similar increase in NADH-stim-ulated H202 flux (not shown). In the presence of rote-none, up to 40% of NADH oxidation resulted in H202synthesis. Since rotenone inhibits electron efflux fromcomplex I through the ubiquinone docking site(s) onthe downstream portion of the complex (Ramsay et al.,1991), the rotenone enhancement of H202 productionimplies a site of oxygen reduction within complex Ibut upstream from the ubiquinone binding site.

Inclusion of PBN with NADH-stimulated mitochon-dna resulted in a 30—40% inhibition of H202 flux witha half-maximal inhibitory concentration of 100 ~zMPBN (Fig. 5A). In experiments where mitochondriawere simultaneously treated with PBN plus NADH inthe presence and absence of rotenone, it was foundthat PBN partially mitigates the rotenone accelerationof H202 flux (Fig. SB).

The H202 biosynthesis illustrated in Figs. 4 and 5represents the enzyme-catalyzed, two-electron reduc-tion of 02 by NADH. Detailed kinetic analysis of 02reduction by complex I is hampered by difficulties inaccurately controlling initial 02 concentrations. In anattempt to partially circumvent this difficulty and fur-ther probe the interaction between PBN and complexI, experiments were undertaken using NBT as an alter-native two-electron acceptor for complex I catalysis.Table 1 summarizes the effects of various tested inhibi-

FIG. 4. Rotenone influence on NADH oxidation and NADH-cou-pled H2O2 flux as measured using the H2DCFDA/HRP system.Error bars indicate SEM of six samples.

FIG. 5. Nitrone effects on NADH-coupled H202 flux in brain mito-chondria. Data were collected as described with respect to Figs.1 and 2. A: Data were collected in a system not exposed tomitochondrial inhibitors. B: H2O2 flux in the absence of rotenone(only NADH and PBN present) was subtracted from the corre-sponding flux in the presence of rotenone to yield the rotenone-dependent component of H202 flux.

tors on NADH dehydrogenase and NADH- or succi-nate-stimulatedNBT reductase activities of brain mito-chondnial preparations. NADH oxidation rate and NBTreduction rate were negligible in the absence of mito-chondria. Furthermore, NBT reduction rate in the pres-ence of mitochondnia was negligible if both NADHand succinate were excluded from the reaction mixture.Combination of NBT with mitochondria and respira-tory substrate (NADH or succinate) caused measur-able reduction of the tetrazolium salt that was linearwith respect to time for —~10 mm. NADH oxidationkinetics were nearly identical to NADH-coupled NBTreduction kinetics, responding similarly to most testedinhibitors except rotenone, which inhibited NADH oxi-dation while leaving NADH-coupled NBT reductionlargely unaffected (30% reduction from control; Table1). As rotenone almost completely inhibited NADHoxidation in the absence of NBT, it must be concludedthat a large fraction of NADH-coupled NBT reductionoccurs within complex I at a site distinct from therotenone-inhibitable portion of the enzyme and at asite more proximal to the flavin nucleotide co-factor.It is conceivable that the NBT reduction site is nearor identical to the oxygen reduction site (describedabove). NBT or 02 reduction need not occur at theflavin center, however, and could conceivably occurat an iron—sulfur center or near the NADH oxidationsite.

Interestingly, succinate proved to be a poor electrondonor for mitochondnia-catalyzed NBT reduction (Ta-

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NITRONE EFFECTS ON MITOCHONDRIA 2553

TABLE 1. Effect of various inhibitors on NADH dehydrogenase and NADH-NBT reducta,se(complex I) activities and succinate-NBT reductase (complex II) activity

in mitochondrial preparations

NADH dehydrogenase NADH-NBT reductase Succinate-NBTactivity (% of activity (% of reductase activity (% of

Inhibitor uninhibited preparation)” uninhibited preparation)h uninhibited preparation)’

Rotenone (25 jiM) 10 ±15 70 ±5 98 ±2Antimycin A (50 pM) 46 ±9 42 ±3 69 ±24Malonate (0.5 mM) 78 ±20 67 ±4 45 ± 10KCN (0.5 mM) 68 ±14 64 ±3 31 ±31DPI(Imfivl) 56±13 67±20 63±15PBN (0.8 mM) 88 ± 10 82 ±3 60 ±12SOD (150U/ml) 87±5 87±3 71 ±10

NBT concentration used was 0.25 mM in all cases. Data represent means ±SD of at least four experiments,each consisting of 6—12 replicate samples.

Actual rates were 10—15 nmol of NADH oxidized/mg/m.in in the absence of inhibitors.Actual rates were 10—15 nmol of NBT reduced/mg/mm in the absence of inhibitors.

‘Actual rates were —‘2—5 nmol of NBT reduced/mg/mm in the absence of inhibitors.

ble 1), a finding consistent with the observation thatsuccinate does not readily facilitate two-electron re-duction of oxygen (described above and in Fig. 2).Succinate-stimulated NBT reduction was completelyinsensitive to rotenone but sensitive to KCN, antimycinA, and DPI (Table 1).

NBT reduction can be facilitated by superoxide orcan be catalyzed by reductase enzymes in an oxygen-independent manner. Given that NADH-coupled NBTreduction rates are almost identical to 02 consumptionand NADH oxidation rates and are 50-fold greater thanH202 efflux rates, it is unlikely that O2~contributesmeaningfully to NBT reduction kinetics in isolatedbrain mitochondnia. To rule out major contributions ofsuperoxide to NBT reduction, superoxide dismutase(SOD) was included in the reaction mixture along withNADH or succinate and mitochondnia. SOD caused asmall but significant 13% decrease in the rate of NBTreduction at levels of SOD above 100 U/ml (0.1 mg/ml) (Table 1). This quantity of SOD was found toinhibit 100% of the O2~production by a xanthine/xanthine oxidase reaction run in KHP at xanthine oxi-dase concentrations sufficient to reproduce the NBTreduction kinetics observed in NADH-stimulated mito-chondnial preparations (data not shown). It was notpossible to conclude that 02~generation occurred inthe mitochondnial preparations, however, since thisquantity of SOD also induced a significant 13% de-crease in NADH oxidation rate (Table 1). The partialSOD inhibition of NADH-stimulated NBT reductionwas therefore more likely a consequence of the unex-pected effect of SOD on enzyme-catalyzed NADH oxi-dation rather than scavenging of superoxide radical.Attempts to detect superoxide in NADH or succinate-stimulated mitochondrial preparations using acetylatedferricytochrome C as a target reductant were unsuc-cessful (data not shown). SOD also inhibited a maxi-mum 30% of the total succinate-stimulated NBT re-sponse, although attempts to measure superoxide using

the cytochrome c reduction assay again demonstratedno O2~presence (data not shown).

PBN addition to stimulated mitochondnia producedsubtle but measurable alterations in the dynamics ofNADH oxidation and NBT reduction. If PBN was in-cluded during NADH-coupled NBT reduction, theformazan formation rate decreased with increasing ni-trone concentration (Fig. 6). PBN was more potentas an inhibitor of succmnate-coupled NBT reduction(Fig. 6), although the absolute rate of succinate-linkedformazan formation was always much less than thecorresponding rate of the NADH-stimulated reaction(Table 1). In contrast to the obvious inhibition of NBTreduction, PBN only weakly inhibited NADH oxida-tion (—-.12% inhibition at 0.8 mM PBN; Fig. 6 andTable 1). Furthermore, if NADH concentration wasdecreased while constant PBN concentration (0.5 mM)was maintained, no evidence of PBN competition withNADH was observed (data not shown). Substitu-

FIG. 6. Inhibition of NBT reduction by PBN. NBT concentrationwas maintained at 0.125 mM, whereas PBN concentration wasincreased. NADH and succinate concentrations were 0.4 mM(initial values). Data indicate means ±SEM of at least four inde-pendent mitochondrial preparations.

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2554 K HENSLEY ET AL.

FIG. 7. Lineweaver— Burk analysis of NBT reduction kinetics (ini-tial reduction rate data) in the presence and absence of PBN(0.5 mM) and in the absence (A) or presence (B) of rotenone(25 pM). At least sixsamples were averaged for each data point.

tion of the nitrone 5,5-dimethyl- 1 -pyrnoline N-oxide(DMPO) for PBN produced only a weak inhibition ofNADH-NBT and succinate-NBT reductase activity athigher DMPO concentrations (p < 0.05 at 0.8 mMDMPO), whereas substitution with salicylate (0—1mM) had virtually no effect on either parameter (notshown). The interaction of PBN with mitochondrialflavin dehydrogenases therefore seems to be a specificfeature of certain nitrone structures rather than a non-specific phenomenon reproducible by substitution ofarbitrary aromatic compounds.

A Lineweaver—Burk analysis of NBT reduction ki-netics is consistent with a model of mostly classiccompetitive inhibition between PBN and NBT (Fig.7). PBN inhibition of NADH-stimulated NBT reduc-tion occurred independently of the presence of rote-none (Fig. 7), indicating that a significant fractionof the PBN-inhibitable NBT reductase activity wasresident within complex I at a site distinct from therotenone binding (ubiquinone docking) site. Inhibitionof NBT reduction by PBN in the presence of rotenone,however, displayed mostly noncompetitive kinetics(Fig. 7B). Regardless of the presence of rotenone,PBN inhibition of NBT reduction was more pro-nounced at low concentrations of NBT (<0.2 mM)and larger concentrations of PBN (0.5—0.8 mM).These concentrations of PBN are similar to concentra-tions reportedly attained in rodent brain after systemicPBN administration (Chen et al., 1990; Cheng et al.,1993).

A final set of experiments were conducted to deter-mine if nitrones interact directly with redox catalyticelements of mitochondrial complex I or II. DPI wassubstituted for NBT as an electron-accepting substrate.Flavin oxidation by DPI results in the formation of areactive phenyl radical that recombines immediatelywith the oxidized flavin co-factor (O’Donnell et al.,1994). If nitrones are accessible to DPI-reactive por-tions of mitochondnial flavin dehydrogenases, then thenitrones should protect the enzymes from DPI inhibi-tion while forming EPR-detectable phenyl spin ad-ducts. DPI inhibits both complex I and complex II as

FIG. 8. PBN inhibition of DPI-mediated complex I inactivation.Reaction mixtures consisted of mitochondria (1.5 mg/mI proteinconcentration),0.4 mMNADH, 0 or 1 mMDPI, 0 or 0.5 mMNBT,and the indicated concentration of PBN. Data are expressed asactivity of incubates containing DPI plus PBN versus the corre-sponding activity of incubates containing PBN but lacking DPI.Reactions were allowed to proceed 15 mm before collection ofkinetic data to allow time for mechanism-based interaction ofDPI with flavin enzymes. Data indicate means ± SEM of sixsamples at each concentration.

indicated by determination of NADH dehydrogenaseor NADH- and succinate-NBT reductase activities(Table 1). Inclusion of PBN with DPI during NADHoxidation could almost completely block DPI inactiva-tion of complex I (Fig. 8; significant protection wasafforded at all nitrone concentrations tested) but hadno discernible effect on DPI inactivation of complexII (not shown).

In the presence of NADH, PBN, DPI, and mitochon-dna, a strong EPR spectrum was obtained (aN = 16.1G, aH = 4.2 G; Fig. 9). An EPR signature could beobserved at PBN concentrations as low as 0.1 mM.

FIG. 9. EPR spectra of PBN—phenyl radical spin adducts formedby mitochondria in the presence of DPI and NADH. k Completesystem (1.5 mg/mI mitochondrial protein, 1 mM PBN, 5 mMNADH, 1 mM DPI). B: Complete system containing 0.5 mMPBN. C: Complete system containing 0.1 mMPBN. D: Completesystem containing 1 mM PBN pIus 25 pM rotenone. E: PBNexcluded from system (DPI, NADH, and mitochondria present).F: DPI excluded from system. G: NADH excluded from system.H: 5 mMsuccinate substituted for NADH in complete system.

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NITRONE EFFECTS ON MITOCHONDRIA 2555

FIG. 10. EPR spectra of DMPO—phenyl radical spin adductsformed by mitochondria in the presence of DPI and NADH. A:Complete system (1.5 mg/mI mitochondrial protein, 1 mMDMPO, 5 mM NADH, 1 mM DPI). B: DPI excluded from com-plete system. C: NADH excluded from complete system. D: 5mM succinate substituted for NADH in complete system.

However, substitution of succinate for NADH failedto produce an EPR spectrum (Fig. 9). Substitution ofthe spin trap DMPO for PBN also allowed detectionof a nitroxide species during NADH stimulation in thepresence of DPI (aN = 16.1 G, aH = 24.5 G; Fig. 10).The hyperfine splitting constants for the spin adductsare consistent with a trapped phenyl radical and withthe presumed mechanism of DPI inactivation of flavinenzymes (Kotake and Janzen, 1988; O’Donnell et al.,1994). Inclusion of rotenone in the reaction mixturecaused —-.50% suppression of the intensity of the ni-trone phenyl adduct (Fig. 9). These EPR data arguethat nitrone interaction with complex I is such thatreactive phenyl radical intermediates formed from en-zyme-catalyzed DPI reduction can be readily trappedand scavenged, thereby preventing phenyl radical at-tack on sensitive enzyme components. Failure of ni-trone spin traps to capture phenyl adducts during succi-nate stimulation of complex II could explain the corre-sponding failure of PBN to protect mitochondriaagainst DPI-mediated complex II inactivation. Effi-cient nitrone trapping of DPI-derived phenyl radicalsformed within complex I of brain mitochondnial prepa-rations suggests that these nitrones may specificallylocalize near a reactive site located along the electronconduit passing through the enzyme complex.

DISCUSSION

Reduced flavin enzymes represent electron sinksthat may, under certain conditions, reduce 02 or othertarget molecules to yield biologically reactive species.The structural complexity of flavin enzymes, particu-larly of the mitochondrial complex I and II dehydroge-nases, has precluded detailed mechanistic studies ofthe electron transfer processes that they catalyze. Thedata presented in the current investigation extend pre-vious work implicating complex I as a major site ofH2O2 generation in brain mitochondnia (Cadenas et al.,1977; Herrero and Barja, 1997) and suggest that therelative contribution of complex I to mitochondnialH2O2 biosynthesis has been greatly underestimated.

Rotenone is a highly specific inhibitor of complexI that binds the enzyme at a point on the cytochromeside of the highest potential Fe—S cluster of the en-zyme but prior to the coenzyme Q site (Horgan et al.,1968; Ramsay et al., 1991). The acceleration ofNADH-coupled H2O2 production by rotenone suggeststhat electron leakage from complex I occurs on orwithin this enzyme, at a location upstream from therotenone binding and ubiquinone reduction sites. Inthis model, rotenone prohibits electron efflux out ofcomplex I, resulting in a buildup of reduced moietiesupstream from the rotenone binding site and driving02 reduction at specific reactive centers. The findingthat NADH-coupled NBT reduction is largely rotenoneinsensitive reaffirms observations made by Slater(1963) and is interpreted within the present contextof understanding mitochondnial ROS production. Theinability of rotenone to inhibit NADH-linked NBT orDPI reduction or to abrogate PBN inhibition of H202flux suggests that the putative electron leak site is pro-miscuous with respect to the number of reduciblesubstrates it may accept. In recent work pertinent tothis chemistry, Herrero and Barja (1997) report thatthiol-reactive agents such as mercunibenzoate inhibitNADH-linked, rotenone-insensitive H202 leakagefrom heart mitochondnia, possibly implicating redox-cycling thiols or iron— sulfur groups within complex Ias an origin for H2O2 synthesis. The existence of sucha putative reactive thiol could explain much of the datareported in the present investigation, although reactionat the flavin co-factor or near the NADH oxidationpoint cannot be excluded.

This study finds that certain nitrone spin-trappingcompounds, particularly PBN, interact with mitochon-dnal flavin dehydrogenases to alter the dynamics ofelectron transit through and leakage from these en-zymes. Such action of nitrones has notbeen previouslydescribed but may explain certain of the antioxidantand antimnflammatory actions of nitrone spin-trappingagents.

Nitrones have often been reported to mitigate oxida-tive stress resulting from disparate experimental ma-nipulations (Hensley et al., 1997a,b). Most notably,nitrone protection against stroke lethality in gerbils isimmediate and persists for several days after dosage(Oliver et al., 1990; Floyd and Carney, 1991; Zhao etal., 1994; Cao and Phillis, 1994; Floyd, 1996). Despitethe relative stability of nitrone radical adducts, mosttested nitrones are actually inefficient free radical-scavenging or chain-breaking antioxidants relative tohindered phenolic compounds. In direct comparisonsof microsomal lipid oxidation inhibition, for instance,inhibitory concentrations of PBN were found to lie inthe millimolar range and were 10—100 times greaterthan those of butylated hydroxytoluene or trolox (Jan-zen et al., 1994). Considering that PBN concentrationsin vivo seldom exceed 500 ~sMand that the half-lifeof the drug in vivo is <120 mm (Chen et al., 1990;Cheng et al., 1993), it is unreasonable to entirely attni-

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2556 K HENSLEY ET AL.

bute the antioxidant effects of PBN to nonspecific freeradical-scavenging action. Contrastingly, site-specificaction of nitrones within microdomains of specificmembrane compartments or at specific enzymes couldoffer a more plausible explanation for the broad anti-oxidant and antiinflammatory action of nitrones.Blockage of electron leakage from mitochondnial fla-yin dehydrogenases, for instance, could reduce steady-state levels of ROS with resultant diminution of proteinand nucleic acid oxidation and cytoprotection.

Alterations in the function of mitochondnial flavindehydrogenases have been associated with increasedoxidative stress during acute inflammation, whereasspecific nitrones have been documented to possess po-tent antiinflammatory actions. In a cecal puncturemodel of sepsis, for example, mitochondnia isolatedfrom liver of septic rats were more efficient in catalyz-ing succinate-stimulated salicylate hydroxylation thanwere mitochondnia isolated from healthy animals, pre-sumably indicating increased mitochondrial generationof hydroxyl radicals during the septic condition (Tay-lor et al., 1995). Similarly, Kantrow et al. (1997) doc-ument increased mitochondrial protein carbonyl levelsand increased salicylate hydroxylation capacity of he-patocytes isolated from septic animals.

PBN as well as a cyclic analogue can rescue rodentsfrom septic shock and mitigate tumor necrosis factor-a and inducible NO synthase induction in vivo afterlipopolysacchanide challenge (Hamburger and McCay,1989; Pogrebniak et a!., 1992; French et al., 1994;Miyajima and Kotake, 1995). Expression of inflam-mation-associated gene products including inducibleNO synthase is regulated largely by the NFKB/Relfamily of transcription factors, which are hyperacti-vated by certain oxidizing conditions (e.g., exposureto H2O2 or depletion of cellular reducing equivalents)(Schreck and Baeuenle, 1991; Adcock et al., 1994; Senand Packer, 1996; Lander, 1997; Suzuki et al., 1997).Alteration in the dynamics of mitochondnial flavoen-zymes by nitrones might therefore affect mitochondnialoxidant production and coupled gene expression.

One particularly relevant action of nitrone com-pounds should be noted within the context of the pres-ent study. PBN as well as DMPO and the nitroneC- (N-oxide-4-pynidyl) -N-tert-butyl nitrone have beendocumented to protect rodents against Parkinson’s dis-ease-like symptoms induced by exposure to 1-methyl-4-phenyl- I ,2,5,6-tetrahydropynidine (MPTP) (Schulzet al., 1995). The neurotoxic effects of MPTP arethought to be mediated by the metabolite 1 -methyl-4-phenylpyridinium (MPP ~) which inhibits complex Iapparently at or near the rotenone binding site (Ram-say et al., 1991 ). Nitrone administration prior to MPTPtreatment prevents dopamine depletion in the caudatenucleus, a phenomenon correlated with increased sali-cylate hydroxylation within the same brain region(Schulz et al., 1995). Nitrone mitigation of MPTPtoxicity, previously interpreted as reflecting bulk freeradical-scavenging action, might be best reinterpreted

within the context of nitrone-inhibited electron leakagefrom MPP + -inhibited complex I.

Study of electron transit through complex I in nor-mal and perturbed states of enzyme function may illu-minate the molecularbasis for oxidative stress in multi-ple pathophysiologic conditions including stroke, sep-sis, and parkinsonism. A more detailed understandingof nitrone action in modulating flavin dehydrogenasefunction could lead to improved pharmacologic strate-gies for these conditions. In future experiments, wewill endeavor to further these goals.

Acknowledgment: Portions of this work were funded bythe National Institutes of Health (NS35747) and the Okla-homa Center for the Advancement of Science and Technol-ogy (H97-067). Additional funding was provided by thePaul F. Glenn Foundation for Medical Research (summerfellowship for F.J.) and a contract from Centaur Pharmaceu-ticals.

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