mitochondrial diaphorases as nad+ donors to segments of the citric acid cycle that support...

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Mitochondrial diaphorases as NAD donors tosegments of the citric acid cycle that supportsubstrate-level phosphorylation yielding ATP duringrespiratory inhibition

Gergely Kiss,* Csaba Konrad,* Issa Pour-Ghaz,* Josef J. Mansour,* Beta Nmeth,*Anatoly A. Starkov, Vera Adam-Vizi,* and Christos Chinopoulos*,1

*Department of Medical Biochemistry, Hungarian Academy of Sciences, SE Laboratory forNeurobiochemistry, Semmelweis University, Budapest, Hungary; and Department of Neurology andNeuroscience, Weill Medical College Cornell University, New York, New York, USA

ABSTRACT Substrate-level phosphorylation medi-ated by succinyl-CoA ligase in the mitochondrial matrixproduces high-energy phosphates in the absence ofoxidative phosphorylation. Furthermore, when theelectron transport chain is dysfunctional, provision ofsuccinyl-CoA by the -ketoglutarate dehydrogenasecomplex (KGDHC) is crucial for maintaining the func-tion of succinyl-CoA ligase yielding ATP, preventing theadenine nucleotide translocase from reversing. Weaddressed the source of the NAD supply for KGDHCunder anoxic conditions and inhibition of complex I.Using pharmacologic tools and specific substrates andby examining tissues from pigeon liver exhibiting nodiaphorase activity, we showed that mitochondrial dia-phorases in the mouse liver contribute up to 81% to theNAD pool during respiratory inhibition. Under theseconditions, KGDHCs function, essential for the provi-sion of succinyl-CoA to succinyl-CoA ligase, is sup-ported by NAD derived from diaphorases. Throughthis process, diaphorases contribute to the maintenanceof substrate-level phosphorylation during respiratoryinhibition, which is manifested in the forward opera-tion of adenine nucleotide translocase. Finally, we show

that reoxidation of the reducible substrates for thediaphorases is mediated by complex III of the respira-tory chain.Kiss, G., Konrad, C., Pour-Ghaz, I., Man-sour, J. J., Nmeth, B., Starkov, A. A., Adam-Vizi, V.,Chinopoulos, C. Mitochondrial diaphorases as NAD

donors to segments of the citric acid cycle that supportsubstrate-level phosphorylation yielding ATP duringrespiratory inhibition. FASEB J. 28, 000000 (2014).www.fasebj.org

Key Words: succinyl-CoA ligase adenine nucleotide translocase DT-diaphorase reducing equivalent

In the absence of oxygen or when the electrontransport chain is impaired, substrate-level phosphory-lation in the matrix is the only means for production ofhigh-energy phosphates in mitochondria. Mitochon-drial substrate-level phosphorylation is almost exclu-sively attributable to succinyl-CoA ligase, an enzyme ofthe citric acid cycle that catalyzes the reversible conver-sion of succinyl-CoA and ADP (or GDP) to CoASH,succinate, and ATP (or GTP) (1). We have shownpreviously that when the electron transport chain iscompromised and F0F1 ATP synthase reverses, pump-ing protons out of the matrix at the expense of ATPhydrolysis, the mitochondrial membrane potential ismaintained, albeit at decreased levels, for as long asmatrix substrate-level phosphorylation is operational,without a concomitant reversal of the adenine nucleo-tide translocase (ANT; ref. 2). This process preventsmitochondria from becoming cytosolic ATP consumers(35). More recently, we have reported that provisionof succinyl-CoA by the -ketoglutarate dehydrogenasecomplex (KGDHC) in respiration-impaired mitochon-

1 Correspondence: Department of Medical Biochemistry,Semmelweis University, 37-47 Tuzolto Street, Budapest 1094,Hungary. E-mail: [email protected]

doi: 10.1096/fj.13-243030This article includes supplemental data. Please visit http://

www.fasebj.org to obtain this information.

Abbreviations: m, membrane potential; 1,2-NQ, 1,2-naphthoquinone; 1,4-NQ, 1,4-naphthoquinone; ANOVA,analysis of variance; ANT, adenine nucleotide translocase;BSA, bovine serum albumin; BQ, p-benzoquinone; cATR,carboxyatractyloside; CBQ, 2-chloro-1,4-benzoquinone; CoQ,coenzyme Q; Cyb5r2, NADH, cytochrome b5 reductase iso-form 2; DCBQ, 2,6-dichloro-1,4-benzoquinone; DCIP, 2,6-dichloroindophenol; diOH-flavone, 7,8-dihydroxyflavone hy-drate; DMBQ, 2,6-dimethylbenzoquinone; DQ, duroquinone;Erev_ANT, reversal potential of adenine nucleotide translocase;FerrCyan, ferricyanide; KGDHC, -ketoglutarate dehydroge-nase complex; MBQ, methyl-p-benzoquinone; MDH, malatedehydrogenase; menadione, vitamin K3; menaquinone, vita-min K2; mitoQ, mitoquinone; NQO, NAD(P)H:quinone oxi-doreductase; PDHC, pyruvate dehydrogenase complex; phyl-loquinone, vitamin K1; SF 6847, tyrphostin-9,RG-50872,malonaben,3,5-di-tert-butyl-4-hydroxybenzylidenemalononi-trile,2,6-di-t-butyl-4-(2=,2=-dicyanovinyl)phenol; TPP, tetraphenyl-phosphonium; WT, wild type

10892-6638/14/0028-0001 FASEB

The FASEB Journal article fj.13-243030. Published online January 3, 2014.

dria is critical to sustaining the succinyl-CoA ligasereaction (6). Mindful of the reaction catalyzed byKGDHC converting -ketoglutarate, CoASH, and NAD

to succinyl-CoA, NADH, and CO2, the question arises asto the source of NAD, under conditions of a dysfunc-tional electron transport chain. It is common knowl-edge that NADH generated in the citric acid cycle isoxidized by complex I, resupplying NAD to the cycle.In the absence of oxygen or when complexes are notfunctional, an excess of NADH in the matrix is ex-pected. Yet, our previous reports showed that withoutNADH oxidation by complex I of the respiratory chain,substrate-level phosphorylation is operational and sup-ported by succinyl-CoA (2, 6), implying KGDHC activ-ity. In the present study we found that during anoxia orpharmacologic blockade of complex I, mitochondrialdiaphorases oxidized matrix NADH supplying NAD toKGDHC, which in turn yields succinyl-CoA, thus sup-porting substrate-level phosphorylation.

In general, diaphorase activity is attributed to aflavoenzyme catalyzing the oxidation of reduced pyri-dine nucleotides by endogenous or artificial electronacceptors called DT-diaphorase because of its reactivitywith both DPNH (NADH) and TPNH (NADPH), iden-tified by Lars Ernster (7, 8), and is now known asNAD(P)H:quinone oxidoreductase (NQO). It has alsobeen identified in parallel by Mrki and Martius (9),called vitamin K reductase, and later confirmed to bethe same enzyme (10). A quinone reductase withproperties similar to the enzyme described by Ernsteralso appears in earlier literature by Wosilait and col-leagues (11, 12), as well as the microsomal TPNH-neotetrazolium diaphorase, described by Williams et al.(13), and a brain diaphorase by Giuditta and Strecker(14, 15). Finally, a vitamin K3 (menadione) reductasehas been reported by Koli et al. (16). DT-diaphorase(EC 1.6.5.2, formerly assigned to EC 1.6.99.2) catalyzes2-electron reductive metabolism [unlike other NAD-(P)linked quinone reductases; ref. 17] detoxifyingquinones and their derivatives (18). Several isoformshave been identified (19, 20); among them, NQO1 andNQO2 have been most extensively characterized (19).A striking difference between these two is that NQO2uses dihydronicotinamide riboside (NRH), while NQO1uses NAD(P)H as an electron donor (21, 22). NQO1has been found to localize, not only in the cytosol, butalso in mitochondria from several tissues (10, 2333).Mitochondrial diaphorase corresponds to 315% oftotal cellular activity (10, 28, 3134) and is localized inthe matrix, since it reacts only with intramitochondrialreduced pyridine nucleotides, but is inaccessible tothose added from the outside (30, 35). However, itmust be emphasized that mitochondrial diaphoraseactivity may not be exclusively due to NQO1. Othermitochondrial enzymes also exhibit diaphorase-like ac-tivity as a moonlighting function; for example, theisolated DLD subunit of KGDHC exhibits diaphoraseactivity, and it is known to exist in the matrix as such,without being part of the KGDHC (3641).

Finally, we scrutinized the pathway responsible forproviding oxidized substrates to the diaphorases andconcluded that reoxidation is mediated by complex IIIof the respiratory chain.

MATERIALS AND METHODS

Animals

Mice were of mixed FVB and C57Bl/6 background, onlyC57Bl/6J, or only C57Bl/6N, as indicated throughout thetext. NADH:cytochrome b5 reductase isoform 2 (Cyb5r2)heterozygous mice on a C57BL/6 background were obtainedfrom the European Mouse Mutant Archive (EMMA) node atthe Medical Research Council (MRC)-Harwell (Harwell, UK)and the European Conditional Mouse Mutagenesis Program(EUCOMM) consortium. They were backcrossed for 5 gener-ations with FVB mice, yielding wild-type (WT), heterozygous,and viable fertile homozygous knockout (KO) Cyb5r2/ mice.The animals used in our study were of either sex and between 2and 3 mo of age. They were housed in a room maintained at2022C on a 12-h light-dark cycle with food and water availablead libitum. Pigeons (Columba livia domestica) were obtained froma local vendor and used on the same or the next day. Allexperiments were approved by the Animal Care and Use Com-mittee of Semmelweis University (Egyetemi llatksrleti Bi-zottsg).

Isolation of mitochondria

Liver mitochondria from all animals were isolated according toa published method (42). Protein concentration was deter-mined with the bicinchoninic acid assay and calibrated withbovine serum standards (43), with a Tecan Infinite 200 PROseries plate reader (Tecan Deutschland GmbH, Crailsheim,Germany). Yields were typically 0.4 ml from 80 mg/ml permouse liver and 0.8 ml from 80 mg/ml per pigeon liver.

Determination of membrane potential (m) in isolatedliver mitochondria

m of isolated mitochondria (1 mg for mouse or pigeonliver per 2 ml of medium, unless stated otherwise) wasestimated fluorometrically with safranin O (44). Traces ob-tained from mouse mitochondria were calibrated to millivolts(2). Fluorescence was recorded in a Hitachi F-7000 spectro-fluorometer (Hitachi High Technologies, Maidenhead, UK)at a 5-Hz acquisition rate, with 495- and 585-nm excitationand emission wavelengths, or at a 1-Hz rate, with the O2k-fluorescence LED2 module of the Oxygraph-2k (OroborosInstruments, Innsbruck, Austria), equipped with an LEDexhibiting a wavelength maximum of 465 25 nm (currentfor light intensity adjusted to 2 mA, i.e., level 4) and a505-nm shortpass excitation filter (dye-based, safranin filterset). Emitted light was detected by a photodiode (range ofsensitivity: 350700 nm), through a 560-nm longpass emis-sion filter (dye-based). Experiments were performed at 37C.m was also estimated from tetraphenylphosphonium (TPP

)ion distribution with a custom-made TPP-selective electrode(45). For these experiments, the assay medium (identical tothat for safranin O experiments) was supplemented with 2

M TPPCl, and the mitochondrial protein concentrationwas 2 mg/ml. The electrode was calibrated by sequentialadditions of TPPCl. Experiments were performed at 37C.

2 Vol. 28 April 2014 KISS ET AL.The FASEB Journal www.fasebj.org

Mitochondrial respiration

Oxygen consumption was performed polarographically withthe Oxygraph-2k. Liver mitochondria (2 mg) was suspendedin 4 ml incubation medium, the composition of which wasidentical to that for m determination (2). Experimentswere performed at 37C. Oxygen concentration and oxygenflux (expressed in picomoles per second per milligram andcalculated as the negative time derivative of oxygen concen-tration, divided by mitochondrial mass per volume andcorrected for instrumental background oxygen flux arisingfrom oxygen consumption of the oxygen sensor and backdiffusion into the chamber) were recorded with DatLabsoftware (Oroboros Instruments).

Determination of pH of the medium

The pH of the suspension medium for mouse liver mitochon-dria was recorded by connecting a glass Ag/AgCl pH micro-electrode (Radiometer Analytical, Lyon, France) to the po-tentiometric channel of the O2k via a BNC connector of theOxygraph-2k. The composition of this medium was 120 mMKCl, 10 mM NaCl, 10 mM mannitol, 1 mM MgCl2, 5 mM Pi,0.01 mM EGTA (K salt), 0.01 mM P1,P5-di(adenosine-5=)pentaphosphate (pH 7.25; titrated with KOH), and 0.5mg/ml bovine serum albumin (BSA). Experiments wereperformed at 37C. The voltage signal output of the electrodewas converted to pH by calibrating with solutions of knownpH values.

Determination of NADH autofluorescence in isolated livermitochondria

NADH autofluorescence was measured using 340- and435-nm excitation and emission wavelengths, respectively.Measurements were performed in a Hitachi F-7000 fluores-cence spectrophotometer at a 5-Hz acquisition rate. Mouseliver mitochondria (1 mg) were suspended in 2 ml incubationmedium, the composition of which was the following: 110mM K-gluconate, 10 mM HEPES (acid free), 10 mM KH2PO4,10 mM mannitol, 10 mM NaCl, 8 mM KCl, 1 mM MgCl2, 0.01mM EGTA, 0.5 mg/ml BSA (essentially fatty acid free), withthe pH adjusted to 7.25 with KOH. Respiratory substrateswere 5 mM glutamate and 5 mM malate. Experiments wereperformed at 37C.

Statistics

Data are presented as averages se. Significant differencesbetween 2 groups were evaluated by Students t test. Signifi-cant differences between 3 groups were evaluated by 1-wayanalysis of variance (ANOVA) followed by Tukeys post hocanalysis. Values of P 0.05 were considered statisticallysignificant. If the normality test failed, ANOVA on ranks wasperformed. Wherever single graphs are presented, they arerepresentative of 3 independent experiments.

Reagents

Standard laboratory chemicals were from Sigma-Aldrich (St.Gallen, Switzerland); tyrphostin-9,RG-50872,malonaben,3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile,2,6-di-t-butyl-4-(2=,2=-dicyanovinyl)phenol (SF 6847) and atpenin A5were from Enzo Life Sciences (ELS AG, Lausen, Switzerland);carboxyatractyloside (cATR) was from Merck (Darmstadt,Germany), and MitoQ was a generous gift from Dr. MikeMurphy (Mitochondrial Biology Unit, Medical Research

Council, Cambridge, UK). Mitochondrial substrate stock so-lutions were dissolved in bidistilled water and titrated to pH7.0 with KOH. ADP was purchased as a K salt of the highestpurity available (Merck) and titrated to pH 6.9.

RESULTS

Identifying mitochondria as extramitochondrial ATPconsumers during anoxia

As elaborated in our prior work (2, 6), to label amitochondrion as an extramitochondrial ATP con-sumer, its m, matrix ATP/ADP ratio, and reversalpotentials of F0F1 ATP synthase and ANT (the mvalues at which there is no ATP generation or hydrolysisfor the former and no net transfer of ADP-ATP acrossthe inner mitochondrial membrane for the latter) mustbe determined, which is an extremely challengingexperimental undertaking. Being mindful that the re-versal potential of the F0F1 ATP synthase is morenegative than that of the ANT (2, 4, 5), meaning thatwhenever the ANT reverses, the ATP synthase works inreverse, too, it is simpler and equally informative toexamine the effect of an ANT inhibitor on m duringADP-induced respiration (2, 4, 5). Since 1 molecule ofATP4 is exchanged for 1 molecule of ADP3 (bothnucleotides being Mg2 free and deprotonated) byANT, the exchange is electrogenic (46). Therefore,during the forward mode of ANT, abolition of itsoperation by a specific inhibitor such as cATR leads toan increase in m, whereas during the reverse modeof ANT, the same condition leads to a loss of m. Thisbiosensor test (i.e., the effect of cATR on safranin Ofluorescence reflecting m) was successfully used inaddressing the directionality of ANT during respiratoryinhibition (2). Further on, it was used in KGDHC-deficient mice (6) to determine the contribution ofKGDHC as a succinyl-CoA provider to the succinyl CoAligase reaction during respiratory inhibition. In thepresent study, we used this method in isolated mito-chondria subjected to true anoxic conditions and/orspecific inhibitors of the electron transport chain, whilesources of NAD for KGDHC were being sought.

Time-lapse recordings of safranin O fluorescencereflecting m while measuring oxygen concentrationin the same sample were obtained by the recentlydeveloped O2k-fluorescence LED2 module of the Oxy-graph-2k (Oroboros Instruments). Mitochondria wereallowed to deplete the oxygen dissolved in the air-sealed chamber and, additions of chemicals through atiny bore hole did not allow reoxygenation of the bufferfrom the ambient atmosphere.

Safranin O is known to increase state 4 respiration(47), decrease maximum Ca2 uptake capacity, andexhibit an appreciable nonspecific binding compo-nent, if used at concentrations above 5 M (48).However, at concentrations below 5 M, calibration ofthe safranin O fluorescence signal to m deviatessignificantly from linearity; therefore, more complexfitting functions are needed, decreasing the faithful-

3DIAPHORASES PROVIDE NAD IN ANOXIA

ness of the conversion. For our experiments, we used 5

M of safranin O at the expense of diminishing therespiratory control ratio by approximately 1 unit (from7.5 to 6.5, when using glutamate and malate), but thesignal-to-noise ratio was optimal, and the calibration ofthe fluorescence signal to m was highly reproduc-ible. TPP appeared to be less toxic than safranin O interms of an effect on mitochondrial respiration; how-ever, the signal-to-noise ratio was not as satisfactory asthat obtained from safranin O, and it could not beimproved by increasing the concentration of TPP

(from 2 to 6 M). The nonspecific binding componentof safranin O to mitochondria is determined by themitochondria/safranin O ratio; by using 5 M ofsafranin O for 2 mg of mitochondria (see below) thenonspecific component is within 10% of the totalsafranin O fluorescence signal, estimated by the in-crease in fluorescence caused by the addition of adetergent to completely depolarized mitochondria(not shown). As such, it was accounted for, during thecalibration of the fluorescence signal to m.

The result of a typical experiment is shown in Fig. 1.Mouse liver mitochondria (2 mg) were added to 4 ml ofbuffer (see Materials and Methods) containing thesubstrates indicated in the panels and were allowed to

fully polarize (solid traces). State 3 respiration wasinitiated by ADP (2 mM), depolarizing mitochondria by25 mV. With a respiration rate of 60 nmol/min/mg, mitochondria run out of oxygen within 56 min, asverified by recording 0 levels of dissolved oxygen in thechamber at 400 s (dotted traces). Anoxia also coin-cided with the onset of additional depolarization, lead-ing to a clamp of m at 100 mV. In mitochondriarespiring on glutamate and malate (i.e., substrates thatsupport substrate-level phosphorylation; ref. 2), subse-quent addition of cATR (Fig. 1A, black solid trace)caused moderate repolarization. This observation im-plies that, at 100 mV, ANT was still operating in theforward mode, in accordance with the ADP-ATP steady-state exchange activity/m relationship shown re-cently (49, 50). In contrast, when the specific F0F1 ATPsynthase inhibitor oligomycin (Fig. 1A, orange solidtrace) was added instead of cATR, immediate depolar-ization was observed, implying that F0F1 ATP synthasewas working in reverse and generated the residualm. This depolarization was complete, since furtheraddition of the uncoupler SF 6847 (250 nM) yielded nofurther depolarization. Obviously, under the condi-tions shown in Fig. 1A, ATP was available in the matrixfrom sources other than ANT. Our previous results

Figure 1. Reconstructed time coursesof safranin O signal calibrated tom (solid traces) and parallel mea-surements of oxygen concentrationin the medium (dotted traces) inisolated mouse liver mitochondria.Effect of cATR (2 M) or oligomycin(oligo, 5 M) on m during anoxia(AC, E) or during compromised re-spiratory chain by poisons (D), in thepresence of different substrate combi-nations. ADP (2 mM) was addedwhere indicated. Substrate concentra-tions were glutamate (glut; 5 mM),malate (mal; 5 mM), succinate (succ;5 mM), and -hydroxybutyrate,(OH; 1 or 2 mM, as indicated).Substrate concentrations were thesame for all subsequent experiments.At the end of each experiment, 1 M

SF 6847 was added to achieve complete depolarization, except for the orange trace in panel A, where 250 nM SF 6847 wasadded.


showed (2, 6) that under this condition, ATP is sup-plied by matrix substrate-level phosphorylation medi-ated by succinyl-CoA ligase. This finding was furthervalidated by the experiments shown in Fig. 1B, C. In Fig.1B, mitochondria respiring on succinate alone (blacktrace) or glutamate plus malate plus succinate (olivegreen trace), both conditions being unfavorable forsubstrate-level phosphorylation by succinyl-CoA ligase(succinate would push this reversible reaction towardATP or GTP hydrolysis), reacted to cATR with animmediate and complete depolarization in anoxia.Likewise, in the presence of 2 M atpenin A5, a specificinhibitor of succinate dehydrogenase (51) causing ac-cumulation of succinate in the matrix, cATR induceddepolarization in mitochondria that had been respiringon glutamate plus malate and were subject to anoxia(Fig. 1C, lilac trace). In the presence of atpenin,however, onset of anoxia was associated with a greaterdepolarization before addition of cATR; thus, it ispossible that the value of m exceeded the value ofthe reversal potential of ANT (Erev_ANT). However,when the electron transport chain was rendered inop-erable by rotenone in lieu of anoxia, m values wereidentical before addition of the ANT inhibitor (Fig. 1D,compare black with green trace), but loss of m,implying ANT reversal in the presence of atpenin A5,was verified by cATR.

From the findings in those experiments, we con-cluded that, in true anoxic conditions, ANT could bemaintained in forward mode, implying active matrixsubstrate-level phosphorylation in isolated mitochon-dria, similar to the previously published paradigms witha poisoned respiratory chain (2, 6). Furthermore, theresults obtained in our earlier study (6) showing thatprovision of succinyl-CoA by KGDHC is critical formatrix substrate-level phosphorylation imply an emerg-ing demand of NAD for KGDHC, a concept that is atodds with the idea that, in anoxia, there is a shortage ofNAD in the mitochondrial matrix. Indeed, Fig. 1Eshows that after elevating the matrix NADH/NAD

ratio by 1 or 2 mM -hydroxybutyrate (leading toNADH and acetoacetate formation through the reac-tion catalyzed by -hydroxybutyrate dehydrogenase),cATR induced slight depolarization, compared to nearrepolarization in Fig. 1A, C, E (black traces). The sameeffect of -hydroxybutyrate was found in mitochondriawith a poisoned respiratory chain (6). These resultsemphasize the importance of NAD for establishingthe conditions for the forward operation of ANT dur-ing anoxia.

Importance of NAD in maintaining the function ofKGDHC during anoxia or respiratory chain inhibition

The negative effect of KGDHC deficiency on matrixsubstrate-level phosphorylation in mitochondria with apoisoned respiratory chain has been demonstratedrecently by our group (6). In the present study, weaddressed the importance of sustaining KGDHC func-tion, requiring a supply of NAD in mitochondria

during anoxia by using arsenite, which enters intactmitochondria in an energy-dependent manner (52)and inhibits pyruvate dehydrogenase complex (PDHC)and KGDHC (53). When mitochondria respire onglutamate plus malate, the effect of arsenite may beattributable to inhibition of KGDHC. Safranin O fluo-rescence and oxygen concentration in the mediumwhere mitochondria underwent anoxia or drug-in-duced respiratory inhibition were recorded. As shownin Fig. 2A, the fully polarized mitochondria, in thepresence of glutamate and malate, were depolarized by25 mV by ADP (2 mM, solid black and green traces),consuming within 6 min the total amount of oxygenpresent in the medium (dotted black and green traces)and leading to additional depolarization to100 mV.Respiration rates are indicated in the figure, in nano-moles per minute per milligram protein (Fig. 2A). Thetransient repolarization on addition of 2 mM sodiumarsenite (NaAsO2) at 700 s was due to the high volumeof the addition (0.08 ml), which contained a significantamount of dissolved oxygen, seen as a minor elevationin oxygen concentration (Fig. 2A, dotted green linenear 700 s) that quickly subsided as it was consumed bythe mitochondria; it was also associated with the rees-tablishment of m to 100 mV. Subsequent addi-tion of cATR to mitochondria treated with NaAsO2(Fig. 2A, green solid trace) initiated a drop in m asopposed to a moderate repolarization observed in theabsence of arsenite (Fig. 2A, black solid trace). cATRalso caused a depolarization when arsenite was presentin the medium before addition of mitochondria (Fig.2A, red solid trace), which, as expected, was associatedwith a diminished rate of respiration (Fig. 2A, reddotted trace) leading to a prolongation until completeanoxia was achieved. cATR also caused a depolarizationwhen arsenite was present in the medium before mito-chondria (Fig. 2B, red trace), in which electron trans-port was halted by inhibiting complex I with rotenone.Subsequent addition of succinate (5 mM) fully restoredm, indicating that mitochondria were capable ofelectron transport from complex II when complex I wasblocked, in the presence of arsenite. Finally, the effectof arsenite was investigated on the rate of acidificationin weakly buffered media, in which mitochondria weretreated with a specific set of inhibitors. The concept ofthis experiment relies on the fact that mitochondria arenet CO2 producers that acidify the medium due to thefollowing equilibria: CO2 H2O H2CO3 H

HCO3

. Depending on the substrates combined withtargeted inhibition of bioenergetic entities, one maydeduce the role of arsenite-inhibitable targets. Mito-chondria were suspended in a weakly buffered medium(see Materials and Methods) containing the substratesshown in Fig. 2C. Bars above the x axis in Fig. 2Cindicate acidification; those below indicate alkaliniza-tion. The sequence of additions (see Fig. 2C) was asfollows: medium (black), mitochondria (2 mg, red),ADP (2 mM, green), rotenone (1 M, yellow), cATR (2

M, blue), oligomycin (5 M, magenta), NaAsO2 (2mM, cyan), and SF 6847 (1 M, gray). With substrate


combinations bypassing PDHC (glutamate plus malate,-ketoglutarate plus malate, or glutamate alone; all at 5mM) arsenite caused a statistically significant decreasein acidification in mitochondria pretreated with rote-none, cATR, and oligomycin. We assumed that, inmitochondria in which complex I is blocked by rote-none, ANT and F0F1 ATP synthase are blocked bycATR and oligomycin, respectively, the arsenite-inhib-itable acidification may only stem from KGDHC gener-ating CO2. The CO2 production by KGDHC in respira-tion-impaired mitochondria suggests the availability ofNAD. Mindful of these results, we sought NAD

sources in mitochondria other than that produced bycomplex I and considered the possibility of NAD

provision by mitochondrial diaphorases.

Effect of diaphorase inhibitors on bioenergeticparameters

As mentioned, diaphorase activity is attributable toflavoproteins designated NQOs (18). Depending onthe organism, several isoforms and their polymor-phisms have been identified (reviewed in refs. 19, 20).Among these, NQO1 and NQO2 have been mostextensively characterized (19). Although NQO1 is notin the list of mouse or human mitochondrial proteins(MitoCarta; ref. 54) and may not localize in mitochon-

dria of certain human cancers (55), it has been foundin mitochondria from different tissues (2325). Toaddress the contribution of mitochondrial diaphorasesto provision of NAD for the KGDHC reaction inanoxia, we used an array of pharmacologic inhibitors;however, all of them exhibit uncoupling properties athigh concentrations (29, 56). The potential uncou-pling effect of an inhibitor would be confounding,because, in its presence, m could become less neg-ative than Erev_ANT, leading to ANT reversal (2). In thiscase, its effect could not be distinguished from agenuine effect on the diaphorases. Therefore, we firstdetermined the concentration range in which theiruncoupling effects were negligible. As shown in Sup-plemental Fig. S1, the dose-dependent effects of 4different NQO1 inhibitorschrysin, 7,8-dihydroxyfla-vone hydrate (diOH-flavone), phenindione, and dicou-marolwere compared to that of a vehicle (Supple-mental Fig. S1, black bars) on m in mitochondriarespiring on glutamate and malate (Supplemental Fig.S1A), after addition of 2 mM ADP (Supplemental Fig.S1B) and after the addition of cATR (SupplementalFig. S1C), while simultaneously, in the same samples,rates of oxygen consumption were recorded (Supple-mental Fig. S1DF, for states 2, 3, and 4c, respectively,induced by cATR). Supplemental Fig. S1 shows theeffects of 8 consecutive additions of chrysin, (2.5 M

Figure 2. A) Reconstructed time courses of safranin O signal calibrated to m(solid traces) and parallel measurements of oxygen concentration in the medium(dotted traces) in isolated mouse liver mitochondria. Effect of cATR (2 M) onm of mitochondria during anoxia in the presence or absence of 2 mM NaAsO2is shown. ADP (2 mM) was added where indicated. Respiration rates in nanomolesper minute per milligram protein are indicated on the dotted lines. B) Recon-structed time courses of safranin O signal calibrated to m in isolated mouse livermitochondria in an open chamber. Effect of cATR (2 M) on m of mitochon-dria treated with rotenone (rot; 1 M, where indicated) in the presence or absenceof 2 mM NaASO2 (2 mM, red trace) is shown. ADP (2 mM) was added whereindicated. At the end of each experiment, 1 M SF 6847 was added to achievecomplete depolarization. C) Rates of acidification in the suspending medium ofmitochondria respiring on the various substrate combinations indicated, onaddition of different bioenergetic poisons. a-Kg, -ketoglutarate; glut, glutamate;mal, maleate; pyr, pyruvate; succ, succinate.


each, red bars); diOH-flavone (5 M each, green bars);phenindione (2.5 M each, yellow bars), and dicouma-rol (1.25 M each, blue bars). From the bar graphs inSupplemental Fig. S1, it is apparent that all diaphoraseinhibitors exhibited a concentration range in whichthey had no significant uncoupling effect (for chrysin,5 M, for phenindione, 20 M, and for dicouma-rol, 5 M). DiOH-flavone showed a significantquenching effect on the safranin O signal during state3 respiration; thus, only its effect on oxygen consump-tion rate was evaluated to establish the safe use at aconcentration below 20 M. Finally, the effect of diaph-orase inhibitors was compared to that of the uncouplerSF 6847 in decreasing NADH signals in intact isolatedmitochondria. Such an experiment (for dicoumarol) isdemonstrated in Supplemental Fig. S1G, H. Mouse livermitochondria (1 mg) were allowed to fully polarize inmedium (detailed in Materials and Methods) contain-ing glutamate and malate. Then, vehicle (control), 1.25

M dicoumarol, or 10 nM SF 6847 was added. As shownin Supplemental Fig. S1G, while SF 6847 dose depend-ently decreased NADH fluorescence, 1.255 M dicou-marol was without effect. The changes in NADH fluo-rescence shown in Supplemental Fig. S1G were largelycontrolled by complex I, because in the presence ofrotenone (Supplemental Fig. S1H), responses to dicou-marol and SF 6847 were almost completely dampened.

Effect of diaphorase inhibitors on ANT directionalityin anoxic or rotenone-treated mitochondria

Having established the concentration range of thediaphorase inhibitors exhibiting no appreciable uncou-pling activity, we wanted to determine their effects inthe biosensor test, which addresses the direction of theoperation of ANT by recording the effect of cATR onm in respiration-impaired mitochondria, when theyare exquisitely dependent on matrix substrate-levelphosphorylation (2). The rationale behind these exper-iments was that diaphorases may be responsible forproviding NAD to KGDHC, which, in turn, is impor-tant for generating succinyl CoA for substrate-levelphosphorylation. The experimental conditions inFig. 3AD were essentially similar to those shown in Fig.1, again demonstrating changes in m in response tocATR. The anoxia also coincided with the onset ofdepolarization, leading to a clamp of m to 100mV. As shown in Fig. 3AD (black solid traces), addi-tion of cATR in mitochondria made anoxic caused arepolarization, implying a forward operation of ANT,despite the lack of oxygen. However, in the presence ofdiaphorase inhibitors (concentration and color-codingdetailed in Fig. 3A), cATR induced depolarization(solid traces) without affecting the rate of respiration(Fig. 3AD, dotted traces), implying ANT reversal.Likewise, in the presence of diaphorase inhibitors(concentration and color-coding detailed in Fig. 3E),rotenone-treated mitochondria (Fig. 3EH, red andorange traces) responded to cATR with depolarization,as compared to their vehicles showing cATR-induced

repolarizations (Fig. 3EH, black and gray traces).From these results, we concluded that diaphorases arelikely to provide NAD to KGDHC that, in turn,support substrate-level phosphorylation via generatingsuccinyl-CoA during anoxia or inhibition of complex Iby rotenone.

To quasi-quantify the extent of contribution of NAD

emanating from the mitochondrial diaphorases that canbe utilized by KGDHC during anoxia, we compared therates of cATR-induced depolarizations (in millivolts persecond) in the presence of diaphorase inhibitors to therate of cATR-induced depolarization in the presence of 2mM NaAsO2. From the data obtained with 20 M diOH-flavone, 5 M dicoumarol, 5 M chrysin, and 20 Mphenindione, we inferred that mitochondrial diaphorasescontributed 26, 41, 37 and 81%, respectively, to the matrixNAD pool during anoxia.

Effect of diaphorase substrates on ANT directionalityof respiration-impaired mitochondria due to anoxiaor rotenone

To strengthen our conclusion, we next performed thebiosensor test in the presence of known diaphorasesubstrates in mitochondria undergoing respiratory in-hibition by anoxia or rotenone. Diaphorase activitymediated by NQO1 exhibits lack of substrate andelectron donor specificity because its active site canaccommodate molecules of various size and structure(57, 58); therefore, various types of quinoid com-pounds and their derivatives can be processed by theisolated enzyme (59). Furthermore, it is able to reactwith different dyes, nitro compounds, and some inor-ganic substances (60). The mitochondrial matrix is aquinone-rich environment, containing several coen-zyme Qs (CoQs) with variable side chains. Of course,NQO1 exhibits unequal affinities for them, but it isreasonable to assume that some CoQs are in themillimolar concentration range and could be substratesfor NQO1. We tested 14 different diaphorase sub-strates: menadione (10 M), vitamin K1 (phylloqui-none; 10 M), vitamin K2 (menaquinone; 10 M),duroquinone (DQ; 10100 M), mitoquinone (mitoQ;0.5 M), p-benzoquinone (BQ; 10 M), methyl-p-ben-zoquinone (MBQ; 10 M), 2,6-dimethylbenzoquinone(DMBQ; 1050 M), 2-chloro-1,4-benzoquinone (CBQ;10 M), 2,6-dichloro-1,4-benzoquinone (DCBQ; 10 M),1,2-naphthoquinone (1.2-NQ; 10 M), 1,4-naphthoqui-none (1.4-NQ; 10 M), 2,6-dichloroindophenol (DCIP;50 M), and 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one (quercetin; 10 M). In concentrationsexhibiting no uncoupling or other side effects on m orrate of respiration (not shown), mouse liver mitochondriawere treated with ADP and cATR, similar to that demon-strated in Fig. 1. As shown in Fig. 4AC and SupplementalFig. S2, when using different substrate combinationssupporting respiration, addition of cATR caused repolar-ization, except when glutamate plus malate plus -hy-droxybutyrate were used (Fig. 4C, black solid trace), asubstrate combination that, as discussed earlier, limits the


availability of NAD during anoxic conditions. In thisparadigm, dose-dependent addition of DQ during anoxialed to cATR-induced repolarization (Fig. 4C, colored solidtraces). Addition of menadione had no effect (Fig. 4A,solid orange trace), whereas mitoQ even abolished thecATR-induced repolarization (Fig. 4B, solid green trace).By contrast, when respiratory inhibition was achieved byrotenone instead of anoxia, DQ, menadione, and mitoQresulted in a strong cATR-induced repolarization (Fig.4DH). This effect of menadione was not shared byphylloquinone and menaquinone, as shown in Fig. 4G, H.The variable effects of a host of other quinones in thisparadigm are shown in Supplemental Fig. S2. Further-more, since safranin O may also be a substrate fordiaphorases due to its structural similarity to Janus greenB, which is a genuine diaphorase substrate (60), redistri-

bution of TPP as an index of m was also measured asan alternative, by using a TPP electrode, (Fig. 4I). Mito-chondria were allowed to polarize from glutamate andmalate, then ADP (2 mM) was added, followed by rote-none, which led to depolarization. Addition of cATRinduced repolarization, as in the above experiment, indi-cating that safranin O is unlikely to be a diaphorasesubstrate. From these results, we concluded that mito-chondrial diaphorases are not saturated by endogenousquinones and most likely provide NAD to KGDHC,which, in turn, yields succinyl-CoA, supporting substrate-level phosphorylation during anoxia or inhibition ofcomplex I by rotenone. Furthermore, there appeared tobe a clear distinction between true anoxia and rotenone-induced respiratory inhibition; in anoxia, menadione andmitoQ were not effective in conferring cATR-induced

Figure 3. Reconstructed time courses of safranin O signal calibrated to m (solid traces) and parallel measurements of oxygenconcentration in the medium (dotted traces) in isolated mouse liver mitochondria supported by glutamate and malate. Effectof diaphorase inhibitors (doses are color-coded in panel A) on cATR-induced changes in m during anoxia (AD) or undercomplex I inhibition by rotenone (EH). Gray traces in panels EH show the effect of vehicles (either DMSO or ethanol). Atthe end of each experiment, 1 M SF 6847 was added to achieve complete depolarization.


Figure 4. AH) Reconstructed time courses of safranin O signal calibrated to m (solid traces) and parallel measurements ofoxygen concentration in the medium (dotted traces) in isolated mouse liver mitochondria, demonstrating the effect ofdiaphorase substrates on cATR-induced changes in m during anoxia (AC) or complex I inhibition by rotenone (rot; DH).Substrate combinations are indicated in the panels. I) Reconstructed time course of TPP signal (in volts) in isolated mouse livermitochondria (mitos) supported by glutamate (glut) and malate (mal). Additions were as indicated by arrows. -Kg,-ketoglutarate; OH, -hydroxybutyrate. At the end of each experiment, 1 M SF 6847 was added to achieve completedepolarization.


repolarization. The reasons for this were investigatedfurther in the experiments outlined below.

Role of complex III in reoxidizing diaphorasesubstrates

To address the discrepancy that emerged from theresults obtained with rotenone-treated vs. anoxia-treated mitochondria, with respect to the effect ofdiaphorase substrates, we inhibited mitochondrial res-piration with stigmatellin, a specific inhibitor of com-plex III. The rationale behind the use of this inhibitorcame from several reports pointing to complex b ofcomplex III as being capable of reoxidizing substratesthat are reduced by mitochondrial diaphorases (29, 32,6164). As shown in Fig. 5AD, in mouse liver mito-chondria respiring on the different substrate combina-

tions indicated in the panels, state 3 respiration initi-ated by ADP (2 mM) was arrested by 0.8 Mstigmatellin, leading to a clamp of m to 100 mV.Subsequent addition of cATR (Fig. 5AC, black solidtraces) conferred depolarization to a variable extent,depending on the substrates used, indicating that func-tional complex III is necessary for the forward opera-tion of ANT when it relies on ATP generated bysubstrate-level phosphorylation. The lack of reoxida-tion of the reduced diaphorase substrate by complex IIIis likely to reflect in the result (see below), showing thatthe presence of menadione (Fig. 5AC, red traces) ormitoQ (Fig. 5D, lilac trace), but not DQ (Fig. 5D, greentrace), conferred a more robust cATR-induced depo-larization. In mitochondria undergoing respiratory ar-rest by anoxia (Fig. 5E), addition of stigmatellin (olivegreen trace) did not result in cATR-induced depolar-

Figure 5. Effect of the diaphorase substrates menadione (10 M; AC), DQ (50 M; D) and mitoQ (0.5 M; D), oncATR-induced changes of m after complex III inhibition by stigmatellin (stigma; E) or complex IV by KCN (F). Reconstructedtime courses of safranin O signal calibrated to m in isolated mouse liver mitochondria and oxygen consumption (E, dottedlines) are shown. Mitochondria respired on different substrates, as shown. -Kg, -ketoglutarate; OH, -hydroxybutyrate;FerrCyan, ferricyanide; glut, glutamate; mal, maleate; Additions were as indicated by the arrows. At the end of each experiment,1 M SF 6847 was added to achieve complete depolarization.


ization. However, while respiratory arrest of mitochon-dria achieved by inhibiting complex IV with KCN (1mM) yielded a very small change in m by cATR (Fig.5F, green trace), the copresence of ferricyanide,K3[Fe(CN)6] (FerrCyan; 1 mM), which can oxidizecytochrome c because of its higher redox potential(400 vs. 247264 mV for cytochrome c, depending onvarious factors; refs. 65, 66) led to cATR-induced repo-larization (Fig. 5F, gray trace). This effect was abolishedby stigmatellin (Fig. 5F, orange trace). When FerrCyanwas used, it was necessary to titrate m back to thesame levels as in its absence; hence, SF 6847 (5 nMboluses) was added where indicated (Fig. 5F, graytrace). From these experiments, we concluded thatstigmatellin negates the beneficial effect of diaphorasesubstrates that assist in cATR-induced repolarization,emphasizing the involvement of complex III in mito-chondria with respiratory inhibition by rotenone oranoxia.

Evidently not all diaphorase substrates assisted inpreventing mitochondria from being extramitochon-drial ATP consumers, which meant that not all of themcould be processed by either the diaphorases, and/orreoxidized by complex III. Relevant to this, it is wellknown that phylloquinone, menaquinone, and Q10 donot react with the isolated diaphorase (10, 67, 68);furthermore, although numerous compounds havebeen shown to react readily with purified diaphorase(10, 26, 59), there is specificity in the oxidation of thereduced quinone by the respiratory chain (29). Indeed,menadione and DQ have been shown to be processedby the mitochondrial diaphorases and reoxidized bycomplex III (29, 62).

Lack of a role of diaphorase in the regeneration ofNAD during anoxia in mitochondria from pigeonliver

The diaphorase activity described in rodent andhuman tissues has been reported to be absent in theliver and breast muscle of pigeons (Columba liviadomestica; refs. 69, 70). We therefore reasoned that inmitochondria obtained from pigeon tissues, the di-aphorase inhibitors and substrates would exert noeffect. As shown in Fig. 6AD (black traces), pigeonliver mitochondria respiring on different substrateswere repolarized by cATR added after ADP androtenone, indicating an ATP generation from theforward operation of ANT. The lack of diaphoraseinvolvement in this effect was confirmed by theresults that menadione failed to cause a more robustcATR-induced repolarization (Fig. 6AC, red traces),and none of the diaphorase inhibitors caused cATR-induced depolarization (Fig. 6D). Accordingly, DQwas without an effect on cATR-induced changes inm of mitochondria during anoxia (Fig. 6E). Theseresults support the conclusion that the effects ofdiaphorase substrates and inhibitors observed inmouse liver mitochondria were mediated throughgenuine diaphorase activity. Furthermore, it is also

apparent that in the absence of a diaphorase, pigeonliver mitochondria were still able to maintain theKGDHC-succinyl-CoA ligase axis sustaining substrate-level phosphorylation. Indeed, in Fig. 6F, it is shownthat the addition of succinate to pigeon mitochon-dria reversed the cATR-induced changes in mduring anoxia from repolarization to depolarization,in accordance with the schemes published recentlyby our group (2, 6). The validity of this scheme isfurther supported in pigeon liver mitochondria fromthe results shown in Fig. 6G, where the addition ofatpenin A5, which is expected to lead to a buildup ofsuccinate in the mitochondrial matrix, also led to acATR-induced depolarization during anoxia (Fig.6G, red trace).

Alternative sources of NAD provision inmitochondria during respiratory arrest

From the above data, it is apparent that mitochondrialdiaphorases are not the sole providers of NAD duringanoxia or respiratory inhibition by poisons. An obviouspossibility for NAD generation would be the malatedehydrogenase (MDH) reaction favoring malate forma-tion from oxaloacetate (71); however, this notion isvery difficult to address. We compared WT C57BL/6Jmice with another strain expressing an isoform ofMDH, which yielded slightly lower activity (MDH2b),but we observed no differences (data not shown). Atransgenic or silencing approach for MDH inherentlysuffers from the pitfall that this enzyme exhibits anextremely high activity compared with that of otherenzymes of the citric acid cycle; therefore, one wouldexpect to require very substantial decreases in activity,to observe an effect on NAD provision. That there areno MDH-specific inhibitors hindered the ability tostudy the extent of contribution of MDH in our proto-cols.

To address the extent of contribution of a proton-translocating transhydrogenase reversibly exchang-ing NADP and NAD for NADH and NADPH,respectively (72), which may serve as a source ofmatrix NAD, we compared mitochondria obtainedfrom C57Bl/6N vs. C57BL/6J mice, because in thelatter strain the gene coding for the transhydroge-nase is absent (73, 74). Although the catalytic site ofthe transhydrogenase for oxidation and reduction ofthe nicotinamide nucleotides is facing the matrix,extramitochondrial pyridine nucleotides are also re-quired; however, those released from broken mito-chondria in our samples may have been sufficient forthe exchange to materialize. As shown in Supplemen-tal Fig. S3, cATR-induced repolarization after anoxiain liver mitochondria from C57Bl/6N mice (blacktrace) was virtually indistinguishable from the mito-chondria obtained from C57BL/6J mice (red trace).From this result, we concluded that, in our isolatedmitochondria preparations, provision of NAD bythe transhydrogenase is not a viable possibility. How-


ever, it remains as an option in vivo, at least inorganisms expressing the transhydrogenase.

DISCUSSION

In earlier work, we have highlighted the critical impor-tance of matrix substrate-level phosphorylation inmaintaining ANT operation in the forward mode (2),thereby preventing mitochondria from becoming cyto-solic ATP consumers during respiratory arrest (35).Succinyl-CoA ligase does not require oxygen for ATPproduction, and it is even activated during hypoxia(75), providing ATP parallel to that by oxidative phos-

phorylation (1, 76, 77). More recently, we showed thatsuccinyl-CoA provision by KGDHC prevents isolated orin situ mitochondria with a dysfunctional electrontransport chain from being dependent on extramito-chondrial ATP (6). Relevant to this, mounting evidencesupports the pronounced conversion of -ketoglutarateto succinate in ischemia and/or hypoxia, implyingKGDHC operability (71, 7893). The question arises asto which metabolic pathways could provide NAD forKGDHC during respiratory arrest when the electrontransport chain is dysfunctional and complex I cannotoxidize NADH. Of course, a rotenone-mediated blockin complex I can be bypassed by succinate or -glycer-

Figure 6. AC) Reconstructed time courses of safranin O signal calibrated to percentage (solid traces; y axes of all panelsare as shown in E) reflecting m in isolated pigeon liver mitochondria, demonstrating the effect of various mitochondrialand diaphorase substrates on cATR-induced changes in m after complex I inhibition by rotenone, with differentrespiratory substrates used, as indicated. D) Diaphorase inhibitors were present as indicated (dicoumarol, 5 M; chrysin,5 M; diOH-flavone, 20 M; and phenindione, 10 M), and mitochondria were supported by glutamate and malate. EG)Oxygen concentrations in the medium (dotted traces) were measured. Effects of DQ (50 M; E), various substrates (F),and inhibitors atpenin A5 and KCN (G) are shown. -Kg, -ketoglutarate; OH, -hydroxybutyrate; AcAc, acetoacetate;glut, glutamate; mal, maleate; rot, rotenone; succ, succinate. At the end of each experiment. 1 M SF 6847 was added toachieve complete depolarization.


ophosphate (94), substrates that generate FADH2; how-ever, succinate disfavors substrate-level phosphoryla-tion by the reversible succinyl-CoA ligase due to massaction, while -glycerophosphate (provided that themitochondrial -glycerophosphate dehydrogenase ac-tivity is sufficiently high; ref. 95) steals endogenousubiquinones from the diaphorases. We considered mi-tochondrial diaphorases and a finite pool of oxidizablequinones as potential sources of NAD generatedwithin the mitochondrial matrix during respiratoryarrest caused by anoxia or poisons of electron transportchain (Fig. 7).

The results presented herein support the notion thata mitochondrial diaphorase, likely encoded by NQO1(10, 96), mediates NAD regeneration in the mito-chondrial matrix during respiratory arrest by anoxia orinhibition of complex I by rotenone. Inexorably, theclassic diaphorase inhibitor dicoumarol (10, 26, 30)suffers from potential specificity problems, in that itacts as a mitochondrial uncoupler (56) in addition toinhibiting other enzymes, such as NADH:cytochromeb5 reductase (97). We have compared the cATR-in-duced effects on m in mitochondria during respira-tory arrest by rotenone or anoxia from WT vs. Cyb5r2-knockout mice, and the results were indistinguishable(not shown). Furthermore, we titrated dicoumarol andother diaphorase inhibitors and used them at concen-trations at which their uncoupling activity was negligi-ble. Finally, none of the diaphorase inhibitors nor anyof the diaphorase substrates had an effect on pigeonliver mitochondria, where DT-diaphorase activity wasabsent (69, 70). On the other hand, pigeon livermitochondria did show robust cATR-induced repolar-izations during respiratory arrest, pointing to alterna-tive mechanisms for providing NAD in the matrix.The absence of diaphorase activity in pigeon liver is notsurprising; 14% of the human population exhibit apolymorphic version of NQO1 that deprives them ofNAD(P)H: quinone oxidoreductase activity (98). It maywell be that pigeons exhibit the same or a similar

polymorphic version of NQO in a much higher per-centage of their population.

We also sought the pathways responsible for provid-ing oxidized substrates to the diaphorases. Althoughvarious CoQ analogues are maintained in reduced formby the DT-diaphorase (99), their availability is finite(100) and is likely to require a means of reoxidation.Such a pathway has been demonstrated in the mito-chondrial matrix; even in the earlier publication byConover and Ernster (29), it was noted that electronsprovided by diaphorase substrates enter the electrontransport chain at the level of cytochrome b, whichbelongs to complex III. Later on, this concept wasentertained by Iaguzhinskiis group (62, 63, 101, 102),which examined the stimulatory effect of various diaph-orase substrates during cyanide-resistant respiration ofisolated mitochondria. Consistent with this, protectionin an ischemia model by menadione was abolished bythe complex III inhibitor myxothiazol (103). In thesame line of investigation, the cytotoxicity caused bycomplex I inhibition by rotenone, but not that causedby complex III inhibition by antimycin, was preventedby CoQ1 or menadione (104). Furthermore, also con-sistent with the substrate selectivity of NQOs in HepG2cells where NQO1 expression is very high (105), bothidebenone and CoQ1, but not CoQ10, partially restoredcellular ATP levels under conditions of impaired com-plex I function, in an antimycin-sensitive manner (68).Cytoprotection by rotenone but not antimycin byCoQ1, mediated by NQO1, has also been shown inprimary hepatocytes (104) and lymphocytes (61). Men-adione has even been shown to support mitochondrialrespiration with an inhibited complex I but not com-plex III before DT-diaphorase was identified (32). Thiswas later confirmed to occur through oxidation ofNADH by the intramitochondrial DT-diaphorase (29).Our results clearly show that the reoxidization of sub-strates being used by the diaphorases for generation ofNAD during respiratory arrest by rotenone or anoxiais mediated by complex III. In the process, complex III

Figure 7. Illustration of the pathway linkingATP production by the succinyl-CoA ligase re-action to KGDHC activity, diaphorase activity,reoxidation of diaphorase substrates by com-plex III, reoxidation of cytochrome c, and rer-eduction of a cytosolic oxidant.


oxidizes cytochrome c (Fig. 7). Therefore, the finite-ness of the reducible amount of cytochrome c wouldcontribute to the finiteness of the oxidizable pool ofdiaphorase substrates. Indeed, addition of FerrCyan ledto cATR-induced repolarization in the presence ofcomplex IV inhibition by cyanide, but not in thepresence of complex III inhibition by stigmatellin.However, the question arises, as to what could oxidizecytochrome c naturally, when oxygen is not available.An attractive candidate is p66Shc, a protein residing inthe intermembrane space of mitochondria (106, 107),which is known to oxidize cytochrome c (107).

In summary, our results point to the importance ofmitochondrial diaphorases in providing NAD for theKGDHC during anoxia, yielding succinyl CoA, which inturn supports ATP production through substrate-levelphosphorylation. In addition, the realization of dia-phorases as NAD providers renders them a likelytarget for cancer prevention, as they may be the meansof energy harnessing in solid tumors with anoxic/hypoxic centers. Finally, since diaphorases are upregu-lated by dietary nutrients such as sulforaphane (108)through the Nrf2 pathway (109) and a gamut of dietaryelements, mainly quinones of plant origin, are sub-strates for this enzyme (110), this may be a convenientway to increase the matrix NAD/NADH ratios thatplay a role in the activation of the mitochondrialNAD-dependent deacetylase sirtuin-3 (111), a majormetabolic sensor.

This work was supported by Orszgos Tudomnyos KutatsiAlapprogram (OTKA) grant 81983 and Hungarian Academyof Sciences grant 02001 (to V.A.-V.); a student grant fromAstellas Pharma Kft.-Kerpel-Fronius dn TehetsggondozProgram (to G.K.); grant AG014930 from the U.S. NationalInstitutes of Health/National Institute on Aging (to A.A.S.);and grants MTA-SE Lendlet Neurobiochemistry ResearchDivision 95003, OTKA NNF 78905, OTKA NNF2 85658, andOTKA K 100918 (to C.C). Cyb5r2 transgenic mice wereprovided by the European Mouse Mutant Archive (EMMA)service project, funded by the EC FP7 Capacities SpecificProgram. The authors declare no conflicts of interest.

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Received for publication October 22, 2013.Accepted for publication December 16, 2013.


mitochondrial diaphorases as nad+ donors to segments of the citric acid cycle that support...

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