NADH Oxidase Activity of Mitochondrial Apoptosis-inducing Factor

Redox activity of AIF

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Mo: 10498

NADH-oxidase activity of mitochondrialapoptosis-inducing factor (AIF)

M. Dolores Miramar1, Paola Costantini2, Luigi Ravagnan2,

Ligia M. Saraiva3, Delphine Haouzi2, Greg Brothers4,

Josef M. Penninger4, M. Luisa Peleato1*,

Guido Kroemer2*, and Santos A. Susin2*

1 Departamento de Bioquímica y Biología Molecular y Celular. Universidad de Zaragoza,

pza San Francisco s/n. 50009 Zaragoza, Spain

2 Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39

rue Camille Desmoulins, F-94805 Villejuif, France;

3 Instituto de Tecnología Química y Biológica. Universidade Nova de Lisboa, rua da

Quinta Grande, 6. Apartado 127. 2780 Oeiras, Portugal;

4 The Amgen Institute and Ontario Cancer Institute, Department of Medical Biophysics

and Immunology, University of Toronto, 620 University Avenue, Suite 706, Toronto,

Ontario M5G 2C1, Canada.

Correspondence to: Dr. Guido KroemerCNRS-UMR 1599Institut Gustave RoussyPavillon de Recherche I39, rue Camille-DesmoulinsF-94805 VillejuifFranceTel. 33-1-42 11 60 46Fax 33-1-42 11 60 47e-mail: [email protected]

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on February 13, 2001 as Manuscript M010498200 by guest on O

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Running title: Redox activity of AIF

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Summary

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein which translocates to the

nucleus during apoptosis and causes chromatin condensation and large scale DNA

fragmentation. Here we report the biochemical characterization of AIF's redox activity.

Natural AIF purified from mitochondria and recombinant AIF purified from bacteria

(AIF∆1-120) exhibit NADH oxidase activity while superoxide anion (O2-) is formed.

AIF∆1-120 is a monomer of 57 kDa containing 1 mol of non covalently bound FAD per

mol of protein. ApoAIF∆1-120, which lacks FAD, has no NADH oxidase activity.

However, native AIF∆1-120, apoAIF∆1-120, and the reconstituted (FAD-containing)

holoAIF∆1-120 protein exhibit a similar apoptosis-inducing potential when microinjected

into the cytoplasm of intact cells. Inhibition of the redox function, by external addition of

superoxide dismutase or covalent derivatization of FAD with diphenyleneiodonium, failed

to affect the apoptogenic function of AIF∆1-120 assessed on purified nuclei in a cell-free

system. Conversely, blockade of the apoptogenic function of AIF∆1-120 with the thiol

reagent para-chloromercuriphenylsulfonic acid did not affect its NADH oxidase activity.

Altogether, these data indicate that AIF has a marked oxidoreductase activity which can be

dissociated from its apoptosis-inducing function.


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Introduction

Mitochondria are considered as central players in apoptosis of mammalian cells (1-

3). Early during the apoptotic process, the outer mitochondrial membrane becomes

permeabilized, and mitochondria release soluble proteins normally confined to the

intermembrane space (4). Such apoptogenic proteins include the caspase activator

cytochrome c (5), procaspases 2, 3 and 9 (6-8), the IAP inhibitor Smac/DIABLO (9, 10),

as well as AIF (11). In contrast to cytochrome c and Smac/DIABLO, AIF is a caspase-

independent death effector, which translocates via the cytosol to the nucleus, where it

causes chromatin condensation and large-scale (50 kbp) DNA fragmentation (12, 13).

Neutralization of the AIF protein by microinjection of a specific antibody into the

cytoplasm of intact cells has revealed AIF to be rate-limiting for apoptotic chromatin

condensation and, in some cases, for mitochondrial membrane permeabilization (11, 14,

15). Conversely, microinjection of AIF may cause full-blown apoptosis with nuclear

condensation, dissipation of the mitochondrial transmembrane potential, release of

cytochrome c, and exposure of phosphatidylserine on the outer plasma membrane leaflet

(11, 14, 15).

The AIF precursor protein (612 aa) contains an N-terminal (first 100 aa) mitochondrial

localization sequence (MLS). The protein is synthesized in cytoplasmic ribosomes and

imported into the mitochondrial intermembrane space, where the MLS is cleaved off (11).

The C-terminal domain of AIF (last 485 aa) shares significant homology with

oxidoreductases from other vertebrates (X. laevis), non-vertebrate animals (C. elegans,

D. melanogaster), plants, fungi, eubacteria, and archaebacteria (16). The mature AIF

protein purifies as a flavoprotein, both from mitochondria and from E.coli used to


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produce recombinant AIF (11). This fact prompted us to investigate the putative electron

transfer (redox) function of AIF, in relation to its apoptogenic activity. Indeed, apoptosis

is accompanied by a general shift of the redox balance characterized by a depletion of

NADH, NADPH, glutathione, as well as by an increase of free radicals, including

superoxide anion, lipid peroxidation products (such as 4-hydroxynonenal), and oxidative

damage of membranes and DNA (17). In several paradigms of apoptosis, culture in

anoxic conditions, treatments with cell-permeable antioxidants, or overexpression of anti-

oxidant enzymes (such as superoxide dismutase, glutathione peroxidase, catalase, the

thioredoxin system) have profound inhibitory effects on cell death (18-20). It appears that

the respiratory chain is a prime source for the generation of oxygen radicals (presumably

derived from uncoupling and/or interruption of electron transfer due to the release of

cytochrome c) (21). Moreover, mitochondrial targeting of anti-oxidant enzymes is

particularly efficient in blocking apoptosis in several models (19, 22).

Here we report the detailed biochemical characterization of the redox function of AIF,

which turns out to be an FAD-containing oxidase capable of oxidizing NAD(P)H while

generating superoxide anion. Interestingly, the electron transfer function of AIF can be

dissociated from its apoptogenic activity, both in cell-free systems and in intact cells.


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Experimental procedures

Cells and culture conditions. HeLa cells were cultured in DMEM supplemented with 2

mM L-glutamine, 1 mM Pyruvate, 100 mM Hepes, 100 U/ml penicillin/streptomycin,

and 10% decomplemented FCS (Life Technologies). These cells were used for the

purification of nuclei and cell free system experiments, as described (23). Rat-1 fibroblast

cells were cultured as above and were used in microinjection experiments.

Recombinant AIF proteins. AIF deletion mutant (∆1-120) and AIF deletion mutant (∆1-

351) (11) were expressed from a Novagen pET32 expression vector and purified from E.

coli. The proteins were stored at -80°C in 50 mM Hepes, pH 7.9, 100 mM NaCl, 2 mM

EDTA, 1 mM DTT and 10% glycerol.

Mass spectroscopy, HPLC and elemental analysis. Molecular mass was measured by

means of a MALDI system from Applied Biosystems at the Services Cientifico-Técnicos,

Barcelona University. Sinapinic acid was used as matrix and bovine serum albumin

(BSA) was used as standard protein. For the experimental analysis, AIF∆1-120 (1.8

mg/ml) was mixed with sinapinic acid (10 mg/ml in H2O:CH3CN 1:1 + 0.3% TFA),

using a 1:6 ratio. Gel filtration on a Superose 12 (Amersham Pharmacia Biotech) was

also used for molecular weight determination. The protein was eluted using 0.3 ml/min of

50 mM Tris-HCl pH 8, 2 mM EDTA, 1 mM DTT and 200 mM NaCl. Semi-quantitative

detection of metals was performed in a Perkin Elmer ELAN 6000 system and semi-

quantitative determination of calcium was performed in a multicanal Thermo Jarred Ash

61E Polyscan following standard protocols.


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SDS-PAGE. Proteins were run in a Phast System from Amersham Pharmacia Biotech,

following the manufacturer's instructions.

Free thiols' content of AIF∆1-120 preparations was determined by using a 50-fold molar

excess of Ellman´s reagent, 5.5´-dithiobis-(2-nitrobenzoate-(Nbs2) (DTNB) (24). The

total thiol content was confirmed in the presence of 8 M urea and excess NaBH4 (25).

AIF was quantified spectrophotometrically on the basis of the extinction coefficient

calculated in this work.

Absorption spectrometry studies were carried out using a Kontron Uvikon 860

spectrophotometer. The molar extinction coefficient of bound FAD at 450 nm was

determined based on the absorption changes detected after releasing the bound FAD from

the enzyme by heating (5 minutes at 90°C) in Tris-HCl 50 mM pH 8. An extinction

coefficient of 11.3 mM-1 cm-1 at 450 nm was assumed for the free FAD (26). Reductive

titrations were performed under anaerobic conditions at 25°C. The anaerobic enzyme

sample, in 50 mM Tris-HCl pH 8 was prepared in an anaerobiosis cuvette by sequential

air evacuation and re-equilibration with oxygen-free argon. Anaerobic NAD(P)H was

prepared identically, introduced by means of the titration syringe. Identical amounts of

NAD(P)H were added to the reference cuvette.

HPLC for identification and quantification of FAD was performed using a C18 Vydac

column. A linear gradient 0-100% of ammonium acetate 0.1 M pH 6 and methanol in 40

minutes was performed using 1ml/min flow rate. FMN, riboflavin and FAD were used as

standards. 1 mg of AIF∆1-120 in 1 ml 50 mM Tris-HCl pH 8, maintained in the dark,

was heated for 10 min at 90°C. After centrifugation, aliquots of the supernatant were

injected into the HPLC, and flavins were detected at 445 nm.


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Phosphorylated residues were determined by dot-blot analysis using mouse monoclonal

antiphosphoserine, antiphosphothreonine and antiphospho-tyrosine antibodies (Sigma)

and an antimouse IgG alkaline phosphatase conjugate (Sigma). The method used was

based on the procedure previously described (27), using PVDF membranes (Immobilon-

P from Millipore).

Visible redox titrations of AIF were performed under anaerobic conditions. Appropriated

mediators that covered a potential range from +11 mV to - 450 mV (1mM) (28) were

added to the protein solution (10 µM) in 50 mM Bis-Tris for pH 6.5 or 100 mM Tris-HCl

for pHs 7.5 and 9. Injection of small volumes of air-free sodium dithionite allowed the

reduction of the protein. A Crison 2002 digital potentiometer was used and spectra were

recorded on a Shimadzu UV-260 spectrophotometer. The reduction potential was

determined by following the absorbance changes at 450 nm.

Potential involvement of cysteinyl redox centers in electron transfers was tested at 25°C,

under anaerobic conditions, following the NADH-DTNB oxidoreductase assay described

by Ohnisni, K. et al. (29). Briefly, 18 µM AIF∆1-120 in 50 mM sodium phosphate pH 7

containing 0.5 mM EDTA was mixed with 0.5 mM NADH, 0.02% BSA. The reaction

was started adding 0.4 mM DTNB and the nitrothiobenzoate anion production was

monitored at 412 nm using an extinction coefficient of 13.6 mM-1 cm-1.

Apoprotein preparation and holoprotein reconstitution. AIF∆1-120 apoprotein was

prepared following the protocol described by Chapman, S.K. and Reid, G.A. (30) by

exhaustive dialysis against 0.1 M Hepes, 2.5 M CaCl2, 1mM DTT, 0.1mM EDTA, 0.1M

guanidine chloride, 17% glycerol (v/v) at pH 7.5, concentration on Centricon 30 K

(Amicon) membranes, and a second round of dialysis against 50 mM Hepes, 100 mM

NaCl, 2mM EDTA, 1mM DTT, 10% glycerol at pH 7.9. The yield of this preparation


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was ~15%. Reconstitution of the holoprotein was performed by incubation with 1000

times molar excess of FAD, acetone precipitation, and repeated ultracentrifugation on

Centricon 30 K membranes to remove non-bound FAD.

Determination of AIF redox activities. Initial velocity studies of the NAD(P)H-oxidase

activity of the flavoprotein followed assay procedures described previously (31). Briefly,

NAD(P)H oxidase activity was measured at 25°C in a total volume of 0.5 ml containing

0.25 mM NAD(P)H in air-saturated 50 mM Tris-HCl pH 8 buffer. The reaction was

initiated by the addition of the enzyme and was followed by the decrease in absorbance at

340 nm. Steady-state kinetic data were obtained by varying NADH concentration. One

unit of activity is defined as the amount of protein required to catalyse the conversion of 1

µmol of NAD(P)H to NAD(P)+ per min at 25°C. NBT reduction and

monodehydroascorbate reductase activity (32, 33), superoxide formed in the reaction of

AIF∆1-120 with oxygen (34) and hydrogen peroxide production (35) were quantified as

described. This latter reaction was carried out coupled with NADH oxidase in a mixture

containing 1mM sodium phosphate buffer pH 6.9, 2.34 mg/ml phenol, 1mg/ml 4-

aminoantipyrine and 0.02 units of horseradish peroxidase in a total volume of 0.5 ml.

The absorbance was measured at 505 nm, and the concentration of H2O2 was calculated

from a calibration curve (36). DCPIP (85 µM) or ferricyanide (2mM) reduction were

assayed as described (37). SOD inhibition was measured by adding the indicated units of

enzyme to the reaction mixtures. The electron transfer between NADH-AIF-

ferredoxin/adrenodoxin-cytochrome c was assayed as described (37), using AIF∆1-120

instead of ferredoxin-NADP+ reductase and adrenodoxin instead of ferredoxin.

AIF western blot. Supernatants obtained from mitochondria undergoing permeability

transition were subjected to a 10% SDS-PAGE and transferred (100 V, 75 min at room


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temperature) to a nitrocellulose membrane. AIF immunoblot analysis was performed

using a rabbit antiserum generated against a mixture of three peptides derived from the

mouse AIF amino acids 151-200 (11).

In situ detection of 2, 2´-Di-p-nitrophenyl-5-5´-diphenyl-3,3´[3-3´-dimetoxy-4-

4´difenilen] tetrazolium chloride (NBT) reduction on native-PAGE was done using the

reaction mixture described by Pez-Huertas, E. L. et al. (32). Briefly, samples obtained

from mitochondria undergoing permeability transition were loaded onto a 10% native-

PAGE. The gel was incubated 20 min in the dark with 2 mM NBT solution. Then, 1 mM

NADH was added to reduce NBT and the reaction was stopped with water after the

appearance of the blue band.

Cell-free systems of nuclear apoptosis. Purified HeLa cell nuclei (103/µl) were exposed

90 min at 37°C to AIF∆1-120 preincubated (15 min at 37°C) with or without NADH,

NADPH, para-chloromercuriphenylsulfonic acid, superoxide dismutase, or

diphenyleneiodonium. For the standard assessment of chromatin condensation, nuclei

were stained with Hoechst 33342 (2 µM, 15 min, room temperature) and analyzed by

fluorescence microscopy (Leica DM IRB). DNA content was determined by staining with

propidium iodide (PI; 10 µg/ml) followed by analysis in a FACS Vantage (Becton-

Dickinson). A minimum of 2500 events were scored.

Microinjection. Rat-1 fibroblasts were microinjected using a computer-controlled

microinjector (pressure 150 hPa; 3 sec; Eppendorf) with buffer only, 7.5 µM of AIF∆1-

120, apoAIF∆1-120, apoAIF∆1-120, FAD-reconstituted holoprotein, and AIF∆1-351.

After microinjection, cells were cultured at 37°C for 180 min and stained with the ∆Ψm-

sensitive dye CMXRos (100 nM, 15 min) and the DNA-intercalating dye Hoechst 33342

(1.5 µM, 15 min) (11). Microinjected viable cells (100 per session, three independent


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sessions of injections) were identified by inclusion of 0.25% (w:v) FITC-dextran (green

fluorescence) in the injectate. Only the blue and the red fluorescence was recorded.

All chemical reagents used in this work were purchased from Sigma.


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Results

Properties of recombinant AIF protein. Recombinant AIF∆1-120 was found to elute as a

single peak on a gel filtration column, with a calculated molecular weight of 57 kDa,

which corresponds to its theoretical molecular mass (57046), indicating that, at near-to

physiological salt concentrations (200 mM NaCl), AIF∆1-120 is a monomer. This result

was confirmed by mass spectroscopy analysis (not shown). It is in contrast with its

apparent molecular weight determined by SDS-PAGE (67.5 kDa, about 10 kDa more

than expected), a migration behaviour reported for other proteins such as ferredoxins

(38). AIF∆1-120 does not contain significant amounts of metals including Ca2+, Co2+,

Cu2+, Mg2+, Fe2+, Se2+ and Zn2+. However, it contains phosphogroups among serine

and threonine residues, as revealed by immunoblotting (not shown). AIF∆1-120 has 3

cysteins in its sequence, and it was found to contain 3.2 accessible thiol groups per

molecule, as determined by derivatization with the Ellman's reagent. Total thiol content

was confirmed after unfolding in 8 M urea. This suggests that none of the three cysteins

contained in the AIF amino acid sequence engages in disulfide links. The flavin moiety of

AIF∆1-120 is non-covalently bound and was identified by HPLC as FAD (not shown).

FAD is the only identified prosthetic group present in AIF∆1-120, at a molar ratio of 1:1.

The absorption spectrum of AIF (Fig. 1) shows the typical features of an oxidized FAD

flavoprotein, with the visible maximum at 378 nm and 450 nm and a shoulder at 467 nm.

The ratio A270nm/A450nm was 7 in pure preparations, and the extinction coefficient for

oxidized AIF at 450 nm was calculated to be 12.12 mM-1 cm-1.

Electron transfer reactions from NADH and NADPH to AIF. The addition of an

equimolar amount of NADH to AIF∆1-120 in anaerobic conditions leads to complete


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flavin reduction, without intermediate semiquinone formation (Fig. 2). Similar titration

curves were obtained using NADPH as reductant, and in both cases the spectral changes

are similar to the reduction of AIF∆1-120 with dithionite (not shown). The reduced form

was stable over several hours, and admission of air to the sample did not lead to the

immediate appearance of the oxidized AIF spectrum. Upon addition of an increasing

molar excess of NADH or NADPH over AIF∆1-120, the appearance of long wavelength

absorbance bands was observed (Fig. 3). These long-wavelength absorbances of the

reduced enzyme were stable at 25°C for hours, even upon exposure to air. They exhibit

the blue-green and green color described for other electron transfer complexes (31). By

analogy to other enzymes (31), these long-wavelength absorptions are likely to

correspond to charge transfer complexes between the reduced FAD and tightly bound

NAD+ or NADP+.

Redox potential of AIF. The spectral titration of AIF∆1-120 with dithionite revealed that

the redox potential of AIF is strongly influenced by the pH (Fig. 4). Assuming a two-

electron reduction step, midpoint redox potentials were determined to be -264 mV+15 mV

at pH 6.5 (Fig. 4A) , -308 mV + 15 mV at pH 7.5 (Fig. 4B), and -373 mV + 15 mV at

pH 9.0 (Fig. 4C). Neither semiquinone formation nor long-wavelength absorbing bands

were detected upon reduction by dithionite. A plot of AIF's redox potential

(FAD/FADH2) versus pH has a slope of -44 mV (not shown). This slope deviates from

that expected for a two-electron reduction involving two protons (58 mV/pH unit) or one

proton (29 mV/pH unit). The deviation from theoretical values indicates the possible

presence of other dissociable groups whose pKa values are linked to the redox state of the

enzyme. Eventhough titrations with dithionite and NAD(P)H showed an uptake of two

electrons, it was considered important to discard the involvement of cysteinyl residues as


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active redox acceptors: in the NADH:DTNB oxidoreductase assay no involvement of

cysteinyl residues in redox transferences was detected (not shown).

AIF redox activities. AIF∆1-120 was found to exhibit NADH- and NADPH-oxidase

activities (Fig. 5A). NAD(P)H oxidation in presence of AIF was followed measuring

initial rates of ∆A340 nm. The apparent Km for NADH was calculated as 99.4+10 µM

and the turnover number 2.09 min-1. When NADPH was used as electron donor, the

apparent Km was 52.9+ 12 µM and the turnover number 2.8 min-1 (Table 1). These

kinetic parameters are very similar to previous values described for other superoxide

forming NADH oxidases (39), and the steady-state kinetic data may be interpreted taking

into account the possible formation of relatively stable charge-transfer complexes.

Addition of exogenous FAD did not stimulate the NADH oxidase activity of AIF∆1-120

(not shown), in contrast to several NADH oxidases from bacteria (29, 35, 40-42). When

oxidizing NADH or NADPH, AIF catalyzed the reduction of the tetrazolium salt NBT

(Fig. 5B). This was due to the AIF(FADH2)-mediated reduction of O2 to O2- (Fig. 6A).

Accordingly, the reduction of NBT was completely abolished by the O2- scavenger

superoxide dismutase (SOD, 40 U/ml). Hydrogen peroxide formation resulting from

superoxide production was also measured (Fig. 6B). AIF did not exhibit any peroxidase

activity (not shown).

AIF∆1-120 was also found to possess a monodehydroascorbate reductase activity with a

specific activity of 8.8 U/mg and a kcat of 0.505 min-1, a feature that is common to

several AIF's homologous described in plants (16). The AIF-monodehydroascorbate

reductase activity was inhibited by SOD, indicating that the reaction occurs via superoxide

radicals (not shown). Cytochrome c reductase activity was also measured, with a Km of

0.46 mM for NADH and a kcat of 21.76 min-1 (Fig. 7A). Again, SOD inhibited the


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cytochrome c reductase activity with an IC50 of ~10 U/ml, indicating that the AIF-

catalyzed cytochrome c reduction occurs via O2- radicals (Fig. 7B). When ferrodoxin

was used as mediator (NADH-AIF-ferredoxin-cytochrome c) it did not modify the

cytochrome c reduction rate (not shown). In contrast, when adrenodoxin was used as

mediator (NADH-AIF-adrenodoxin-cytochrome c), cytochrome c reduction was

increased (Fig. 7C). The kinetic parameters calculated from Figure 7C gave an apparent

Km for adrenodoxin of 1.08 µM with a kcat of 18.58 min-1.

AIF∆1-120 was found to transfer electrons at low rate from NADH (but not from

NADPH) to DCPIP or ferricyanide, two typical electron acceptors of NAD(P)H

dehydrogenases. From NADH, a two electron donor, AIF∆1-120 was able to reduce

DCPIP, a two electron acceptor, with a kcat of 21.6 min-1 and a calculated Km for the

NADH of 3.4 + 0.4 mM (Table 1). SOD did not affect the electron transfer rate indicating

that the diaphorase activity of AIF is not mediated by O2-. When the diaphorase activity

was assayed using a one-electron acceptor, ferricyanide, the catalytic efficiency for

NADH was reduced by half approximately.

In summary, AIF exhibits NADH oxidase activity and is able to transfer one electron (to

molecular oxygen and ferricyanide) or two electrons (to DCPIP), as summarized in Table

1.

AIF is the dominant NADH oxidase released through the mitochondrial outer

membrane. Purified mitochondria can be induced to undergo the so-called permeability

transition, a manipulation that leads to osmotic swelling of the matrix and physical rupture

of the outer membrane, causing the release of soluble intermembrane proteins (4). Upon

induction of permeability transition with Ca2+ or arsenite, immunodetectable AIF was

found in the mitochondrial supernatant. The release of AIF was inhibited by cyclosporin


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A, a specific inhibitor of the permeability transition pore (Fig. 8). Separation of proteins

via native PAGE, followed by the in situ detection of an NADH oxidase activity causing

the reduction of NBT, yielded one single blue band (Fig. 8A). This band also reacted

with a specific anti-AIF antiserum and co-migrated with recombinant AIF∆1-120 (Fig.

8B). Altogether, these data confirm that natural (mitochondrial) AIF possesses a NADH

oxidase/NBT reductase activity and indicate that AIF is the quantitatively most important

NADH oxidase/NBT reductase contained in the mitochondrial intermembrane space.

Apoptotic versus oxidoreductase activity of AIF. The FAD moiety of AIF∆1-120 can be

removed by dialysis in the presence of guanidine chloride (a manipulation which

generates apoAIF∆1-120) and may be reconstituted by external addition of FAD (a

manipulation which generates reconstituted holoAIF∆1-120). ApoAIF lacked any

detectable NADH oxidase-NBT reductase activity, whereas the reconstituted holoAIF had

a NADH oxidase-NBT reductase activity undistinguishable from native AIF∆1-120 (Fig.

9A). Unmanipulated AIF∆1-120, apoAIF∆1-120, and reconstituted holoAIF∆1-120 were

micro-injected into the cytoplasm of Rat-1 fibroblasts. All three protein preparations

induced a similar level of nuclear chromatin condensation, as well as a dissipation of the

mitochondrial transmembrane potential (Fig. 9B, C). A deletion mutant of AIF∆1-351,

which lacks part of the oxidoreductase domain, yielded negative results in this system

(Fig. 9C). The nuclear effects of AIF were recapitulated in a cell-free system, in which

AIF∆1-120 was added to purified HeLa nuclei. In such a system, AIF∆1-120 caused

marked peripheral chromatin condensation (Fig. 10A) as well as a loss in DNA content

(Fig. 10B). Addition of NADH or NADPH failed to enhance the apoptogenic activity of

AIF∆1-120 (Fig. 10C). Moreover, inhibition of the oxidoreductase activity by external

addition of SOD or diphenyleneiodonium, an inhibitor of flavonoid-containing enzymes

covalently reacting with FAD (43-45), failed to modify the apoptogenic activity of AIF


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(Fig. 10D). In contrast, addition of para-chloromercuriphenylsulfonic acid, a thiol-

reactive agent, did abolish the apoptogenic activity of AIF∆1-120, yet did not affect its

NTB reductase activity (Fig. 10D).

Altogether, these data show that the apoptogenic and oxidase functions of AIF can be

dissociated.


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Discussion

The results from this work indicate that AIF has a marked NADH oxidase activity.

According to the classification by Massey (46), AIF may belong to the electron-

transferase class of NADH reductases, because it reacts rapidly with oxygen, forming

O2- and the flavoprotein neutral radical as products. No semiquinone intermediate could

be detected although such a neutral radical is expected to be produced after the transfer of

a single electron. In each of the activities described, the reaction could be initiated by the

reduction of AIF by the two electron donors NAD(P)H, in the absence of exogenous

electron acceptor. The reduced form of AIF reacted with one electron (molecular oxygen

and ferricyanide) or two electron (DCPIP) acceptors. Kinetic analysis of the AIF steady-

state data are rather difficult to interpret given the formation of relatively stable charge

transfer complexes.

Several NADH oxidases from bacterial sources have been isolated and characterized (31,

35). The putative role of those enzymes is to maintain the cellular redox balance under

aerobic conditions, by converting NADH to NAD+ (42, 47). Also, several poorly

characterized superoxide (O2-) forming NADH oxidases have been detected in animal

cells, namely in heart mitochondria (48), liver mitochondria (49), microsomes from

bovine myocytes (50), and endothelial cells (51). However, AIF is not similar to any of

the previously described NADH oxidases. First it is monomeric, whereas bacterial

NADH oxidases are usually dimeric or tetrameric (52). Second, AIF transfers electrons

without the involvement of cysteinyl groups, whereas other NADH oxidases rely on a

redox-active disulphide center constituted by two vicinal cysteine residues (35, 42).

Third, AIF oxidizes NADH via a mechanism that does not require the addition of


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exogenous FAD whereas several NADH oxidases from bacteria do require FAD (29, 35,

40-42). Fourth, in contrast to several NADH oxidases (42), AIF does not function as a

hydrogen peroxide scavenger.

AIF protein is present in the mitochondria of all mouse tissues that have been assessed,

and has also been found in a panel of 60 human cancer cell lines (53), suggesting that

AIF may fulfill important metabolic functions. However, based on the present data, it is

difficult to understand what the physiological function of AIF in normal (non-apoptotic)

conditions may be. Since AIF is the only NADH oxidase detected in the intermembrane

space, it is tempting to speculate that AIF accounts for the mitochondrial superoxide anion

or hydrogen peroxide-generating NADH oxidase activity (54, 55), which is lost from

mitochondria, once cells have been induced to die (55). Clearly, an NADH oxidase

activity causing the collateral generation of superoxide anion radicals would be of no

advantage for the cell. It thus may be speculated that the true, yet-to-be-discovered

substrates of the AIF oxidoreductase compete for endogenous NADH and/or that AIF is

normally inactivated by local inhibitory factors within the intermembrane space. If AIF

acted as a superoxide-generating NADH reductase outside of mitochondria, after its

apoptotic release, what might be the contribution of this enzymatic activity to the apoptotic

process? Apoptosis is notoriously associated with a massive depletion of NADH/NADPH

(56), as well as an increase in the generation of superoxide anions (57), at both

mitochondrial and extramitochondrial localizations (17). Furthermore, it should be

mentioned that, in isolated mitochondria, the permeability transition pore complex

(PTPC) is tightly regulated by the oxidation-reduction state of the pyridine nucleotide

pool, with oxidation causing an increase in the pore opening probability (58). Although

these changes in the redox potential may be explained by a variety of factors, including

uncoupling/blockade of the respiratory chain (21) and activation of PARP (59), it will be


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interesting to study the contribution of AIF to this process, for instance in embryonic

stem cells in which the AIF gene is ablated.

It appears that the known apoptogenic functions of AIF and its novel oxidoreductase

activity can be dissociated from each other, based on three arguments. First, removal of

the prosthetic FAD group (which obviously abolishes the oxidoreductase function of

AIF) does not curtail the apoptogenic effects of AIF on mitochondria and nuclei of

microinjected cells. Second, addition of NADH, addition of SOD, or covalent

derivatization of FAD failed to modulate the capacity of AIF to induce nuclear apoptosis

in a cell-free system. Third, inhibition of the apoptogenic effect of AIF by means of para-

chloromercuriphenylsulfonic acid failed to affect its NADH oxidase activity. These data

are similar to those obtained for cytochrome c in the sense that the apoptogenic activity of

cytochrome c does not depend on its redox status. Exchange of the Fe2+ by Co2+ within

the heme prosthetic group of cytochrome c (a manipulation which abolishes the electron

transfer function of heme) fails to alter its caspase-activatory functions (60), whereas

certain amino acid substitutions which do not affect its redox function do abrogate

cytochrome c-mediated caspase activation (60, 61). In conclusion, both cytochrome c and

AIF thus appear to be bifunctional molecules with clearly dissociable redox and

apoptogenic activities.

Acknowledgments

We would like to thank Dr. Rita Bernhartd (Universität des Saarlandes,

Saarbrucken, Germany) for kindly providing adrenodoxin.


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Footnotes

# This work has been supported by a special grant of the Ligue Nationale contre le Cancer, as well as by

grants from Agence Nationale de Recherches sur le Sida, Fondation pour la Recherche Medicale (FRM)

and European Union (to GK). M.D.M. receives a short fellowship from Caja de Ahorros de la Inmaculada

(CAI). P.C. receives a Postdoctoral fellowship from FRM, L.R. receives a PhD fellowship from the

French Ministry of Science & Technology.

* M.L.P., G.K., and S.A.S. share senior co-authorship.

1 The abbreviations used are: AIF, apoptosis inducing factor; Cyt-c, cytochrome c; ∆Ψm, mitochondrial

transmembrane potential; DPI, diphenyleneiodonium; DTNB, 5.5´-dithiobis-(2-nitrobenzoate-(Nbs2) ;

NBT, 2,2´-Di-p-nitrophenyl-5-5´-diphenyl-3,3´[3-3´-dimetoxy-4-4´difenilen] tetrazolium chloride;

PCMPS, para-chloromercuriphenylsulfonic acid; PT, permeability transition; PTPC, permeability

transition pore complex; SMAC, second mitochondria-derived activator of caspases; SOD, superoxide

dismutase.


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Figure legends

Fig. 1: Absorption spectrum of recombinant AIF∆1-120. The concentration used was 13

µM.

Fig. 2: NADH titration of AIF∆1-120. The top line shows the spectrum of the oxidized

enzyme (47 µM) before (a) and after addition of 0.12 (b), 0.35 (c), 0.59 (d), 0.83 (e),

1.07 (f), 1.31 (g), 1.57 (h), and 1.79 (i) equivalents of NADH/FAD. The inset shows

the absorbance at 450 nm versus added NADH equivalents.

Fig. 3: Anaerobic titration of AIF∆1-120 with an excess of NAD(P)H. Long wavelength

absorbance changes observed in oxidized AIF∆1-120 (22 µM) without (a) or after

addition of NADH (b: molar excess NADH/AIF was 3:1 and c: molar excess was 6:1,

with maxima at 637 nm and 774 nm, respectively) (A). Same as A using NADPH instead

of NADH, with appearance of maxima at 740 nm (b, molar excess NADPH/AIF, 3:1)

and 769 nm (c, molar excess 6:1) (B).

Fig. 4: pH effect on the redox potential of AIF∆1-120. Titration curves obtained

following absorbance variations at 450 nm of oxidized AIF∆1-120 (10 µM) upon

dithionite addition. Solid curves calculated from a dielectronic Nernst equation of -264

mV at pH 6.5 (A), -308 mV at pH 7.5 (B) and -373 mV at pH 9.0 (C).

Fig. 5: NAD(P)H oxidation by AIF∆1-120. NADH (•) and NADPH (o) oxidation

measured following absorbance variation at 340 nm, after addition of different amounts

of NAD(P)H. AIF was added at a concentration of 3 µM (A). AIF∆1-120 induced NBT


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reduction with NADH (•) or NADPH (o) as electron donors. AIF was added at a

concentration of 95 nM (B).

Fig. 6: NADH (•) and NADPH (o) oxidase activities of AIF result in generation of

superoxide anion (A) and hydrogen peroxide (B). AIF concentration was 95 nM in A and

3 µM in B.

Fig. 7: Cytochrome c reduction mediated by superoxide anion, using NADH as electron

donor (A). Specific inhibition of superoxide anion-mediated Cyt c reduction by SOD (B).

Adrenodoxin enhances Cyt c reduction (C). AIF concentrations were 72 nM in A and B,

and 90 nM in C. NADH concentration was 3.4 mM in B and C.

Fig. 8: AIF is the dominant NADH-oxidase released from mitochondria undergoing

permeability transition. AIF release induced by 100 µM Ca2+ and 2 mM sodium arsenite

measured by in situ NBT detection (A). AIF detection by immunoblot of mitochondrial

intermembrane proteins (B). As a control of permeability transition induction,

mitochondria were pretreated with 1 µM of cyclosporine A to inhibit the release of the

intermembrane mitochondrial proteins.

Fig. 9: Apoptotic activity of apoAIF∆1-120 and reconstituted holoAIF∆1-120 in a cell

free system and in microinjection assays. Spectral characteristics of apoAIF and

reconstituted holoAIF are shown in A. The inset shows the recovery of the NADH-

oxidase activity of apoAIF after FAD reconstitution measured by in situ NBT detection

(as in Fig. 8A). Rat-1 cells were microinjected with the indicated protein (7.5 µM of

AIF∆1-120, apoAIF∆1-120 and reconstituted holoAIF∆1-120), and stained with Hoechst

33342 (blue fluorescence) and the ∆Ψm-sensitive dye CMXRos (red fluorescence).


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Representative phenotypes obtained 3 h after injection are shown in (B). Quantification of

nuclear apoptosis and ∆Ψm reduction induced by microinjection (100-150 Rat-1 cells per

session) of AIF∆1-120, apoAIF∆1-120, reconstituted holoAIF∆1-120 or AIF∆1-351

(determined as in B) (C; X +/- SE of three experiments).

Fig. 10: Independence between the redox and apoptotic activities of AIF. Apoptotic

nuclear features induced by AIF∆1-120 or AIF∆1-120+NADH in a cell-free system.

HeLa nuclei were exposed 120 min to 100 µg/ml of AIF∆1-120 and/or 2 mM NADH

followed by staining with Hoechst 33342 (A). In addition, nuclei were stained with

propidium iodide and the percentage of hypoploid nuclei was measured by flow

cytometry (B). Dose-response of the apoptogenic effect of AIF∆1-120 in the presence or

absence of 2 mM NADH or NADPH measured as in B (C). Comparison of AIF redox

and apoptotic activity obtained after pretreatment of AIF∆1-120 (100 µg/ml, 15 min at

37°C) with NADH (2 mM), NADPH (2 mM), SOD (40 U/ml), diphenyleneiodonium

(DPI, 250 µM), or PCMPS (30 µM) ( D; X +/-SE of five experiments).


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Table 1.- Steady-state kinetic parameters of AIF 1-120.

NAD(P)H OXIDASE ACTIVITY

Specific activity (U/mg) kcat (min-1) Km (µM) kcat/Km (µM-1 min-1)

NADH 0.036 2.09 99.4 + 10 0.021

NADPH 0.098 2.84 52.9 + 12 0.054

NBT REDUCTASE ACTIVITY

Specific activity (U/mg) kcat (min-1) Km (mM) kcat/Km (mM-1 min-1)

NADH 38.733 2244.2 2.2 + 0.3 1020

NADPH 0.383 22.1 1.5 + 0.1 14.8

SUPEROXIDE PRODUCTION


NADH 5.559 322.1 2.2 + 0.2 146.4

NADPH 0.79 45.6 1.5 + 0.1 30.4

PEROXIDE PRODUCTION

Specific activity (U/mg) kcat (min-1) Km (µM) kcat/Km (mM-1 min-1)

NADH 0.462 26.7 19 + 3 1.4

NADPH n.d. n.d. n.d.

DIAPHORASE ACTIVITY (DCPIP)


NADH 0.375 21.6 3.4 + 0.4 6.3

NADPH n.d. n.d. n.d.

n.d.: non detected


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Santos A. SusinHaouzi, Greg Brothers, Josef M. Penninger, M. Luisa Peleato, Guido Kroemer and

M. Dolores Miramar, Paola Costantini, Luigi Ravagnan, Ligia M. Saraiva, DelphineNADH-oxidase activity of mitochondrialapoptosis-inducing factor (AIF)

published online February 13, 2001J. Biol. Chem.

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NADH Oxidase Activity of Mitochondrial Apoptosis-inducing Factor

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