Full Paper

Electrochemistry of Mitochondria: A New Way to UnderstandTheir Structure and Function

Jing Zhao,a Fanben Meng,a Xiaoli Zhu,a Kun Han,a Shanli Liu,b Genxi Lia*a Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,P. R. China

b School of Life Science and Shanghai Key Laboratory of Bio-Energy Crops, Shanghai University, Shanghai 200444, P. R. China*e-mail: genxili@nju.edu.cn

Received: November 26, 2007Accepted: March 19, 2008

AbstractIn this article, electrochemistry of mitochondria is achieved. Cyclic voltammograms of freshly prepared mitochondriawere obtained by immobilizing mitochondria together with glutaraldehyde and bovine serum albumin on the surfaceof a pyrolytic graphite electrode. Two pairs of redox peaks could be observed which were ascribed to the electrontransfer reactions of cytochrome c and FAD/FADH2. Study of submitochondrial particles was also conducted, whichcould confirm the results of the study of the entire mitochondria. The redox wave of NADH could be obtained due tothe destruction of the membrane of mitochondria. We have also checked the function of succinate in mitochondria byemploying the electrochemical method. This work is not only the first to be able to obtain the direct electrochemistryof mitochondria, but is also beneficial to the further understanding of the structure and function of mitochondria invitro.

Keywords: Bioelectrochemistry, Chemically modified electrode, Mitochondria, Submitochondrial particles, Succinate

DOI: 10.1002/elan.200704205

1. Introduction

As is well known, mitochondrion plays a crucial role in livecell, so the investigation of mitochondrial structure andfunction is always a hot topic of biology [1 – 4]. Mitochond-rion carries out a series of oxidative reactions for energygeneration to create the majority of ATP which is necessaryto support all cellular functions. Meanwhile, a lot ofmetabolic reactions, such as the metabolism of lipids, aminoacids, ketone bodies, are carried out inmitochondria.On theother hand, it has been known that the mitochondrialrespiratory chain, as the Aproduct lineB of ATP, is composedof four critical protein complexes embedded in the innermembrane, which communicate via two membrane-associ-ated electron carriers, cytochrome c in the intermembranespace and ubiquinone in the inner membrane. Electronsfrom the product ofmetabolism, such as succinate, enter therespiratory chain and are passed along the four proteincomplexes as the potential energy falls gradually.With the rapid development of surface functionalization

and protein electrochemistry, electrochemical method hasbeen proven to be a power tool for the studies of electrontransfer reactions. The technique has been able to beemployed for the studies of not only the relatively smallmolecules but also some biological macromolecules [5, 6].However, study of intact organelle with electrochemicalmethod has not been performed, although the electro-chemistry of some critical proteins in them, such as

cytochrome c [7], has been reported. Recently, some proteincomplexes in lipid film, such as Photosystem I [8] andPhotosystem II [9] in chloroplast, the Complex I [10] andComplex IV [11] in mitochondria, and a few live cell [12]and bacteria [13] are studied by electrochemical technique.And, a kind of organelle, chloroplast, has been used for thepreparation of an electrochemical biosensor [14]. Never-theless, electrochemistry of mitochondria has not beenachieved. In this paper, we will not only report the electro-chemistry of mitochondria, but also present the observationof the function of succinate in mitochondria and the processof electron-transfer in respiratory chain, whichwill be a newway to understand the structure and function of mitochon-dria.

2. Experimental

2.1. Materials

Cytochrome c from chicken heart, albumin from bovineserum (BSA), flavin-adenine dinucleotide (FAD), phenyl-menthanesulfonyl fluoride (PMSF), and glutaraldehydesolution (25% in water) were all purchased from Sigma.Other chemicals were all of analytical grade. All solutionswere prepared with doubly distilled water, which waspurified with a Milli-Q purification system (Branstead,USA) to a specific resistance of >18 MW cm.

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2.2. Mitochondria Preparation

Mitochondria were isolated from the liver of guinea pig,according to the method of differential centrifugation [15].The guinea pig hadbeen starved overnight before killing. 5 gof livers were minced ,washed and homogenized on ice inextraction buffer (1�MS) containing 210 mM mannitol,70 mM sucrose, 5 mM Tris, and 1 mM Ethylenediaminete-traaceticacid (EDTA), which had been adjusted to pH 7.4.Homogenate was centrifuged at 800 g for 10 min, and theresulting pellets, which contained nuclei and undisruptedcells, were discarded. Supernate was centrifuged at 9500 gfor 20 min, and the resulting pellets were suspended in 1�MS buffer. The two centrifugation steps were repeatedtwice, and the final pellet was resuspended by 1.5 mL ofextraction buffer as the mitochondria suspension. Allcentrifugation steps were carried out at 4 8C.A kind of mitochondria analogue was further prepared.

Submitochondrial particles were obtained by sonic irradi-ation of the mitochondria suspension in a sonic dismem-brator at 30 s intervals for a total time of 5 min [16].

2.3. Preparation of Mitochondria Modified Electrode

The substrate pyrolytic graphite (PG) electrode was pre-pared by inserting a PG rod into a glass tube and fixing itwith epoxy resin. Electrical contact was made by adhering acopperwire to the rodwith the help ofWoodsAlloy. ThePGelectrode was first polished on rough and fine sand papers.Then its surface was polished to mirror smoothness with analumina (particle size of about 0.05 mm)/water slurry on silk.Eventually, the electrode was thoroughly washed by ultra-sonicating in both doubly distilled water and ethanol forabout 5 min.The prepared intact mitochondria suspension or mito-

chondria analogue was mixed with an isometric solutioncontaining 10% glutaraldehyde and 0.4 mg/mL BSA [14].30 mL of this resulting mixture was evenly spread onto thesurface of the PG electrode immediately. The electrodesurfacewas coveredwith anEppendorf tube for the first twohours to prepare uniform films, and dried overnight in theair. Then themodified electrode was thoroughly rinsed withpure water and dried again.

2.4. Electrochemical Measurements

Electrochemical experiments were carried out with amodel263A potentiostat/galvanostat (EG&G) and a three-elec-trode system. The working electrode was the mitochondria/glutaraldehyde/BSA modified PG electrode. A saturatedcalomel reference electrode (SCE) and a platinum wireauxiliary electrode were used for the measurements simul-taneously.All experiments were performed at room temperature of

about 20 8C. The pH 7.4 PBS (100 mM) buffer was used astest solution, which was first bubbled thoroughly with highpurity nitrogen for 5 min. Then a stream of nitrogen wasblown gently across the surface of the solution in order tomaintain the solution anaerobic throughout the experiment.Cyclic voltammetry was carried out in the potential rangefrom�500 mV to 500 mV, or�800 mV to 0 mV, or 0 mV to1000 mV, with the scan rate of 200 mV/s.

3. Results and Discussion

3.1. Electrochemistry of Mitochondria

Based on our previous studies on some redox proteins andthe obtained experimental results by employing variouselectrochemical systems for the study of mitochondria, wehave found that electrochemistry of mitochondria can beachieved by using glutaraldehyde/BSA/mitochondria filmmodified electrode, which has been depicted in Scheme 1.Figure 1 shows the cyclic voltammograms (CVs) obtained atthemodified electrode for an anaerobic PBS buffer solutionwith pH 7.4, at a scan rate of 200 mV s�1. As is shown inFigure 1, no electrochemical response can be observed inthe potential scan range from �500 to 500 mV in the firstcycle scan. However, with the modified electrode beingscanned, an apparent CV wave appears. The anodic andcathodic peaks are separately located at 60 mV and�10 mV. Further studies reveal that the peaks currentsincrease gradually and will keep unchanged ultimately. Theplot of the peak current versus scan rate is linear from10 mVs�1 to 1000 mV s�1, indicating that this is a surface controlledelectron-transfer process.

Scheme 1. Schematic drawing of electrode immobilization and reactions of mitochondrion. GPDH: glycerol 3-phosphate dehydrogen-ase, Cytc: cytochrome c.

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It has beenknown that the structure of outermembrane inmitochondria is very simple and the only exited redoxprotein is monoamine oxidase containing the copper redoxcenter, whose electrochemical response is very difficult tobe obtained [17]. We have also tried to obtain the directelectrochemistry of monoamine oxidase with this electro-chemical system, but cannot obtain any electrochemicalresponse. There are abundant redox proteins in the inter-membrane space, inner membrane and matrix of mitochon-dria. However, the outer membrane of mitochondria isnonconductive lipid film, and the electron transfer betweenthese redox proteins and electrode is unachievable. There-fore, the redox waves in the CVs observed in our experi-ments should be from the electroactive molecules in theintermembrane space, which may leak through the outermembrane under the effect of potential scanning. As is wellknown, cytochrome c, containing hemeas active center, is animportant electron carrier in respiratory chain of mitochon-dria. Abundant cytochrome c is in the intermembrane spaceas the electron carrier between cytochrome bc1 complex/coenzyme Q-cytochrome c oxidoreductase (Complex III)and cytochrome c oxidase (Complex IV). Direct electro-chemical response of cytochrome c have been achievedbefore and the formal potential of the protein is at around0 mV [7, 18] We have also studied the electrochemistry ofcytochrome c by immobilizing this protein in Glutaralde-hyde-BSA film, same as that for the study of mitochondria.It is observed that the redox potential of cytochrome c isnearly the same as that for mitochondria in this potentialscan range between �500 mV and 500 mV. These resultsindicate that the pair of peaks of mitochondria in thispotential scan range should be caused by the redox reactionof cytochrome c, which leaks from the intermembrane spacedue to the potential scanning effect.The outer membrane has permeability to the molecules,

theMWofwhich is less than 1000 D. Therefore, cytochrome

c (MW 13000 D) cannot cross the outer membrane toachieve the electron-exchange with electrode in normalcondition. However, since the membrane structure will beaffected by external electric field, the selective permeabilityof the membrane will be changed. So, cytochrome c will beallowed to leaveout of the inner part ofmitochondria, and toachieve the electron transfer with electrode. Consequentlyredox peaks are obtained. With the potential scan going on,the amount of released cytochrome c increases graduallyand reaches maximum ultimately.It should be mentioned that a pair of redox waves can be

observed, even at the first scan, if the potential scan range isbetween �800 and 0 mV, i.e., in the negative potentialrange. As is shown in Figure 2, the peaks are very welldefined. The anodic and cathodic peaks are located at�490 mVand �520 mV, respectively. We propose that thispair of redoxwaves is ascribed to the redox reaction of FAD/FADH2. As is well known, FAD/FADH2 and NAD/NADHare quite important coenzyme in mitochondria, and thereduced coenzymes are the initial electron-donators in thetwo routes of respiratory chain of mitochondria. FAD/FADH2 locates on the inner and outer side of the innermembrane as the coenzymeof succinate dehydrogenase andglycerol 3-phosphate dehydrogenase, while NAD/NADHlocates only on the inner side of the inner membrane as thecoenzyme ofNADHoxidoreductase.As electroactive smallmolecules, FAD/FADH2 can leave out of the inner part andget to the outside of mitochondria, to exchange electronswith the electrode. So, electrochemical response can beobserved.The electrochemical responses of FAD/FADH2 have

been obtained by several different groups. Base on theprevious reports, we have first proposed that the pair ofredox waves located at �500 mV are due to the redoxreactions of FAD/FADH2, and have then checked the redoxpotentials by employing this electrochemical system. Ex-perimental results show that the redox peaks potentialsobtained at FAD/FADH2 and glutaraldehyde-BSA co-modified film electrode are nearly the same as that obtainedby employing mitochondria (data not shown). Therefore,the pair of redox waves in the CV of Figure 2 is due to theredox reaction of FAD/FADH2, which moves to the outsideof mitochondria from the inner part.Above studies have also revealed that the bilayer

membrane structure ofmitochondria has not been damagedduring the process of its immobilization onto the PGelectrode surface. Nevertheless, since the outer membraneof mitochondria is nonconductive, electron transfer be-tween the redox species inside mitochondria and electrodecannot be achieved, which makes it difficult to probe intothe organelle with electrochemical method. Because of therequirement for the further experiments and affected byexternal electric field, the membrane structure is somewhatdamaged, and the selective permeability of themembrane isthus changed, which allows the species with a MW higherthan 1000 D, such as cytochrome c, to cross the membrane.Consequently, electrochemical studies on mitochondriabecome possible.

Fig. 1. Cyclic voltammograms obtained at a mitochondria modi-fied electrode for a 100 mM PBS buffer solution with pH 7.4, atthe first, second, third, and fourth scan in the potential scan rangebetween �500 and 500 mV. Scan rate: 200 mV s�1.

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3.2. Electrochemistry of Submitochondrial Particles

In order to further confirm the above proposals, we havealso studied the electrochemistry of mitochondria byemploying submitochondrial particles, which are obtainedby sonic irradiation ofmitochondria suspension [16]. Similarelectrochemical experiments are conducted by using sub-mitochondrial particles as that with mitochondria. Two pairof redox peaks assigned to cytochrome c and FAD/FADH2,which is similar to that of an entire mitochondrion, can beobtained.However, steady redox peaks of cytochrome c canbe observed at the first scan. This should be reasonable,because the outer membrane of mitochondria have beendestroyed completely by sonic irradiation. Therefore, cyto-chrome c is exposed, and the response of this protein can beobserved in the first cycle scan. It has also been observedthat the redox potentials for FAD/FADH2 will shiftpositively, compared with the potentials for the entiremitochondria, as is shown in Figure 3. This is also reason-able, since FAD/FADH2 will be on the surface aftermitochondria have been sonic irradiated. So, the trans-membrane process can be removed when electron transferbetween FAD/FADH2 and electrode is proceeded. Thus apositive shift in potentials occurs.Figure 4 shows that an oxidation peak can be obtained for

the submitochondrial particles and the value of the peakcurrent decreases gradually with the scanning if thepotential scan range is between 0 mV and 1000 mV. Thisshould be ascribed to the redox reaction of anotherimportant coenzyme, NADH. This coenzyme is on theinner side of the inner membrane of mitochondria, so it isnot possible for the species to interact with electrode.Therefore, no response of NADH can be obtained formitochondria. However, in submitochondrial particles,NADH is on the surface, Thus it can be electrochemicallyoxidized directly. On the other hand, since NADH is

oxidized to NAD irreversibly, the oxidation peak decreasesgradually, and disappears ultimately, as is shown in Figure 4.

3.3. Electrochemical Study on the Function of Succinate

We have further studied the function of succinate inmitochondria by employing electrochemical method. Suc-cinate is an intermediate of tricarboxylic acid cycle inmitochondrial matrix, and electrons derived from succinateare passed directly to FAD in succinate dehydrogenase(Complex II). Succinate can get across the outer and innermitochondrial membrane to matrix freely. Unfortunately,experimental results reveal that succinate is an electro-chemically inactive species. Nevertheless, the function ofsuccinate in mitochondria can be still clearly observed byelectrochemical technique.As is shown inFigure 5, after the

Fig. 2. Cyclic voltammogram obtained at a mitochondria modi-fied electrode for a 100 mM PBS buffer solution with pH 7.4, inthe potential scan range between 0 and �800 mV. Scan rate:200 mV s�1.

Fig. 3. Cyclic voltammogram obtained at a submitochondrialparticles modified electrode. Others same as in Figure 2.

Fig. 4. Cyclic voltammograms obtained at a submitochondrialparticles modified electrode at the first, second, third, and fourthscan in the positive potential scan range between 0 and 1000 mV.Others same as Figure 3.

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addition of succinate into the test buffer solution, a newreduction peak appears at about �300 mV. The moresuccinate is added, the higher this reduction peak is.However, in an aerobic condition, for instance, if the testsolution has not been previously bubbled thoroughly withhigh purity nitrogen, the reduction peak located at about�300 mV cannot be obtained. Nevertheless, another re-duction peak will be observed at about�650 mV character-izing the direct reduction of O2 on the surface of theelectrode (data not shown).O2 is the final electron-accepter in the respiratory chain. If

succinate is added into the test buffer solution, succinatewilldiffuse into the intermembrane space and matrix ofmitochondria freely, donate electrons to FAD, and reduceO2 ultimately. In an aerobic condition, O2 will acceptelectrons from electrode directly, so a reduction peaklocated at �650 mV can be observed. In an anaerobiccondition, only a little O2 exists in themitochondrial matrix,which will accept the electrons from the respiratory chain inthe intermembrane space, and the reactive oxygen species(ROS) can be formed [19]. Therefore, the activation energyfor the reduced reaction is decreased significantly, and thelocation of the reduced peak is far more positive, which islocated at �300 mV. As the concentration of succinate isincreased, more ROS are produced, so the peak will behigher and higher. It should bementioned that the reductionprocess is irreversible, so the value of the reduction peakcurrent in the second scan will decrease significantly, if acertain number of succinate has been added in the testsolution, as is shown in Figure 6.

4. Conclusions

Mitochondrion is an important organelle in cells, whichplays crucial roles in many vital activities, such as metabo-lism, respiration, apoptosis, and so on. So the study of

mitochondria is necessary and significant in the fields ofboth biology and chemistry, and lots of technologies havebeen employed. In this work, we have in the first timestudied mitochondria by using electrochemical method,which provides another way to understand their structureand function. This work may be also extended for furtherstudies on mitochondria in the future with electrochemicalmethod to obtain more information from this importantorganelle. For instance, since the electrochemical responseof cytochrome c inside mitochondria has been obtaineddirectly, it may be used to detect apoptosis of the cellsbecause cytochrome c is the mark protein in the program ofapoptosis. Therefore, more and more researches on mito-chondria will be carried out with electrochemical techniquein the future.

5. Acknowledgements

This work is supported by the National Natural ScienceFoundation of China (Grants 90406005, 20575028) and theProgram for New Century Excellent Talents in University,the Chinese Ministry of Education (NCET-04-0452).

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Fig. 5. Cyclic voltammograms obtained at a mitochondria modi-fied electrode for a 100 mM PBS buffer solution, containingdifferent concentrations of succinate. Others same as Figure 3.

Fig. 6. Cyclic voltammograms obtained at a mitochondria modi-fied electrode for a 100 mM PBS buffer solution, containing 4 mMsuccinate at the a) first and b) second scan. Others same asFigure 5.

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