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Understanding the mechanism of direct electrochemistry of mitochondria-modified electrodes from yeast, potato and bovine sources at carbon paper electrodes
Electrochimica Acta 110 (2013) 112–119
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Understanding the mechanism of direct electrochemistry of mitochondria-modified electrodes from yeast, potato and bovine sources at carbon paper electrodes Fabien Giroud a , Tera A. Nicolo a , Sara J. Koepke a , Shelley D. Minteer a,b,∗ a b
Department of Chemistry, University of Utah, Salt Lake City, UT 84112, United States Department of Materials Science and Engineering, University of Utah, Salt Lake City, UT 84112, United States
a r t i c l e
i n f o
Article history: Received 30 November 2012 Received in revised form 15 February 2013 Accepted 18 February 2013 Available online 26 February 2013 Keywords: Mitochondria Bioelectrocatalysis Cytochrome c Quinone pool Electrochemistry
a b s t r a c t Although mitochondria have been used for bio-electrochemistry for over 5 years, little is known about their direct electrochemistry mechanism. This paper focuses on developing a better understanding of the electron transfer mechanism of mitochondria from three different organisms at carbon electrodes. Yeast, potato and bovine mitochondria have been successfully isolated and immobilized onto Toray paper electrodes via vapor deposited silica. Organelle-modified electrodes were first characterized using cyclic voltammetry. Similar electrochemical signals were obtained for all organisms. Direct electron transfer was observed when a metabolite of the Krebs cycle was present in the buffer solution. Control experiments based on the immobilization of two electron carriers contained in mitochondria, cytochrome c and a quinone (coenzyme Q10 ), tend to show the electron transfer mechanism to the carbon material comes from the quinone pool of the organelles. As quinones are known to be pH-dependent, we further investigated the response of the electrochemical signal of the three isolated mitochondria and the two electron carriers separately. The half wave potentials obtained from the organelles appeared to be pH-dependent and their variations are comparable to coenzyme Q10 rather than cytochrome c. Finally, extraction of both the cytochrome c and the quinone pool from intact mitochondria was performed to validate our hypothesis that direct electrochemistry of mitochondria happens via the quinone pool. Electrochemistry of immobilized quinone-depleted mitochondria validated the hypothesis that the mitochondria are communicating with the electrodes through the quinone pool. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Mitochondria are organelles found in living cells that produce energy by breaking down pyruvate and fatty acids to carbon dioxide through the Krebs cycle and possess the complexes responsible for the electron transport chain. All of the Krebs cycle enzymes are located inside the matrix of the mitochondria. On the other hand, the four complexes taking part in the electron transport chain are found in the inner membrane. Complexes I and II collect electrons from NADH and succinate, both of which are produced by the Krebs cycle. Then, the two complexes transfer their electrons to complex III via electron carriers called the quinone pool situated inside the inner membrane. Finally, cytochrome c (a protein loosely attached to the outer part of the inner membrane) transfers the electron from complex III to complex IV for molecular oxygen reduction.
∗ Corresponding author at: Department of Chemistry, University of Utah, Salt Lake City, UT 84112, United States. Tel.: +1 801 587 8325; fax: +1 801 581 8181. E-mail address: [email protected] (S.D. Minteer). 0013-4686/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.electacta.2013.02.087
Mitochondria have gathered more attention from the scientific community over the last 5 years especially in bio-electrochemistry. Their use was essentially focused on deep oxidation of different substrates such as pyruvate, fatty acids [1–4] and more recently amino acids [5], but they have been used for the reduction reaction at the cathode as well [6]. This biocatalyst was utilized mainly, because this organelle contains all the necessary enzymes for the fuel oxidation in their matrix and they offer better substrate transport through their membranes than whole microbes. Furthermore, due to the presence of the membrane that encapsulates the enzymes, mitochondrial use and stability are greatly improved compared to enzymatic bio-electrodes reproducing metabolic pathways [7]. Consequently, mitochondria were first immobilized on the electrode in order to generate power from the conversion of the chemical energy of a biofuel to electrical energy contained in an organelle-based biofuel cell [8]. Since then, the aim of mitochondria-based biofuel cells have evolved from basic biofuel cells to include their use as self-powered biosensors. The Minteer group studied and developed several sensors based on mitochondrial use and assembly in a fuel cell. In these cases,
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the mitochondria electrode is the anode with an air-breathing platinum cathode [9–12]. However, the electron transfer mechanism between the mitochondria and the electrode was not well studied. Few examples have been found dealing with organelle wiring to the electrode. Arechederra’s study postulated that organelles could undergo mediated electron transfer via the use of a polyazine layer or direct electron transfer (DET) to the electrode through the cytochrome c of the mitochondria due to similarities between mitochondrial modified electrode cyclic voltammetry and cytochrome c cyclic voltammetry [8]. Another group showed that in certain conditions, DET can be obtained at pyrolitic graphite electrodes through another cofactor contained in the inner membrane [4]. The authors were able to detect a reversible peak from mitochondria from guinea pig liver located at −500 mV vs. SCE, which they ascribed to flavine adenine dinucleotide (FAD/FADH2 ). However, work on the mammalian protein cytochrome P450, another membrane-bound protein located in the inner membrane of mitochondria, showed a reversible redox signal at the same potential [13]. Thus, the electron pathway from the mitochondria to the electrode is still unclear and electrons could come from one unique electron carrier or from different redox proteins depending on the preparation of the mitochondria dispersion (i.e. the isolation and purification procedure). This paper presents the direct electrochemistry of three different types of mitochondria (yeast, potato and bovine sources). All of the organelles show the same redox behavior. In anaerobic conditions, well-defined redox signals were observed near 100 mV vs. SCE (oxidation) and −350 mV vs. SCE. This paper attempts to identify the source of the electron leakage from the mitochondrial electron transport chain. It is more likely this leakage is coming from electron carriers (small molecule mediators) rather than the complexes themselves. Cytochrome c (located on one side of the inner membrane) was the best candidate, but cyclic voltammetry data showed the quinone pool (coenzyme Q) could be the origin too. Thus, we compared electrochemistry of organelleand electron carrier- modified electrodes in different pH conditions. Finally, cyclic voltammetry was carried out on cytochrome c-depleted mitochondrial-modified electrodes and quinone pooldepleted mitochondrial-modified electrodes and compared to intact mitochondrial-modified electrodes. Results indicate that immobilized organelles are communicating with the electrode via the quinone pool.
2. Materials and methods 2.1. Material Yeast cells from Saccharomyces cerevisiae were purchased from ATCC (# 9080, strain ID 4228) (USA). Organic Russet potatoes were obtained from a local farmer. Fresh beef heart was purchased from a local slaughterhouse. Yeast extract and tetramethyl orthosilicate (TMOS) were purchased from Fluka (USA). Pentane was purchased from Fisher Scientific (USA). Toray carbon paper (TGPH-60, non-wet-proof) was purchased from Fuel Cell Earth (USA). All other reagents used were purchased from Sigma (USA). Solutions were prepared with 18 M cm de-ionized water from a Milli-Q system.
2.2. Yeast growth Lactate media was prepared by the protocol outlined by Leister and Hermann [14]. S. cerevisiae cells were inoculated into lactic
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acid media pH 5.5 and allowed to grow with agitation at 30 ◦ C for approximately 2 days. 2.3. Mitochondria extraction from yeast, potato and bovine sources Yeast mitochondria were isolated from S. cerevisiae according to the procedure in reference [14]. Potato mitochondria were isolated from Russet potatoes according to Moore’s procedure [15]. Bovine mitochondria were isolated from a beef heart according to Palloti’s procedure with slight modification [16]. The basic procedure is as follows: 200 g of bovine heart tissue was rinsed in 400 mL of 250 mmol L−1 sucrose, 10 mmol L−1 Tris–Cl at pH 7.8 solution (rinse solution). The tissue was placed into 400 mL of isolation solution (rinse solution with 0.2 mmol L−1 EDTA, 1 mol L−1 PMSF dissolved in 0.5 mL ethanol added directly before use) and the pH was adjusted with KOH. The tissue was blended while pH was maintained at 7.8. The blended tissue was centrifuged for 20 min at 1200 × g and the supernatant was re-centrifuged for 15 min at 26,000 × g. After discarding the supernatant, the pellet was re-suspended in 10 mL of isolation solution and homogenized in two 5 mL volumes with a Kontes size 21 5 mL Potter-Elvehjem homogenizer and Craftsman Evolv 17217 5.2 amp corded 3/8 drill on the middle setting for two passes of 5 s each. This homogenate was diluted to 180 mL in isolation solution, pH adjusted, and centrifuged at 26,000 × g for 15 min. The pellet was re-suspended in 60 mL of isolation solution and centrifuged at 12,000 × g for 30 min. All final pellets for the three different organisms were re-suspended in rinse solution to a wet weight of 200 mg mL−1 . 2.4. Cytochrome c and coenzyme Q depletion of mitochondria The cytochrome c extraction procedure was the same as used by Jacobs et al. [17]. Suspensions of freshly isolated mitochondria were suspended in 15 mmol L−1 KCl (hypotonic medium for whole mitochondria) at a protein concentration of 200 mg mL−1 . Mitochondria were allowed to swell for 10 min on ice before centrifugation at 105,000 × g for 15 min. The supernatant was kept and stored at 4 ◦ C. The pellet was re-suspended in 150 mmol L−1 KCl (isotonic medium for whole mitochondria). The suspension was kept on ice for 10 min and then centrifuged at 105,000 × g for 15 min. The resulting supernatant was kept at 4 ◦ C. The extraction procedure is repeated twice in the isotonic medium, as described above. The cytochrome c was extracted into the isotonic supernatant. The final pellet of cytochrome cdepleted mitochondria is re-suspended in 250 mmol L−1 sucrose and 10 mmol L−1 pH 7.5 Tris–acetate buffer and stored at −80 ◦ C until use. The coenzyme Q extraction procedure was the same as used by Szarkowska et al. [18]. The mitochondrial preparation was thawed on ice and diluted to a final protein concentration of 200 mg ml−1 in 250 mmol L−1 sucrose and 10 mmol L −1 Tris–HCl at pH 7.5. The suspension was centrifuged at 35,000 × g for 10 min. The pellet was re-suspended in 150 mmol L−1 KCl, frozen in liquid nitrogen, and lyophilized. The extraction of CoQ was realized in pentane (1 mL pentane/1 mg lyophilized mitochondria). The mixture was homogenized in a Potter-Elvejhem homogenizer with Teflon pestle and then centrifuged at 5000 × g for 10 min. The same extraction–homogenization was repeated 4 times. Finally, pentane was removed from the extracted mitochondria under nitrogen flow for 4 h. Extracted mitochondria were homogenized in 250 mmol L−1 sucrose and 10 mmol L−1 Tris–HCl buffer at pH 7.5, centrifuged at 35,000 × g for 10 min at 4 ◦ C, and re-suspended in the same buffer. This suspension was frozen in liquid nitrogen and stored at −80◦ C for later use.
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2.5. Immobilization of organelles, quinones and cytochrome c onto the electrodes
3. Results and discussion 3.1. Electrochemical detection of immobilized organelles
Prior to modification, Toray paper electrodes were cleaned by sonication in three methanol baths (10 min each), rinsed thoroughly in DI water and dried at room temperature. The wet mitochondria precipitates were used directly. Wet precipitates were re-suspended in the washing buffer to give a 200 mg mL−1 solution. 35 L of the mitochondria suspension was drop cast onto a 1 cm2 unmodified Toray paper electrode and allowed to dry for 1 h. Once dry, mitochondria were immobilized by a new layer of silica. Briefly, tetramethyl orthosilicate (TMOS) was hydrolyzed by ambient water vapor giving the layer of vapor-deposited silica according to the procedure described in References [19,20]. Electrodes were placed at 4 ◦ C overnight. Prior to use, organelle electrodes were soaked for 1 h in pH 7.40 phosphate buffer that contained 100 mmol L−1 nitrate, 100 mmol L−1 pyruvate and 1 mg mL−1 ADP. Coenzyme Q10 was dissolved in ethanol at a concentration of 2 mmol L−1 . Subsequently, 30 L of this solution was drop cast on the 1 cm2 Toray paper electrode. Cytochrome c from equine heart was dissolved in deionized water at a concentration of 10 mg mL−1 and 30 L of this solution was drop cast on the Toray paper electrodes. Both electrode types were covered with the TMOS layer, using the procedure described above.
2.6. Determintation of NADH leaching In order to determine if any NADH leached out of the mitochondria membrane once immobilized onto the Toray paper with the TMOS silica layer, an NADH leaching assay was performed. NADH leakage could indicate that mitochondrial membrane lysis had occurred. For the assay, mitochondria-modified electrodes as described above were soaked in 3 mL disposable plastic cuvettes filled with a pH 7.40 phosphate buffer solution. An initial absorbance measurement was taken with a UV spectrophotometer at a wavelength of 340 nm at time zero. Then the cuvettes were allowed to sit for 2 h and the measurement was taken again to determine if NADH had leached into the solution.
Mitochondria from three different species were isolated and immobilized on the electrode by drop casting, followed by the vapor deposition of a TMOS layer. After immobilization, separate experiments were investigated to determine if mitochondria were still intact and active. An NADH assay using UV–vis spectroscopy was performed to determine if the different mitochondria used were lysed during the extraction. Absorbance measured at 340 nm immediately after soaking the immobilized-organelle electrodes in the buffer solution was compared to the absorbance of the same solution after 2 h. The control electrodes showed a change of absorbance of 0.0015 ± 0.0008, which was not statistically different from the organelle-modified electrodes (yeast: 0.0021 ± 0.0008, potato: 0.0010 ± 0.0007, bovine: 0.0019 ± 0.0008). The small variation in absorbance indicated the NADH was not leaching from the mitochondria, suggesting their membranes were still intact. Separately, a Carbon-13 NMR experiment was performed to verify that immobilized mitochondria were still active and results were compared to spectra obtained from free mitochondria in suspension. Carbon-13 labeled pyruvate was used as the natural substrate of the mitochondria. The results indicated that only the pyruvate peaks were found for the controls experiments (no mitochondria immobilized onto the electrodes or without inoculation of organelle suspension) (peaks at 26 ppm: RCH3 , 170 ppm: RCOOH, 204 ppm: RR’CO). In the presence of mitochondria (both immobilized or in suspension), all the peaks previously mentioned decreased in intensity while new peaks appeared (48 ppm: RR’CH2 , 73 ppm: RR’COH, 135 ppm: C C, 182 ppm: RCOOH) which correspond to by products (succinate, fumarate and malate) released from the mitochondria matrix in exchange of phosphate required for the oxidative phosphorylation [21]. After the Toray paper electrodes were modified with the organelles, cyclic voltammetry experiments were carried out in order to characterize the direct electrochemistry of mitochondria at the carbon electrodes. Toray paper electrodes were preferred due to their high surface area compared to glassy carbon electrodes. Fig. 1 presents representative cyclic voltammograms of
2.7. Carbon-13 NMR study of pyruvate oxidation A Carbon-13 NMR experiment was carried out by using a 100 mM Carbon-13 labeled pyruvate fuel solution in pH 7.4 phosphate/nitrate buffer. Mitochondria were first immobilized onto Toray paper electrodes as previously described. Mitochondriamodified electrodes were allowed to soak in the labeled pyruvate solution for 24 h. NMR samples were made by mixing 350 L of carbon-13 labeled pyruvate solution with equal volume of deuterated water. The results were compared to NMR spectra obtained using mitochondria directly inoculated into the solution. Controls were performed using bare Toray paper electrodes.
2.8. Electrochemical measurements The electrodes were tested using cyclic voltammetry on a CH Instruments potentiostat model 650A (TX, USA), Biologic VSP 150 (Claix, France) or Digi Ivy DY2300 (TX, USA) with a platinum mesh counter and a calomel reference electrode. The scan window was 0.5 to −0.7 V. Potentials are referenced to the saturated calomel reference electrode (SCE). All experiments were conducted under nitrogen flow unless specified.
Fig. 1. Cyclic voltammetry of mitochondria from different sources immobilized on Toray paper electrode with TMOS in 100 mmol L−1 phosphate, 100 mmol L−1 nitrate and 100 mmol L−1 pyruvate at pH 7.4 recorded at 10 mV s−1 . Yeast (thick solid line), potato (dashed line), bovine (dotted line). Bare Toray paper electrode control (thin solid line).
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Fig. 2. Cyclic voltammograms of mitochondria from different sources immobilized on Toray paper electrode and control experiments without (solid line) and with pyruvate (dashed line) present in the electrolyte indicating a direct electron transfer between the organelle and the electrode. (A) unmodified Toray paper, (B) yeast-modified electrode, (C) potato-modified electrode and (D) bovine-modified electrode.
the modified electrodes. Compared to the bare Toray paper electrodes, the three mitochondria-modified electrodes displayed two well-defined oxidation and reduction peaks. Oxidation peaks were located at 186 ± 14 mV, 240 ± 3 mV and 98 ± 9 mV vs. SCE for the yeast, potato and bovine mitochondria electrodes, respectively, at 50 mV s−1 . The reduction peaks were detected at −357 ± 24 mV, −442 ± 5 mV and −335 ± 26 mV vs. SCE for the same organisms. The half wave potential for each type of mitochondria was 87 ± 7 mV, −156 ± 6 mV and −152 ± 14 mV for yeast, potato and bovine mitochondria, respectively. It is important to mention here that redox signals obtained at our mitochondria-modified electrode with this isolation and purification procedure are different than Arechederra’s or Zhao’s work [4,8]. This shows that the isolation and purification procedures are a critical step in the production of mitochondrial-modified electrodes and that it affects the electrochemistry observed. In order to assess that the whole mitochondria was performing direct electron transfer at the carbon electrode, cyclic voltammetry experiments were accomplished in the absence and presence of a metabolite participating in the Krebs cycle. Fig. 2 presents the voltammograms obtained at mitochondria-modified electrodes in pH 7.4 phosphate/nitrate buffer before and after immersion of the
electrodes in 100 mM pyruvate solution. For all organisms, cyclic voltammograms presented an increase in current near the oxidation peak potential, which is indicative of a catalytic behavior of the immobilized mitochondria. At more negative potential, the reduction currents increased too, which is not common in a typical electrocatalytic process. Electrocatalytic processes generally involve only one electrochemical reaction (for instance oxidation OR reduction). However, in the case of mitochondria, catalysis can be established from either the ubiquinone or the cytochrome c. Both electron carriers are involved in a whole catalytic process, from NADH or succinate oxidation in order to reduce oxygen. Thus, in the more positive potential region, we observed DET in catalysis of the oxidation of the pyruvate through the Krebs cycle; whereas in the more negative potential region, the same electron carrier is catalyzing the O2 reduction. This explains how mitochondria have been used for both biocathodes and bioanodes. To determine if the electrochemical reaction observed at the mitochondria electrodes were coming from the mitochondria themselves or from the presence of the pyruvate, cyclic voltammetry at different scan rates was performed. The peak current intensities for both the oxidation and reduction reaction were
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Fig. 3. Peak current versus the scan rate (left) and the square root of the scan rate (right) for yeast (square), potato (triangle) and bovine (empty square) mitochondrialmodified electrodes in 100 mmol L−1 phosphate, 100 mmol L−1 nitrate and 100 mmol L−1 pyruvate solution. Scan rates varied from 50, 25, 10 and 5 mV s−1 . Linear fit obtained for yeast (solid line), potato (dashed line) and bovine mitochondria (dotted line).
plotted against the scan rate and the square root of the scan rate (Fig. 3). The linear correlation between the intensity and the scan rate tends to indicate the reaction is surface limited [22] and thus confirms the direct electrochemistry of mitochondria at the electrode.
3.2. Immobilization of cytochrome c and coenzyme Q Cytochrome c is located in the inter-membrane space of mitochondria whereas the quinone pool is found in the inner membrane. Thus, we hypothesized that the electrode leakage from the mitochondria could come from one of these compounds. To further understand and determine the origin of the redox signal, cytochrome c (from equine heart) and coenzyme Q10 (a quinone known to be part of the quinone pool in some organisms [23,24]) were immobilized onto Toray paper electrodes with a layer TMOS. As depicted in Fig. 4, cytochrome c-modified electrodes did not show any electrochemical signal. On the contrary, voltammograms obtained for coenzyme Q10 (CoQ10 ) had oxidation and reduction potentials close to those obtained for mitochondria with a half wave potential of −189 ± 1 mV vs. SCE. Because the electrochemical response of the quinone was more similar to the organelle
electrodes, additional experiments were performed to compare their electrochemical properties.
3.3. pH-dependence of organelle-modified electrodes As presented earlier, cytochrome c was not detected once immobilized on Toray paper electrodes. Hence, as the electrochemistry of quinones is pH dependent, study of the half wave potential (E1/2 ) variation has been performed in the case of yeast, potato, and bovine mitochondria and compared to our quinone reference, CoQ10 . Cyclic voltammograms were recorded in different buffers at pH 5.0, 6.0, 7.4 and 8.0. Results obtained at the organelle electrodes are shown in Fig. 5. The oxidation and reduction peak potentials were affected depending on the pH used during the cyclic voltammetry. A negative shift was observed in all cases when the pH increased. By plotting the half wave potential against the pH of the solution (Fig. 6), we found that the E1/2 changed linearly with respect to the pH value of the solution (yeast: −34 ± 2 mV pH units−1 , potato: −39 ± 4 mV pH units−1 , bovine: −47 ± 3 mV pH units−1 ). Moreover, the same experiments with CoQ10 resulted in a similar trend (Q10 : −49 ± 1 mV pH units−1 ). As CoQ10 is a widely found quinone in bovine and other mitochondria [24], this report
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Fig. 4. Cyclic voltammetry obtained at a cytochrome c-immobilized electrode (A) and a coenzyme Q10 -immobilized electrode (B) compared to the signal obtained at yeast mitochondrial-modified electrodes (dashed line) in 100 mmol L−1 phosphate, 100 mmol L−1 nitrate and 100 mmol L−1 pyruvate solution at 10 mV s−1 .
Fig. 5. Cyclic voltammetry of potato (left), yeast (center) and bovine (right) mitochondria immobilized on Toray paper in 100 mmol L−1 phosphate, 100 mmol L−1 nitrate and 100 mmol L−1 pyruvate at 50 mV s−1 at different pHs.
supports the theory of electron transfer from the organelle to the electrode via the quinone pool. 3.4. Cytochrome c and coenzyme Q depletion of mitochondria To further confirm the theory of electron leakage from the quinone pool of mitochondria, cytochrome c and coenzyme Q were
Fig. 6. E1/2 versus pH for electrodes modified with coenzyme Q10 and mitochondria isolated from yeast, potato and bovine.
extracted from mitochondria without denaturing the organelles to identify if the redox signal changed when each electron carrier was removed. During the cytochrome c extraction procedure, mitochondria were first swollen in a hypotonic solution of KCl (0.015 mM). Cytochrome c was subsequently extracted with isotonic solution of KCl (0.15 mM). The resulting cytochrome c-depleted mitochondria pellet was re-suspended in sucrose/Trisacetate buffer and cast onto clean Toray paper electrodes. Fig. 7 shows a representative cyclic voltammogram of the cytochrome c-depleted potato mitochondria-modified electrode compared to a cyclic voltammogram previously collected for potato mitochondria containing cytochrome c and modified onto the same electrode material. For the three different species, the peaks were still distinguishable, but appeared to be shifted to lower voltages. The oxidation peaks of cytochrome c-depleted mitochondria were found to be 81 ± 2 mV, 175 ± 14 mV and 79 ± 1 mV, while the oxidation peak for whole mitochondria were found at 186 ± 14, 240 ± 3 mV and 98 ± 9 mV for yeast, potato and bovine sources, respectively. The reduction peaks were −485 ± 8 mV, −391 ± 14 mV and −374 ± 3 mV for mitochondria without cytochrome c and −357 ± 24 mV, −442 ± 5 mV, −335 ± 26 mV for intact mitochondria given in the same order as aforementioned. Although the redox peaks seem to be located at slightly different potentials, the E1/2 values for both unextracted and extracted cytochrome c are in the same range (E1/2 cyt c-depleted potato mitochondria = −167 ± 15 mV,
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Fig. 7. Representative cyclic voltammograms of (A) Yeast mitochondria (B) potato mitochondria and (C) bovine mitochondria. CVs were carried out in 100 mmol L−1 phosphate, 100 mmol L−1 nitrate and 100 mmol L−1 pyruvate solution at pH 7.4 at 50 mV s−1 . Undepleted mitochondria (solid line), cytochrome c-depleted mitochondria (dashed line) and CoQ-depleted mitochondria (dotted line).
E1/2 potato mitochondria = −156 ± 6 mV) and match the E1/2 obtained for the quinone. Nevertheless, the peak currents were significantly lower in the cytochrome-c depleted mitochondria than for the intact mitochondria. Although this decrease in current may suggest that the redox couple could be cytochrome c, because the peak currents only decreased once the cytochrome was removed from the organelle, we previously demonstrated that immobilized cytochrome c was not able to connect electrochemically to the electrode (Fig. 4A). Thus, the decrease of the redox signal should not be attributed to the cytochrome c, but rather to a change of the mitochondria membrane properties after the cytochrome c extraction. Another explanation could be the production of reactive oxygen species (ROS) produced at complex I or III with the increase of O2 level in the matrix of mitochondria [25]. The presence of ROS may damage the mitochondria and change its electron transport chain properties. The coenzyme Q pool was removed from the mitochondria using pentane. As reported by Norling et al., this procedure gives mitochondria where activities of its complexes remain intact, but the quinone pool is removed [26]. From a lyophilized powder, the mitochondria were suspended in pentane, homogenized and CoQs were collected in the supernatant by centrifugation. After the resulting CoQ-depleted mitochondrial pellet was dried by removal of the pentane under nitrogen flow, it was re-suspended in sucrose buffer and cast onto the Toray paper electrode. Cyclic voltammetry was carried out on the CoQ-depleted mitochondria electrodes and the voltammograms are presented in Fig. 7 by dotted lines. Yeast and bovine representative cyclic voltammograms did not show any oxidation or reduction peak in the potential ranges where whole organelles had their respective redox processes. For potato mitochondria, the apparent oxidation located near 0.1 V vs. SCE was not attributed to electrochemical reaction from the mitochondria, but rather to the carbon electrode fingerprint, which appears clearly on the same graph for intact potato organelle or on control electrodes (Fig. 1). The absence of reduction peak for the CoQdepleted potato electrodes confirms this supposition. 4. Conclusion We have demonstrated the origin of the direct electrochemistry of three different types of mitochondria on carbon electrodes. To the best of our knowledge, this paper describes for the first evidence of the direct electrochemistry of an organelle through the quinone pool. While cytochrome c was not observed at the electrode, the half wave potential of our “reference coenzyme” coenzyme Q10 was comparable to E1/2 obtained for yeast, potato and bovine organelle-modified electrodes. Cyclic
voltammetry in different pH solutions along with cyclic voltammetry on cytochrome c-depleted or coenzyme Q-depleted mitochondria confirmed our hypothesis. This work gives important information on the electrochemistry of immobilized mitochondria and could help to orient research to develop mediatorless organelle-based biosensors. Acknowledgements The authors would like to thank the Utah Science and Technology Research Initiative and the US Army Engineer Research and Development Center (ERDC) for funding. T.A.N. would also like to thank the NASA Space Grant and the University of Utah Undergraduate Research Opportunities Program for her fellowships. References [1] J.B. Robinson, P.A. Srere Jr., Organization of Krebs tricarboxylic acid cycle enzymes in mitochondria, The Journal of Biological Chemistry 260 (1985) 10800. [2] V.M. Darley-Usmar, D. Rickwood, M.T. Wilson, Mitochondria a Practical Approach, IRL, Press, Oxford, Washington, DC, 1987. [3] R.L. Arechederra, K. Boehm, S.D. Minteer, Mitochondrial bioelectrocatalysis for biofuel cell applications, Electrochimica Acta 54 (2009) 7268. [4] J. Zhao, F. Meng, X. Zhu, K. Han, S. Liu, G. Li, Electrochemistry of mitochondria: a new way to understand their structure and function, Electroanalysis 20 (2008) 1593. [5] D. Bhatnagar, S. Xu, C. Fischer, R.L. Arechederra, S.D. Minteer, Mitochondrial biofuel cells: expanding fuel diversity to amino acids, Physical Chemistry Chemical Physics 13 (2011) 86. [6] A.M. Sastry, M.V. Inamdar, C.W. Wang, M. Philbert, J.A. Miller, Biological battery or fuel cell utilizing mitochondria, United States Patent Application #20080261085, (2008). [7] R.L. Arechederra, S.D. Minteer, Complete oxidation of glycerol in an enzymatic biofuel cell, Fuel Cells 9 (2009) 63. [8] R. Arechederra, S.D. Minteer, Organelle-based biofuel cells: immobilized mitochondria on carbon paper electrodes, Electrochimica Acta 53 (2008) 6698. [9] S.L. Maltzman, S.D. Minteer, Mitochondrial-based voltammetric sensor for pesticides, Analytical Methods 4 (2012) 1202. [10] R.L. Arechederra, A. Waheed, W.S. Sly, S.D. Minteer, Electrically wired mitochondrial electrodes for measuring mitochondrial function for drug screening, The Analyst 136 (2011) 3747. [11] M.N. Arechederra, C.N. Fischer, D.J. Wetzel, S.D. Minteer, Evaluation of the electron transport chain inhibition and uncoupling of mitochondrial bioelectrocatalysis with antibiotics and nitro-based compounds, Electrochimica Acta 56 (2010) 938. [12] M.N. Germain, R.L. Arechederra, S.D. Minteer, Nitroaromatic actuation of mitochondrial bioelectrocatalysis for self-powered explosive sensors, Journal of the American Chemical Society 130 (2008) 15272. [13] D. Johnson, S. Norman, R.C. Tuckey, L.L. Martin, Electrochemical behaviour of human adrenodoxin on a pyrolytic graphite electrode, Bioelectrochemistry 59 (2003) 41. [14] D. Leister, J. Herrmann, Mitochondria: Practical Protocols, Humana Press, Totowa, NJ, 2007.
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Understanding the mechanism of direct electrochemistry of mitochondria-modified electrodes from yeast, potato and bovine sources at carbon paper electrodes Fabien Giroud a , Tera A. Nicolo a , Sara J. Koepke a , Shelley D. Minteer a,b,∗ a b
Department of Chemistry, University of Utah, Salt Lake City, UT 84112, United States Department of Materials Science and Engineering, University of Utah, Salt Lake City, UT 84112, United States
a r t i c l e
i n f o
Article history: Received 30 November 2012 Received in revised form 15 February 2013 Accepted 18 February 2013 Available online 26 February 2013 Keywords: Mitochondria Bioelectrocatalysis Cytochrome c Quinone pool Electrochemistry
a b s t r a c t Although mitochondria have been used for bio-electrochemistry for over 5 years, little is known about their direct electrochemistry mechanism. This paper focuses on developing a better understanding of the electron transfer mechanism of mitochondria from three different organisms at carbon electrodes. Yeast, potato and bovine mitochondria have been successfully isolated and immobilized onto Toray paper electrodes via vapor deposited silica. Organelle-modified electrodes were first characterized using cyclic voltammetry. Similar electrochemical signals were obtained for all organisms. Direct electron transfer was observed when a metabolite of the Krebs cycle was present in the buffer solution. Control experiments based on the immobilization of two electron carriers contained in mitochondria, cytochrome c and a quinone (coenzyme Q10 ), tend to show the electron transfer mechanism to the carbon material comes from the quinone pool of the organelles. As quinones are known to be pH-dependent, we further investigated the response of the electrochemical signal of the three isolated mitochondria and the two electron carriers separately. The half wave potentials obtained from the organelles appeared to be pH-dependent and their variations are comparable to coenzyme Q10 rather than cytochrome c. Finally, extraction of both the cytochrome c and the quinone pool from intact mitochondria was performed to validate our hypothesis that direct electrochemistry of mitochondria happens via the quinone pool. Electrochemistry of immobilized quinone-depleted mitochondria validated the hypothesis that the mitochondria are communicating with the electrodes through the quinone pool. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Mitochondria are organelles found in living cells that produce energy by breaking down pyruvate and fatty acids to carbon dioxide through the Krebs cycle and possess the complexes responsible for the electron transport chain. All of the Krebs cycle enzymes are located inside the matrix of the mitochondria. On the other hand, the four complexes taking part in the electron transport chain are found in the inner membrane. Complexes I and II collect electrons from NADH and succinate, both of which are produced by the Krebs cycle. Then, the two complexes transfer their electrons to complex III via electron carriers called the quinone pool situated inside the inner membrane. Finally, cytochrome c (a protein loosely attached to the outer part of the inner membrane) transfers the electron from complex III to complex IV for molecular oxygen reduction.
∗ Corresponding author at: Department of Chemistry, University of Utah, Salt Lake City, UT 84112, United States. Tel.: +1 801 587 8325; fax: +1 801 581 8181. E-mail address: [email protected] (S.D. Minteer). 0013-4686/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.electacta.2013.02.087
Mitochondria have gathered more attention from the scientific community over the last 5 years especially in bio-electrochemistry. Their use was essentially focused on deep oxidation of different substrates such as pyruvate, fatty acids [1–4] and more recently amino acids [5], but they have been used for the reduction reaction at the cathode as well [6]. This biocatalyst was utilized mainly, because this organelle contains all the necessary enzymes for the fuel oxidation in their matrix and they offer better substrate transport through their membranes than whole microbes. Furthermore, due to the presence of the membrane that encapsulates the enzymes, mitochondrial use and stability are greatly improved compared to enzymatic bio-electrodes reproducing metabolic pathways [7]. Consequently, mitochondria were first immobilized on the electrode in order to generate power from the conversion of the chemical energy of a biofuel to electrical energy contained in an organelle-based biofuel cell [8]. Since then, the aim of mitochondria-based biofuel cells have evolved from basic biofuel cells to include their use as self-powered biosensors. The Minteer group studied and developed several sensors based on mitochondrial use and assembly in a fuel cell. In these cases,
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the mitochondria electrode is the anode with an air-breathing platinum cathode [9–12]. However, the electron transfer mechanism between the mitochondria and the electrode was not well studied. Few examples have been found dealing with organelle wiring to the electrode. Arechederra’s study postulated that organelles could undergo mediated electron transfer via the use of a polyazine layer or direct electron transfer (DET) to the electrode through the cytochrome c of the mitochondria due to similarities between mitochondrial modified electrode cyclic voltammetry and cytochrome c cyclic voltammetry [8]. Another group showed that in certain conditions, DET can be obtained at pyrolitic graphite electrodes through another cofactor contained in the inner membrane [4]. The authors were able to detect a reversible peak from mitochondria from guinea pig liver located at −500 mV vs. SCE, which they ascribed to flavine adenine dinucleotide (FAD/FADH2 ). However, work on the mammalian protein cytochrome P450, another membrane-bound protein located in the inner membrane of mitochondria, showed a reversible redox signal at the same potential [13]. Thus, the electron pathway from the mitochondria to the electrode is still unclear and electrons could come from one unique electron carrier or from different redox proteins depending on the preparation of the mitochondria dispersion (i.e. the isolation and purification procedure). This paper presents the direct electrochemistry of three different types of mitochondria (yeast, potato and bovine sources). All of the organelles show the same redox behavior. In anaerobic conditions, well-defined redox signals were observed near 100 mV vs. SCE (oxidation) and −350 mV vs. SCE. This paper attempts to identify the source of the electron leakage from the mitochondrial electron transport chain. It is more likely this leakage is coming from electron carriers (small molecule mediators) rather than the complexes themselves. Cytochrome c (located on one side of the inner membrane) was the best candidate, but cyclic voltammetry data showed the quinone pool (coenzyme Q) could be the origin too. Thus, we compared electrochemistry of organelleand electron carrier- modified electrodes in different pH conditions. Finally, cyclic voltammetry was carried out on cytochrome c-depleted mitochondrial-modified electrodes and quinone pooldepleted mitochondrial-modified electrodes and compared to intact mitochondrial-modified electrodes. Results indicate that immobilized organelles are communicating with the electrode via the quinone pool.
2. Materials and methods 2.1. Material Yeast cells from Saccharomyces cerevisiae were purchased from ATCC (# 9080, strain ID 4228) (USA). Organic Russet potatoes were obtained from a local farmer. Fresh beef heart was purchased from a local slaughterhouse. Yeast extract and tetramethyl orthosilicate (TMOS) were purchased from Fluka (USA). Pentane was purchased from Fisher Scientific (USA). Toray carbon paper (TGPH-60, non-wet-proof) was purchased from Fuel Cell Earth (USA). All other reagents used were purchased from Sigma (USA). Solutions were prepared with 18 M cm de-ionized water from a Milli-Q system.
2.2. Yeast growth Lactate media was prepared by the protocol outlined by Leister and Hermann [14]. S. cerevisiae cells were inoculated into lactic
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acid media pH 5.5 and allowed to grow with agitation at 30 ◦ C for approximately 2 days. 2.3. Mitochondria extraction from yeast, potato and bovine sources Yeast mitochondria were isolated from S. cerevisiae according to the procedure in reference [14]. Potato mitochondria were isolated from Russet potatoes according to Moore’s procedure [15]. Bovine mitochondria were isolated from a beef heart according to Palloti’s procedure with slight modification [16]. The basic procedure is as follows: 200 g of bovine heart tissue was rinsed in 400 mL of 250 mmol L−1 sucrose, 10 mmol L−1 Tris–Cl at pH 7.8 solution (rinse solution). The tissue was placed into 400 mL of isolation solution (rinse solution with 0.2 mmol L−1 EDTA, 1 mol L−1 PMSF dissolved in 0.5 mL ethanol added directly before use) and the pH was adjusted with KOH. The tissue was blended while pH was maintained at 7.8. The blended tissue was centrifuged for 20 min at 1200 × g and the supernatant was re-centrifuged for 15 min at 26,000 × g. After discarding the supernatant, the pellet was re-suspended in 10 mL of isolation solution and homogenized in two 5 mL volumes with a Kontes size 21 5 mL Potter-Elvehjem homogenizer and Craftsman Evolv 17217 5.2 amp corded 3/8 drill on the middle setting for two passes of 5 s each. This homogenate was diluted to 180 mL in isolation solution, pH adjusted, and centrifuged at 26,000 × g for 15 min. The pellet was re-suspended in 60 mL of isolation solution and centrifuged at 12,000 × g for 30 min. All final pellets for the three different organisms were re-suspended in rinse solution to a wet weight of 200 mg mL−1 . 2.4. Cytochrome c and coenzyme Q depletion of mitochondria The cytochrome c extraction procedure was the same as used by Jacobs et al. [17]. Suspensions of freshly isolated mitochondria were suspended in 15 mmol L−1 KCl (hypotonic medium for whole mitochondria) at a protein concentration of 200 mg mL−1 . Mitochondria were allowed to swell for 10 min on ice before centrifugation at 105,000 × g for 15 min. The supernatant was kept and stored at 4 ◦ C. The pellet was re-suspended in 150 mmol L−1 KCl (isotonic medium for whole mitochondria). The suspension was kept on ice for 10 min and then centrifuged at 105,000 × g for 15 min. The resulting supernatant was kept at 4 ◦ C. The extraction procedure is repeated twice in the isotonic medium, as described above. The cytochrome c was extracted into the isotonic supernatant. The final pellet of cytochrome cdepleted mitochondria is re-suspended in 250 mmol L−1 sucrose and 10 mmol L−1 pH 7.5 Tris–acetate buffer and stored at −80 ◦ C until use. The coenzyme Q extraction procedure was the same as used by Szarkowska et al. [18]. The mitochondrial preparation was thawed on ice and diluted to a final protein concentration of 200 mg ml−1 in 250 mmol L−1 sucrose and 10 mmol L −1 Tris–HCl at pH 7.5. The suspension was centrifuged at 35,000 × g for 10 min. The pellet was re-suspended in 150 mmol L−1 KCl, frozen in liquid nitrogen, and lyophilized. The extraction of CoQ was realized in pentane (1 mL pentane/1 mg lyophilized mitochondria). The mixture was homogenized in a Potter-Elvejhem homogenizer with Teflon pestle and then centrifuged at 5000 × g for 10 min. The same extraction–homogenization was repeated 4 times. Finally, pentane was removed from the extracted mitochondria under nitrogen flow for 4 h. Extracted mitochondria were homogenized in 250 mmol L−1 sucrose and 10 mmol L−1 Tris–HCl buffer at pH 7.5, centrifuged at 35,000 × g for 10 min at 4 ◦ C, and re-suspended in the same buffer. This suspension was frozen in liquid nitrogen and stored at −80◦ C for later use.
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2.5. Immobilization of organelles, quinones and cytochrome c onto the electrodes
3. Results and discussion 3.1. Electrochemical detection of immobilized organelles
Prior to modification, Toray paper electrodes were cleaned by sonication in three methanol baths (10 min each), rinsed thoroughly in DI water and dried at room temperature. The wet mitochondria precipitates were used directly. Wet precipitates were re-suspended in the washing buffer to give a 200 mg mL−1 solution. 35 L of the mitochondria suspension was drop cast onto a 1 cm2 unmodified Toray paper electrode and allowed to dry for 1 h. Once dry, mitochondria were immobilized by a new layer of silica. Briefly, tetramethyl orthosilicate (TMOS) was hydrolyzed by ambient water vapor giving the layer of vapor-deposited silica according to the procedure described in References [19,20]. Electrodes were placed at 4 ◦ C overnight. Prior to use, organelle electrodes were soaked for 1 h in pH 7.40 phosphate buffer that contained 100 mmol L−1 nitrate, 100 mmol L−1 pyruvate and 1 mg mL−1 ADP. Coenzyme Q10 was dissolved in ethanol at a concentration of 2 mmol L−1 . Subsequently, 30 L of this solution was drop cast on the 1 cm2 Toray paper electrode. Cytochrome c from equine heart was dissolved in deionized water at a concentration of 10 mg mL−1 and 30 L of this solution was drop cast on the Toray paper electrodes. Both electrode types were covered with the TMOS layer, using the procedure described above.
2.6. Determintation of NADH leaching In order to determine if any NADH leached out of the mitochondria membrane once immobilized onto the Toray paper with the TMOS silica layer, an NADH leaching assay was performed. NADH leakage could indicate that mitochondrial membrane lysis had occurred. For the assay, mitochondria-modified electrodes as described above were soaked in 3 mL disposable plastic cuvettes filled with a pH 7.40 phosphate buffer solution. An initial absorbance measurement was taken with a UV spectrophotometer at a wavelength of 340 nm at time zero. Then the cuvettes were allowed to sit for 2 h and the measurement was taken again to determine if NADH had leached into the solution.
Mitochondria from three different species were isolated and immobilized on the electrode by drop casting, followed by the vapor deposition of a TMOS layer. After immobilization, separate experiments were investigated to determine if mitochondria were still intact and active. An NADH assay using UV–vis spectroscopy was performed to determine if the different mitochondria used were lysed during the extraction. Absorbance measured at 340 nm immediately after soaking the immobilized-organelle electrodes in the buffer solution was compared to the absorbance of the same solution after 2 h. The control electrodes showed a change of absorbance of 0.0015 ± 0.0008, which was not statistically different from the organelle-modified electrodes (yeast: 0.0021 ± 0.0008, potato: 0.0010 ± 0.0007, bovine: 0.0019 ± 0.0008). The small variation in absorbance indicated the NADH was not leaching from the mitochondria, suggesting their membranes were still intact. Separately, a Carbon-13 NMR experiment was performed to verify that immobilized mitochondria were still active and results were compared to spectra obtained from free mitochondria in suspension. Carbon-13 labeled pyruvate was used as the natural substrate of the mitochondria. The results indicated that only the pyruvate peaks were found for the controls experiments (no mitochondria immobilized onto the electrodes or without inoculation of organelle suspension) (peaks at 26 ppm: RCH3 , 170 ppm: RCOOH, 204 ppm: RR’CO). In the presence of mitochondria (both immobilized or in suspension), all the peaks previously mentioned decreased in intensity while new peaks appeared (48 ppm: RR’CH2 , 73 ppm: RR’COH, 135 ppm: C C, 182 ppm: RCOOH) which correspond to by products (succinate, fumarate and malate) released from the mitochondria matrix in exchange of phosphate required for the oxidative phosphorylation [21]. After the Toray paper electrodes were modified with the organelles, cyclic voltammetry experiments were carried out in order to characterize the direct electrochemistry of mitochondria at the carbon electrodes. Toray paper electrodes were preferred due to their high surface area compared to glassy carbon electrodes. Fig. 1 presents representative cyclic voltammograms of
2.7. Carbon-13 NMR study of pyruvate oxidation A Carbon-13 NMR experiment was carried out by using a 100 mM Carbon-13 labeled pyruvate fuel solution in pH 7.4 phosphate/nitrate buffer. Mitochondria were first immobilized onto Toray paper electrodes as previously described. Mitochondriamodified electrodes were allowed to soak in the labeled pyruvate solution for 24 h. NMR samples were made by mixing 350 L of carbon-13 labeled pyruvate solution with equal volume of deuterated water. The results were compared to NMR spectra obtained using mitochondria directly inoculated into the solution. Controls were performed using bare Toray paper electrodes.
2.8. Electrochemical measurements The electrodes were tested using cyclic voltammetry on a CH Instruments potentiostat model 650A (TX, USA), Biologic VSP 150 (Claix, France) or Digi Ivy DY2300 (TX, USA) with a platinum mesh counter and a calomel reference electrode. The scan window was 0.5 to −0.7 V. Potentials are referenced to the saturated calomel reference electrode (SCE). All experiments were conducted under nitrogen flow unless specified.
Fig. 1. Cyclic voltammetry of mitochondria from different sources immobilized on Toray paper electrode with TMOS in 100 mmol L−1 phosphate, 100 mmol L−1 nitrate and 100 mmol L−1 pyruvate at pH 7.4 recorded at 10 mV s−1 . Yeast (thick solid line), potato (dashed line), bovine (dotted line). Bare Toray paper electrode control (thin solid line).
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Fig. 2. Cyclic voltammograms of mitochondria from different sources immobilized on Toray paper electrode and control experiments without (solid line) and with pyruvate (dashed line) present in the electrolyte indicating a direct electron transfer between the organelle and the electrode. (A) unmodified Toray paper, (B) yeast-modified electrode, (C) potato-modified electrode and (D) bovine-modified electrode.
the modified electrodes. Compared to the bare Toray paper electrodes, the three mitochondria-modified electrodes displayed two well-defined oxidation and reduction peaks. Oxidation peaks were located at 186 ± 14 mV, 240 ± 3 mV and 98 ± 9 mV vs. SCE for the yeast, potato and bovine mitochondria electrodes, respectively, at 50 mV s−1 . The reduction peaks were detected at −357 ± 24 mV, −442 ± 5 mV and −335 ± 26 mV vs. SCE for the same organisms. The half wave potential for each type of mitochondria was 87 ± 7 mV, −156 ± 6 mV and −152 ± 14 mV for yeast, potato and bovine mitochondria, respectively. It is important to mention here that redox signals obtained at our mitochondria-modified electrode with this isolation and purification procedure are different than Arechederra’s or Zhao’s work [4,8]. This shows that the isolation and purification procedures are a critical step in the production of mitochondrial-modified electrodes and that it affects the electrochemistry observed. In order to assess that the whole mitochondria was performing direct electron transfer at the carbon electrode, cyclic voltammetry experiments were accomplished in the absence and presence of a metabolite participating in the Krebs cycle. Fig. 2 presents the voltammograms obtained at mitochondria-modified electrodes in pH 7.4 phosphate/nitrate buffer before and after immersion of the
electrodes in 100 mM pyruvate solution. For all organisms, cyclic voltammograms presented an increase in current near the oxidation peak potential, which is indicative of a catalytic behavior of the immobilized mitochondria. At more negative potential, the reduction currents increased too, which is not common in a typical electrocatalytic process. Electrocatalytic processes generally involve only one electrochemical reaction (for instance oxidation OR reduction). However, in the case of mitochondria, catalysis can be established from either the ubiquinone or the cytochrome c. Both electron carriers are involved in a whole catalytic process, from NADH or succinate oxidation in order to reduce oxygen. Thus, in the more positive potential region, we observed DET in catalysis of the oxidation of the pyruvate through the Krebs cycle; whereas in the more negative potential region, the same electron carrier is catalyzing the O2 reduction. This explains how mitochondria have been used for both biocathodes and bioanodes. To determine if the electrochemical reaction observed at the mitochondria electrodes were coming from the mitochondria themselves or from the presence of the pyruvate, cyclic voltammetry at different scan rates was performed. The peak current intensities for both the oxidation and reduction reaction were
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Fig. 3. Peak current versus the scan rate (left) and the square root of the scan rate (right) for yeast (square), potato (triangle) and bovine (empty square) mitochondrialmodified electrodes in 100 mmol L−1 phosphate, 100 mmol L−1 nitrate and 100 mmol L−1 pyruvate solution. Scan rates varied from 50, 25, 10 and 5 mV s−1 . Linear fit obtained for yeast (solid line), potato (dashed line) and bovine mitochondria (dotted line).
plotted against the scan rate and the square root of the scan rate (Fig. 3). The linear correlation between the intensity and the scan rate tends to indicate the reaction is surface limited [22] and thus confirms the direct electrochemistry of mitochondria at the electrode.
3.2. Immobilization of cytochrome c and coenzyme Q Cytochrome c is located in the inter-membrane space of mitochondria whereas the quinone pool is found in the inner membrane. Thus, we hypothesized that the electrode leakage from the mitochondria could come from one of these compounds. To further understand and determine the origin of the redox signal, cytochrome c (from equine heart) and coenzyme Q10 (a quinone known to be part of the quinone pool in some organisms [23,24]) were immobilized onto Toray paper electrodes with a layer TMOS. As depicted in Fig. 4, cytochrome c-modified electrodes did not show any electrochemical signal. On the contrary, voltammograms obtained for coenzyme Q10 (CoQ10 ) had oxidation and reduction potentials close to those obtained for mitochondria with a half wave potential of −189 ± 1 mV vs. SCE. Because the electrochemical response of the quinone was more similar to the organelle
electrodes, additional experiments were performed to compare their electrochemical properties.
3.3. pH-dependence of organelle-modified electrodes As presented earlier, cytochrome c was not detected once immobilized on Toray paper electrodes. Hence, as the electrochemistry of quinones is pH dependent, study of the half wave potential (E1/2 ) variation has been performed in the case of yeast, potato, and bovine mitochondria and compared to our quinone reference, CoQ10 . Cyclic voltammograms were recorded in different buffers at pH 5.0, 6.0, 7.4 and 8.0. Results obtained at the organelle electrodes are shown in Fig. 5. The oxidation and reduction peak potentials were affected depending on the pH used during the cyclic voltammetry. A negative shift was observed in all cases when the pH increased. By plotting the half wave potential against the pH of the solution (Fig. 6), we found that the E1/2 changed linearly with respect to the pH value of the solution (yeast: −34 ± 2 mV pH units−1 , potato: −39 ± 4 mV pH units−1 , bovine: −47 ± 3 mV pH units−1 ). Moreover, the same experiments with CoQ10 resulted in a similar trend (Q10 : −49 ± 1 mV pH units−1 ). As CoQ10 is a widely found quinone in bovine and other mitochondria [24], this report
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Fig. 4. Cyclic voltammetry obtained at a cytochrome c-immobilized electrode (A) and a coenzyme Q10 -immobilized electrode (B) compared to the signal obtained at yeast mitochondrial-modified electrodes (dashed line) in 100 mmol L−1 phosphate, 100 mmol L−1 nitrate and 100 mmol L−1 pyruvate solution at 10 mV s−1 .
Fig. 5. Cyclic voltammetry of potato (left), yeast (center) and bovine (right) mitochondria immobilized on Toray paper in 100 mmol L−1 phosphate, 100 mmol L−1 nitrate and 100 mmol L−1 pyruvate at 50 mV s−1 at different pHs.
supports the theory of electron transfer from the organelle to the electrode via the quinone pool. 3.4. Cytochrome c and coenzyme Q depletion of mitochondria To further confirm the theory of electron leakage from the quinone pool of mitochondria, cytochrome c and coenzyme Q were
Fig. 6. E1/2 versus pH for electrodes modified with coenzyme Q10 and mitochondria isolated from yeast, potato and bovine.
extracted from mitochondria without denaturing the organelles to identify if the redox signal changed when each electron carrier was removed. During the cytochrome c extraction procedure, mitochondria were first swollen in a hypotonic solution of KCl (0.015 mM). Cytochrome c was subsequently extracted with isotonic solution of KCl (0.15 mM). The resulting cytochrome c-depleted mitochondria pellet was re-suspended in sucrose/Trisacetate buffer and cast onto clean Toray paper electrodes. Fig. 7 shows a representative cyclic voltammogram of the cytochrome c-depleted potato mitochondria-modified electrode compared to a cyclic voltammogram previously collected for potato mitochondria containing cytochrome c and modified onto the same electrode material. For the three different species, the peaks were still distinguishable, but appeared to be shifted to lower voltages. The oxidation peaks of cytochrome c-depleted mitochondria were found to be 81 ± 2 mV, 175 ± 14 mV and 79 ± 1 mV, while the oxidation peak for whole mitochondria were found at 186 ± 14, 240 ± 3 mV and 98 ± 9 mV for yeast, potato and bovine sources, respectively. The reduction peaks were −485 ± 8 mV, −391 ± 14 mV and −374 ± 3 mV for mitochondria without cytochrome c and −357 ± 24 mV, −442 ± 5 mV, −335 ± 26 mV for intact mitochondria given in the same order as aforementioned. Although the redox peaks seem to be located at slightly different potentials, the E1/2 values for both unextracted and extracted cytochrome c are in the same range (E1/2 cyt c-depleted potato mitochondria = −167 ± 15 mV,
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Fig. 7. Representative cyclic voltammograms of (A) Yeast mitochondria (B) potato mitochondria and (C) bovine mitochondria. CVs were carried out in 100 mmol L−1 phosphate, 100 mmol L−1 nitrate and 100 mmol L−1 pyruvate solution at pH 7.4 at 50 mV s−1 . Undepleted mitochondria (solid line), cytochrome c-depleted mitochondria (dashed line) and CoQ-depleted mitochondria (dotted line).
E1/2 potato mitochondria = −156 ± 6 mV) and match the E1/2 obtained for the quinone. Nevertheless, the peak currents were significantly lower in the cytochrome-c depleted mitochondria than for the intact mitochondria. Although this decrease in current may suggest that the redox couple could be cytochrome c, because the peak currents only decreased once the cytochrome was removed from the organelle, we previously demonstrated that immobilized cytochrome c was not able to connect electrochemically to the electrode (Fig. 4A). Thus, the decrease of the redox signal should not be attributed to the cytochrome c, but rather to a change of the mitochondria membrane properties after the cytochrome c extraction. Another explanation could be the production of reactive oxygen species (ROS) produced at complex I or III with the increase of O2 level in the matrix of mitochondria [25]. The presence of ROS may damage the mitochondria and change its electron transport chain properties. The coenzyme Q pool was removed from the mitochondria using pentane. As reported by Norling et al., this procedure gives mitochondria where activities of its complexes remain intact, but the quinone pool is removed [26]. From a lyophilized powder, the mitochondria were suspended in pentane, homogenized and CoQs were collected in the supernatant by centrifugation. After the resulting CoQ-depleted mitochondrial pellet was dried by removal of the pentane under nitrogen flow, it was re-suspended in sucrose buffer and cast onto the Toray paper electrode. Cyclic voltammetry was carried out on the CoQ-depleted mitochondria electrodes and the voltammograms are presented in Fig. 7 by dotted lines. Yeast and bovine representative cyclic voltammograms did not show any oxidation or reduction peak in the potential ranges where whole organelles had their respective redox processes. For potato mitochondria, the apparent oxidation located near 0.1 V vs. SCE was not attributed to electrochemical reaction from the mitochondria, but rather to the carbon electrode fingerprint, which appears clearly on the same graph for intact potato organelle or on control electrodes (Fig. 1). The absence of reduction peak for the CoQdepleted potato electrodes confirms this supposition. 4. Conclusion We have demonstrated the origin of the direct electrochemistry of three different types of mitochondria on carbon electrodes. To the best of our knowledge, this paper describes for the first evidence of the direct electrochemistry of an organelle through the quinone pool. While cytochrome c was not observed at the electrode, the half wave potential of our “reference coenzyme” coenzyme Q10 was comparable to E1/2 obtained for yeast, potato and bovine organelle-modified electrodes. Cyclic
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