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The reaction of cytochrome c from different species with cytochrome c oxidase immobilized in an electrode supported lipid bilayer membrane
Journal of Electroanalytical Chemistry 534 (2002) 143 /150 www.elsevier.com/locate/jelechem
The reaction of cytochrome c from different species with cytochrome c oxidase immobilized in an electrode supported lipid bilayer membrane Melissa C. Rhoten a, James D. Burgess b, Fred M. Hawkridge c,* a
Department of Natural Sciences, Longwood University, Farmville, VA 23909, USA Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA c Department of Chemistry, Virginia Commonwealth University, Box 842006, Richmond, VA 23284, USA b
Received 28 June 2002; received in revised form 6 August 2002; accepted 14 August 2002
Abstract In past work the direct electron transfer reactions of bovine cytochrome c oxidase in an electrode-supported lipid bilayer membrane have been studied. Its reaction with cytochrome c in solution was also studied and found to be consistent with previous solution studies. In this work it is shown that the electron transfer reactions of cytochrome c oxidase in this electrode-supported lipid bilayer membrane depend on the source of cytochrome c . This property has also been widely studied for solution samples. The differences in the electron transfer reaction rates correlate with the differences in the amino acid sequence for the cytochrome c molecules studied. Electrochemical results suggest that the dissociation of the cytochrome c /cytochrome c oxidase reaction complex is the rate-controlling step in this electron transfer mechanism for cytochrome c from some sources. Moreover, the electron transfer reaction mechanism exhibits biphasic reaction kinetics, which is consistent with earlier work on reactions between solubilized cytochrome c oxidase/cytochrome c samples. These results indicate that the cytochrome c oxidase modified electrodes described herein could be used to distinguish amino acid sequence variations in proteins such as cytochrome c , and this has potential relevance as a diagnostic for disease states. # 2002 Published by Elsevier Science B.V. Keywords: Immobilized cytochrome c oxidase; Lipid bilayer membrane; Cytochrome c ; Enzyme kinetics; Proteomics
1. Introduction The electron transfer reactions of proteins and enzymes at electrodes can now be studied and controlled under a variety of conditions that mimic some of their native reaction properties [1]. Electron transfer communication between more structurally and functionally complex enzymes with electrodes has been facilitated by redox mediators, electron relays, and chemically modified electrodes [2]. Many redox enzymes are membrane-bound and function within the confines of a phospholipid bilayer. Embedding or adsorbing integral membrane enzymes in or on electrode-supported lipid bilayer membranes facilitates direct electron trans* Corresponding author. Tel.: /1-804-828-7505; fax: /1-804-8288599 E-mail address: [email protected] (F.M. Hawkridge).
fer and stabilizes the enzyme against denaturation [3 /8]. ˚, The dimensions of these structures, approximately 50 A also minimize mass transfer concerns involving analytes, substrates, mediators, counter ions, and detected redox active products in micron thick films containing enzymes on an electrode. Opportunities to exploit the native catalytic activity and selectivity of an enzyme can be envisioned in sensor applications, biomass conversion reactors, or in driving fuel cell applications when combined with an electrode held at a controlled potential. In this work cytochrome c oxidase is immobilized in an electrode-supported lipid bilayer membrane [6]. Fig. 1 shows a model of the electrode-supported lipid bilayer membrane with an embedded oxidase molecule. The diameter of the oxidase is known to be approximately 80 ˚ , and the larger hydrophilic end of the molecule A ˚ into the aqueous phase. This model protrudes 50 /80 A
0022-0728/02/$ - see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 0 2 2 - 0 7 2 8 ( 0 2 ) 0 1 1 3 8 - 5
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Fig. 1. Model of a cytochrome c oxidase-modified electrode (not drawn to scale).
mimics the in vivo case for the enzyme in the inner mitochondrial membrane where the cytochrome c binding site protrudes into the intermembrane space [9 /14]. Phase contrast tapping mode-scanning force microscopy [15] has confirmed the structure shown in Fig. 1. This structure is thought to enable direct electron transfer between the electrode and cytochrome c oxidase by holding the heme a3 end close to the electrode surface. It is also important to recognize that this structure holds cytochrome c oxidase so that its cytochrome c binding site is exposed to solution. This is the same sort of configuration as for cytochrome c oxidase in the intermembrane space of the mitochondria in vivo that allows for its reaction with reduced cytochrome c. Briefly, this structure is built using a polycrystalline, vapor-deposited, gold quartz crystal microbalance (QCM) electrode. The gold quartz crystal electrode is pretreated by electrochemically depositing 1.6 monolayers of silver [16] for reasons described earlier [6]. This surface is then derivatized with octadecyl mercaptan (OM) using the QCM to monitor and stop the reaction when one-half monolayer is formed [6,16]. The enzymecontaining bilayer membrane is then formed by deoxycholate dialysis [4,6,17]. Again, cytochrome c oxidase is able to transfer electrons to the metal surface without added mediators or chemical modification of the enzyme [4,16]. Moreover, it has been shown by voltammetry and spectroelectrochemistry [4] and amperometry under sample flow conditions [5,6] that the cytochrome c oxidase is necessary to couple the oxidation of reduced cytochrome c in solution to the electrode in this system. In addition, cytochrome c oxidase has been shown to undergo a transition from the resting to the pulsed kinetic state upon turnover [5] as originally described by
Antonini et al. [18], and this transition can be reversibly induced through control of the electrode potential. Cytochrome c oxidase is a complex respiratory enzyme located in the inner mitochondrial membrane that has been extensively studied under solution-solubilized conditions. It is responsible for the four-electron reduction of molecular oxygen to water, and it also serves to pump protons from the matrix to the intermembrane space. The Gibbs energy associated with this gradient ultimately produces the driving force necessary for oxidative phosphorylation [19 /21]. Bovine cytochrome c oxidase, studied here, is comprised of 13 subunits and has an approximate molar mass of 200,000 Da [19,20]. The four redox centers (heme a, heme a3, CuA, and CuB) which reside in subunits I and II [9] are thought to enable electron transfer as follows: cyt. c 0/ CuA 0/heme a 0/heme a3/CuB site (O2 binding site) [22]. Its reaction partner, cytochrome c, is a small, soluble protein that delivers electrons from cytochrome c reductase [23]. Cytochrome c proteins have molar masses that range from 12,400 to 12,700 Da [24] with a single polypeptide chain containing from 103 to 113 amino acids [25]. Extensive work has been conducted to establish the region of the cytochrome c surface involved in binding to cytochrome c oxidase before electron transfer [26 / 29]. Alteration in the amino acid composition in this region changes its electron transfer kinetics with cytochrome c oxidase. Ferguson-Miller et al. mono-functionalized cytochrome c with carboxydinitrophenyl (CDNP) at eight lysine residues (8, 13, 27, 39, 60, 72, 87, and 22/99). The reactions of CDNP-modified cytochrome c with cytochrome c oxidase [28], cytochrome c reductase [29], and cytochrome c peroxidase [30] were consistent with the binding region involving several lysine residues surrounding the exposed heme edge of cytochrome c. Modification of lysines 13 and 72 resulted in highly inhibited kinetics, 8, 27, and 87 inhibited the reaction to a lesser extent, and 22, 60, 99, and 39 had a minimal effect on the kinetics of the reaction. The latter group is not located in close proximity to the exposed heme edge. The results given in this study indicate that lysines 8, 13, 27, and 72, which are in close proximity to the exposed heme edge on cytochrome c, are the sites of interaction with cytochrome c oxidase [28]. Chemical modification studies [31,32], site-directed mutagenesis [33 /37], computational methods [38], and the crystal structure of cytochrome c oxidase [9,19] reveal that there is patch of negatively charged residues located on subunit II of the oxidase that forms the putative cytochrome c binding site. Electrostatic binding interactions between cytochrome c and cytochrome c oxidase at these sites on both reaction partners are also controlled by ionic strength. For example, the optimum electron transfer
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rate between cytochrome c and cytochrome c oxidase was observed at an ionic strength of 50 /110 mM for Paracoccus denitrificans [35] and bovine sources [39], respectively. NMR has also been used in cases where structures are known to probe ‘soft-docking’ conformational changes attendant to binding for related protein/ enzyme systems [40,41]. The primary structure of cytochrome c varies with different organisms [42]. While cytochrome c from various organisms exhibits a high degree of sequence homology on the interior of the protein [43 /45], differences in amino acid composition do occur on the protein surface. For example, there are 19 differences in amino acid composition between horse and tuna cytochrome c [42], 11 differences between horse and rhesus monkey [46], three differences between horse and bovine [42], and one difference between horse and chicken cytochrome c [47]. Ferguson-Miller et al. [48] showed that in cases where the affinity of cytochrome c for oxidase was dramatically lowered, the rate of electron transfer from the cytochrome c to the oxidase also decreased. Amino acid substitutions that occur between cytochromes from different species can contribute to how effectively the protein binds and releases electrons to cytochrome c oxidase from a given species. The electron transfer reaction between cytochrome c from various species and the bovine cytochrome c oxidase immobilized on electrodes in this work were found to show subtle but reproducible differences. In earlier work cytochrome c from primate species (human, baboon, rhesus monkey) exhibited low electron transfer activity with bovine cytochrome c oxidase [49 /52]. Kinetic studies on cytochrome c from rhesus monkey and bovine species showed that they have identical binding rates with bovine cytochrome c oxidase. However, the rate of rhesus monkey cytochrome c dissociation from the oxidase is much lower, which accounts for the lowered activity [50]. The surface of the rhesus monkey cytochrome c surrounding the heme edge is more hydrophobic compared to the bovine protein [50,53]. The increase in hydrophobic character of this surface area of the rhesus monkey cytochrome may facilitate hydrophobic interactions with the surface of the oxidase causing the dissociation rate of the oxidase /cytochrome complex to be diminished [50]. In mammalian cytochrome c the net charge on this protein at neutral pH varies between /8.5 and /10 depending on its origin. The dipole moment of cytochrome c is large and results from the asymmetric charge distribution on the surface of the protein with values of 312 and 300 Debye for the oxidized and reduced forms of the protein, respectively [54]. The strong dipolar character of the molecule is thought to orient the cytochrome c molecule so that it can most easily react with either of its redox partners [49], i.e. the
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distance of electron transfer is minimized. Therefore, substitutions in the primary structure of surface residues can affect the dipole moment of the molecule thereby affecting its rate of electron transfer with cytochrome c oxidase from a specific source, i.e. bovine [55]. In this work fixed potential amperometry under flow conditions has been used to observe the oxidation of reduced cytochrome c from various species at oxidasemodified electrodes [5,6]. Reported here are the current responses which result from oxidase mediated electron transfer from solution-resident, reduced cytochrome c from various sources to the gold electrode. The current responses show that the rate of mediated electron transfer varies with the primary structure of the cytochrome delivering electrons. Cytochrome c from horse, rabbit, pig, pigeon, and tuna has been investigated. Horse cytochrome c exhibited the largest current response while tuna cytochrome c gave the smallest current response of the cytochrome c molecules studied. These data illustrate that the electrode-immobilized oxidase is sensitive to differences in the primary structure of the cytochrome under investigation.
2. Experimental Horse, rabbit, pig, pigeon, and tuna heart cytochrome c were all purchased from Sigma Chemical Company. These cytochromes c were purified, reduced, and desalted as described earlier [6]. The concentrations of the cytochrome c solutions used in these experiments were determined spectrophotometrically using a HP 8452A UV /vis diode array spectrophotometer. The molar extinction coefficient (l /550 nm) used for reduced horse heart cytochrome c was 29,500 M 1 cm 1 whereas the value for the other reduced cytochromes c mentioned above was taken to be 28,000 M 1 cm 1 [42,56]. The instrumentation, cell design, preparation of the oxidase-modified electrodes, and flow injection conditions have been reported previously [4 / 6,16]. The water used in all experiments was deionized and further purified using a Milli-Q system (Millipore Corporation) to exhibit a resistivity of 17/18 MV cm 1 upon delivery. The buffer used in all experiments was sodium phosphate (100 mM, pH 7.4, ACS reagent grade). Bovine cytochrome c oxidase was isolated from fresh beef hearts following the procedure of Soulimane and Buse [57] or Yoshikawa et al. [58]. In all flow injection experiments, the electrode was allowed to equilibrate with sodium phosphate buffer (0.1 M, pH 7.4) at a flow rate of 0.5 ml/min for approximately 30 min with the electrode poised at a potential of 472 mV versus SHE. Cytochrome c was then injected into the cell for approximately 100 s (0.83 ml total volume) followed by sodium phosphate buffer (0.1 M, pH 7.4) until the baseline current was re-
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established. This procedure was repeated three times for each cytochrome c and for each concentration used to confirm that the current responses were reproducible for the oxidase-modified electrode under investigation.
3. Results The electron transfer reaction of reduced cytochrome c in solution at the electrode (i.e. mediated electron transfer via the oxidase) has been investigated versus the concentration of cytochrome c to determine whether the kinetics of the reaction are mono- or biphasic. Fig. 2 (data given in Table 1, vide infra) shows a plot of steady state current response versus cytochrome c concentration in 100 mM, pH 7.4 phosphate buffer. The concentration of cytochrome c in this experiment ranges from 0.25 to 150 mM. A double reciprocal plot (1/ current vs. 1/[cyt. c]) of the data shown in Fig. 2 resulted in two lines of differing slopes indicating typical nonhyperbolic kinetics [59]. At low cytochrome c concentrations (0.5 /10 mM) a Km value of 109/6 mM was calculated while at higher concentrations of cytochrome c (18 /150 mM) a Km value of 1169/55 mM was obtained. Table 1 shows the experimental and theoretical limiting currents obtained in this investigation. The theoretical limiting currents were calculated using Eq. (1), which describes the limiting diffusion current for a wall jet electrode [60,61]. This equation predicts the limiting current obtained at an electrode where the flow is normal to the surface and the diameter of the flow stream is less than that of the electrode: Il 0:898nFcD2=3 n 5=12 a 1=2 A3=8 U 3=4
(1)
where n is the number of electrons, F is the Faraday constant (96,500 C mol 1), c is the concentration (mol cm 3), D is the diffusion coefficient (1.12 /10 6 cm2 s 1 for reduced horse heart cytochrome c), n is the
Fig. 2. Steady state current response of varying concentrations of reduced cytochrome c reacting at an oxidase-modified electrode. The electrode area is 0.2 cm2, the flow rate is 0.5 ml min 1, the buffer is 0.1 M phosphate (pH 7.4), and the applied potential is 472 mV vs. SHE.
Table 1 Summary of experimental and theoretical limiting currents obtained at an oxidase-modified electrode [cyt. c ]/mM
Il (exp.)/nA
Il (theor.)/nA
0.25 0.5 1.1 2.3 4.5 9.4 18 38 75 150
0.5 1.2 2.4 4.4 8.1 14 24 39 69 120
1 2 4 8 16 33 62 131 259 415
kinematic viscosity (0.01 cm2 s 1), a is the diameter of the wall jet (0.0794 cm), A is the active area of the electrode (0.2 cm2), and U is the average volume flow rate (0.0085 cm3 s 1). From the phase contrast tapping mode scanning force microscopy (TM-SFM) data reported earlier [15] the surface coverage of oxidase was estimated to be approximately 20%. If the electrode functions as a microelectrode array with overlap of the spherical diffusion profiles of each oxidase site and semi-infinite linear diffusion applies, then the entire geometric area of the electrode would be effectively active. The fit between experimental and theoretical limiting currents (Eq. (1)) at low concentrations of reduced cytochrome c (about 10 mM) is linear but with approximately one-half the slope. However, above about 10 mM the experimental currents are no longer linearly dependent on concentration indicating that the reaction is kinetically limited (i.e. nonhyperbolic kinetics). The data shown here are representative of replicate experiments conducted at more than 10 separate oxidase-modified electrodes. The limiting currents obtained from the oxidation of reduced cytochrome c at oxidase-modified electrodes depends on the history of the electrode, i.e. age of the electrode and the number of injections in a given time period. Therefore, an error of approximately 15% is associated with limiting currents reported for each concentration of cytochrome c shown in Table 1. The reproducibility of the limiting currents from oxidase-modified electrode to oxidase-modified electrode is approximately 30%. Despite the variability in the current responses from electrode to electrode, the data exhibit the same general trend (i.e. biphasic kinetics). Under aerobic conditions the current responses obtained for solutions of reduced cytochrome c from different sources vary. Cyclic voltammetric (CV) characterization was conducted on each oxidase-modified electrode used in this investigation to confirm that the enzyme is in the oxidized form at the potential used in
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these experiments (i.e. 472 mV vs. SHE). The concentration of all of the cytochrome c solutions used was 10 mM unless otherwise specified. Upon injection of horse heart cytochrome c , a sharp increase in oxidation current is observed. Between each injection of cytochrome c , the electrode is exposed to phosphate buffer alone (0.1 M, pH 7.4) until the baseline current response is obtained. The increase in oxidation current is due to electrons flowing from the reduced cytochrome c in solution to the electrode via the immobilized oxidase (i.e. mediated electron transfer). Control experiments at lipid bilayer membranes containing no oxidase show little or no electron transfer from cytochrome c in solution to the electrode (data not shown) confirming that the electrode immobilized oxidase mediates electron transfer [4,6]. Fig. 3 shows representative current responses obtained at an oxidase-modified electrode upon injection of: (a) horse; (b) pig; (c) rabbit; (d) pigeon; and (e) tuna heart cytochrome c . The oxidase used in this experiment was isolated following the method of Yoshikawa et al. [58]. The current responses for the various cytochromes used are smaller (approximately 33%) compared to the responses obtained at electrodes using oxidase isolated from the procedure of Soulimane and Buse [57]. This suggests that the oxidase isolated using the Yoshikawa preparation is less active than that obtained from the method of Soulimane and Buse or possibly a smaller amount of oxidase is immobilized on the electrode surface. Despite the smaller responses obtained with the Yoshikawa preparation, the trends in the data are still the same (vide infra). The data shown in Fig. 3 are representative of replicate experiments conducted at five separate oxidase-modified electrodes.
Fig. 3. FIA data of: (a) horse; (b) pig; (c) rabbit; (d) pigeon; and (e) tuna heart cytochrome c reacting at an oxidase-modified electrode. Each injection has a volume of 583 ml of 10 mM reduced cytochrome c . The electrode area is 0.2 cm2, the flow rate is 0.5 ml min 1, the buffer is 0.1 M phosphate (pH 7.4), and the applied potential is 472 mV vs. SHE.
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4. Discussion There are several explanations for nonhyperbolic kinetics with cytochrome c oxidase: (a) two active sites [62]; (b) a regulatory site mechanism [63,64]; (c) a negative cooperativity mechanism [65]; and (d) two conformations with differing affinities for substrate [59,66]. Michel and Bosshard [67] determined that there is a single cytochrome c binding site per heme aa3, and that the kinetics of the reaction between cytochrome c and oxidase are always biphasic, independent of the aggregation state of the enzyme. The results of this work are not consistent with mechanisms 1 /3 but support the conformational transition mechanism. Briefly, this mechanism states that the enzyme cycles between two conformations, E1 and E2, to accomplish proton pumping. Each of these conformations has a different affinity for cytochrome c that results in typical biphasic kinetics, i.e. the electron transfer rate becomes limited by the conformational transition. The results of the work presented here indicate that the kinetics of the cytochrome c /cytochrome c oxidase reaction are biphasic; however, the mechanism responsible for this behavior is not known. The amino acid sequence of pig, rabbit, pigeon, and tuna heart cytochrome c differs from that of horse. The number of amino acid substitutions is as follows: two (horse /pig), six (horse /rabbit), nine (horse /pigeon), and 19 (horse /tuna) and the positions of these substitutions are known as well [47]. As indicated in Fig. 3, there is a discernible difference in the current responses obtained for the cytochromes used here. On reviewer recommendation experiments have now been conducted at oxidase-modified electrodes to evaluate and compare the current responses upon injection of reduced bovine heart and horse heart cytochrome c (10 mM). There are three differences in the amino acid primary sequences between these two cytochromes, and the current responses obtained are not experimentally distinguishable from the results shown in Fig. 3 (data not shown). The steady state current responses obtained here decrease as the number of amino acid substitutions increases with the most dramatic difference in current response observed between horse and tuna heart cytochrome c. As mentioned above, the primary structure of tuna cytochrome c differs from that of horse cytochrome c by 19 amino acid residues. Given the differences that exist in the amino acid sequences between tuna and horse cytochrome c, the effect that a specific amino acid substitution has on the rate of electron transfer has not been explicitly assessed. However, the difference in the rates of electron transfer observed in this investigation may be explained by variation in the hydrophobicity of the two proteins resulting from amino acid substitutions. Earlier work in this laboratory has shown that cytochromes c from
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various species have very similar reaction center entropy changes (DS rc ) at pH 7.0 indicating that these amino acid differences do not substantially alter the structure of the reaction center [47]. The differences in amino acid composition between horse and tuna heart cytochrome c do result in a change in the dipolar character of the molecule [47]. Molecular modeling calculations were used to compare the hydrophobicity of the two proteins. From these calculations it is evident that the substitutions occurring on the surface of the tuna cytochrome c result in a more hydrophobic molecule. Of the 19 changes in primary structure, only four occur in the interior of the protein [47]. These changes, which are located near the bottom of the heme crevice, result in a more hydrophilic environment around the heme. The most stable arrangement for a protein is one in which the hydrophobic residues are buried and the hydrophilic residues are on the surface where they can interact with solvent. The differences in amino acid composition between horse and tuna cytochrome c create a less stable structure for the tuna heart protein [47]. As mentioned above, rhesus monkey and bovine cytochrome c exhibited the same binding rates (109 / 1010 M 1 s 1) with bovine cytochrome c oxidase [49,50]. However, the equilibrium dissociation constant for the bovine/bovine case (10 8 M) is about a factor of 10 greater than the value observed for the rhesus monkey/bovine case. There are nine differences in amino acid composition between bovine and rhesus monkey cytochrome c . Four of these changes (11, 12, 15, and 83) cause a decrease in the polar nature of the molecule around the exposed heme edge. Ferguson-Miller et al. [50] attributed the decrease in dissociation constant to increased hydrophobic bonding between the rhesus cytochrome c and the bovine oxidase. Two interpretations of the results obtained in this study are presented. The hydrophobic nature of the surfaces of tuna, pigeon, and rabbit cytochrome c could affect the orientation of the molecule with respect to the binding site on the oxidase. From molecular modeling calculations [47], it is clear that tuna cytochrome c is comparatively more hydrophobic than horse cytochrome c . The more hydrophobic surface residues found in tuna cytochrome c could cause the dipolar character of the molecule to be diminished thereby affecting its binding capacity with the bovine oxidase. The dipolar nature of the cytochrome serves to orient the molecule so that binding with the oxidase results in electron transfer as noted earlier. An increase in surface hydrophobicity could change the cytochrome binding. If this were the case, then binding of the cytochrome to the oxidase would limit the kinetics of electron transfer. Alternatively, because cytochromes from different species have similar or identical binding rates with bovine oxidase [49,50], the difference in current response
obtained in this study could be due to a difference in the rate of dissociation of the cytochrome from the oxidase. The increase in surface hydrophobicity for tuna cytochrome relative to the horse cytochrome could cause the tuna protein to remain bound to the oxidase longer due to hydrophobic interactions with the surface of the oxidase [50]. It is clear from the results presented that the immobilized bovine oxidase is capable of distinguishing between cytochromes from different species. However, the shape of the current response shown in Fig. 3e for the tuna case is more consistent with the latter interpretation. Note the decrease in oxidation current observed at longer times (approximately 100 s) compared with the initial maximum current (approximately 70 s). This suggests that the tuna cytochrome c molecules remain bound to the active site of the immobilized bovine cytochrome c oxidase molecules longer due to the increased surface hydrophobicity of the tuna cytochrome c compared with the horse cytochrome c .
5. Conclusions The electron transfer kinetics of the cytochrome c oxidase/cytochrome c reaction studied here are biphasic in agreement with previous literature reports. The current responses measured following injections of reduced cytochrome c from different sources at cytochrome c oxidase immobilized in electrode-supported lipid bilayer membranes vary. The oxidase electron transfer reactions depend on the primary structure of the cytochrome c molecules studied here. The differences in the current responses correlate with amino acid sequence changes that impact the surface hydrophobicity of the cytochrome c molecule. The ability to use the native selectivity of the cytochrome c oxidase enzyme system coupled to an electrode to distinguish between protein structure is exciting. Experiments aimed at further delineating the factors that contribute to this enzymatic selectivity are underway. Specifically, horse heart cytochrome c with varying degrees of lysine acetylation are being studied to gauge further the role of electrostatic interactions in promoting electron transfer from cytochrome c to the oxidase. Myocardial damage induced during ischemia and reperfusion decreases oxidative phosphorylation, in part, through generation of reactive oxygen species and oxidative damage to the components of the mitochondrial electron transport chain. The cytochrome c binding site of cytochrome c reductase has been identified as a site of ischemic injury [68] and decreased cytochrome c oxidase activity is also observed [69]. Recent studies suggest that oxidative damage to cytochrome c may also contribute to decreased electron transport after reperfusion [69]. The oxidase-modified electrodes described
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herein should be useful for characterizing the electron transfer reactions of cytochrome c isolated from ischemic and control hearts.
Acknowledgements Dr Bertha C. King, Dr Zoia Nikolaeva, and Professor Mikhail Smirnov are gratefully acknowledged for the isolation of the cytochrome c oxidase. We also acknowledge the National Science Foundation (Grant NSF CHE-0071777) for support of this research.
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The reaction of cytochrome c from different species with cytochrome c oxidase immobilized in an electrode supported lipid bilayer membrane Melissa C. Rhoten a, James D. Burgess b, Fred M. Hawkridge c,* a
Department of Natural Sciences, Longwood University, Farmville, VA 23909, USA Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA c Department of Chemistry, Virginia Commonwealth University, Box 842006, Richmond, VA 23284, USA b
Received 28 June 2002; received in revised form 6 August 2002; accepted 14 August 2002
Abstract In past work the direct electron transfer reactions of bovine cytochrome c oxidase in an electrode-supported lipid bilayer membrane have been studied. Its reaction with cytochrome c in solution was also studied and found to be consistent with previous solution studies. In this work it is shown that the electron transfer reactions of cytochrome c oxidase in this electrode-supported lipid bilayer membrane depend on the source of cytochrome c . This property has also been widely studied for solution samples. The differences in the electron transfer reaction rates correlate with the differences in the amino acid sequence for the cytochrome c molecules studied. Electrochemical results suggest that the dissociation of the cytochrome c /cytochrome c oxidase reaction complex is the rate-controlling step in this electron transfer mechanism for cytochrome c from some sources. Moreover, the electron transfer reaction mechanism exhibits biphasic reaction kinetics, which is consistent with earlier work on reactions between solubilized cytochrome c oxidase/cytochrome c samples. These results indicate that the cytochrome c oxidase modified electrodes described herein could be used to distinguish amino acid sequence variations in proteins such as cytochrome c , and this has potential relevance as a diagnostic for disease states. # 2002 Published by Elsevier Science B.V. Keywords: Immobilized cytochrome c oxidase; Lipid bilayer membrane; Cytochrome c ; Enzyme kinetics; Proteomics
1. Introduction The electron transfer reactions of proteins and enzymes at electrodes can now be studied and controlled under a variety of conditions that mimic some of their native reaction properties [1]. Electron transfer communication between more structurally and functionally complex enzymes with electrodes has been facilitated by redox mediators, electron relays, and chemically modified electrodes [2]. Many redox enzymes are membrane-bound and function within the confines of a phospholipid bilayer. Embedding or adsorbing integral membrane enzymes in or on electrode-supported lipid bilayer membranes facilitates direct electron trans* Corresponding author. Tel.: /1-804-828-7505; fax: /1-804-8288599 E-mail address: [email protected] (F.M. Hawkridge).
fer and stabilizes the enzyme against denaturation [3 /8]. ˚, The dimensions of these structures, approximately 50 A also minimize mass transfer concerns involving analytes, substrates, mediators, counter ions, and detected redox active products in micron thick films containing enzymes on an electrode. Opportunities to exploit the native catalytic activity and selectivity of an enzyme can be envisioned in sensor applications, biomass conversion reactors, or in driving fuel cell applications when combined with an electrode held at a controlled potential. In this work cytochrome c oxidase is immobilized in an electrode-supported lipid bilayer membrane [6]. Fig. 1 shows a model of the electrode-supported lipid bilayer membrane with an embedded oxidase molecule. The diameter of the oxidase is known to be approximately 80 ˚ , and the larger hydrophilic end of the molecule A ˚ into the aqueous phase. This model protrudes 50 /80 A
0022-0728/02/$ - see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 0 2 2 - 0 7 2 8 ( 0 2 ) 0 1 1 3 8 - 5
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Fig. 1. Model of a cytochrome c oxidase-modified electrode (not drawn to scale).
mimics the in vivo case for the enzyme in the inner mitochondrial membrane where the cytochrome c binding site protrudes into the intermembrane space [9 /14]. Phase contrast tapping mode-scanning force microscopy [15] has confirmed the structure shown in Fig. 1. This structure is thought to enable direct electron transfer between the electrode and cytochrome c oxidase by holding the heme a3 end close to the electrode surface. It is also important to recognize that this structure holds cytochrome c oxidase so that its cytochrome c binding site is exposed to solution. This is the same sort of configuration as for cytochrome c oxidase in the intermembrane space of the mitochondria in vivo that allows for its reaction with reduced cytochrome c. Briefly, this structure is built using a polycrystalline, vapor-deposited, gold quartz crystal microbalance (QCM) electrode. The gold quartz crystal electrode is pretreated by electrochemically depositing 1.6 monolayers of silver [16] for reasons described earlier [6]. This surface is then derivatized with octadecyl mercaptan (OM) using the QCM to monitor and stop the reaction when one-half monolayer is formed [6,16]. The enzymecontaining bilayer membrane is then formed by deoxycholate dialysis [4,6,17]. Again, cytochrome c oxidase is able to transfer electrons to the metal surface without added mediators or chemical modification of the enzyme [4,16]. Moreover, it has been shown by voltammetry and spectroelectrochemistry [4] and amperometry under sample flow conditions [5,6] that the cytochrome c oxidase is necessary to couple the oxidation of reduced cytochrome c in solution to the electrode in this system. In addition, cytochrome c oxidase has been shown to undergo a transition from the resting to the pulsed kinetic state upon turnover [5] as originally described by
Antonini et al. [18], and this transition can be reversibly induced through control of the electrode potential. Cytochrome c oxidase is a complex respiratory enzyme located in the inner mitochondrial membrane that has been extensively studied under solution-solubilized conditions. It is responsible for the four-electron reduction of molecular oxygen to water, and it also serves to pump protons from the matrix to the intermembrane space. The Gibbs energy associated with this gradient ultimately produces the driving force necessary for oxidative phosphorylation [19 /21]. Bovine cytochrome c oxidase, studied here, is comprised of 13 subunits and has an approximate molar mass of 200,000 Da [19,20]. The four redox centers (heme a, heme a3, CuA, and CuB) which reside in subunits I and II [9] are thought to enable electron transfer as follows: cyt. c 0/ CuA 0/heme a 0/heme a3/CuB site (O2 binding site) [22]. Its reaction partner, cytochrome c, is a small, soluble protein that delivers electrons from cytochrome c reductase [23]. Cytochrome c proteins have molar masses that range from 12,400 to 12,700 Da [24] with a single polypeptide chain containing from 103 to 113 amino acids [25]. Extensive work has been conducted to establish the region of the cytochrome c surface involved in binding to cytochrome c oxidase before electron transfer [26 / 29]. Alteration in the amino acid composition in this region changes its electron transfer kinetics with cytochrome c oxidase. Ferguson-Miller et al. mono-functionalized cytochrome c with carboxydinitrophenyl (CDNP) at eight lysine residues (8, 13, 27, 39, 60, 72, 87, and 22/99). The reactions of CDNP-modified cytochrome c with cytochrome c oxidase [28], cytochrome c reductase [29], and cytochrome c peroxidase [30] were consistent with the binding region involving several lysine residues surrounding the exposed heme edge of cytochrome c. Modification of lysines 13 and 72 resulted in highly inhibited kinetics, 8, 27, and 87 inhibited the reaction to a lesser extent, and 22, 60, 99, and 39 had a minimal effect on the kinetics of the reaction. The latter group is not located in close proximity to the exposed heme edge. The results given in this study indicate that lysines 8, 13, 27, and 72, which are in close proximity to the exposed heme edge on cytochrome c, are the sites of interaction with cytochrome c oxidase [28]. Chemical modification studies [31,32], site-directed mutagenesis [33 /37], computational methods [38], and the crystal structure of cytochrome c oxidase [9,19] reveal that there is patch of negatively charged residues located on subunit II of the oxidase that forms the putative cytochrome c binding site. Electrostatic binding interactions between cytochrome c and cytochrome c oxidase at these sites on both reaction partners are also controlled by ionic strength. For example, the optimum electron transfer
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rate between cytochrome c and cytochrome c oxidase was observed at an ionic strength of 50 /110 mM for Paracoccus denitrificans [35] and bovine sources [39], respectively. NMR has also been used in cases where structures are known to probe ‘soft-docking’ conformational changes attendant to binding for related protein/ enzyme systems [40,41]. The primary structure of cytochrome c varies with different organisms [42]. While cytochrome c from various organisms exhibits a high degree of sequence homology on the interior of the protein [43 /45], differences in amino acid composition do occur on the protein surface. For example, there are 19 differences in amino acid composition between horse and tuna cytochrome c [42], 11 differences between horse and rhesus monkey [46], three differences between horse and bovine [42], and one difference between horse and chicken cytochrome c [47]. Ferguson-Miller et al. [48] showed that in cases where the affinity of cytochrome c for oxidase was dramatically lowered, the rate of electron transfer from the cytochrome c to the oxidase also decreased. Amino acid substitutions that occur between cytochromes from different species can contribute to how effectively the protein binds and releases electrons to cytochrome c oxidase from a given species. The electron transfer reaction between cytochrome c from various species and the bovine cytochrome c oxidase immobilized on electrodes in this work were found to show subtle but reproducible differences. In earlier work cytochrome c from primate species (human, baboon, rhesus monkey) exhibited low electron transfer activity with bovine cytochrome c oxidase [49 /52]. Kinetic studies on cytochrome c from rhesus monkey and bovine species showed that they have identical binding rates with bovine cytochrome c oxidase. However, the rate of rhesus monkey cytochrome c dissociation from the oxidase is much lower, which accounts for the lowered activity [50]. The surface of the rhesus monkey cytochrome c surrounding the heme edge is more hydrophobic compared to the bovine protein [50,53]. The increase in hydrophobic character of this surface area of the rhesus monkey cytochrome may facilitate hydrophobic interactions with the surface of the oxidase causing the dissociation rate of the oxidase /cytochrome complex to be diminished [50]. In mammalian cytochrome c the net charge on this protein at neutral pH varies between /8.5 and /10 depending on its origin. The dipole moment of cytochrome c is large and results from the asymmetric charge distribution on the surface of the protein with values of 312 and 300 Debye for the oxidized and reduced forms of the protein, respectively [54]. The strong dipolar character of the molecule is thought to orient the cytochrome c molecule so that it can most easily react with either of its redox partners [49], i.e. the
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distance of electron transfer is minimized. Therefore, substitutions in the primary structure of surface residues can affect the dipole moment of the molecule thereby affecting its rate of electron transfer with cytochrome c oxidase from a specific source, i.e. bovine [55]. In this work fixed potential amperometry under flow conditions has been used to observe the oxidation of reduced cytochrome c from various species at oxidasemodified electrodes [5,6]. Reported here are the current responses which result from oxidase mediated electron transfer from solution-resident, reduced cytochrome c from various sources to the gold electrode. The current responses show that the rate of mediated electron transfer varies with the primary structure of the cytochrome delivering electrons. Cytochrome c from horse, rabbit, pig, pigeon, and tuna has been investigated. Horse cytochrome c exhibited the largest current response while tuna cytochrome c gave the smallest current response of the cytochrome c molecules studied. These data illustrate that the electrode-immobilized oxidase is sensitive to differences in the primary structure of the cytochrome under investigation.
2. Experimental Horse, rabbit, pig, pigeon, and tuna heart cytochrome c were all purchased from Sigma Chemical Company. These cytochromes c were purified, reduced, and desalted as described earlier [6]. The concentrations of the cytochrome c solutions used in these experiments were determined spectrophotometrically using a HP 8452A UV /vis diode array spectrophotometer. The molar extinction coefficient (l /550 nm) used for reduced horse heart cytochrome c was 29,500 M 1 cm 1 whereas the value for the other reduced cytochromes c mentioned above was taken to be 28,000 M 1 cm 1 [42,56]. The instrumentation, cell design, preparation of the oxidase-modified electrodes, and flow injection conditions have been reported previously [4 / 6,16]. The water used in all experiments was deionized and further purified using a Milli-Q system (Millipore Corporation) to exhibit a resistivity of 17/18 MV cm 1 upon delivery. The buffer used in all experiments was sodium phosphate (100 mM, pH 7.4, ACS reagent grade). Bovine cytochrome c oxidase was isolated from fresh beef hearts following the procedure of Soulimane and Buse [57] or Yoshikawa et al. [58]. In all flow injection experiments, the electrode was allowed to equilibrate with sodium phosphate buffer (0.1 M, pH 7.4) at a flow rate of 0.5 ml/min for approximately 30 min with the electrode poised at a potential of 472 mV versus SHE. Cytochrome c was then injected into the cell for approximately 100 s (0.83 ml total volume) followed by sodium phosphate buffer (0.1 M, pH 7.4) until the baseline current was re-
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established. This procedure was repeated three times for each cytochrome c and for each concentration used to confirm that the current responses were reproducible for the oxidase-modified electrode under investigation.
3. Results The electron transfer reaction of reduced cytochrome c in solution at the electrode (i.e. mediated electron transfer via the oxidase) has been investigated versus the concentration of cytochrome c to determine whether the kinetics of the reaction are mono- or biphasic. Fig. 2 (data given in Table 1, vide infra) shows a plot of steady state current response versus cytochrome c concentration in 100 mM, pH 7.4 phosphate buffer. The concentration of cytochrome c in this experiment ranges from 0.25 to 150 mM. A double reciprocal plot (1/ current vs. 1/[cyt. c]) of the data shown in Fig. 2 resulted in two lines of differing slopes indicating typical nonhyperbolic kinetics [59]. At low cytochrome c concentrations (0.5 /10 mM) a Km value of 109/6 mM was calculated while at higher concentrations of cytochrome c (18 /150 mM) a Km value of 1169/55 mM was obtained. Table 1 shows the experimental and theoretical limiting currents obtained in this investigation. The theoretical limiting currents were calculated using Eq. (1), which describes the limiting diffusion current for a wall jet electrode [60,61]. This equation predicts the limiting current obtained at an electrode where the flow is normal to the surface and the diameter of the flow stream is less than that of the electrode: Il 0:898nFcD2=3 n 5=12 a 1=2 A3=8 U 3=4
(1)
where n is the number of electrons, F is the Faraday constant (96,500 C mol 1), c is the concentration (mol cm 3), D is the diffusion coefficient (1.12 /10 6 cm2 s 1 for reduced horse heart cytochrome c), n is the
Fig. 2. Steady state current response of varying concentrations of reduced cytochrome c reacting at an oxidase-modified electrode. The electrode area is 0.2 cm2, the flow rate is 0.5 ml min 1, the buffer is 0.1 M phosphate (pH 7.4), and the applied potential is 472 mV vs. SHE.
Table 1 Summary of experimental and theoretical limiting currents obtained at an oxidase-modified electrode [cyt. c ]/mM
Il (exp.)/nA
Il (theor.)/nA
0.25 0.5 1.1 2.3 4.5 9.4 18 38 75 150
0.5 1.2 2.4 4.4 8.1 14 24 39 69 120
1 2 4 8 16 33 62 131 259 415
kinematic viscosity (0.01 cm2 s 1), a is the diameter of the wall jet (0.0794 cm), A is the active area of the electrode (0.2 cm2), and U is the average volume flow rate (0.0085 cm3 s 1). From the phase contrast tapping mode scanning force microscopy (TM-SFM) data reported earlier [15] the surface coverage of oxidase was estimated to be approximately 20%. If the electrode functions as a microelectrode array with overlap of the spherical diffusion profiles of each oxidase site and semi-infinite linear diffusion applies, then the entire geometric area of the electrode would be effectively active. The fit between experimental and theoretical limiting currents (Eq. (1)) at low concentrations of reduced cytochrome c (about 10 mM) is linear but with approximately one-half the slope. However, above about 10 mM the experimental currents are no longer linearly dependent on concentration indicating that the reaction is kinetically limited (i.e. nonhyperbolic kinetics). The data shown here are representative of replicate experiments conducted at more than 10 separate oxidase-modified electrodes. The limiting currents obtained from the oxidation of reduced cytochrome c at oxidase-modified electrodes depends on the history of the electrode, i.e. age of the electrode and the number of injections in a given time period. Therefore, an error of approximately 15% is associated with limiting currents reported for each concentration of cytochrome c shown in Table 1. The reproducibility of the limiting currents from oxidase-modified electrode to oxidase-modified electrode is approximately 30%. Despite the variability in the current responses from electrode to electrode, the data exhibit the same general trend (i.e. biphasic kinetics). Under aerobic conditions the current responses obtained for solutions of reduced cytochrome c from different sources vary. Cyclic voltammetric (CV) characterization was conducted on each oxidase-modified electrode used in this investigation to confirm that the enzyme is in the oxidized form at the potential used in
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these experiments (i.e. 472 mV vs. SHE). The concentration of all of the cytochrome c solutions used was 10 mM unless otherwise specified. Upon injection of horse heart cytochrome c , a sharp increase in oxidation current is observed. Between each injection of cytochrome c , the electrode is exposed to phosphate buffer alone (0.1 M, pH 7.4) until the baseline current response is obtained. The increase in oxidation current is due to electrons flowing from the reduced cytochrome c in solution to the electrode via the immobilized oxidase (i.e. mediated electron transfer). Control experiments at lipid bilayer membranes containing no oxidase show little or no electron transfer from cytochrome c in solution to the electrode (data not shown) confirming that the electrode immobilized oxidase mediates electron transfer [4,6]. Fig. 3 shows representative current responses obtained at an oxidase-modified electrode upon injection of: (a) horse; (b) pig; (c) rabbit; (d) pigeon; and (e) tuna heart cytochrome c . The oxidase used in this experiment was isolated following the method of Yoshikawa et al. [58]. The current responses for the various cytochromes used are smaller (approximately 33%) compared to the responses obtained at electrodes using oxidase isolated from the procedure of Soulimane and Buse [57]. This suggests that the oxidase isolated using the Yoshikawa preparation is less active than that obtained from the method of Soulimane and Buse or possibly a smaller amount of oxidase is immobilized on the electrode surface. Despite the smaller responses obtained with the Yoshikawa preparation, the trends in the data are still the same (vide infra). The data shown in Fig. 3 are representative of replicate experiments conducted at five separate oxidase-modified electrodes.
Fig. 3. FIA data of: (a) horse; (b) pig; (c) rabbit; (d) pigeon; and (e) tuna heart cytochrome c reacting at an oxidase-modified electrode. Each injection has a volume of 583 ml of 10 mM reduced cytochrome c . The electrode area is 0.2 cm2, the flow rate is 0.5 ml min 1, the buffer is 0.1 M phosphate (pH 7.4), and the applied potential is 472 mV vs. SHE.
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4. Discussion There are several explanations for nonhyperbolic kinetics with cytochrome c oxidase: (a) two active sites [62]; (b) a regulatory site mechanism [63,64]; (c) a negative cooperativity mechanism [65]; and (d) two conformations with differing affinities for substrate [59,66]. Michel and Bosshard [67] determined that there is a single cytochrome c binding site per heme aa3, and that the kinetics of the reaction between cytochrome c and oxidase are always biphasic, independent of the aggregation state of the enzyme. The results of this work are not consistent with mechanisms 1 /3 but support the conformational transition mechanism. Briefly, this mechanism states that the enzyme cycles between two conformations, E1 and E2, to accomplish proton pumping. Each of these conformations has a different affinity for cytochrome c that results in typical biphasic kinetics, i.e. the electron transfer rate becomes limited by the conformational transition. The results of the work presented here indicate that the kinetics of the cytochrome c /cytochrome c oxidase reaction are biphasic; however, the mechanism responsible for this behavior is not known. The amino acid sequence of pig, rabbit, pigeon, and tuna heart cytochrome c differs from that of horse. The number of amino acid substitutions is as follows: two (horse /pig), six (horse /rabbit), nine (horse /pigeon), and 19 (horse /tuna) and the positions of these substitutions are known as well [47]. As indicated in Fig. 3, there is a discernible difference in the current responses obtained for the cytochromes used here. On reviewer recommendation experiments have now been conducted at oxidase-modified electrodes to evaluate and compare the current responses upon injection of reduced bovine heart and horse heart cytochrome c (10 mM). There are three differences in the amino acid primary sequences between these two cytochromes, and the current responses obtained are not experimentally distinguishable from the results shown in Fig. 3 (data not shown). The steady state current responses obtained here decrease as the number of amino acid substitutions increases with the most dramatic difference in current response observed between horse and tuna heart cytochrome c. As mentioned above, the primary structure of tuna cytochrome c differs from that of horse cytochrome c by 19 amino acid residues. Given the differences that exist in the amino acid sequences between tuna and horse cytochrome c, the effect that a specific amino acid substitution has on the rate of electron transfer has not been explicitly assessed. However, the difference in the rates of electron transfer observed in this investigation may be explained by variation in the hydrophobicity of the two proteins resulting from amino acid substitutions. Earlier work in this laboratory has shown that cytochromes c from
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various species have very similar reaction center entropy changes (DS rc ) at pH 7.0 indicating that these amino acid differences do not substantially alter the structure of the reaction center [47]. The differences in amino acid composition between horse and tuna heart cytochrome c do result in a change in the dipolar character of the molecule [47]. Molecular modeling calculations were used to compare the hydrophobicity of the two proteins. From these calculations it is evident that the substitutions occurring on the surface of the tuna cytochrome c result in a more hydrophobic molecule. Of the 19 changes in primary structure, only four occur in the interior of the protein [47]. These changes, which are located near the bottom of the heme crevice, result in a more hydrophilic environment around the heme. The most stable arrangement for a protein is one in which the hydrophobic residues are buried and the hydrophilic residues are on the surface where they can interact with solvent. The differences in amino acid composition between horse and tuna cytochrome c create a less stable structure for the tuna heart protein [47]. As mentioned above, rhesus monkey and bovine cytochrome c exhibited the same binding rates (109 / 1010 M 1 s 1) with bovine cytochrome c oxidase [49,50]. However, the equilibrium dissociation constant for the bovine/bovine case (10 8 M) is about a factor of 10 greater than the value observed for the rhesus monkey/bovine case. There are nine differences in amino acid composition between bovine and rhesus monkey cytochrome c . Four of these changes (11, 12, 15, and 83) cause a decrease in the polar nature of the molecule around the exposed heme edge. Ferguson-Miller et al. [50] attributed the decrease in dissociation constant to increased hydrophobic bonding between the rhesus cytochrome c and the bovine oxidase. Two interpretations of the results obtained in this study are presented. The hydrophobic nature of the surfaces of tuna, pigeon, and rabbit cytochrome c could affect the orientation of the molecule with respect to the binding site on the oxidase. From molecular modeling calculations [47], it is clear that tuna cytochrome c is comparatively more hydrophobic than horse cytochrome c . The more hydrophobic surface residues found in tuna cytochrome c could cause the dipolar character of the molecule to be diminished thereby affecting its binding capacity with the bovine oxidase. The dipolar nature of the cytochrome serves to orient the molecule so that binding with the oxidase results in electron transfer as noted earlier. An increase in surface hydrophobicity could change the cytochrome binding. If this were the case, then binding of the cytochrome to the oxidase would limit the kinetics of electron transfer. Alternatively, because cytochromes from different species have similar or identical binding rates with bovine oxidase [49,50], the difference in current response
obtained in this study could be due to a difference in the rate of dissociation of the cytochrome from the oxidase. The increase in surface hydrophobicity for tuna cytochrome relative to the horse cytochrome could cause the tuna protein to remain bound to the oxidase longer due to hydrophobic interactions with the surface of the oxidase [50]. It is clear from the results presented that the immobilized bovine oxidase is capable of distinguishing between cytochromes from different species. However, the shape of the current response shown in Fig. 3e for the tuna case is more consistent with the latter interpretation. Note the decrease in oxidation current observed at longer times (approximately 100 s) compared with the initial maximum current (approximately 70 s). This suggests that the tuna cytochrome c molecules remain bound to the active site of the immobilized bovine cytochrome c oxidase molecules longer due to the increased surface hydrophobicity of the tuna cytochrome c compared with the horse cytochrome c .
5. Conclusions The electron transfer kinetics of the cytochrome c oxidase/cytochrome c reaction studied here are biphasic in agreement with previous literature reports. The current responses measured following injections of reduced cytochrome c from different sources at cytochrome c oxidase immobilized in electrode-supported lipid bilayer membranes vary. The oxidase electron transfer reactions depend on the primary structure of the cytochrome c molecules studied here. The differences in the current responses correlate with amino acid sequence changes that impact the surface hydrophobicity of the cytochrome c molecule. The ability to use the native selectivity of the cytochrome c oxidase enzyme system coupled to an electrode to distinguish between protein structure is exciting. Experiments aimed at further delineating the factors that contribute to this enzymatic selectivity are underway. Specifically, horse heart cytochrome c with varying degrees of lysine acetylation are being studied to gauge further the role of electrostatic interactions in promoting electron transfer from cytochrome c to the oxidase. Myocardial damage induced during ischemia and reperfusion decreases oxidative phosphorylation, in part, through generation of reactive oxygen species and oxidative damage to the components of the mitochondrial electron transport chain. The cytochrome c binding site of cytochrome c reductase has been identified as a site of ischemic injury [68] and decreased cytochrome c oxidase activity is also observed [69]. Recent studies suggest that oxidative damage to cytochrome c may also contribute to decreased electron transport after reperfusion [69]. The oxidase-modified electrodes described
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herein should be useful for characterizing the electron transfer reactions of cytochrome c isolated from ischemic and control hearts.
Acknowledgements Dr Bertha C. King, Dr Zoia Nikolaeva, and Professor Mikhail Smirnov are gratefully acknowledged for the isolation of the cytochrome c oxidase. We also acknowledge the National Science Foundation (Grant NSF CHE-0071777) for support of this research.
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