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Mitochondrial Inner Membrane Biomimic for the Investigation of Electron Transport Chain Supercomplex Bioelectrocatalysis Lindsey N Pelster, and Shelley D. Minteer ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b00950 • Publication Date (Web): 22 Jun 2016 Downloaded from http://pubs.acs.org on June 23, 2016

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Mitochondrial Inner Membrane Biomimic for the Investigation of Electron Transport Chain Supercomplex Bioelectrocatalysis Lindsey N. Pelster and Shelley D. Minteer* Department of Chemistry, University of Utah, Salt Lake City, UT 84112

ABSTRACT Researchers have proposed that the efficiency of the electron transport chain is due to the synergy between Complex I, III, and IV in the membrane. In this paper, the enzymes of the supercomplex were isolated together, reconstituted into lipids that mimic the inner membrane of mitochondria and immobilized in a tethered lipid bilayer on a gold electrode. The supercomplex enzymes retained their activity with the addition of their substrates with high currents and were inhibited by their respective toxins. The bioelectrocatalytic studies indicate the interdependency of the activity of the different complexes in the bioelectrocatalysis of the electron transport chain supercomplex.

These fundamental studies provide a starting point to consider the use of

supercomplexes and enzyme cascades for bioenergy conversion applications and biosensing through the regulation of the activity by inhibition.

KEYWORDS: electron transport chain, metabolon, bioelectrocatalysis, tethered lipid bilayer, cytochrome c, enzyme cascade

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INTRODUCTION

The electron transport chain (ETC) is a group of five membrane enzymes that are the driving force for the conversion of energy in mitochondria. The ETC enzymes shuttle electrons through their substrates (NADH, ubiquinone, and cytochrome c), producing a proton gradient along the inner membrane of the mitochondria that is used to produce ATP.1 NADH is received from the citric acid cycle in the mitochondrial matrix, where Complex I (E.C. 1.6.5.3) oxidizes it to NAD+, passing the electrons to ubiquinone to form ubiquinol. Ubiquinol is oxidized by Complex III (E.C. 1.10.2.2) and electrons are passed to oxidized cytochrome c to be reduced. Reduced cytochrome c diffuses in the intermembrane space to nearby Complex IV (E.C. 1.9.3.1), where it is oxidized and the electrons passed to reduce molecular oxygen to water. The original enzyme organization model was based on the fluid state of enzymes in the membrane.2 As the individual complexes were purified and studied, Complexes I and III and Complexes III and IV were found to be associated in purified fractions.3 These new developments led to an evolution of the model from a fluid to solid description, where the enzyme complexes aggregate and associate together in the membrane.4 Three ETC enzymes form the ETC supercomplex, where Complex I, dimer Complex III, and various copies of Complex IV are electrostatically, hydrophobically, structurally, and catalytically connected in a stoichiometric assembly referred to as a supercomplex.5 The ETC supercomplex was first studied by detergent solubilization of mitochondria and blue native gel electrophoresis by Schagger et al.,6 leading to a general description of the complexes and a theory on the protein-protein interactions responsible for the supercomplex formation. Multiple studies on kinetic analysis, gene knockouts, and electron microscopy have provided more insight into the specific interactions, stability, and catalytic enhancement of the enzymes together.7 This


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enzyme cascade can be described as a metabolon, which has protein-protein and electrostatic interactions responsible for transferring the substrate intermediate from one enzyme active site to the next, with minimal substrate diffusion into the bulk solution. This substrate channeling of ubiquinone and cytochrome c increases efficient electron transfer between ETC complexes in the membrane and intermembrane space,8 leading to higher overall enzymatic catalytic rates for cellular respiration.5d, 9 For kinetic studies, the electron transport chain was examined by metabolic control analysis, where the effect of individual enzyme activity on the overall flux through the complex is evaluated. Potato tuber mitochondria showed that Complex IV had a much higher flux control coefficient than beef heart mitochondria.7b

The ETC supercomplex has been purified and

reconstituted with amphiphols for cryo-transmission electron microscopy to provide a structural view of the supercomplex,3,6a,10 although many characteristics of the operation of the ETC supercomplex are unanswered by in vitro studies. Therefore, electrochemically studying the ETC supercomplex in a membrane biomimic structure will allow more insights into the electron transport properties of these important bioenergetic enzymes. The lessons learned from this important enzyme chain can provide fundamental knowledge for utilizing multi-enzyme cascades for complete oxidation of substrates in the bioelectrochemistry field and discover more about the biological energy conversion in cells that can be used as an inspiration for biofuel cell energy applications or other bioelectronics devices. While soluble enzymes can be easily immobilized on electrodes, integral membrane enzymes need the structural support of a lipid bilayer to retain their activity. Advancements have been made to incorporate membrane-bound enzymes into lipid bilayers by supported, tethered, and polymer cushioned membrane architectures on solid surfaces.11,12 With increased interest in


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studying metabolic and cellular processes in an in-vivo state, tethered lipid bilayers permit in depth studies of membrane enzyme redox complexes and bioelectrocatalysis that are important to cellular energy. Recent studies have detailed the bioelectrocatalytic processes of the individual ETC complexes on various electrode structures13 and structural, kinetic, and genetic evidence prove the formation of the ETC supercomplex,7b, 7c, 14 characterization of the bioelectrocatalysis of the three mitochondrial complexes together on an electrode has not been explored to our knowledge. Scheme 1 depicts the architecture and the reactions of a supercomplex immobilized in a tethered lipid bilayer. By isolating and reconstituting the ETC complexes into a tethered lipid bilayer on a gold electrode, we have constructed a mitochondrial inner membrane biomimic and characterized the unique bioelectrocatalysis of the electron transport chain enzymes with its substrates.

SCHEME 1. Tethered lipid bilayer scheme for ETC metabolon bioelectrocatalysis. The scheme depicts the preferred orientation of the metabolon into the tethered bilayer with NADH oxidation and oxygen reduction taking place in the bulk solution while cytochrome c oxidation/reduction takes place in the hydrated space of the tethered bilayer near the electrode surface. RESULTS AND DISCUSSION


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A isolation procedure for the mitochondrial ETC metabolon was developed by combining multiple literature procedures to perform a sucrose density gradient centrifugation followed by gel filtration column chromatography to achieve an isolated supercomplex in the active state. The purifications of the metabolon were monitored by native PAGE assays in conjunction with enzymatic activity and protein quantification assays. Figure S1 shows the native gel of the column purification and the subsequent enzymatic activity staining of NADH for Complex I and cytochrome c for Complex III and IV. The native gels demonstrate the dynamic nature of the supercomplexes during gel electrophoresis, where Complex I is found in multiple bands with and without Complex III and IV. Assay data can be seen in the Supporting Information. The purification of the ETC supercomplex was monitored by enzymatic activity assays for Complex I via NADH oxidation, Complex III via cytochrome c reduction, and Complex IV via cytochrome c oxidation. The principle of the supercomplex solid state theory describes increased activity of the enzymes while complexed together. Table S1 displays the activity of each fraction of the sucrose gradient with high activity for all three complexes in the first several fractions. Fractions 1-6 were concentrated to remove the sucrose and purified by a gel filtration column. The first fractions of the isolated supercomplexes were reconstituted into lipids mimicking the inner membrane composition for maximum activity and used for electrochemical analysis. The importance of the inner membrane lipid cardiolipin and substrate ubiquinone are demonstrated in the assays of Table S2. These assays were performed with enzymes that were reconstituted without cardiolipin or ubiquinone, respectively. The assays show that the reconstituted enzymes lost a large amount of activity in Complexes III and IV with the removal of the lipid cardiolipin. Complex IV has 50% less activity without ubiquinone substrate for Complex III, but is not as affected compared to the removal of cardiolipin, which eliminates 98%


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of the activity. This demonstrates the established link between cardiolipin and ETC enzyme stabilization, which has been postulated in the literature.15 Different assay conditions were utilized to investigate the supercomplex formation. A low ionic strength can change the diffusion and collisions of cytochrome to improve the movement between Complex III and IV. Figure S2 depicts the activity of the purified fractions with a low ionic strength buffer (12 mM) with 25 µM oxidized and reduced cytochrome c, as well as inhibition with sodium azide (Complex IV) and azoxystrobin (Complex III) and solubilization with β-docedyl maltoside (DDM). The addition of azoxystrobin shows a slight increase in cytochrome c oxidation from Complex IV. The addition of azide shows increased cytochrome c reduction for the activity of Complex III, showing the presence of both Complex III and IV. Fractions 4 and 5 have low activity for Complex III and high activity for Complex IV, which would demonstrate a difference in the turnover of the complexes and different concentrations. Solubilization with DDM leads to a decrease in overall oxidase activity, showing that the detergent breaks some of the protein-protein interactions of this supercomplex. The addition of potassium chloride, to increase the ionic strength, increases the oxidase activity showing that ionic strength will increase the transport of cytochrome c in the buffer and therefore the number of collisions with the complexes.16 These assays show the presence of the three complexes in their respective fractions and how their activities are dependent on each other in different buffer environments with ionic strengths and crowding reagents. The gold disc electrodes were modified with a mixed monolayer of β-mercaptoethanol and cysteamine monolayers to make the gold surface hydrophilic and provide an amine functional group for the PEGylated cholesterol N-hydroxysuccinimide. The PEGylated cholesterol becomes the tether for the proteoliposomes to fuse with, forming a bilayer. Gold disc


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electrodes were characterized by cyclic voltammetry as a bare gold electrode, the self-assembled monolayer, bilayer, and a protein modified bilayer in buffer shown in Figure S3. Control electrodes were made by modifying an electrode with liposomes without enzymes to determine the current response with oxidized cytochrome c in solution, which is the electron shuttle between Complex III and IV. The liposomes were allowed to evaporate to decrease the inner volume of the vesicles, collapse and then be rehydrated with a low ionic strength buffer to induce fusion into a tethered bilayer.17 It is assumed that the tethered bilayer formed on the surface is not perfectly insulating and will have defects that allow cytochrome c to freely diffuse between the lipid bilayer and the hydrated space next to the electrode surface.18 The cyclic voltammograms with oxidized cytochrome c in solution shows a surface confined reversible redox response with a E1/2 at 0.035 V (vs. Ag|AgCl), indicating adsorption on the monolayer surface or being trapped in the hydrated space of the cholesterol tether in Figure S4. The response was linear with concentrations of cytochrome c until ~5 µM where the current response levels out, indicating possible saturation of the cytochrome c at the surface of the electrode. Metabolon proteoliposomes were immobilized onto the electrode by drop casting onto the electrode surface by the same procedure as the control liposomes. In the absence of cytochrome c, no redox peaks were observed from Complex III heme c1 or Complex IV binuclear CuA center, which would be closest to the electrode surface assuming the orientation suggested in Scheme 1. With the maximum surface concentration of the enzymes on the electrode, any possible redox peaks would be buried beneath the capacitance and the orientation and spacing of the cofactors would prevent electron tunneling or electron hopping. The bioelectrocatalytic response of the ETC metabolon in the presence of increasing concentrations of oxidized cytochrome c is shown in Figure 1, which displays a sigmoidal catalytic reduction


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current response demonstrating that the enzyme kinetics are rate limiting.19 The onset potential of the catalytic reduction at ~0.150 V (vs. Ag|AgCl), corresponding to the potential of cytochrome c in the bilayer control, exhibiting mediated bioelectrocatalysis with cytochrome c. The cyclic voltammetry response in the prescence of oxidized cytochrome c demonstrated Michaelis-Menten behavior and non-linear regression presented an Imax of 231 ± 18 nA and a KM of 4.9 ± 0.7 µM for oxidized cytochrome c. Additions of high concentrations of NADH to the buffer without cytochrome c in solution did not produce a catalytic response in the cyclic voltammetry in the limiting window with a thiol monolayer. Additions of NADH with cytochrome c in solution did result in a minor decrease the catalytic current as shown in Figure S5. This could mean that NADH is consumed by Complex I and the electrons could be lost to the pool of ubiquinone/ubiquinol, where it does not yield an immediate effect on the catalytic current.

Figure 1. Representative ETC metabolon cyclic voltammograms with increasing oxidized cytochrome c concentrations (0-5 µM). Arrow indicates the increasing catalytic cathodic current


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with increasing concentrations of cytochrome c. Experiments were performed in nitrogen purged 10 mM potassium phosphate, pH 7.5, 70 mM potassium chloride at a scan rate of 5 mVs-1. Previous reports have shown cytochrome c can mediate Complex IV oxygen reduction reaction, since the enzyme active is not close enough or orientated for direct electron transfer.20 The potentials of Complex IV’s CuA subunit (-0.005 V vs Ag|AgCl)21 and Complex III’s heme c1 (-0.045 V vs Ag|AgCl)21 are within 50 mV of each other and cytochrome c (0.035 V vs. Ag AgCl) electrochemical mediation is facile, being the natural substrate and electron transfer molecule in the intermembrane space. The electrochemical reaction reduces the cytochrome c where the electrons are passed to Complex IV to reduce oxygen to water. Although the buffer solution was purged by nitrogen, the small amounts of oxygen left would be enough for Complex IV to consume in the buffer on the electrode. Because the cyclic voltammograms do not show a redox peak for any of the enzymes, the surface concentration of the electroactive species cannot be determined. The current differences between the metabolon and Complex IV electrodes cannot be compared without enzymatic concentrations and catalytic turnover rates of the enzymes in the lipid bilayer on the electrode. I Inhibition of enzymatic activity was used to determine the responsible enzyme(s) contributing to the current response in this system. Complex IV is competitively inhibited by sodium azide, where azide reversibly binds to the heme a3-CuB complex inhibiting oxygen reduction to water. The addition of sodium azide in the presence of oxidized cytochrome c lowers the catalytic current, indicating the inhibition of Complex IV in the metabolon, limiting the acceptance of electrons. Sodium azide concentrations produce a diffusion limited response with the redox peak corresponding to cytochrome c as shown in Figure S6A. By replacing the


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buffer and addition of 5 and 10 µM oxidized cytochrome c, the catalytic response was partially recovered but dramatically reduced as shown in Figure S6B. Complex IV proteoliposomes were examined as a positive control for the metabolon bioelectrocatalysis, shown in Figure 2A. In the absence of reduced cytochrome c, the response was the same as the metabolon, where no redox peaks were present from Complex IV. With the addition of reduced cytochrome c, the current response is the same as the metabolon with oxidized cytochrome c, presenting a sigmoidal catalytic reduction current. This provides evidence that Complex IV is part of the bioelectrocatalysis in the metabolon complex, where cytochrome c is oxidized by Complex IV diffusing to the electrode surface or being caught between the membrane and the electrode surface. Figure 2B shows the Michaelis Menten response of Complex IV in the prescence of reduced cytochrome c with an Imax of 298 ± 6 nA and a KM of 0.64 ± 0.04 µM. This kinetic response is a tight affinity for cytochrome c with Complx IV, where the metabolon kinetics had less affinity, suggesting possible increased movement of cytochrome c between the complexes. The cyclic voltammetry response of the metabolon electrodes demonstrated Michaelis-Menten behavior and non-linear regression presented an Imax of 231 ± 18 nA and a KM of 4.9 ± 0.7 µM for oxidized cytochrome c.

A

B


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Figure 2. (A) Cyclic voltammetry comparison for metabolon electrodes with oxidized cytochrome c (solid) and Complex IV electrodes with reduced cytochrome c (dot) and cytochrome c control bilayer electrodes (dash dot). (B) Non-linear regression for the Michaelis Menten kinetics of metabolon (circles) and Complex IV (squares) modified electrodes and cytochrome c control bilayer electrodes (triangles). Experiments were performed in nitrogen purged 10 mM potassium phosphate buffer, pH 7.5, 70 mM potassium chloride and at a scan rate 5 mVs-1. Error bars represent one standard deviation (n=3). Amperometry was performed to understand kinetics with more control and decrease the capacitive backgeound. The amperometric experiments were held at an Eappl of -0.100 V vs. Ag|AgCl. Raw amperometric data with the metabolon sample, with injections of oxidized cytochrome c in the buffer, showed a gradual increase in the current response before stabilizing, as shown in Figure S7. Figure 3A displays the enzymatic catalytic curve obtained by the amperometry experiments. The metabolon response had an Imax of 310 ± 13 nA and a KM of 6.3 ± 0.5 µM oxidized cytochrome c, similar to the cyclic voltammetry responses. The inhibition of the metabolon sample with injections of axozystrobin showed a decrease in current, but does not completely level out to a steady state to effectively quantitate the inhibition kinetics. Injections with sodium azide produced the same effect as cytochrome c oxidase and was more sensitive for the metabolon sample, with almost complete inhibition by 100 µM sodium azide, as shown in Figure 3B and S8. The kinetic analysis of the metabolon enzymes resulted with an Imax of inhibition at 163 ± 3 nA and a Ki of 42 ± 2 µM for sodium azide. The azide had a stronger inhibiting effect on the catalytic current, decreasing the current by ~70% compared to 50% with Complex IV. This shows that the metabolon is more sensitive to the inhibition of Complex IV, because Complex IV could exert control over the activity of the enzyme and protein-protein


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interactions and the possible terminal source of the bioelectrocatalytic response. Complex IV had the same amperometric response with an Imax of 189 ± 2 nA and a KM of 0.55 ± 0.02 µM for reduced cytochrome c. For inhibition with sodium azide, Complex IV bioelectrocatalysis produced an inhibition Imax of 143 ± 3 nA and a Ki of 130 ± 7 µM sodium azide.

A

B

Figure 3. (A) Non-linear regression for the Michaelis-Menten amperometry substrate kinetics of the ETC metabolon with oxidized cytochrome c (circles) and Complex IV with reduced cytochrome c (squares) and inactive enzyme control bilayer with oxidized cytochrome c (triangles). (B) Non-linear regression for the Michaelis-Menten amperometry inhibition kinetics of the ETC metabolon with oxidized cytochrome c (circles) and Complex IV with reduced cytochrome c (squares) and inactive enzyme control bilayer with oxidized cytochrome c (triangles). Experiments were performed in nitrogen purged 10 mM potassium phosphate buffer, pH 7.5, 70 mM potassium chloride and at a Eappl=-0.100 V. Error bars represent one standard deviation (n=3). CONCLUSIONS


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The mitochondrial ETC enzymes have been isolated, reconstituted into lipids, and immobilized onto an electrode in a tethered lipid bilayer. This mitochondrial inner membrane biomimic has allowed us to study the bioelectrocatalysis of the ETC metabolon enzymes on the electrode together. With inhibition by azoxystrobin and sodium azide, both Complex III and Complex IV catalytic contributions to the current response in the metabolon can be observed, where azide decreases the catalytic current by 70%. The enzymes show high mediated catalytic currents with additions of oxidized and reduced cytochrome c. The positive control of Complex IV showed the same response of the ETC metabolon enzymes indicating that Complex IV is mediated by cytochrome c, where the catalytic current produced is influenced by the flux of the entire enzymatic chain. This is the first report of the bioelectrocatalytic characterization of the mitochondrial electron transport chain metabolon enzymes in a tethered lipid bilayer. We found that cytochrome c has tighter binding affinity with cytochrome c than the metabolon chain. The low affinity, or high KM, of oxidized cytochrome c indicates the possible continued movement of cytochrome c as it travels between Complex III and IV. Isolating the ETC metabolon enzymes and immobilizing the enzyme complexes on an electrode allows for the investigation of the protein-protein interactions and the overall electron flux of the chain. This metabolic and bioenergetics enzyme cascade can be investigated to learn more about bioenergy implications with different inhibitors or drugs, poisons, and toxins. The outcome of this study can enable detailed studies for membrane enzymatic pathways for determination of substrate channeling by the bioelectrocatalysis of the entire enzyme chain. These findings can be translated into other enzymatic cascade applications for biosensors and enzymatic fuel cell applications. ASSOCIATED CONTENT


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Supporting Information. The following files are available free of charge. Materials, Experimental Methods, Spectroscopic assay data, Cyclic voltammograms of controls and Amperometric data are available in the Supporting Information. AUTHOR INFORMATION Corresponding Author *[email protected] ACKNOWLEDGMENT The authors acknowledge the Air Force Office of Scientific Research for funding of this research. REFERENCES 1.

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TABLE OF CONTENTS FIGURE


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