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Department of Biochemistry and Molecular Biology Faculty of Medicine University of Debrecen BIOCHEMISTRY PRACTICE STUDIES ON THE MITOCHONDRIAL ELECTRON TRANSPORT AND ATP SYNTHESIS Theoretical Background János Kádas, Ph.D. 2015 The development of this curriculum was sponsored by TÁMOP 4.1.1.C-13/1/KONV-20140001. The project is supported by the European Union and co-financed by the European Social Fund. 1 STUDIES ON THE MITOCHONDRIAL ELECTRON TRANSPORT AND ATP SYNTHESIS – THEORETICAL BACKGROUND The structure and function of the mitochondria Mitochondria are double membrane bounded organelles mostly found in all eukaryotic cells. Structurally in the mitochondria the outer membrane; inner membrane with the convolutions (cristae); intermembrane space is bordered with the two membranes, and the mitochondrial matrix is bordered with the inner membrane can be perceptible. The outer membrane structure is simple; the inner membrane is more complex which can be characterized by about 75% protein content. The outer membrane is permeable to small molecules and ions. Transmembrane channels composed of porin proteins allow smaller molecules to pass through the membrane. Translocase systems are responsible for the transportation of higher molecules into the mitochondria. The intermembrane space contains high number of cytochrome c molecules and has a role in forming a proton gradient. The inner membrane is highly impermeable to all (small) molecules and ions (including protons), for the passage through specific transporters are required. The surface of the inner membrane is very large due to the membrane convolutions. These convolutions are the cristae. The inner membrane contains the mitochondrial electron transport chains, adenosine nucleotide (ADP-ATP) translocases and the ATP synthase complexes. Inner membrane of the liver mitochondria may have more than 10.000 sets of electron transport systems. Besides the several metabolic intermediates, mitochondrial DNA, and ribosomes, the mitochondrial matrix contains the pyruvate dehydrogenase enzyme complexes, the enzymes of citric acid cycle reactions, amino acid and fatty acid oxidation. Primarily the mitochondrion is responsible for the energy production of the cells since the center of energy production tightly connected to other metabolic pathways. In addition the mitochondria are also involved in signaling, calcium storage, heat production, differentiation and cell growth, cell death, hem synthesis, steroid synthesis and aging processes (Figure 1.). Figure 1. Structural elements of the mitochondrion. 2 The biological oxidation Carbon atoms of organic molecules are oxidized during the biological oxidation, while oxygen is reduced. Carbon atoms are oxidized to carbon dioxide (CO2), the oxygen is reduced to water during the terminal oxidation. Catabolism of nutrients from food intake and mobilized storages occurs in the three stages of the cellular respiration (Figure 2.). In the first stage the oxidation of glucose, fatty acids and amino acids yields acetylCoA. In the citric acid cycle (TCA or tricarboxylic acid cycle), during the second stage the acetyl group of acetyl-CoA is oxidized to carbon dioxide, and the released energy is conserved in reduced electron carrier molecules (NADH and FADH2). The third stage is the oxidation of reduced electron carrier molecules in the terminal oxidation leading to the release of protons and electrons. In the last stage the electrons reduce oxygen to water; the released energy drives the ATP synthesis during the process of oxidative phosphorylation. Figure 2. Stages of biological oxidation in the cell. Reduced electron carrier molecules ultimately release their hydrogens and electrons in the terminal oxidation. While reduced electron carrier molecules are formed in the citric acid cycle and during the reaction catalyzed by pyruvate dehydrogenase enzyme directly oxidized in the mitochondrion; for the transportation to the mitochondrial matrix of NADH is produced in the glycolysis in the cytosol during the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase specific transport mechanism is necessary. Primarily the malate – aspartate and glycerol phosphate shuttle are responsible for the mitochondrial transport. With the help of malate – aspartate shuttle the NADH formed in the cytosol connected to the electron transport chain in a form of mitochondrial NADH. During the process the malate dehydrogenase enzyme converts malate to oxaloacetate in the intermembrane space in addition to the oxidation of NADH. The resulting malate is transferred to the mitochondrial matrix by the involvement of malate – α-ketoglutarate antiporter. In the matrix the malate is transformed to oxaloacetate with the reduction of NAD cofactor of the malate – dehydrogenase enzyme. Aspartate, which is formed in a transaminase reaction (with a conversion of glutamate to α-ketoglutarate) from oxaloacetate, is transferred to intermembrane space with the involvement of glutamate – aspartate antiporter. Oxaloacetate reproduced due to the repeated transaminase reaction in the intermembrane 3 space from the aspartate, can be involved into subsequent transportation cycle of the NADH. The reduction reaction is catalyzed by the malate-dehydrogenase enzyme (Figure 3.). Figure 3. The malate - aspartate shuttle. In case of the glycerol-3-phosphate dehydrogenase shuttle the glycerol-3-phosphate dehydrogenase converts dihydroxyacetone phosphate to glycerol-3-phosphate in the cytosol, while the NADH is oxidized. After access to the mitochondria, the glycerol-3-phosphate is converted back to dihydroxyacetone phosphate by the mitochondrial isoform of the enzyme with the reduction of its FAD cofactor. With this shuttle mechanism the NADH produced in the cytosol enters into the respiratory chain as FADH2 (Figure 4.). Both shuttle mechanisms are suitable for the transportation of reducing equivalents. While the malate – aspartate shuttle is reversible, and is activated in case of high cytosolic NADH concentration, the operation of glycerol-3-phosphate dehydrogenase shuttle is irreversible and independent from the concentration conditions. Figure 4. The glycerol - 3 - phosphate shuttle. 4 During the biological oxidation, oxidation of one NADH molecule is resulted in the phosphorylation of 3 ADP molecules, while oxidation of one molecule succinate generates 2 molecules of ATP. The two NADH molecules formed during the glycolysis can be utilized for the synthesis of 4 or 6 ATP depending on which transport system is used for entering into the mitochondrial matrix. Under aerobic conditions total oxidation of one molecule of glucose can lead to 36 or 38 ATP formation. It is important to note that the under anaerobic circumstances the pyruvate is utilized for the synthesis of lactate, and only 2 molecules of ATP are synthesized. The mitochondrial electron transfer chain In terminal oxidation, on the mitochondrial electron transfer chain the hydrogen is oxidized and the oxygen is reduced to water. Connected to the process an oxidative phosphorylation occurs whereby the ADP is phosphorylated to ATP. The terminal oxidation and the oxidative phosphorylation are spatially and temporally linked processes. The mitochondrial electron transport system is a complex structure of four supramolecular organizations connected to the fifth element of the ATP synthase complex, ubiquinone and cytochrome c are also part of the system. The oxidation of reduced electron carrier molecules occurs via a coordinated action of the four respiratory complexes (Figure 5.). Figure 5. Elements of the mitochondrial electron transfer chain and the ATP synthase. The Complex I. is the NADH - ubiquinone oxidoreductase or also known as the NADH dehydrogenase. Proteins containing iron – sulfur (Fe-S) or flavin mononucleotide (FMN) prosthetic groups are involved as a part of the complex. The complex oxidizes the NADH, and carries the electrons to ubiquinone, while pumps protons into the intermembrane space. Next member is the Complex II., succinate-ubiquinone oxidoreductase complex a flavin adenine dinucleotide (FAD) prosthetic group containing enzyme complex. In fact, it is a combination of succinate dehydrogenase and a hydrogen transferase. During the operation the ubiquinone is reduced by this complex. Electrons released from succinate – fumarate conversion are transferred to FAD of succinate dehydrogenase in the first step, and then enter 5 into the electron transport chain and are delivered to ubiquinone. The succinate dehydrogenase also a part of the citrate cycle as the one membrane-bound enzyme of the system. The complex II., in contrast with the other complexes, has no proton pump activity. Ubiquinone is the oxygen supplier in the respiratory chain. It is long, hydrophobic molecule consists of isoprene subunits, is located in the membrane and it is diffusible. It is an important electron transporter of the respiratory chain, which is not bounded to other proteins, but moving inside the membrane and connects the first and second complexes with the respiratory complex III. The Complex III. is the ubiquinone-cytochrome c oxidoreductase, the second enzyme complex, which pumps protons into the intermembrane space. Electrons that are carried by the ubiquinone are moved to cytochrome c through the complex III. The cytochrome c transfers the electrons to complex IV. The complex contains cytochrome b and c1 molecules with heme prosthetic groups. Cytochrome c is not a membrane bounded protein. It has heme prosthetic group, and it is moving between the two membranes and connects to complex III. and IV. Last element of the mitochondrial electron transport system is the IV., Cytochrome oxidase complex. Contains cytochrome a and a3, proteins with heme – iron groups. In addition, two copper ions can be found in the complex. The cytochrome oxidase oxidizes the cytochrome c and reduces oxygen to water molecules in the matrix. The complex works as a proton pump too (Figure 6.). Figure 6. Complexes of the mitochondrial electron transfer chain. Mechanism of the ATP synthesis Electron transfer components of terminal oxidation are localized in the inner membrane of the mitochondrion, and interconnected spatially and temporally to structural components that are responsible for the ATP synthesis. Since the ATP synthesis is closely related to terminal oxidation it is called oxidative phosphorylation. 6 The chemiosmotic theory The mitochondrial electron transport is connected to proton transportation, and produces both chemical and an electrical gradient. In the respiratory chain a proton gradient is generated between mitochondrial matrix and intermembrane space due to the operation of the complexes with proton pumping activity. Figure 7. Schematic representation of the electron transfer, and the ATP synthesis. According to Peter Mitchell’s chemiosmotic theory during mitochondrial electron transport the energy of electrons is transformed into the generation of H+ concentration difference (proton gradient) between two surfaces of the inner membrane. Complexes are pumping protons into the intermembrane space during their operation and make difference in proton concentration and that is resulted in pH change. Membrane potential is also generated, and the matrix will be negatively charged, while the intermembrane space becomes positively charged. The proton motive force that drives the ATP synthesis is provided by electrochemical potential and hydrogen concentration difference (Figure 7.). The oxidative phosphorylation Energy source of the ATP synthesis is the proton gradient connected to the electron flow and electrochemical potential difference that leads to the formation of ATP macroergic phosphate bounds. For the protons that have been pumped out into intermembrane space, specific channels (F0) of ATP synthase complex provides opportunity to flow back into the mitochondrial matrix. Energy from the concentration difference and membrane potential is utilized for ATP synthesis in the equalization. The ATP synthase complex contains F1 and F0 units. Four integral protein subunits of the F0 unit build the proton channel, while the matrixfacing F1 unit is responsible for the ATP synthesis. ATP molecules that have been synthetized in the mitochondrion are exported by the ADP-ATP translocase, while an inner membrane transport system carries the ADP and anorganic phosphate that are necessary for ATP synthesis in the matrix. 7 The acceptor control and P/O ratio The electron transport system and the oxidative phosphorylation processes are tightly connected. This phenomenon can be explained by the acceptor control regulatory mechanism. Electrons and hydrogens that are necessary for the oxidative phosphorylation are provided by reduced electron carrier molecules, while anorganic phosphate and ADP are also required for the generation of ATP. The concentration of ADP is decisive in the aspect of ATP synthesis. At high concentrations of ADP the ATP synthesis is increased. The accelerating effect of ADP on reaction speed is called acceptor control or respiratory control (RC). In the absence or at a low level of ADP the proton flow rate is decreased due to the decreased speed of the phosphorylation. If the proton flow through the F1-F0 complex into the matrix is inhibited, the proton concentration is increased significantly in the intermembrane space and an increased energy is required for keeping the proton pumping ability of the complexes against the increased gradient. This energy exceeds the energy that is released during the electron transfer and it consequently stops the electron flow. The increased proton gradient stops not only the terminal oxidation, but also inhibits the citric acid cycle due to the increased NADH concentration. Quantity of the anorganic phosphate incorporation for the use of one oxygen atom, i.e. production of one molecule of water in the terminal oxidation can be experimentally determined, and this value is the P/O ratio. In the course of succinate - fumarate conversion, the Complex II. (succinateubiquinone oxidoreductase) transfers the hydrogens to FAD of the succinate dehydrogenase (FADH2 is formed), and then reduces the ubiquinone, and the end of the electron transfer the ATP synthesis will be started. The malate can get through the inner membrane by the help of malate α-ketoglutarate transporter. It is converted into oxaloacetate by malate-dehydrogenase enzyme with the generation of NADH+H+. The NADH+H+ is oxidized by the Complex I. (NADH-ubiquinone oxidoreductase), which subsequently reduces the ubiquinone. Synthesis of one molecule ATP requires 4 protons. In case of an oxidation of one NADH+H+, 10 protons are pumped out into the intermembrane space due to the proton pump activity of the mitochondrial electron transport complexes , and 6 protons are pumped out during one FADH2 oxidation. Thus, if 4 protons are necessary for the synthesis of one molecule ATP, the P/O ratio is 2.5 (10/4) for NADH+H+, while 1.5 (6/4) for FADH2. In case of NADH+H+ the 3 and in case of FADH2 the 2 is commonly used as P/O value in the literature. Inhibitors of the mitochondrial electron transfer chain and ATP synthesis Functionality and the operational order of the elements of electron transport chain, terminal oxidation and oxidative phosphorylation can be tested and verified with experimental methods. The different reactions that are catalyzed by each complex can be determined and can be measured by the examination of the fractions containing different respiratory chain complexes that were isolated from mitochondrial fraction with chromatography methods. The operation sequence of the elements of the electron transport chain can be determined by examining the effects of the different electron transfer inhibitors on the oxidation state and kinetics of each transporter. Different steps of the electron transport can be blocked specifically with various inhibitors (Figure 8.). 8 Figure 8. Inhibitors of the mitochondrial electron transfer chain and ATP synthesis. Some well-known inhibitors and effects on the terminal oxidation and oxidative phosphorylation (Figure 9.): Cyanid (CN-) and Carbon monoxide (CO): inhibitors of the cytochrome oxidase (Complex IV.), inhibit Cytochrome a and a3 oxidation, and electron flow to the oxygen. Antimycin A: inhibits the electron transition between Cytochrome b and Cytochrome c1 in the Complex III. Malonate: competitive inhibitor of the succinate – dehydrogenase (Complex II.). Rotenone: inhibits the electron transfer from Fe-S center to ubiquinone in NADH – ubiquinone oxidoreductase (Complex I.). Oligomycin: specific inhibitor of the ATP synthase, it is responsible for the inhibition of F0 and CF1. Other agents that interfere with oxidative phosphorylation: Dinitrophenol (DNP): protonophore, uncoupling agent, which makes the membrane permeable to protons, so the protons will not flow back through the ATP synthase to the matrix. It uncouples the electron transport chain and oxidative phosphorylation. Atractyloside: specific inhibitor of ATP-ADP (adenine nucleotide) translocase inhibits adenine nucleotide exchange between the two sides of the inner membrane. 9 Figure 9. Effect of different inhibitors on the electron transport chain. In the presence of a given inhibitor all components of the chain prior to the inhibited point become reduced, while all subsequent components are remained in oxidized form, and the electron transport and oxygen uptake are stopped (Figure 10.). Figure 10. Blocking effect of different inhibitors on the electron transport chain. In certain physiological conditions uncoupling of the ATP synthesis (oxidative phosphorylation) and electron transport chain may be biologically useful. The Thermogenin is a physiological uncoupling protein (UCP) typically occurs in inner membrane of brown fat tissue mitochondria. Due to the operation of thermogenin, the protons that flow back into the matrix flow through thermogenin pores instead of channel F0, which results in thermogenesis instead of ATP production (Figure 11.). 10 Figure 11. Role and function of the uncoupling protein (UCP, Thermogenin). Measurement of the oxygen consumption in practice, determination of P/O ratio Under adequate experimental conditions, terminal oxidation, the properties of the electron transport chain and the effect of various agents can be tracked by measuring oxygen consumption using isolated mitochondrial suspension. Determination of dissolved oxygen concentration with Clark-type polarographic oxygen electrode is a suitable method for the monitoring of oxygen consumption (Figure 12.). This device is a bipolar electrochemical oxygen sensor composed of a platinum cathode and a larger silver anode covered with silver-chloride. Electrodes are immersed in saturated potassium chloride (KCL). The reaction vessel is separated from the electrodes by a specific membrane which permits dissolved gases, like oxygen but impermeable for components of the reaction solution (e.g. water, ions, etc.). Polarization voltage between the two electrodes is 0.6-0.7 V. Due to the voltage that is applied on the electrode, the platinum electrode (cathode) surface becomes negatively charged, oxygen diffuses to the cathode through the membrane, which is permeable for dissolved gases and reduced; the silver is oxidized on the anode and silver chloride (AgCl) is precipitated. Current generated by the electrode processes becomes directly proportional to the concentration of oxygen reduced at the cathode. Figure 12. Elements of the equipment are used during the practical experiment. 11 The mixture in the reaction vessel should be maintained at a constant temperature, since changes in temperature affect the solubility of oxygen. Continuous mixing of the reaction mixture with magnetic stirrer is needed for equipartition of the mitochondrial suspension, substrates and the inhibitors in the reaction chamber. No air bubbles are allowed during the addition of reagents in order to avoid additional oxygen dissolution the reaction chamber should be closed. This ensures the constant oxygen concentration; besides application of permanent voltage the oxygen amount measured by the electrode will be proportional to the concentration of the oxygen in the reaction sample. Since the membrane is not permeable to the dissolved substances, such as corresponding substrates and inhibitors that were added to the mitochondria suspension, therefore these materials do not affect processes on the electrodes. When the inhibitor affect is formed after the addition of agent, restart of the respiration can be checked with application of uncoupling agent (DNP). Relatively to a given temperature, the saturation concentration of oxygen can be determined (the temperature should be kept at an approximately constant rate). A zero oxygen concentration can be also determined by the addition of sodium dithionite, which removes oxygen from the reaction mixture and it also helps to demonstrate that the respiration is not slowed or stopped due the lack of oxygen. During experiments oxygen consumption is monitored as a function of time and the changes in the current is recorded using computer system. Current recorded at these conditions is used to calculate oxygen level during respiration (Figure 13 and 14.). Figure 13. Recorded curves in different buffers during the experiment. Effects on the oxygen consumption. 12 Figure 14. Effects of different compounds on the oxygen consumption during the experiments. Due to the operation of the respiratory chain ATP synthesis occurs, the released amount of oxygen can be determined experimentally and the experimental data can be used to calculate the P / O ratio. In the absence of ADP the rate respiration is slow and the O2 consumption is low, while the addition of ADP increases O2 consumption. After adding of ADP parallel with the O2 uptake a stoichiometric amount of ATP is generated, and this coefficient gives the P/O ratio. The P/O ratio is the number of ATP synthesis / consumed oxygen atoms, namely the number of ATP molecules generated after every 2 electrons that are transferred from NADH to O2 (synthesized ATP molecules / 2 electrons). The P/O ratio can be determinable based on the known saturation concentration of oxygen and the amount of ADP used in the reaction (Figure 15.). Figure 15. Calculation of the P/O ratio. 13 References Imre Törő: Az élet alapjai (in Hungarian). Gondolat Kiadó, Budapest 1989. Studies on the Coupling of Mitochondrial Electron Transport by Proton Motive Force to ATP Synthesis. Medical Biochemistry and Molecular Biology: Practical Manual. Semmelweis University, Department of Medical Biochemistry, Budapest, 2014. Biochemistry of the Mitochondria, Biochemistry I. lecture presentation. Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, 2014. 14