Electron Transport System Supplemental Reading Key Concepts - PETER MITCHELL'S CHEMIOSMOTIC THEORY - THE ELECTRON TRANSPORT SYSTEM IS A SERIES OF COUPLED REDOX REACTIONS Complex I: NADH-ubiquinone oxidoreductase Complex II: Succinate dehydrogenase Complex III: Ubiquinone-cytochrome c oxidoreductase Cytochrome C Complex IV: Cytochrome c oxidase - BASIS FOR THE ATP CURRENCY EXCHANGE RATIOS OF NADH AND FADH 2 KEY CONCEPT QUESTIONS IN THE ELECTRON TRANSPORT SYSTEM: What is the Chemiosmotic Theory and how does it explain proton motive force? What is the role of coenzyme Q (ubiquinone) in the electron transport system? Biochemical Application of the Electron Transport System Hydrogen cyanide is a deadly gas that kills cells by blocking electron transfer from cytochrome oxidase in complex IV to oxygen. Other electron transport inhibitors are rotenone, a poison, and amytal, a barbiturate, both of which block electron transfer from iron-sulfur centers. The Electron Transport System, also called the Electron Transport Chain, converts redox energy available from oxidation of NADH and FADH 2, into proton-motive force which is used to synthesize ATP through conformational changes in the ATP synthase complex through a process called oxidative phosphorylation. The relationship of the electron transport system and oxidative phosphorylation to other metabolic pathways in illustrated in figure 1. Figure 1. PETER MITCHELL'S CHEMIOSMOTIC THEORY Oxidation of NADH and FADH 2 in the mitochondrial matrix by the electron transport system links redox energy to ATP synthesis by oxidative phosphorylation (mitochondrial ATP synthesis) through the establishment of a proton (H + ) gradient across the mitochondrial inner membrane. This "chemiosmotic" process was first proposed by Peter Mitchell, a British biochemist, and involves the outward pumping of H + from the mitochondrial matrix by three protein complexes in the electron transport system (complexes I, III, IV). The protons flow back down the gradient through the membranebound ATP synthase complex in response to a chemical (H + concentration) and electrical (separation of charge) differential. This same chemisomotic process generates the bulk of ATP used by bacterial cells, which are thought to be the ancestors of eukaryotic mitochondria and is used to synthesize ATP in plant chloroplasts which utilize sunlight energy rather than redox energy to establish a H + gradient across the thylakoid membrane. The electron transport system consists of the linked redox reactions that occur sequentially in a set of protein complexes imbedded in the inner mitochondrial membrane. The net result of 1 of 10 pages
these redox reactions is the coupled oxidation of NADH and reduction of molecular oxygen ( 1 2 O 2) to form NAD + and H 2 O. The difference in reduction potential (ΔEº') between the NADH/NAD + conjugate redox pair (Eº' = -0.32V), and that of the 1 2 O 2/H 2 O conjugate redox pair (Eº' = +0.82V), provides a huge amount of free energy for ATP synthesis (220 kj/mol). Figure 2 summarizes the electron transport system and the process of chemiosmosis as it applies to mitochondria. Figure 2. The basic idea of the chemiosmotic theory (now generally accepted as being correct) is that energy from redox reactions or light is translated into vectorial energy through the coupling of electron transfer to membrane bound proton pumps that transverse a proton impermeable membrane and thereby establish an Figure 3. electrochemical proton gradient. A "proton circuit" is established when the protons respond to the chemical and electrical gradient across the membrane by flowing back across the membrane through the ATP synthase protein complex to catalyze ATP synthesis as shown in figure 3. Note that the vectorial H + pumping results in both a chemical gradient across the membrane represented by ΔpH, and an electrical gradient due to the separation of charge which can be measured as a membrane potential Δψ. The separation of charge is due to the build-up of positively-charged protons (H + ) on one side of the membrane and accumulation of negative charges on the other side of the membrane (OH - ). Note that in mitochondria, the contribution of Δψ to ΔG is actually greater than that of ΔpH (the ΔpH across the mitochondrial membrane is only 1 ph unit), whereas in chloroplasts, the ΔpH contribution to ΔG is much more significant with ΔpH close to 3 ph units. 2 of 10 pages
Figure 4. Bioc 460 - Dr. Miesfeld Spring 2008 As shown in figure 4, a critical feature of the mitochondrion is the extensive surface area of the inner mitochondrial membrane which forms the protonimpermeable barrier required for chemiosmosis. This highly convoluted membrane is the location of electron transport system proteins and the ATP synthase complex, and therefore maximum surface area is required to accommodate as much protein as possible. Electron microscopy studies have shown that the inner mitochondrial membrane forms structures called cristae which have been estimated to cover as much as 3,000 µ 2 per cell (~5 µ 2 per mitochondrion). Mitchell's chemiosmotic theory was eventually validated using biochemical approaches. This was done using "inside-out" membrane vesicles that could Figure 5. be shown to pump protons into the interior of the vesicle when oxidizable substrate was made available, leading to the production of ATP at the surface of the vesicle exactly as predicted by Mitchell's theory. As illustrated in figure 5, one type of experiment used synthetic membrane vesicles consisting of a 4:1 mixture of phosphatidylethanolamine and phosphatidylcholine that were reconstituted in the presence of purified bacteriorhodopsin protein from the photosynthetic bacteria Halobacterium halobium and bovine heart mitochondrial membrane fractions consisting of the intact ATP synthase complex. When the vesicles were exposed to light, proton pumping by the bacteriorhodopsin protein resulted in both inward proton pumping and ATP synthesis on the vesicle surface. The bacteriorhodopsin proton pump is analogous to the electron transport system, except proton pumping is initiated by light rather than oxidation of NADH and FADH 2. We are now ready to start our journey through the metabolic forest by answering our four key questions about the "pathway" that links the electron transport system to oxidative phosphorylation: 1. What does the electron transport system/oxidative phosphorylation accomplish for the cell? Generates ATP derived from oxidation of metabolic fuels accounting for 28 out of 32 ATP (88%) obtained from glucose catabolism. Tissue-specific expression of uncoupling protein-1 (UCP1) in brown adipose tissue of mammals short-circuits the electron transport system and thereby produces heat for thermoregulation. 2. What is the overall net reaction of NADH oxidation by the coupled electron transport and oxidative phosphorylation pathway? 2 NADH + 2 H + + 5 ADP + 5 P i + O 2 2 NAD + + 5 ATP +2 H 2 O 3 of 10 pages
3. What are the key enzymes in the electron transport and oxidative phosphorylation pathway? ATP synthase complex the enzyme responsible for converting proton-motive force (energy available from the electrochemical proton gradient) into net ATP synthesis through a series of proton-driven conformational changes. NADH dehydrogenase also called complex I or NADH-ubiquinone oxidoreductase. This enzyme catalyzes the first redox reaction in the electron transport system in which NADH oxidation is coupled to FMN reduction and pumps 4 H + into the inter-membrane space. Ubiquinone-cytochrome c oxidoreductase - also called complex III, translocates 4 H + across the membrane via the Q cycle and has the important role of facilitating electron transfer from a two electron carrier (QH 2 ), to cytochrome c, a mobile protein carrier that transfers one electron at a time to complex IV. Cytochrome c oxidase - also called complex IV pumps 2 H + into the intermembrane space and catalyzes the last redox reaction in the electron transport system in which cytochrome a 3 oxidation is coupled to the reduction of molecular oxygen to form water ( 1 O 2 2 + 2 e- + 2 H + --> H 2 O). 4. What is an example of the electron transport system? Cyanide binds to the heme group in cytochrome a 3 of complex IV and blocks the electron transport system by preventing the reduction of oxygen to form H 2 O. Hydrogen cyanide gas is the lethal compound produced in prison gas chambers when sodium cyanide crystals are dropped into sulfuric acid. San Quentin gas chamber. THE ELECTRON TRANSPORT SYSTEM IS A SERIES OF COUPLED REDOX REACTIONS Early work using purified mitochondria showed by fractionation methods and biochemical assays that five enzyme complexes contained within the inner mitochondrial membrane were required for oxidative phosphorylation. These components were named Complex I; NADH-ubiquinone oxidoreductase (NADH dehydrogenase), Complex II; succinate dehydrogenase, Complex III; Ubiquinone-cytochrome c oxidoreductase, and Complex IV; cytochrome c oxidase. The fifth enzyme fraction contained the F 1 F o ATP synthase complex (actually purified as an ATP hydrolyzing activity) consisting of a large multisubunit complex which could be further fractionated into a membrane bound "stalk" (F o ) and a spherical "head" (F 1 ) encoding the catalytic subunit. Using specific redox reaction inhibitors such as rotenone, antimycin A and cyanide, and based on the known reduction potentials (Eº') of conjugate redox pairs, it was possible to order the four electron transport system complexes as shown in figure 6. Figure 6. 4 of 10 pages
Metabolic fuel in the form of NADH and FADH 2 feed into the electron transport system from the citrate cycle and fatty acid oxidation pathways. Pairs of electrons (2 e-) are donated by NADH and FADH 2 to complex I and II, respectively, and flow through the electron transport system until they are used to reduce oxygen to form water ( 1 2 O 2 + 2 e- + 2 H + --> H 2 O). The two mobile electron carriers in this series of reactions are coenzyme Q (Q), also called ubiquinone, and cytochrome c which transfer electrons between various complexes. The stoichiometry of "proton pumping" is 4 H+ in complex I, 4 H+ in complex III, and 2 H+ in complex IV (10 H + /NADH and 6 H + /FADH 2 ). The donation of 2 e- from NADH in the form of a hydride ion (:H + ) to FMN initiates a series of sequential redox reactions involving as many as 20 discrete electron carriers, culminating in the reduction of molecular oxygen to form water. Pairs of electrons as hydrogen atoms (2H + + 2e-) can also enter the electron transport system through oxidation of FADH 2 molecules covalentlyattached to enzymes associated with either the cytosolic side of the inner mitochondrial membrane (mitochondrial glycerol-3-phosphate dehydrogenase), or that belong to metabolic pathways located within the mitochondrial matrix (succinate dehydrogenase and ETF-Q oxidoreductase). The four functional components of the electron transport system are: Three large multisubunit protein complexes, I, III and IV, that transverse the inner mitochondrial membrane and function as proton "pumps". Coenzyme Q (Q), also called ubiquinone, a small hydrophobic electron carrier that diffuses laterally within the membrane to donate electrons to complex III. Three membrane-associated FAD-containing enzymes (succinate dehydrogenase, electrontransferring flavoprotein; ETF, and glycerol-3-phosphate dehydrogenase) that pick up electrons from linked metabolic pathways and donate them to coenzyme Q. Cytochrome c, a small water-soluble protein that associates with the cytosolic side of the membrane and carries electrons one at a time from complex III to complex IV. Figure 7 below shows the reduction potentials for many of the electron carriers in this system. How is the energy released by redox reactions used to "pump" protons into the inter-membrane space? The answer is not yet completely understood but it is thought to involve, 1) a redox loop mechanism in which there is a separation of the H + and e- on opposite sides of the membrane; 5 of 10 pages
the Q cycle in complex III uses this mechanism to translocate protons across the membrane, and 2) redox-driven conformational changes in the protein complex that "pump" protons across the membrane by altering pk a values of functional groups located on the inner and outer faces of the membrane. Both complexes I and IV have properties that are consistent with such a proton pumping mechanism. It is likely that for some electron transport proteins both a redox loop mechanism and protein conformational changes are involved in net proton movement across the membrane. Figure 8 illustrates how these two mechanisms could be operating. Figure 8. Complex I: NADH-ubiquinone oxidoreductase Complex I is the largest of the four protein complexes in the mitochondrial electron transport system consisting of as many as 42 polypeptide chains having a combined molecular mass of ~850 kda. Because of its complexity and size, the structure of complex I is not yet known at the molecular level, however, electron microscopy methods have provided a low resolution map of its physical contour which looks a bit like a sideways "L" as shown in figure 9. The function of complex I is to pass 2 e- obtained from the oxidation of NADH to Q using a coupled reaction mechanism that results in the net movement of 4 H + across the membrane. Complex I contains a covalently bound flavin mononucleotide (FMN) that accepts the two electrons from NADH, as well as at least six different iron-sulfur centers (Fe-S) that carry one electron at a time from one end of the complex to the other. The poison rotenone blocks electron transfer within complex I by preventing a redox reaction between two Fe-S centers. Complex I passes the two electrons obtained from NADH as a hydride ion (:H - ) to Q (ubiquinone) which has three critical roles in Figure 9. the electron transport system,.1) Q serves as a mobile electron carrier that transports electrons laterally in the membrane from complex I to complex III, 2) Q is the entry point into the electron transport system for electron pairs (2 e-) obtained from the citrate cycle, fatty acid oxidation and the enzyme glycerol-3p dehydrogenase, and 3) Q has the important task of converting a 2 e- transport system into a 1 e- transport system which passes electrons one at time to the mobile 6 of 10 pages
carrier protein cytochrome c. This conversion process (2 e- --> 1 e- + 1 e-) is accomplished by the Q cycle as described later. Complex II: Succinate dehydrogenase Succinate dehydrogenase is a citrate cycle enzyme Figure 10. we first encountered in lecture 28. It catalyzes an oxidation reaction that converts succinate to fumarate in a coupled redox reaction involving FAD. This enzyme was co-purified along with other polypeptides that together constitute complex II of the electron transport system. The 2 e- extracted from succinate in the citrate cycle is passed through the other protein subunits in the complex to Q as shown in figure 10. No protons are translocated across the inner mitochondrial membrane by complex II. The electron pair donated by succinate to FAD ultimately leads to the translocation of four fewer H + than NADH because complex II is not a proton pump. This is why the ATP currency exchange ratio for FADH 2 oxidation is lower than it is for NADH, giving rise to only ~1.5 ATP/FADH 2 instead of ~2.5 ATP/NADH. The same holds true for electron pairs extracted from the FADH 2 moiety of glycerol-3p dehydrogenase and ETF-Q oxidoreductase, both of which donate 2 e- to Q. Complex III: Ubiquinone-cytochrome c oxidoreductase Complex III is the docking site for QH 2 (ubiquinol) and consists of 11 protein subunits in each of two monomer subunits. Figure 11 shows a diagram of complex III emphasizing the relative position of the electron carriers and the presence of two distinct binding sites for ubiquinone called Q P and Q N which play a crucial role in diverting one electron at a time to cytochrome c via the Q cycle. The terms Q P and Q N refer to the proximity of the sites to the positive (inter-membrane space) and negative (matrix) sides of the membrane. The molecular structure of a complex III monomer is also shown in figure 11 illustrating its orientation in the inner mitochondrial membrane. Figure 11. 7 of 10 pages
The four steps in the Q cycle are shown in figure 12 and can be summarized as: 1. Oxidation of QH 2 at the Q P site results in transfer of one electron to the Rieske Fe-S center which is transferred to cytochrome c 1 and then passed off to Cyt c. The second electron is transferred to cytochrome b L which "stores" it temporarily. The oxidation of QH 2 in this first step contributes 2 H + P to the inter-membrane space. 2. The oxidized Q molecule moves from the Q P site to the Q N site through a proposed substrate channel within the protein Figure 12. complex. This stimulates electron transfer from b L to b H which then reduces Q in the Q N site to form the semiquinone Q - intermediate. 3. A new QH 2 molecule binds in the vacated Q P site and is oxidized in the same way as step 1 such that one electron is transferred to cytochrome c 1 and then to a new molecule of Cyt c. Oxidation of this second QH 2 molecule translocates another 2 H + P into the intermembrane space (4 H + P total) and the resulting Q molecule is released into the membrane (the Q N site is occupied with Q - ). 4. The second electron from the QH 2 oxidation in step 3 is passed directly from b L to b H and then used to reduce the semiquinone Q - intermediate already sitting in the Q N site which uses 2 H + N to regenerate a QH 2 molecule. To see how the Q cycle accomplishes the 2 e- --> 1 e- + 1 e- conversion process is to write out two separate QH 2 oxidation reactions and then sum them to get the net reaction for complex III: QH 2 + Cyt c (oxidized) ---> Q - + 2 H + P + Cyt c (reduced) QH 2 + Q - + 2 H + N + Cyt c (oxidized) ---> Q + QH 2 + 2 H + P + Cyt c (reduced) QH 2 + 2 H + N + 2 Cyt c (oxidized) ---> Q + 4 H + P + 2 Cyt c (reduced) Note that the Q cycle reactions require that 2H + N from the matrix be used to regenerate QH 2, even though 4H + P are translocated. However, this apparent imbalance of 2H + N is corrected by the redox reactions of complex IV where 2H + N are required to reduce oxygen to water and 2H + N are pumped across the membrane. Therefore, the net translocation of protons across the membrane in the combined redox reactions of complexes III and IV becomes 6 H + N --> 6 H + P. Cytochrome C Cytochrome c (Cyt c) is a small protein of ~13 kda that associates with the cytosolic side of the inner mitochondrial membrane and is responsible for transporting an electron from complex III to complex IV using an iron-containing heme prosthetic group. Oxidized Cyt c contains ferric iron 8 of 10 pages
Figure 14. Bioc 460 - Dr. Miesfeld Spring 2008 (Fe 3+ ) in the heme group and reduced Cyt c contains ferrous iron (Fe 2+ ). The heme group of Cyt c is covalently attached to the protein through cysteine residues and Figure 13. lies within a pocket surrounded by three helices. The molecular structure of a typical Cyt c protein is shown in figure 13. A version of the Cyt c molecular structure is used in the Bioc460 website header. Complex IV: Cytochrome c oxidase Complex IV accepts electrons one at a time from Cyt c and donates them to oxygen to form water. In the process, two net H + are pumped across the membrane using a conformational-type mechanism similar to complex I. The mitochondrial complex IV protein consists of two monomers of ~200 kda that each contain 13 polypeptides, two copper centers (Cu A and Cu B ) and two heme groups (cytochrome a and cytochrome a 3 ). As shown in figure 14, Cyt c docks on the P side of the membrane to complex IV near Cu A which accepts the electron leading to oxidation of the heme group in Cyt c (Fe 2+ --> Fe 3+ ). The reduced Cu A passes the electron to an iron atom in the heme of cytochrome a which then transfers it to cytochrome a 3. Finally, the e- is passed to Cu B which donates it to oxygen. BASIS FOR THE ATP CURRENCY EXCHANGE RATIOS OF NADH AND FADH 2 Considering that 3 H + P are required to synthesize 1 ATP when they flow back down the electrochemical proton gradient through the ATP synthase complex, and 1 H + P is needed to transport each negatively-charged P i molecule into the matrix (see figure 15), we can now see where the ATP currency exchange ratios of ~2.5 ATP/NADH and ~1.5 ATP/FADH 2 come from. Namely, oxidation of NADH by complex I leads to 10 H + P/4 H + N = 2.5 ATP, whereas, oxidation of FADH 2 by complex II yields 6 H + P/4 H + N = 1.5 ATP for FADH 2. Figure 15. 9 of 10 pages
ANSWERS TO KEY CONCEPT QUESTIONS IN THE ELECTRON TRANSPORT SYSTEM: Bioc 460 - Dr. Miesfeld Spring 2008 The Chemiosmotic Theory was first proposed by Peter Mitchell in 1961 and states that energy captured by coupled redox reactions in the electron transport system is used to translocate (pump) protons across the inner mitochondrial membrane creating a proton-motive force (ΔpH + Δψ) that drives ATP synthesis by proton flow through the F 1 F o ATP synthase complex. The H + are pumped across the membrane by complex I (4 H + ), complex III (4 H + ) and complex IV (2 H + ). The return of these 10 H + to the mitochondrial matrix in response to proton-motive force results in the production of 2.5 ATP/NADH. This ATP currency exchange ratio is based on the assumption that 3 H + must pass through the F 1 F o ATP synthase complex for every ATP generated, and that 1 H + is required to import P i into the matrix through the phosphate translocase (10 H + /4 H + = 2.5). Coenzyme Q (ubiquinone) is a hydrophobic molecule that functions as a mobile electron carrier that transports a pair of electrons (2 e-) in its reduced form of QH2 (ubiquinol), from either complex I, or from membrane-bound FADH2-containing enzymes such as succinate dehydrogenase or glycerol-3-phosphate dehydrogenase, to complex III. The oxidation of NADH by complex I results in the extraction of 2 e- that pass through a series of Fe-S centers in the complex until being donated to Q to form QH 2 which then travels to complex III and donates one electron at a time to cytochrome c using via the Q cycle. Oxidation of FADH 2 by Q bypasses complex I and therefore results in only 6 H+ being pumped across the membrane in the reduction of 1 2 O 2 to form H 2 O (4 H+ from complex III and 2 H+ from complex IV). The difference of 1 ATP in the ATP currency exchange ratio of NADH and FADH2 is the result of direct oxidation of Q downstream of complex I (loss of 4 H+ = 1 ATP). 10 of 10 pages