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Unformatted text preview: Electron Transport Chain and Oxida4ve Phosphoryla4on Bioc 460 Spring 2010 Lectures 27 and 28 Dr. Lisa Rezende How is redox energy converted to chemical energy in the form of ATP? How is ATP synthesized during oxida4ve phosphorya4on? How is respira4on controlled? How can it be disrupted? Learning Objec-ves Describe the relationship between the reduction potential difference (Eo) and reduction-oxidation (redox) reactions and the free energy difference (Go) of such a reaction. Calculate E'0 and G' for a given redox reaction. Discuss the essential features of oxidative phosphorylation and role of redox reactions in the electron transport (respiratory) chain. Explain how energy from the respiratory chain complexes is conserved and which produce sufficient energy to make ATP. Explain how pH and charge gradients are formed across the mitochondrial inner membrane. Discuss oxidative phosphorylation in relation to the chemiosmotic model. Given the substrate and tissue, calculate the ATP yield of complete oxidation. Identify the components of the ATP synthase complex, and describe their roles. Explain how electron shuttles generate energy from the NADH produced by glycolysis. Define respiratory control and uncoupling, and describe the physiological importance of these processes. Reading Chapter 18 Problems: 1 (a-d), 4. 5, 8, 14, 15, and 22 plus problem set Image courtesy of Dr. R.L. Miesfeld Redox Reac-on Review Reduc4on-oxida4on (redox) couple: pair of molecules of which one is reduced and the other is oxidized (e.g., lactate-pyruvate, NADH-NAD+, and FADH2-FAD) each pair cons4tutes a half reac4on the reductant of one pair donates electrons and the oxidant of the other pair accepts the electrons Red1 + Ox2 Ox1 + Red2 Mnemonics anyone? Oil Rig = Oxida4on is loss of electrons; reduc4on is gain of electrons LEO says GER = Loss of electrons is oxida4on; gain of electrons is reduc4on What is a reduction potential? Reduc4on poten4al (EO): how well one substance reduces another (donates electrons). EO: standard reduc4on poten4al difference between two half reac4ons similar to the GO (standard free energy difference) EO is posi4ve for favorable reac4ons (GO is nega4ve for favorable reac4ons) Four Different Modes of Electron Transfer in Oxidation-Reduction Reactions 1. Directly as Electrons. This mode is common for electron transport proteins with metal ligands. Fe2+ + Cu2+ Fe3+ + Cu+ 2. As Hydrogen Atoms. A hydrogen atom consists of an H+ ion and an electron. This mode is common when FAD or FMN are involved as electron carriers, and when carbon-carbon bonds are being oxidized. AH2 A + 2e- + 2H+ AH2 + B A + BH2 3. As a Hydride Ion ( :H-). A hydride ion has two electrons. This type of reaction occurs when NAD is an electron carrier, and is common when an oxygen-containing functional group is oxidized. 4. Through Direct Combination with Oxygen. Oxygen combines with a reactant and is covalently incorporated into the product. R-CH3 + 1/2 O2 R-CH2-OH Conjugate redox pairs . Each half reac4on consists of a conjugate redox pair represented by a molecule with and without an electron (e-). Fe2+/Fe3+ is a conjugate redox pair in which the ferrous ion (Fe2+) is the reductant that loses an e- during oxida4on to generate a ferric ion (Fe3+) the oxidant: Fe2+ <--> Fe3+ + e- Similarly, the reductant cuprous ion (Cu+) can be oxidized to form the oxidant cupric ion (Cu2+) plus an e- in the reac4on: Cu+ <--> Cu2+ + e- Two half reac-ons combine to form a redox reac-on Two half reac4ons are combined to form a redox reac4on. Fe2+ <--> Fe3+ + e- Cu2+ + e- <--> Cu+ Fe2+ + Cu2+ <--> Fe3+ + Cu+ The Fe was oxidized and the Cu was reduced in a redox reac4on in which the e- was the shared intermediate. What do we need to think about when setting up redox equations? Measuring Redox Potential Standard reduc4on poten4als are expressed as half reac4ons and wriden in the direc4on of a reduc4on reac4on. Redox pairs with a posi-ve E' have a higher affinity for electrons than redox pairs with a nega4ve E'. Electrons move from the redox pair with the lower E' (more nega4ve) to the redox pair with the higher E' (more posi4ve). The hydrogen half reac0on is set as the standard with a E' = 0 Volts. Standard reduc4on poten4als are valuable tools for calcula4ng and predic4ng the direc4on of metabolic reac4ons involving electron transfer. E' = (E'e- acceptor) - (E'e- donor) The E' for a coupled redox reac4on is propor4onal to the change in free energy G' as described by the equa4on (n is the number of e-): G' = -nFE' If E' > 0, then the reac4on is favorable since G' will be nega4ve. A coupled redox reac4on is favorable when the reduc4on poten4al of the e- acceptor is more posi0ve than that of the e- donor. Let's try one we've seen: Reduction potential of the lactate/ pyruvate and NADH/NAD+ redox pairs. The reaction: Pyruvate + NADH + H+ -> Lactate + NAD+ Can be broken into the half reactions: Pyruvate + 2 H+ + 2e- -> lactate NAD+ + H+ + 2e- -> NADH REMEMBER
Electron Acceptor Pyruvate NAD+ Electron donor Lactate NADH Eo 0.19 V 0.32 V E'0 = -0.19 V E'0 = -0.32 V For the coupled reaction: E' = (E'e- acceptor) - (E'e- donor) Eo = (-0.19 V) - (-0.32 V) = +0.13 V Now how do we calculate the free energy change for this reac4on? Electron transport and Oxidative phosphorylation take place at the inner mitochondrial membrane -Citrate cycle and fatty acid oxidation Occurs in the matrix -Electrons transport oxidizes NADH and FADH2 using complexes In the inner mitochondrial membrane -ATP is synthesis by oxidative Phosphorylation occurs at the inner Mitochondrial membrane -Movement of ions, protons, and small molecules mediated by membrane transport proteins Chemiosmo-c theory explains how redox energy powers ATP synthesis Oxida4on of NADH and FADH2 in the mitochondrial matrix by the electron transport system links redox energy to ATP synthesis. Chemiosmosis involves the outward pumping of H+ from the mitochondrial matrix through three protein complexes in the electron transport system (ETS complexes I, III, IV). H+ flow back down the proton gradient through the membrane-bound ATP synthase complex in response to a chemical (H+ concentra4on) and electrical (separa4on of charge) differen4al. Note the key role played by the inner mitochondrial membrane in these processes! Energy Conversion Requires the Proton Circuit Chemiosmo-c Theory Energy from redox reac4ons or light is translated into a proton circuit. H+ pumping results in chemical gradient (pH) and a membrane poten4al (psi) Separa4on of charge is due to build-up of posi4vely-charged protons (H+) and nega4ve hydroxyl ions (OH-) Overview of Chemiosmo-c Theory Electron Transport System FADH2 Ox Phos ATP synthase complex Courtesy of Dr. RL Miesfeld Chemiosmo-c theory explains ATP synthesis during both respira-on and photosynthesis Change in free energy (G) for a membrane transport process is the sum of the ion concentra4on (RTln(C2/C1)) and the membrane poten4al (ZFV) In mitochondria, the ZFV term makes a larger contribu4on than does RTln (C2/C1). Explain why this is oPen called the "proton-mo-ve force." What happens to the energy released? Generates ATP derived from oxida4on of metabolic fuels accoun4ng for 28 out of 32 ATP (88%) obtained from glucose catabolism. Some released as heat in an controlled fashion. In brown adipose 4ssue of mammals short-circuits the electron transport system and thereby produces heat for thermoregula4on. The Electron Transport System Is A Series Of Coupled Redox Reac-ons The electron transport system consists of five large protein complexes: 1. Complex I; NADH-ubiquinone oxidoreductase (NADH dehydrogenase 2. Complex II; succinate dehydrogenase (citrate cycle enzyme) 3. Complex III; Ubiquinone-cytochrome c oxidoreductase 4. Complex IV; cytochrome c oxidase 5. F1F0 ATP synthase complex consis4ng of a "stalk" (F0) and a spherical "head" (F1) Electrons flow spontaneously in this direc4on Metabolic Fuel for Electron Transport NADH and FADH2 feed into the electron transport system from the citrate cycle and fady acid oxida4on pathways. Pairs of electrons (2 e-) are donated by NADH and FADH2 to complex I and II, respec4vely Pairs of electrons flow through the electron transport system un4l they are used to reduce oxygen to form water (O2 + 2 e- + 2 H+ H2O). The two mobile electron carriers in this series of reac4ons are coenzyme Q (Q), also called ubiquinone, and cytochrome c which transfer electrons between various complexes. Electrons carried by NADH enters at Complex I Complex 1 passes along 2 e- obtained from the oxida-on of NADH to Q using a coupled reac4on mechanism that results in the net movement of 4 H+ across the membrane. Electrons from FADH2 enter at complex II The 2 e- extracted from succinate in the citrate cycle is passed through the other protein subunits in the complex to Q as shown below. No protons are translocated across the inner mitochondrial membrane by complex II. How do the differences between complex one and complex 2 explain the difference In ATP conversion from NADH and FADH2? Ubiquinone-cytochrome c oxidoreductase - also called complex III, translocates 4 H+ across the membrane via the Q cycle and has the important role of facilita4ng electron transfer from a two electron carrier (QH2), to cytochrome c, a mobile protein carrier that transfers one electron at a 4me to complex IV. Cytochrome c oxidase - also called complex IV pumps 2 H+ into the inter- membrane space and catalyzes the last redox reac4on in the electron transport system in which cytochrome a3 oxida4on is coupled to the reduc4on of molecular oxygen to form water ( O2 + 2 e- + 2 H+ H2O). Cytochrome Oxidase extracts 8 protons from the mitochondrial matrix -4 are pumped, adding to the proton gradient - 4 are used to form water -Overall reac-on 4 Cyt cred + 8 H+ + O2 -> 4 Cyt cox + 2 H2O + 4 H+ Summary of Electron Trasport Complexes Complex designation I NADH-Q reductase II Succinate-Q reductase Functional groups FMN (flavin mononucleotide); Fe-S FAD; Fe-S Function oxidizes NADH to NAD+; transfers electrons to coenzyme Q oxidizes succinate to fumarate with reduction of FAD to FADH2; electron transfer to CoQ transfers electrons between coenzyme Q and cytochrome C (C becomes reduced) oxidizes cytochrome C; reduces O2 to H 2O Antimycin A Poisons Rotenone III Cytochrome reductase IV Cytochrome C oxidase
Courtesy of Dr. M. Tischler heme b; heme c1; Fe-S heme a-a3; Cu Carbon monoxide Cyanide Proton-mo-ve force generated by ETC is used to synthesize ATP ATP synthase: the final step A mul4subunit protein machine Consis4ng of two major domains: F1 (the cataly4c domain) and F2 (the proton channel) Both domains composed of mul4ple subunits and can be divided into three main components: 1.the rotor rotates as protons enter and exit the ring 2.the cataly4c head piece contains the enzyme ac4ve site in each of the three beta subunits 3.the stator consists of the subunit imbedded in the membrane which contains two half channels for protons to enter and exit, and a stabilizing arm. Why so many different subunits? Many different proteins work together to convert the poten4al energy of the electrochemical gradient to mechanical energy, and finally to chemical energy in the form of ATP Proton flow though the F0 alters the affinity of F1 for ATP This is known as the binding change mechanism for ATP synthesis Image credit PDB hdp://www.rcsb.org/pdb/sta4c.do?p=educa4on_discussion/molecule_of_the_month/pdb72_1.html Anima4ons 3D view movie A 3D view of alpha3-beta3-gamma. UC Berkeley site for ATP synthase movies hdp://nature.berkeley.edu/~hongwang/Project/ ATP_synthase/ A 3D view of alpha3-beta3-gamma A top view of alpha3-beta3-gamma A cross-sec4on view of alpha-beta-gamma The binding change mechanism The subunit directly contacts all three subunits, however, each of these interac4ons are dis4nct giving rise to three different subunit conforma4ons. The ATP binding affini4es of the three beta subunit conforma4ons are defined as: T, 4ght; L, loose; and O, open. As protons flow through F0, the subunit rotates such that with each 120 rota4on, the subunits sequen4ally undergo a conforma4onal change from O --> L -- > T --> O --> L --> etc. The binding change mechanism model predicts that one full rota4on of the subunit should generate 3 ATP. Boyer's model predicts that ATP hydrolysis by the F1 headpiece should reverse the direc:on of the subunit rotor. Movie available from Yoshida lab: hdp://www.res.4tech.ac.jp/~seibutu/ How does proton movement through the c subunit ring cause rota-on of the subunit? Model based on the yeast mitochondrial c subunit ring that was found to contain 10 iden4cal subunits. Since the concentra4on of H+ on the P side (posi4ve side; inter-membrane space) is higher than it is on the N side (nega4ve side; matrix), a H+ will readily enter the half channel in the a subunit where it then comes in contact with a nega4vely charged aspartate residue (D61) in the nearby c subunit. For ATP synthesis to occur, ADP and Pi must be transported into the mitochondrial matrix ATP/ADP Translocase for every ADP molecule that is imported from the cytosol, an ATP molecule is exported from the matrix Phosphate Translocase translocates one Pi and one H+ into the matrix Note that the phosphate translocase is using the proton gradient to power transport- how will this affect total ATP yields? ATP Currency Exchange Ra-os Taking into account the requirement of 3 H+/ATP synthesized, and the use of 1 H+ to translocate ADP, the total is 4 H+/ATP. Oxida:on of NADH star:ng at complex I yields: 10 H+/4 H+ = 2.5 ATP Oxida:on of FADH2 star:ng at complex II yields: 6 H+/4 H+ = 1.5 ATP for FADH2 How does cytosolic NADH reach the ETC? Numerous dehydrogenase reac4ons in the cytosol generate NADH, one of which is the glycoly4c enzyme glyceraldehyde-3-phosphate dehydrogenase. However, cytosolic NADH cannot cross the inner mitochondrial membrane, instead the cell uses an indirect mechanism that only transfers the electron pair (2 e-), from the cytosol to the matrix using two different "shudle" systems. Muscle uses the glycerol phosphate shudle How does transferring electrons to FAD rather than NAD+ affect ATP yields? The net yield of ATP from glucose oxida-on in liver and muscle cells Let's add everything up to see how one mole of glucose can be used to generate 32 ATP in liver cells via the malate-aspartate shujle, or 30 ATP in muscle cells which use the glycerol-3-phosphate shujle. Courtesy of Dr. RL Miesfeld Control of Cell Respira-on Be able to discuss each control step in respira4on Studying Respiratory Control Isolated mitochondria can be used to study respiratory control and other aspects of electron transport and oxida4ve phosphoryla4on. ETC and oxida4ve phosphoryla4on can be blocked By metabolic poisons Dinitrophenol (DNP) dissipates the proton gradient by carrying H+ across the inner mitochondrial membrane through simple diffusion-mediated transport The result is that carbohydrate and lipid stores are depleted in an adempt to make up for the low energy charge in cells resul4ng from decreased ATP synthesis; DNP short-circuits the proton circuit. Courtesy of Dr. R.L. Miesfeld Compare the affects of oligomycin (blocks proton channels) to DNP (an uncoupler) Brown adipose -ssue express uncoupling proteins that disrupt the proton gradient Why would a cell produce uncoupling proteins? ...
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