Ch15 Oxidative Phosphorylation PDF

Ch15 Oxidative Phosphorylation Do you remember… Living organisms obey the laws of thermodynamics (Section 1.3). *Transporters obey the laws of thermodynamics, providing a way for solutes to move down their concentration gradients or using ATP to move substances against their gradients (Sect...

Ch15 Oxidative Phosphorylation Do you remember… Living organisms obey the laws of thermodynamics (Section 1.3). *Transporters obey the laws of thermodynamics, providing a way for solutes to move down their concentration gradients or using ATP to move substances against their gradients (Section 9.1). Coenzymes such as NAD+ and ubiquinone collect electrons from compounds that become oxidized (Section 12.2). A reaction that hydrolyzes a phosphoanhydride bond in ATP occurs with a large negative change in free energy (Section 12.3). Ch15 Oxidative Phosphorylation Learning Objectives 1. Summarize the thermodynamics of oxidation– reduction reactions. 2. Map the path of electrons through the redox groups of the electron transport pathway. 3. Explain how the protonmotive force links electron transport and ATP synthesis. 4. Describe the structure and operation of ATP synthase. Section 15.1 The Thermodynamics of Oxidation- Reduction Reactions Learning Objective 1. Outline the thermodynamics of oxidation-reduction reactions Predict the direction of electron transfer in a mixture of two substances. Harvesting of free energy ▪ Metabolic fuels such as glucose, fatty acids, and amino acids, as well as the oxidation of acetyl carbons to CO2 via the citric acid cycle, yield the reduced cofactors NADH and ubiquinol (QH 2), which get reoxidized, ultimately releasing free energy ▪ That free energy is harvested to synthesize ATP, a phenomenon called oxidative phosphorylation Recap of Oxidation One reactant is in its oxidized state while the other is in its reduced state. Loss of electrons = oxidation Gain of electrons = reduction  Oxidative phosphorylation in context ▪ ATP synthesis is not directly coupled to a single discrete chemical reaction, but rather, oxidative phosphorylation is a more indirect process of energy transformation via the flow of electrons from reduced compounds such as NADH and QH2 to an oxidized compound such as O2. The reduced cofactors NADH and QH2, which are generated in the oxidative catabolism of amino acids, monosaccharides, and fatty acids, are reoxidized by a process that requires molecular oxygen. The energy of this process is conserved in a manner that powers the synthesis of ATP from ADP + Pi. Recap of oxidation–reduction ▪ In the reactions involving the cofactor FADH2 the two electrons are transferred as H atoms ▪ When NAD+ is involved, the electron pair takes the form of a hydride ion (H–) ▪ In biological systems, electrons usually travel in pairs, although, they may also be transferred one at a time. Reduction potential ▪ The tendency of a substance to accept electrons (to become reduced) is described by its standard reduction potential (εo′) at 1 M, 1 atm, pH 7, and 25˚C ▪ It is customary to consider just one substance at a time, that is, a half-reaction, such as reduction of ubiquinone: Q + 2 H+ + 2 e− ⇌ QH2 (a complete oxidation–reduction reaction is just a combination of two half-reactions)  Half-reaction Ɛ °′ (V) Standard ​1/2​ ​O​2​ + 2 ​H+​ ​ + 2 e​ − ⇌ ​H2​ ​O​ 0.815 ​SO​42−​ + 2 ​H+​ ​ + 2 e​ − ⇌ ​SO​32−​ + ​H​2​O​ 0.48 reduction ​NO​3−​ + 2 ​H​+​ + 2 e− ⇌ ​NO​2−​ + ​H​2​O​ 0.42 Cytochrome a3 (Fe3+) + e− ⇌​ cytochrome a3 (Fe2+) potentials of Cytochrome a (Fe3+) + e− ⇌​ cytochrome a (Fe2+) 0.385 0.29 Cytochrome c (Fe3+) + e− ⇌​ cytochrome c (Fe2+) 0.235 some Cytochrome c1 (Fe3+) + e− ⇌​ cytochrome c1 (Fe2+) 0.22 Cytochrome b (Fe3+) + e− ⇌​ cytochrome b (Fe2+) (mitochondrial) 0.077 biological Ubiquinone + 2 H+ + 2 e− ⇌​ ubiquinol 0.045 Fumarate− + 2 H+ + 2 e− ⇌​ succinate− 0.031 substances FAD + 2 H+ + 2 e− ⇌​ FADH2 (in flavoproteins) ~ 0. Oxaloacetate− + 2 H+ + 2 e− ⇌​ malate− − 0.166 Pyruvate− + 2 H+ + 2 e− ⇌​ lactate− − 0.185 Acetaldehyde + 2 H+ + 2 e− ⇌​ ethanol − 0.197 S + 2 H+ + 2 e− ⇌​ H2S − 0.23 Lipoic acid + 2 H+ + 2 e− ⇌​ dihydrolipoic acid − 0.29 NAD+ + H+ + 2 e− ⇌​ NADH − 0.315 NADP+ + H+ + 2 e− ⇌​ NADPH − 0.320 Acetoacetate− + 2 H+ + 2 e− ⇌​ 3-hydroxybutyrate− − 0.346 Acetate− + 3 H+ + 2 e− ⇌​ acetaldehyde + H2O − 0.581 The actual reduction potential depends on the actual concentrations of oxidized and reduced species. The Nernst Equation R = Gas Constant = 8.3145 J · mol-1 · K–1 T = temperature in Kelvin n = # of electrons ℱ = Faraday’s constant = 96,485 J · V–1 · K–1 ▪ Like a ΔG value, the actual reduction potential depends on the actual concentrations of the oxidized and reduced species. The free energy change can be calculated from the change in reduction potential  Overview of mitochondrial electron transport Passing electrons to a molecule “below” (ie, more positive) on this scale has a L1G< 0, or is favourable. NADH is the first electron donor on this scale, and O2 is the final electron acceptor, and it is reduced to H2O The reduction potentials of the key electron carriers are indicated. The oxidation–reduction reactions mediated by Complexes I, III, and IV release free energy. Mitochondrial Electron Transport ▪ The redox potential energy of NADH and and FADH2 is released stepwise via the electron transport chain Review ▪ Explain why an oxidation–reduction reaction must include both an oxidant and a reductant. ▪ When two reactants are mixed together, explain how you can predict which one will become reduced and which one will become oxidized. ▪ Select the two half-reactions from the table on Slide 9 that would be most likely to form a freely reversible (near-equilibrium) redox reaction. 14 Section 15.2 Mitochondrial Electron Transport Learning Objective 2. Map the path of electrons through the redox groups of the electron transport pathway Explain why the mitochondrion includes a variety of transport systems Identify the sources of electrons for Complexes I, III, and IV Describe the mechanisms for transporting protons across the mitochondrial membrane Explain why respiratory complexes may not actually form a chain Cellular respiration ▪ In aerobic organisms, the NADH and ubiquinol produced by glycolysis, the citric acid cycle, fatty acid oxidation, and other metabolic pathways are ultimately reoxidized by molecular oxygen, a process called cellular respiration. ▪ The standard reduction potential of +0.815 V for the reduction of O2 to H2O indicates that O2 is a more effective oxidizing agent than any other biological compound. ▪ Electrons are shuttled from NADH to O2 in a multistep process called the respiratory electron transport chain (ETC).  Electron transport takes place in the mitochondrion This is where the citric acid cycle happens! The relatively impermeable inner mitochondrial membrane encloses the protein- rich matrix. The intermembrane space has an ionic composition similar to that of the cytosol. Experimental imaging helps us know what mitochondria look like Conventional electron Three-dimensional Fluorescence micrograph of micrograph showing reconstruction of a cultured human cells. The cristae as a system of mitochondrion by electron cytosol contains a network of planar baffles. tomography, showing irregular mitochondria (red). The tubular cristae. nucleus is blue. Cofactors transfer electrons to the ETC ▪ Much of the cell’s NADH and QH2 is generated by the citric acid cycle inside mitochondria. Fatty acid oxidation also takes place largely inside mitochondria and yields NADH and QH2. ▪ These reduced cofactors transfer their electrons to the protein complexes of the respiratory electron transport chain, which are tightly associated with the inner mitochondrial membrane. ▪ NADH produced by glycolysis and other oxidative processes in the cytosol cannot directly reach the respiratory chain and instead use “reducing equivalents” The malate-aspartate shuttle system is an example of “reducing equivalents” Cytosolic oxaloacetate is reduced to malate for transport into mitochondria. Malate is then reoxidized in the matrix. The net result is the transfer of “reducing equivalents” from the cytosol to the matrix. Mitochondrial oxaloacetate can be exported back to the cytosol after being converted to aspartate by an aminotransferase. Mitochondrial transport system for ATP, ADP and Pi ▪ ATP translocase: The ADP/ATP carrier binds either ATP or ADP and changes its conformation to release the nucleotide on the opposite side of the inner mitochondrial membrane. This translocase can therefore export ATP and import ADP. ▪ A symport protein permits simultaneous movement of Pi and H+ into the mitochondria l matrix. Summary of mitochondrial electron transport ▪ The electron transport chain is associated with the mitochondrial inner membrane Complex I transfers electrons from NADH to ubiquinone NADH + H+ + Q ⇌ ​ ​ NAD+ + QH2 ▪ Complex I is the largest of the electron transport proteins in the mitochondrial respiratory chain ▪ Electron transport takes place in the peripheral arm of Complex I, which includes several prosthetic groups, or redox centers, that undergo reduction as they receive electrons and become oxidized as they give up their electrons to the next group. ▪ The electrons travel from one redox center to another of increasing reduction potential.  Structure of bacterial Complex I The 16 subunits are shown in different colors, with Arrangement of the redox centers the redox centers in red (FMN) and orange (Fe–S in Complex I. Atoms are color clusters). The horizontal portion is embedded in the coded: C gray, N blue, O red, P membrane (represented by black lines) and the orange, Fe gold, and S yellow. “arm” projects into the cytosol in bacteria (or Electrons flow through the groups mitochondrial matrix in eukaryotes). from upper left to lower right. Flavin mononucleotide (FMN) ▪ The two electrons donated by NADH are first picked up by flavin mononucleotide (FMN) near the far end of the Complex I arm. This noncovalently bound prosthetic group, which is similar to FAD, then transfers the electrons, one at a time, to a second type of redox center, an iron–sulfur (Fe–S) cluster. Iron-sulfur clusters ▪ Unlike the electron carriers we have introduced so far, Fe–S clusters are one-electron carriers. ▪ Electrons travel between several Fe–S clusters before reaching ubiquinone. Complex I function ▪ As electrons are transferred from NADH to ubiquinone, Complex I transfers four protons from the matrix to the intermembrane space via a proton wire (a series of hydrogen-bonded protein groups plus water molecules that form a chain through which a proton can be rapidly relayed). ▪ Note that the protons taken up from the matrix are not the same ones that are released into the intermembrane space. Reactions that contribute to the ubiquinol pool ▪ The reduced quinone product of the Complex I reaction joins a pool of quinones that are soluble in the inner mitochondrial membrane. ▪ The pool of reduced quinones is augmented by the activity of other oxidation–reduction reactions. ▪ One of these is catalyzed by succinate dehydrogenase Complex II): succinate + Q ⇌ fumarate + QH2 Complex III transfers electrons from ubiquinol to cytochrome c ▪ Ubiquinol is reoxidized by Complex III, also called ubiquinol:cytochrome c oxidoreductase or cytochrome bc1, which transfers electrons to the peripheral membrane protein cytochrome c. ▪ Cytochromes are proteins with heme prosthetic groups. ▪ The flow of electrons through Complex III is complicated because the two electrons donated by ubiquinol must split up in order to travel through a series of one-electron carriers (the 2Fe– 2S cluster of the iron–sulfur protein, cytochrome c1, and cytochrome b). The Fe-S protein must change its conformation by rotating and moving about 22 Å in order to pick up and deliver an electron. The heme group of a b cytochrome The heme groups of cytochromes undergo reversible one-electron reduction, with the central Fe atom cycling between the Fe3+ (oxidized) and Fe2+ (reduced) states The planar porphyrin ring surrounds a central Fe atom, shown here in its oxidized (Fe3+) state. The heme substituent groups that are colored blue differ in the a and c cytochromes. The heme group of a b cytochrome Cytochromes are proteins with heme prosthetic groups.  Unlike the heme groups in hemoglobin and myoglobin, heme in cytochrome c undergoes reversible one- electron transfers.  The central iron atom is either oxidized (Fe3+) or reduced (Fe2+).  Structure of mammalian Complex III Backbone model. Eight transmembrane helices Arrangement of prosthetic groups. The two in each monomer of the dimeric complex are heme groups of each cytochrome b (blue) and contributed by cytochrome b (light blue with the heme group of cytochrome c1 (purple), heme groups dark blue). The iron–sulfur along with the iron–sulfur clusters (Fe atoms protein (green with Fe–S clusters orange) and orange), provide a pathway for electrons cytochrome c1 (pink with heme groups purple) between ubiquinol (in the membrane) and project into the intermembrane space. cytochrome c (in the intermembrane space). The Q cycle (the route of electrons from ubiquinol to cytochrome c) – round 1 The Q cycle – round 2 ▪ The net result of the Q cycle is that two electrons from QH2 reduce two molecules of cytochrome c. In addition, four protons are translocated to the intermembrane space, two from QH2 in the first round of the Q cycle and two from QH2 in the second round. QH2 + 2 cytochrome c (Fe3+) ⇋ Q + 2 cytochrome c (Fe2+) + 2 H+ Complex III function Cytochrome c ▪ Cytochrome c, transfers electrons, one at a time, between Complexes III and IV. ▪ Four electrons delivered by cytochrome c are consumed in the reduction of molecular oxygen to water and four additional protons are relayed from the matrix to the intermembrane space: 4 cytochrome c (Fe2+) + O2 + 4H+ → 4 cytochrome c (Fe3+) + 2 H2O The protein is shown as a gray transparent surface over its ribbon backbone. The heme group (pink) lies in a deep pocket. Structure of Complex IV(cytochrome c oxidase) The heme groups (C atoms gray, N blue, O red, and Fe gold) and copper ions (brown) The 13 subunits in each monomeric half of the from one half of the complex mammalian complex comprise 28 are shown in space-filling transmembrane α helices. form. Complex IV function For every two electrons donated by cytochrome c, two protons are translocated to the intermembrane space Two protons from the matrix are also consumed in the reaction: ½ O2 ➔ H2O Review ▪ Describe the compartments of a mitochondrion. ▪ List the transport proteins that occur in the inner mitochondrial membrane. ▪ Draw a simple diagram showing the electron-transport complexes and the mobile carriers that link them. ▪ List the different types of redox groups in the respiratory electron transport chain and identify them as one- or two-electron carriers. ▪ Explain why O2 is the final electron acceptor in the chain. ▪ Describe the operation of a proton wire. ▪ Write an equation to describe the overall redox reaction carried out by each mitochondrial complex. ▪ Compare the arrangement of an electron transport chain and a supercomplex. 39 Section 15.3 Chemiosmosis Learning Objective 3. Explain how the protonmotive force links electron transport and ATP synthesis Describe the formation of the proton gradient Relate the pH difference of the proton gradient to the free energy change How much energy is available from electron transport? Complex I: NADH → QH2 ΔG°′ = –69.5 kJ mol–1 Complex III: QH2 → cytochrome c ΔG°′ = –36.7 kJ mol–1 Complex IV: cytochrome c → O2 ΔG°′ = –112.0 kJ mol–1 NADH → O2 ΔG°′ = –218.2 kJ mol–1 ▪ Using the ΔG values calculated from the standard reduction potentials of the substrates and products of Complexes I, III, and IV, we can see that each of the three respiratory complexes theoretically releases enough free energy to drive the endergonic phosphorylation of ADP to form ATP (ΔG°′ = +30.5 kJ·mol−1). Generation of a proton gradient Generation of a proton gradient ▪ The proton-translocating activity of the electron transport complexes in the inner mitochondrial membrane generates a proton gradient across the membrane, a source of energy, that can drive the activity of an ATP synthase. ▪ The protons cannot diffuse back into the matrix because the membrane is impermeable to ions ▪ This proton-motive force is a and the imbalance creates source of free energy and will protonmotive force. be what drives ATP synthesis! Protonmotive force for driving the phosphorylation of ADP ▪ The free energy from passage of the proton back into the matrix ΔG°′ = –20 kJ · mol−1 would not be enough to drive the phosphorylation of ADP (ΔG°′ = +30.5 kJ·mol−1). ▪ However, the 10 protons translocated for each pair of electrons transferred from NADH to O2 have an associated protonmotive force of 218.2 kJ · mol−1, enough to drive synthesis of several molecules of ATP. Review ▪ Describe the importance of mitochondrial structure for generating the protonmotive force. ▪ Identify the source of the protons for the transmembrane gradient. ▪ Explain why the proton gradient has a chemical and an electrical component. 45 Section 15.4 ATP Synthase Learning Objective Describe the structure and operation of ATP synthase Recognize the structural components of ATP synthase. Identify the energy transformations that occur in ATP synthase. Describe the binding change mechanism. Explain why P:O ratios are nonintegral. Explain why oxidative phosphorylation is coupled to electron transport.  Oxidative Phosphorylation  As protons move back into the matrix through a special transmembrane protein, the energy stored in this electrochemical gradient is used to make ATP  The chemiosmotic hypothesis earned Peter Mitchell the Nobel Prize in 1978 Components of ATP synthase ▪ The protein that taps the electrochemical proton gradient to phosphorylate ADP is known as the F-ATP synthase (or Complex V). ▪ The Fo part functions as a transmembrane channel that permits H+ to flow back into the matrix, following its gradient. ▪ The F1 component catalyzes the reaction ADP + Pi → ATP + H2O Components of ATP synthase https://youtu.be/kXpzp4RDGJI?si=o4kpxO4JYTLLUoPc  Structure of ATP synthase ▪ In all species, proton transport through ATP synthase requires rotation of the c ring past the stationary a subunit. ▪ The carboxylate side chain of a highly conserved aspartate or glutamate residue on each c subunit serves as a proton binding site. Diagram of the mammalian enzyme. The α and β subunits are connected via a central shaft to the membrane- embedded ring of 8 c subunits. A peripheral stalk links the a subunit to the catalytic domain. Mechanism of proton transport by ATP synthase ▪ A c subunit takes up a proton from the intermembrane space and the binding neutralizes the carboxylate group, freeing it from the electrostatic attraction of a positively charged arginine residue on the a subunit. ▪ The protonated c subunit moves away, and this slight rotation of the c ring brings another c subunit into position so that it can release its bound proton into the matrix. Structure of the F1 component of ATP synthase ▪ The three αβ pairs change their conformations as the γ subunit rotates. ▪ As each proton moves across the membrane, the c ring and γ subunit rotate. ▪ The αβ hexamer itself does not rotate, since it is held in place by the peripheral stalk b that is anchored to the a subunit. ▪ α = blue β = green γ = purple Production of ATP ▪ ATP Synthase uses mechanical energy (rotation) to form a chemical bond (the attachment of a phosphoryl group to ADP) ▪ Rotation-driven conformational changes alter the affinity of each catalytic β subunit for an adenine nucleotide (although all six subunits can bind adenine nucleotides, only the β subunits have catalytic activity). At any moment, each catalytic site has a different conformation (and binding affinity), referred to as the open, loose, or tight state. Regulation of ATP synthase in eukaryotes ▪ In eukaryotes, ATP synthase itself is regulated by a small protein called inhibitory factor 1 (IF1). Different forms of IF1 are intrinsically disordered at relatively high matrix pH values, when the electron transport chain is pumping protons into the intermembrane space and the proton gradient is steep. ATP synthase dimers ▪ When the pH drops, IF1 dimerizes and forms extended α helices that insert in between the α and β subunits of F1 and contact the γ shaft. ▪ IF1 binding prevents ATP synthase from carrying out the binding change mechanism. Powering Human Muscles ▪ Cells cannot stockpile ATP ▪ Phosphocreatine plays a role? Babies can’t shiver ▪ When kids say they aren’t cold …  Where else does uncoupling happen? ▪ Hibernating nammals 1) Increases in membrane and transport proteins during hibernation allow for an increase in fatty acid handling in BAT mitochondria. 2) Along with fatty acid handling, an increase in mitochondrial metabolism associated with-oxidation and TCA cycle are present during hibernation. 3) The increase in reducing equivalents shuttling through the ETC is indicated by enhanced respiration rates through the various complexes of the ETC during hibernation. 4) The enhancements of all the various mitochondrial aspects (nos. 1-3) allow for an increase in heat production via UCP1 during hibernation. 2,4-Dinitrophenol (DNP) It was first discovered to induce wight loss during World War I when it was noticed that French munitions workers who were exposed to dinitrophenol during the synthesis of dynamite (trinitrotoluene, TNT) rapidly lost weight. In the 1930s it was prescribed by physicians for weight loss and was also available over-the-counter, but because people suffered significant side effects, such as cataracts, blindness, kidney and liver damage and death, it was banned for medical use in United States after a congressional investigation. Review ▪ Draw a simple diagram of ATP synthase and indicate which parts are stationary and which rotate. ▪ Explain how ATP synthase dissipates the proton gradient. ▪ Recount how the three conformational states of the β subunits of ATP synthase are involved in ATP synthesis. ▪ Explain how ATP synthase could operate in reverse to hydrolyze ATP. ▪ Explain why the number of protons translocated per ATP synthesized varies among species. ▪ Explain why the availability of reduced substrates is the primary mechanism for regulating oxidative phosphorylation. 60