Mitochondrial Structure and Function PDF

Mitochondrial Structure and Function • The early Earth was populated by anaerobes, which captured and utilized energy by oxygen-independent metabolism like glycolysis and fermentation • Oxygen accumulated in the primitive atmosphere after cyanobacteria appeared, which carried out a new type of photo...

Mitochondrial Structure and Function • The early Earth was populated by anaerobes, which captured and utilized energy by oxygen-independent metabolism like glycolysis and fermentation • Oxygen accumulated in the primitive atmosphere after cyanobacteria appeared, which carried out a new type of photosynthetic process in which water molecules were split apart and molecular oxygen was released • Aerobes evolved to use oxygen to extract more energy from organic molecules, and they eventually gave rise to all of the oxygen-dependent prokaryotes and eukaryotes living today • In eukaryotes, the utilization of oxygen as a means of energy extraction takes place in a specialized organelle, the mitochondrion. Mitochondrial Structure :How do they look like? Mitochondria: characteristic morphologies despite variable appearance. Typical mitochondria are bean-shaped organelles but may be round or threadlike. Size and number of mitochondria reflect the energy requirements of the cell. Fig 5.1 Elongated mitochondria of fibroblast Transmission electron micrograph Mitochondria in the sperm mid-piece (50-75 mitochondria in a sperm cell)-why so many- needs energy to reach the egg Mitochondrial Structure • Mitochondria can fuse with one another, or split in two. • The balance between fusion and fission is likely a major determinant of mitochondrial number, length, and degree of interconnection. Fig 5.2 b 3D model of contacts between ER and mitochondria © 2013 John Wiley & Sons, Inc. All rights reserved. Mitochondrial Structure Fig 5.2 a Fission is accomplished by proteins (Drp1 in mammals) that assemble into helices around the outer surface of the mitochondria (step2). Fig 5.2 c: Model for mitochondrial fission: ER tubules and Drp1 mediate mitochondrial constriction. https://www.youtube.com/watch?v=CIXY-Ns5vks Mitochondrial Structure: Mitochondrial Membranes • The outer boundary of a mitochondrion contains two membranes: – The outer mitochondrial membrane and – The inner mitochondrial membrane • The outer mitochondrial (50 % protein) membrane serves as its outer boundary Fig 5.3a-Scanning electron micrograph of a macerated mitochondrion  The outer membrane contains a large pore-forming protein called porin.  The inner mitochondrial membrane (75 % protein) is divided into two major domains that have different proteins and carry out distinct functions :  Inner boundary membrane  Cristae: where the machinery for ATP is located  The inner membrane contains cardiolipin but not cholesterol (just like bacterial membranes) Mitochondrial Structure: Mitochondrial Membranes – The inner boundary membrane domain, along with the outer membrane, forms a double-membrane outer envelope • The other domain lies in the interior of the organelle as a series of invaginated membranous sheets (large surface area), called cristae, which houses the machinery needed for aerobic respiration and ATP formation • The inner boundary membrane and internal cristal membranes are joined to one another by cristae junctions Mitochondria contain 37 genes and all are essential for normal mitochondrial function while 13 genes are involved in oxidative phosphorylation Fig 5.3c. Schematic diagrams showing the 3D internal structure and a thin section of a mitochondrion from bovine heart tissue Mitochondrial Structure: Mitochondrial Membranes • The membranes of the mitochondrion divide the organelle into two aqueous compartments: • One within the interior of the mitochondrion, called the matrix, with gel-like consistency Image taken from :https://www.quia.com/files/quia/users/lmcgee/Biology-Learning-Center-upload-10-2105/cell_processes/cellular_respiration/assets/graphics/cells_and_parts/mitochondrialabeled1.gif • A second between the outer and inner membrane, called the intermembrane space • The inner membrane is impermeable to even small molecules, virtually all molecules and ions require special membrane transporters to gain entrance to the matrix. Mitochondrial Structure: Mitochondrial Matrix • The mitochondrial matrix contains ribosomes and several molecules of circular DNA to manufacture their own RNAs and proteins • The DNA encodes a small number of mitochondrial polypeptides (13 in humans) that are tightly integrated into the inner mitochondrial membrane. – The other polypeptides are encoded by genes residing within the nucleus http://www.bio.unipd.it/~bubacco/assets/images/mitochondrion2a.jpg • Mitochondrial DNA (mtDNA) is a relic thought to be the legacy from a single aerobic bacterium that took up residence in the cytoplasm of a primitive cell that ultimately became an ancestor of all eukaryotic cells. How mitochondria get DNA? Bacteria survived within the eukaryotic cell, becoming an endosymbiont, which over time became mitochondria. All eukaryotes (including us) are descended from the mitochondriacontaining eukaryote. This event is thought to have occurred around 2 billion years ago. Eukaryotic cells engulfed an aerobic prokaryote which then formed an endosymbiotic relationship with the host eukaryote and gradually develop into a mitochondrion. Summary Slide: Mitochondria Structure and Functions Intermembrane space Small space to quickly accumulate protons Inner membrane Rich in protein (75%) and contains ETC and ATP synthase for oxidative phosphorylation Matrix Cristae Contains enzymes and a suitable pH to facilitate reactions Highly folded structure and increases SA: Vol ratio Outer membrane (Porins) and transport proteins called pyruvate translocase to transport pyruvate into the mitochondria Q) What is the difference between cytosol and cytoplasm? Cytosol is a fluid present in the cell membrane. It is composed of soluble ions, water, water-soluble proteins and molecules Cytoplasm is a cell component present inside the cell membrane. It is composed of enzymes, water, lipids, carbohydrates, nucleic acids and inorganic ions. Mitochondrial Structure and Function Summary of Eukaryotic Carbohydrate Metabolism Glycolysis takes place in (cytosol) TCA cycle takes place in (mitochondria) Coupling cytosolic glycolysis and pyruvate production to the mitochondrial TCA cycle and ATP formation Oxidative Metabolism in the Mitochondrion An overview Glycolysis • The first steps in oxidative metabolism are carried out in glycolysis in the cytosol; it is an anaerobic process (9/10 steps not use oxygen) • Glycolysis produces two pyruvate, two NADH, and two (net) molecules of ATP per glucose • Most of the energy remains in pyruvate! • Aerobic organisms use O2 to extract more ATPs from pyruvate and NADH • The ATP molecules are produced via substrate-level phosphorylation here Oxidative Metabolism in the Mitochondrion An overview Glycolysis To produce two Pyruvate molecules: ATP produced= 2x2=4 ATP used=2 Net gain of ATP=4-2=2 glucose+ fructose Acetyl CoA Formation from Pyruvate What did you notice? First CO2 is removed from the original glucose molecule Pyruvate is transported across the inner membrane (into the matrix) and decarboxylated to form acetyl CoA, which enters the next stage Thioester bond Joining to CoA results in acetyl CoA by thioester link. It is a high-energy bond and very unstable. Q) How Acetyl CoA forms from Pyruvate? Pyruvate formed is transported into the into matrix by oxidative decarboxylation of pyruvate and form acetyl CoA (electrons are transferred to NAD+ to NADH)used later to generate ATP Q) What is the end product of glycolysis? 2 pyruvate, 2 NADH and 2 ATP molecules Oxidative Metabolism in the Mitochondrion: The TCA Cycle For two pyruvate molecules: CO2 produced=6 NADH produced= 8x3=24 FADH2 produced=2x2=4 GTP= 2 Total= 30 ATP For one pyruvate molecule: CO2 produced=3 NADH produced= 4 FADH2 produced=1 GTP= 1 8.Dehydrogenation 1.Condensation 7.Hydration 2.Dehydration/Rehydration 5 paors of elctrons (from hydrogen atoms of substrate) to be used In ATP production 3. Oxidative decarboxylation 6.Dehydration X 5. Substrate level phosphorylation 4.Oxidative decarboxylation Oxidative Metabolism in the Mitochondrion: The TCA Cycle • Acetyl-CoA is fed into the TCA cycle where it’s oxidized and its energy conserved • Other than succinate dehydrogenase, which is bound to the inner membrane, all the enzymes of the TCA cycle reside in the soluble phase of the matrix • The first step in the TCA is the condensation of the 2-carbon acetyl group of CoA with the fourcarbon oxaloacetate to form a six-carbon citrate • During the cycle, two carbons are oxidized to CO2, regenerating the four-carbon oxaloacetate needed to continue the cycle • Four reactions in the cycle transfer a pair of electrons to NAD+ to form NADH, or to FAD to form FADH2. • NAD+ and FAD are coenzymes Citric acid cycle (Krebs cycle or TCA cycle) succinate dehydrogenase, which is bound to the inner membrane, all the enzymes of the TCA cycle reside in the soluble phase of the matrix Four reactions in the cycle transfer a pair of electrons to NAD+ to form NADH, or to FAD to form FADH2. Some steps are combine in the next slide Q) How many enzymes catalyzes the TCA cycle? Can you name them? 1.Citrate synthase 2.Aconitase 3.Isocitrate dehydrogenase 4.α-ketoglutarate dehydrogenase 5.Succinyl-CoA synthetase 6.Succinate dehydrogenase 7.Fumarase 8.Malate dehydrogenase Q) How many total carbons from an original glucose molecule will enter into the TCA cycle? a) 2 b) 3 c) 4 d) 6 Q) How many total carbons from an original glucose molecule will enter into the TCA cycle in the absence of oxygen? a) 2 b) 3 c) 4 d) 0 Recall or Summary succinyl-CoA is hydrolyzed with release of free energy. That energy is conserved in the substrate phosphorylation of GDP with phosphate and form GTP Why GTP ? 5. Substrate level phosphorylation Where do you see GTP hydrolysis and ATP hydrolysis? GTP hydrolysis is common in signal transduction, whereas ATP hydrolysis is used for force generation or other energy requiring process. What happened with the lactate produced in the body? Under limited supply of oxygen body temporarily converts pyruvate into a substance called lactate that convert into pyruvate and produce glucose by a process called gluconeogenesis (formation of glucose from non-carbohydrate sources is called gluconeogenesis) Q) How TCA cycle keeps going on? Q) From where TCA cycle is getting oxaloacetate (starter of the TCA cycle)? Q) Why it does not deplete? Catabolic pathways generate compounds that are fed into the TCA cycle  Oxidation of fatty acids and activation to thio (SH) group of coenzyme A  Input of amino acids into the TCA cycle Oxidative Metabolism in the Mitochondrion: The TCA Cycle Input of amino acids into the TCA cycle Acetyl CoA Reaction intermediates in the TCA cycle are common compounds generated in other catabolic reactions making the TCA cycle the central metabolic pathway of the cell https://www.youtube.com/watch?v=JPCs5pn7UNI https://www.youtube.com/watch?v=p-k0biO1DT8 Fig 5.8: Catabolic pathways generate compounds that are fed into the TCA cycle Oxidative Metabolism in the Mitochondrion What happens to NADH molecules produced during glycolysis? How NADH imports into the mitochondrial matrix from cytosol? • Mitochondria are not able to import the NADH formed in the cytosol during glycolysis ( 2 NADH/glucose molecule) • There are two main ways that cells can make use of the NADH molecules produced in the cytosol: 1. Malate-Aspartate Shuttle 2. Glycerol Phosphate Shuttle Summary Slide 1. Malate-Aspartate Shuttle MAD Commute Malate in. Alpha-ketoglutarate and D(Aspartate) out. 2. Glycerol Phosphate Shuttle GLYCOLSIS- Step 5: Isomerization of dihydroxyacetone phosphate (DHAP) into glyceraldehyde 3phpsphate https://www.ncbi.nlm.nih.gov/books/NBK22470/ Oxidative Metabolism in the Mitochondrion Malate-aspartame shuttle Malate dehydrogenase is present in two forms in the shuttle system: mitochondrial malate dehydrogenase and cytosolic malate dehydrogenase. 1. In cytosol, malate dehydrogenase catalyzes the reaction of oxaloacetate and produces malate (require 2 electrons and H+ to form malate) – NADH is oxidized to NAD+ in cytosol and now can enter into the mitochondrial matrix. 2. Now antiporter imports the malate from cytosol and exports alpha-ketoglutarate from the matrix into the cytosol simultaneously. 3. Once Malate reaches the mitochondrial matrix, Mitochondrial malate dehydrogenase catalyzes the reaction into oxaloacetate (NAD+ is reduced with two electrons to form NADH). 4. Oxaloacetate is then transformed into aspartate by mitochondrial aspartame aminotransferase and transport into the cytosol. 5. Aspartame needs an amino radical to form oxaloacetate which is supplied by glutamate. Is it symport or antiporter? What are the two forms of nicotinamide adenine dinucleotide (NAD) NAD+ and NADH NAD+ forms NADH by accepting a hydride atom (a hydrogen atom with and extra electrons or two electrons H-) Reduction Oxidation Oxidized form NAD+ or NADP+ Reduced form NADH or NADPH Remember: NAD+ is an electron carrier and provides a stable shuttle for the movement of electrons in the internal environment of the cell. Oxidative Metabolism in the Mitochondrion The Glycerol phosphate shuttle • The reduced coenzymes FADH2 and NADH are the primary products of the TCA cycle • Mitochondria are not able to import the NADH formed in the cytosol during glycolysis ( 2 NADH/glucose molecule) • It can be done by cytosolic glycerol 3-phosphate dehydrogenase and mitochondrial 3- phosphate dehydrogenase enzymes. 1. Electrons are transferred from NADH to dihydroxyacetone phosphate (DHAP) to form glycerol 3-phosphate (G3P). • DHAP + 2e- + H+  G3P • G3P moves into intermembrane space and gets oxidized by G3P dehydrogenase, reverting back to DHAP • G3P dehydrogenase reduces FAD into FADH2 by taking two electrons 2. These electrons are indirectly fed into the mitochondrial electron-transport chain and used for ATP formation NADH formed during glycolysis enters the mitochondria via the glycerol phosphate shuttle Fig 5.9: Electron transfer from NADH to DHAP to form glycerol 3phosphate, then to FAD to form FADH2 Oxidative Metabolism in the Mitochondrion Summary of oxidative phosphorylation The Importance of Reduced Coenzyme in the Formation of ATP in the Electron Transport Chain • Step 1:High-energy electrons from NADH and FADH2 are passed to electron carriers of ETC, energy is released, which is coupled to energy-required conformational changes, leading to H+ being pumped out across the inner membrane Inner membrane Inter membrane space • Step 2: ATP is formed by the controlled movement of H+ back across the membrane through the ATP-synthesizing enzyme • Coupling of H+ translocation to ATP synthesis is called chemiosmosis • Tally: • 3 molecules of ATP are formed from each pair of electrons donated by NADH • 2 molecules of ATP are formed from each pair of electrons donated by FADH2. • Per glucose molecule, the net gain is about 36 ATP molecules https://www.youtube.com/watch?v=VER6xW_r1vc Fig 5:10- Two step process of oxidative phosphorylation: Formation and harnessing of the proton gradient Oxidative Metabolism in the Mitochondrion Summary of oxidative phosphorylation Glycolysis To produce two Pyruvate molecules: ATP produced= 2x2=4 NADH produced= 2x3=6 ATP used=2 Total= 8 TCA cycle For two pyruvate molecules: CO2 produced=6 NADH produced= 8x3=24 FADH2 produced=2x2=4 Total= 28 ATP ATP is formed by the controlled movement of H+ through the ATP synthesizing enzymechemiosmosis The Human Perspective: The Role of Anaerobic and Aerobic Metabolism in Exercise • ATP hydrolysis increases 100-fold during exercise, quickly exhausting ATP available. • Muscles used stored creatine phosphate (CrP) to rapidly generate but must rely on aerobic or anaerobic synthesis of new ATP for sustained activity. CrP + ADP  Cr + ATP Oxidative Phosphorylation in the Formation of ATP • Mitochondria extract energy from organic materials and store it, temporarily, in the form of electrical energy (ionic gradient) • Mitochondria utilize an ionic gradient across their inner membrane to drive numerous energy-requiring activities, the synthesis of ATP • When ATP formation is driven by energy that is released from electrons removed during substrate oxidation, the process is called oxidative phosphorylation • Oxidative phosphorylation accounts for the production of more than 2x1026 (>60) kg of ATP in our bodies per day! Redox Refresher • Redox Reactions: It is an electron transfer reactions where reduction and oxidation occurs simultaneously • In terms of oxygen: • Oxidation is the gain of oxygen • Reduction is the loss of oxygen • In terms of hydrogen: • Oxidation is the loss of hydrogen • Reduction is the gain of hydrogen • In terms of electrons: • Oxidation is the loss of electrons • Reduction is the gain of electrons Loss of electron OXIDATION Gain of electron REDUCTION A is oxidized because it lost electrons B is reduced because it gains electrons (REDUCING AGENT) (OXIDIZING AGENT) A e e Oxidation A Oxidized Reducing agent B Oxidizing agent Reduction B Reduced e e Oxidative Phosphorylation in the Formation of ATP Oxidation Reduction Potentials • Strong oxidizing agents have a high affinity for electrons; strong reducing agents have a weak affinity for electrons • Reducing agents are ranked according to electron-transfer potential • A high electron-transfer potential means a strong reducing agent • Oxidizing and reducing agents occur as couples (see next slide), such as NAD+ and NADH, which differ in their number of electrons • Strong reducing agents are coupled with weak oxidizing agents and vice versa. For e.g. NAD + (of the NAD + -NADH couple) is a weak oxidizing agent-loss electrons, whereas O 2 (of the O 2 -H 2 O couple) is a strong oxidizing agent-gains electrons. • The transfer of electrons between a couple causes charge separation that can be measured as an oxidation-reduction, or redox, potential by instruments that detect voltage. • Redox reactions are accompanied by a decrease in free energy. Oxidative Phosphorylation in the Formation of ATP Oxidation Reduction Potentials  Standard conditions are 1 .0M for solutes and ions and 1 atm pressure for gases (e.g., H2 ) at 25°C.  The standard redox potential for the oxidation–reduction reaction involving hydrogen ( 2 H+ + 2 electrons H2 ) is 0 .00V  An electric circuit is formed by connecting the half-cells through a voltmeter and a salt bridge that provides a path for counter ions to move between the half-cells. What is the voltmeter reading? What did you observe?  Electrons flowing from the sample half-cell to the reference halfcell result in negative redox potential  If electrons flow in the opposite direction, the standard redox potential of the sample couple is positive. Fig 5.11: Measuring the standard oxidation–reduction potential in half-cells using a voltmeter and salt bridge Oxidative Phosphorylation in the Formation of ATP Oxidation Reduction Potentials  Redox potential is the potential for a species to acquire or to donate electrons  At pH 7, the standard redox potential is indicated by Eo’ rather than Eo.  Those couples whose reducing agents are better donors of electrons are assigned more negative redox potentials (NAD+ - NADH couple is -0.32)  Acetaldehyde is a stronger reducing agent than NADH , Acetate-acetaldehyde couple has a standard redox potential of -0.58V.  The couples whose oxidizing agents are better electron acceptors than NAD + have greater affinity for electrons than NAD+ and have more positive redox potentials. Redox potential of some reaction couples Summary: Oxidative Phosphorylation in the Formation of ATP • Electrons are transferred to NAD+ (or FAD) within the mitochondrion from substrates of the TCA cycle: isocitrate, malate, α-ketoglutarate, and succinate • Most have redox potentials of relatively high negative values, sufficiently high to transfer electrons to NAD+ Example: • What does this mean? (use H2 as a reference) • Couples whose reduced form has a lower affinity to electrons than does H2, will have negative redox-potentials • Couples whose reduced form has a higher affinity to electrons than does H2, will have positive redox-potentials Oxidative Phosphorylation in the Formation of ATP Electron Transport  Five of the reactions from glycolysis to TCA cycle is catalyzed by dehydrogenases, enzymes that transfer pairs of electrons from substrates to coenzymes (such as NAD+ and FAD): Pyruvate dehydrogenase 1.Citrate synthase – Coenzymes: are organic molecules that bind 2.Aconitase 3.Isocitrate dehydrogenase loosely to the active site of an enzyme and 4.α-ketoglutarate aid in substrate recruitment. dehydrogenase – Cofactors: a non-protein compound 5.Succinyl-CoA synthetase (inorganic) that is needed for an enzymes 6.Succinate dehydrogenase biological activity 7.Fumarase 8.Malate dehydrogenase  Four of these reactions generate NADH and one produces FADH2  High-energy electrons associated with NADH or FADH2 are transferred through a series of specific electron carriers that constitute the electron-transport chain of the inner mitochondrial membrane What are cofactors and coenzymes Enzyme Inorganic (Metal ions) Non protein component Cofactors Organic Not tightly bound with enzyme and release after catalysis the catalysis called coenzyme Mg+2 Holoenzyme Holoenzyme Tightly bound organic factor is called a prosthetic group Apoenzyme Coenzymes are derived from vitamins Examples: NADH, NADPH, FAD etc. Q) Name the active form of enzyme Holoenzyme Q)Name the inactive form of enzyme Apoenzyme Select True/ False Without coenzymes or cofactors, enzymes cannot catalyze reactions effectively. True What do you called an enzyme when it loses its cofactor? apoenzyme Oxidative Phosphorylation in the Formation of ATP Types of Electron Carrier • In the ETC, there are 5 types of membrane-bound electron carriers: • Flavoproteins • Cytochromes • Copper atoms • Ubiquinone • Iron-sulfur proteins • All their redox centres are prosthetic groups, except for ubiquinone • Most of these carriers are parts of larger complexes in the ETC Oxidative Phosphorylation in the Formation of ATP Types of Electron Carrier 1. Flavoproteins are polypeptides bound to one of two prosthetic groups: • Flavin adenine dinucleotide (FAD) or • Flavin mononucleotide (FMN) • Prosthetic group is derived from Vit B2 (riboflavin) • FMN and FAD are capable of accepting and donating two protons and two electrons.  Major flavoproteins of the mitochondria are NADH dehydrogenase of the electron-transport chain and succinate dehydrogenase of the TCA cycle. H+ + e- H+ + e- Quinone Semiquinone Hydroquinone Fig 5.12 a Oxidative Phosphorylation in the Formation of ATP Types of Electron Carrier 2. Cytochromes contain heme prosthetic groups bearing Fe or Cu metal ions.  The iron atom of a heme undergoes reversible transition between the Fe3+ and Fe2+ oxidation states as a result of the acceptance and loss of a single electron  There are three distinct cytochrome types (a, b, and c) present in the electron-transport chain, which differ from one another by substitutions within the heme group Cytochrome c is a soluble protein in the intermembrane space Fig 5.12 b Oxidative Phosphorylation in the Formation of ATP Types of Electron Carrier 3. Copper atoms all located within a single protein complex of the inner mitochondrial membrane accept and donate a single electron as they alternate between the Cu 2+ and Cu 1+ states. Flow of electrons: four redox centers of cytochrome oxidase-iron atoms are red and copper atoms are yellow: cytochrome c  CuA  Heme group of cytochrome a  heme group of cytochrome a3  CuB Fig 5.20 Oxidative Phosphorylation in the Formation of ATP Types of Electron Carrier 4. Ubiquinone (coenzyme Q) is a lipid-soluble molecule.  Each ubiquinone is able to accept and donate two electrons and two protons  The partially reduced molecule is the free radical ubisemiquinone, and the fully reduced molecule is ubiquinol ( UQH 2 ). H+ + eUbiquinone Ubisemiquinone H+ + e- Ubiquinol Fig 5.12 c Oxidative Phosphorylation in the Formation of ATP Types of Electron Carrier 5. Iron-sulfur proteins are iron-containing proteins in which the iron atoms are not located within a heme group but instead are linked to inorganic sulfide ions as part of an iron-sulfur center • The most common centers contain either two or four atoms of iron and sulfur—designated [2Fe-2S] and [4Fe4S]—linked to the protein at cysteine residues • Even though a single center may have several iron atoms, the entire complex is capable of accepting and donating only a single electron. Fig 5.13: Iron-sulfur centres Types of Electron Carrier 1.Flavoproteins (accept and donate 2e- and 2 H+) – derived from Vit B2 2. Cytochromes (accept and donate 1e- ) – soluble protein in the intermembrane space 3. Copper atoms (accept and donate 1e- ) 4.Ubiquinone (accept and donate 2e- and 2 H+) – lipid soluble molecule 5. Iron-sulfur proteins (accept and donate 1e- ) – iron atoms are linked Oxidative Phosphorylation in the Formation of ATP Types of Electron Carrier How do these carriers arrange?  The carriers of the electron-transport chain are arranged in order of increasingly positive redox potential (i.e they have progressively higher affinity for electrons)  Each carrier is reduced by the gain of electrons from the preceding carrier in the chain and is subsequently oxidized by the loss of electrons to the carrier following  Electrons lose energy as they move “downhill” along the chain  The final acceptor is O2, which accepts the energy-depleted electrons and is reduced to water. Fig 5.14: Arrangement of several carriers in the electron-transport chain Oxidative Phosphorylation in the Formation of ATP How Did We Figure Out the Sequence of Events of the ETC?  The specific sequence of carriers that constitute the electron-transport chain was worked out using a variety of inhibitors that blocked electron transport at specific sites along the route.  After an inhibitor was added to cells, the oxidation state of the various electron carriers in the inhibited cells was determined.  Thus, by identifying reduced and oxidized components in the presence of different inhibitors, the sequences of the carriers could be determined. Reduced state Oxidized state Fig 5.15: Inhibitors to determine the ETC carrier sequence Practice Question Q) You are trying to figure out an electron transport pathway including the following electron transport molecules: B, K, T, Q and X. You do so by employing inhibitors for various steps in the process. When you do, you get the following results: Inhibitor Ticin Digitin Estin Lucin Electron Transport Molecules Trapped in Reduced Form Q&K K T, K, Q & B Q, K & T What is the order of the molecules (the pathway) in the electron transport chain suggested by the above data from the most reduced to the least reduced molecule? a) K —> T —> B —> Q —> X b) K —> X —> B —> Q —> T c) K —> Q —> T —> B —> X d) X —> B —> T —> Q —> K e) T —> B —> K —> Q —> X Electron-Transport Complexes Main function=? transports 4 H+ per pair of electrons Transfer of electrons to O2 and transfer 2 H+ per pair of electrons Transfer of electrons without transport of H+ transports 4 H+ per pair of electrons NADH dehydrogenase Complex III (cytochrome bc1) succinate dehydrogenase Complex IV (cytochrome c oxidase) Fig 5.17 a: The electron-transport chain of the inner mitochondrial membrane © 2013 John Wiley & Sons, Inc. All rights reserved. Electron-Transport Complexes  Disrupting the inner mitochondrial membrane by detergents reveals that various electron carriers exist as part of four distinct membrane spanning complexes:  Complex I (NADH dehydrogenase) catalyzes transfer of electrons from NADH to ubiquinone and transports four H+ per pair of electrons  Complex II (succinate dehydrogenase) catalyzes transfer of electrons from succinate to FAD to ubiquinone without transport of H+ • Only enzyme that is used in both the TCA and ETC!  Complex III (cytochrome bc1) catalyzes the transfer of electrons from ubiquinone to cytochrome c and transports four H+ per pair of electrons  Complex IV (cytochrome c oxidase) catalyzes transfer of electrons to O2 and transports two H+ per pair of electrons across the inner membrane. The metabolic poisons CO, N3–, and CN– bind catalytic sites in Complex IV.  cytochrome c (soluble in the intermembrane space)and ubiquinone (dissolved in lipid bilayer) are not part of any of the four complexes Electron-Transport Complexes Complex I (NADH Dehydrogenase)  Complex I is the gateway to the ETC, catalyzing the transfer of a pair of electrons from NADH to ubiquinone (UQ) to form ubiquinol (UQH2)  This complex carries out the two different activities that are required of the ETC: – electron transfer – proton translocation Image is taken from Wikipedia: Oxidative phosphorylation Electron transfer occurs within the hydrophilic portion of the complex: NADH  FMN  7 Fe-S complexes  UQ The passage of a pair of electrons from NADH to ubiquinone is paired with moving four protons from the matrix into the intermembrane space Dysfunction in complex 1 has been linked to neurological diseases Electron-Transport Complexes Complex II (Succinate Dehydrogenase)  Complex II consists of four polypeptides: – Two hydrophobic subunits that anchor the protein in the membrane and – Two hydrophilic subunits that comprise succinate dehydrogenase. Image is taken from Wikipedia: Oxidative phosphorylation Complex II provides a pathway for feeding lower-energy electrons from succinate to FAD (becomes FADH2) to ubiquinone.  Electron transfer occurs to ubiquinone through three iron-sulfur cluster and it contains a heme group.  Electron transfer through complex II is not accompanied by proton translocation. FADH2  3 FE-S complexes  UQ Electron-Transport Complexes Complex III (Cytochrome bc1)  Complex III catalyzes the transfer of electrons from ubiquinol (reduced from of ubiquinone) to cytochrome c, with four protons translocated across the membrane for every pair of electrons transferred. Image is taken from Wikipedia: Oxidative phosphorylation It contains three cytochromes: one cytochrome c1 and two b cytochromes Protons are released into the intermembrane space in two separate steps:  Two protons are derived from the molecule of ubiquinol that entered the complex,  Two other protons are pumped from the matrix and translocated to intermembrane space. UQH2 Cyt b  Fe-S  Cyt c1  Cyt c Underlined text are the actual components of complex III Electron-Transport Complexes Complex IV (Cytochrome c oxidase)  The final step of electron transport in a mitochondrion is the successive transfer of electrons from reduced cytochrome c to oxygen.  O2 reduction is catalyzed by complex IV, a huge assembly of subunits known as cytochrome c oxidase Image is taken from Wikipedia: Oxidative phosphorylation  For every molecule of O2 reduced, 8 protons are taken up from the matrix. Four are consumed to form two molecules of water, and the other four are translocated to the intermembrane space. • Cyt c  Cyt a  Cyt a3  Copper (Fe-Cu)  O2 • Underlined text are the actual components of complex IV  The copper in this complex plays a critical role on holding onto O2 until the requisite four electrons have been transferred to O2 Q) How many complexes are involved in electron transport in the electron transport chain? Can you name them? Q) Which of the complex is not involved in the transport of H+ ions? Summary of Electron-Transport Complexes Fig: 5.17: The electron-transport chain of the inner mitochondrial membrane Summary of Electron-Transport Complexes Summary Slide Becker’s World of the Cell 8th ed Summary Slide What we have learned so far?  Mitochondria-Look at its structure and function  Glycolysis- cytosol- generates  The first steps in oxidative metabolism are carried out in glycolysis in the cytosol; it is an anaerobic process (9/10 steps not use oxygen)  Glycolysis produces two pyruvate, two NADH, and two (net) molecules of ATP per glucose  Acetyl CoA Formation from Pyruvate by removal of CO2 and use of Coenzyme A? Thioester bond that CoA by thioester link  TCA cycle –mitochondrion-succinate dehydrogenase, substrate phosphorylation of GDP with phosphate and formation of GTP, 4 NADH and 1 FADH2 production Summary Slide What we have learned so far?  Pyruvate transfer into the mitochondria – discuss shuttle system (Malate-aspartame and Glycerol phosphate shuttle)  ATP production in exercise (creatinine phosphate form)  Electron transfer chain  What are oxidizing and reducing agents – couple reaction more negative strong reducing agent (loss electrons)  Types of membrane-bound electron carrier protein in ETC ( 5 types) –Ubiquinone (lipid soluble) and cytochrome c is a soluble protein in the intermembrane space  Electrons lose energy downhill fashion (exp of inhibitor)  Electron transport complexes (Complex 1, II, III, IV) – Complex I linked to neurological diseases Establishment of a Proton-Motive Force  Two components of the proton gradient: • Concentration gradient of hydrogen ions between matrix and intermembrane space creates a pH gradient (ΔpH). • Separation of charge across the membrane creates an electric potential (Ψ).  Energy present in both components of the proton electrochemical gradients is called the proton-motive force (Δp). Fig 5.21: Visualizing the proton-motive force in active mitochondria with the fluorescent, cationic dye rhodamine Treatment of cells with lipid-soluble agents, 2, 4-dinitrophenol (DNP) uncouples glucose oxidation and ATP formation by increasing the permeability of the inner membrane to H+, thus eliminating the proton gradient. DNP is used to inhibit ATP formation in labs. In the 1920s, Physicians prescribed DNP as a diet pill—people died—why? https://publichealthmatters.blog.gov.uk/2018/08/13/deadly-dnp/ This drug can uncouple oxidation and phosphorylation because it is lipid soluble and the electron transport chain requires an electrochemical gradient. The consumption of drug result to send protons back into the matrix and the gradient generated by the electron transport chain is dissipated and leading to the release of energy as heat - cause excessive heat production and organ failure-death  Different people have different metabolism rates: Differences in endogenous uncoupling proteins (UCPs) account for these differences. People have natural (endogenous) uncouplers found in brown adipose tissues of hibernating mammals and serve as a source of heat production. Also infants (when exposed to cold). White adipose tissues Brown adipose tissues DNP Victim- Kill People https://www.bbc.com/news/uk-england-44388389 The Structure of ATP Synthase Fig 5.22:Electron micrograph of a mitochondrion, with spherical particles attached by a stalk to the cristae membranes Fig 5.23: ATP formation in membrane vesicles reconstituted with the Na+/K+-ATPase  Fernandez-Moran discovered a layer of spheres projecting into the matrix.  Isolation of coupling factor 1, or F1, showed that it hydrolyzed ATP, and under experimental conditions, it behaves as an ATPase. • Remember that enzymes can catalyze both forward and reverse reactions (depends on prevailing conditions)  Led to conclusion that an ionic gradient establishes a proton-motive force to phosphorylate ADP. The Structure of ATP Synthase Fig 5.24 a, b: Schematic diagram and 3D structure of the bacterial ATP synthase. The enzyme consists of two major portions, called F 1 and F0  The F1 particle is the catalytic subunit, and contains three catalytic sites α3β 3γ for ATP synthesis. (The five subunits of F1 are 3 alpha, 3 beta, 1 delta, 1 epsilon and 1 gamma)  The F0 particle attaches to the F1 and is embedded in the inner membrane and contains a channel through which protons are conducted from the intermembrane space to the matrix.  (Fo subunits are 1a, 2b, 10-14c subunits)- F0 stands for oligomycin- a toxin that binds to the Fo unit  The number of subunits in the c ring is 10–14 because structural studies have revealed that this number can vary depending on the source of the enzyme. Q) How does a proton electrochemical gradient provide the energy required to drive the synthesis of ATP? Paul Boyer of UCLA published an innovative hypothesis in 1979, called the binding change mechanism The Binding Change Mechanism for ATP Formation Components of the Binding Change Hypothesis The binding change mechanism states the following: 1) The energy released by the movement of protons is not used to drive ADP phosphorylation directly but principally to change the binding affinity of the active site for the ATP product. 2) Each active site progresses successively through three distinct conformations that have different affinities for substrates and product. 3) ATP is synthesized by rotational catalysis in which one part of the ATP synthase rotates relative to another part. The Binding Change Mechanism for ATP Formation Components of the Binding Change Hypothesis 1) The energy released by the movement of protons is not used to drive ADP phosphorylation directly but principally to change the binding affinity of the active site for the ATP product. • The reaction can occur spontaneously without the input of energy. enzyme - bound ADP + enzyme - bound Pi enzyme - bound ATP + H2O • This does not mean that ATP can be formed from ADP without energy expenditure, rather energy is required for the release of the tightly bound product from the catalytic site, rather than the phosphorylation event itself. The Binding Change Mechanism for ATP Formation Components of the Binding Change Hypothesis 2) Each active site progresses successively through three distinct conformations that have different affinities for substrates and product. Fig 5.27 a • Each active site progresses successively through three distinct conformations that have different affinities for substrates and product - protons induce a shift in the conformation. • At any given instant, • The first site is in the “loose” or L conformation in which ADP and Pi are loosely bound; • The second site is in the “tight” or T conformation in which nucleotides (ADP + Pi substrates, or ATP product) are tightly bound; • The third site is in the “open” or O conformation, which, because it has a very low affinity for nucleotides, allows release of ATP. • Each one of those three catalytic sites goes through the cycle of LTO sequentially • Electrical energy stored in the proton gradient is transduced into mechanical energy of a rotating stalk (next slide), which is transduced into chemical energy stored in ATP. The Binding Change Mechanism for ATP Formation Components of the Binding Change Hypothesis 3) ATP is synthesized by rotational catalysis in which one part of the ATP synthase rotates relative to another part. Fig 5.27 b  The γ subunit is , so at any instant, its different faces interacts with the different β subunits, causing them to adopt different (L, T, and O) conformations.  As it rotates in steps of 120°, each binding site on the subunit interacts successively with the three subunits of F1.  During a single catalytic cycle, the subunit rotates a full 360°, causing each catalytic site to pass sequentially through the L, T, and O conformations.  Condensation of ADP and Pi to form ATP occurs while each subunit is in the T conformation Mitochondrial F0 F1 Complex –Video Using the Proton Gradient What is the path taken by protons as they move through the Fo complex, and how does this movement lead to the synthesis of ATP? • It had been postulated that: 1. The c subunits of the Fo base are assembled into a ring that resides within the lipid bilayer. 2. The c ring is physically bound to the γ subunit of the stalk. 3. The “downhill” movement of protons through the membrane drives the rotation of the ring of c subunits. 4. The rotation of the c ring of Fo provides the twisting force (torque) that drives the rotation of the attached γ subunit, leading to the synthesis and release of ATP by catalytic subunits of the F1 ring. Fig 5.29: A model of the proton diffusion coupled to rotation of c ring in the F0 complex Using the Proton Gradient The Role of the Fo portion of ATP Synthase in ATP Synthesis Movement of the ring is driven by the conformational changes associated with the sequential protonation and deprotonation of an acidic residue in each c subunit. Fig 5.29: A model of the proton diffusion coupled to rotation of c ring in the F0 complex Peroxisomes Peroxisomes The Cell: A Molecular Approach. 2nd edition.  Peroxisomes are small, membrane-enclosed organelles that contain at least 50 enzymes involved in a variety of metabolic reactions, including several aspects of energy metabolism.  Peroxisomes are oxidative organelles and contain digestive enzymes that break downs toxic material in the cell and oxidative enzymes for the catabolic activities.  Peroxisomes originally were defined as organelles that carry out oxidation reactions leading to the production of hydrogen peroxide.  Hydrogen peroxide is harmful to the cell, peroxisomes also contain the enzyme  catalase, which decomposes hydrogen peroxide either by converting it to water or by using it to oxidize another organic compound.  Zellweger syndrome (ZS) is a rare inherited disease characterized by a variety of neurologic, visual, and liver abnormalities leading to death during early infancy. (patients lacking peroxisomes –problem they can synthesize peroxisomeal enzymes- enzymes fail to incorporate into the peroxisomes and remain largely in the cytosol and cannot carry out their normal function- How we know- found empty membranous structures called “ghosts” The Human Perspective: Diseases that Result from Abnormal Mitochondrial or Peroxisomal Function • Mitochondria • A variety of disorders are known that result from abnormalities in mitochondria structure & function. • Majority of mutations linked to mitochondrial diseases are traced to mutations in mtDNA. • Mitochondrial disorders are inherited maternally. Degenerating muscle shows red-staining “blotches” due to abnormal proliferation of mitochondria a b Mitochondrial DNA (mtDNA)- circular DNA has many special features such as a high copy number in cell, maternal inheritance, and a high mutation rate which have made it attractive to scientists from many fields. © 2013 John Wiley & Sons, Inc. All rights reserved. Electron micrograph showing crystalline structures within the mitochondrial matrix The Human Perspective: Diseases that Result from Abnormal Mitochondrial or Peroxisomal Function A premature-aging phenotype caused by increased mutations in mtDNA. The defective nuclear gene encodes for the DNA polymerase responsible for mtDNA replication • It is speculated that accumulations of mutations in mtDNA is a major cause of aging. • In mice encoding a mutation in their mtDNA, signs of premature aging • Additional findings suggest that mutations in mtDNA may cause premature aging but are not sufficient for the normal aging process. © 2013 John Wiley & Sons, Inc. All rights reserved. Chapter 5 Q1) Why mitochondria is so important to study? Explain with three examples Q2) Explain the net reaction of glycolysis? How many ATP are produced and consumed during this process? Q3) Which product of glycolysis is responsible to start TCA cycle? Explain how it happens? Q4) What are the two main ways that cells can make the use of the NADH molecules produced during glycolysis pathway? Briefly explain it. Q5) Explain the net reaction of TCA cycle? a) Explain the step which reduces FAD to FADH2 b) Explain the steps which reduces NAD+ to NADH c) How many carbon dioxide generates in TCA cycle? Can you identify the steps where carbon dioxide generates? d) Which step in the TCA cycle is an example of substrate level phosphorylation? Q6) What is chemiosmosis? Where do you see? Q7) What are the two main steps in ATP formation? Q8)How muscle cope up with rapid ATP supply during exercise? Q9) Based on the redox potential values of a given reaction (Table 5.1) can you identify which one is strong oxidizing or reducing agent why is it? Q10) What is the difference a) between coenzyme and cofactor? b) Apoenzyme and holoenzyme Q11) What are the types of electron carriers? How many electrons they accept or donates? Q12) What is the primary role of complex IV? Q13) What is a proton-motive force? Q14) What does uncoupling mean and why was it tried as a weight-loss strategy? Q15) Describe the basic structure of ATP synthase. Q16) What is the binding change hypothesis? Q17) What are key characteristics of peroxisomes? What diseases it can cause?