Book notes chapter 12
Book notes chapter 12 BIOL4100
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Cell Biology Spring 2016 Chapter 12 The energy to drive ATP synthesis from AP derives primarily from two sources: the energy in the chemical bonds of nutrients and the energy of sunlight. The two processes primarily responsible for converting these energy sources into ATP are o Aerobic respiration: occurs in the mitochondria in nearly all eukaryotic cells. o Photosynthesis: occurs in chloroplasts only in leaf cells of plants and certain single-cell organisms. o Glycolysis and the Citric Acid Cycle are also important sources of ATP in both plant and animal cells. Aerobic respiration: o Breakdown products of sugars and fatty acids are converted by oxidation with O2 to carbon dioxide and water. The energy released from this overall reaction is transformed into the chemical energy of phosphoanhydride bonds in ATP. o 02 is consumed to generate CO2 and H2O. Photosynthesis: o The energy of light is absorbed by pigments such as chlorophyll and used to make ATP and carbohydrates (sucrose and starch). o Uses CO2 as a substrate and generates O2 and carbohydrates in plants. Mitochondria, bacteria, and chloroplasts all use the same mechanism to generate ATP known as chemiosmosis. In chemiosmosis, a proton electrochemical gradient is first generated across a membrane, driven by energy released as electrons travel down their electrical potential gradient through an electron transport chain. The energy stored in the proton electrochemical gradient is called the proton-motive force. The proton motive force is then used to power the synthesis of ATP or other energy requiring processes. 12.1 First step of harvesting energy from glucose: glycolysis Advantages of aerobic respiration: o By dividing the energy conversion process into multiple steps that generate several energy-carrying intermediates, chemical bond energy is efficiently channeled into the synthesis of ATP with less energy lost as heat. o Different fuels (sugars and fatty acids) are reduced to common intermediates that can then share subsequent pathways for combustion ant ATP synthesis. o Because the total energy stored in the bonds of the initial fuel molecule is substantially greater than that required to drive the synthesis of a single ATP molecule, many ATP molecules are produced. Electron Transport Chain: series of redox reactions during respiration to produce to CO2 and water. o The combination of these redox reactions with phosphorylation of ADP to form ATP is called oxidative phosphorylation. Occurs in the mitochondria of nearly all eukaryotic cells Aerobic Respiration: When oxygen is the final recipient of the electrons transported via the electron transport chain. o Efficient way to maximize conversion of nutrient energy to ATP b/c oxygen is a strong oxidant. Anaerobic Respiration: Some molecule other than oxygen (sulfate or nitrate) is the final recipient of the electrons in the Electron Transport Chain. The complete aerobic oxidation of one molecule of glucose yields 6 molecules of CO2 and the energy released is couple to the synthesis of as many as 30 ATP molecules. 4 steps of glucose oxidation in eukaryotic cells 1. Glycolysis: in the cytosol; 6-carbon glucose is converted into two 3-carbon pyruvate molecules; yields 2 ATP molecules for each glucose molecule. 2. Citric Acid Cycle: mitochondria; pyruvate oxidation to CO2 is coupled to the generation of high energy electron carriers NADH and FADH2, which store the energy for later use. 3. Electron Transport Chain: High energy electrons flow down their electric potential gradient form NADH and FADH2 to )2 via membrane proteins that convert the energy released into a proton-motive force (H+ gradient) 4. ATP Synthesis: The proton motive force powers synthesis of ATP as protons flow down their concentration and voltage gradients through the ATP synthesis enzyme. For each original glucose molecule, an estimated 28 additional ATPs are produced by this oxidative phosphorylation. Glycolysis: takes place in the cytosol; does not require (O2), form of catabolism; or the breakdown of complex substances into simpler ones; involves 10 enzymes constituting the glycolytic pathway. During the glycolytic pathway, the metabolic intermediates are phosphorylated and water soluble. Generates 4 ATP molecules, but uses 2 ATP molecules to start the process. This ATP is produced by substrate level phosphorylation. After glycolysis, only a fraction of the available energy has been extracted from glucose. The ability to convert the remaining energy to ATP depends on the presence of O2. (aerobic vs anaerobic) o Glycolysis and the citric acid cycle are regulated primarily by allosteric mechanisms of 3 allosteric enzymes; Hexokinase: inhibited by its reaction product, glucose 6- phosphate Pyruvate kinase: inhibited by ATP Phosphofructokinase-1: principal rate-limiting enzyme. This enzyme is inhibited by ATP and activated by AMP. o Glucose metabolism is controlled differently in various mammalian tissues to meet the metabolic needs of the organism as a whole. During carbohydrate starvation, the liver will release glucose into the bloodstream by converting glycogen to glucose 6- phosphate Most eukaryotes can generate some ATP by anaerobic metabolism. o In the absence of O2, yeasts utilize fermentation to convert pyruvate to CO2 and ethanol. o Fermentation in animal cells produces lactic acid instead of alcohol. When oxygen within the muscle tissue is limited, glycolysis can occur and cells convert pyruvate to two molecules of lactic acid by reduction reaction that oxidizes two NADHs to two NAD+. Once the lactic acid is passed to the blood, the liver will reoxidize it to pyruvate and either further metabolized to CO2 aerobically or converted back to glucose. o Fermentation is a much less efficient way to generate ATP than aerobic oxidation. o In the presence of O2, pyruvate formed by glycolysis is transported into mitochondria where it is oxidized by O2 to CO2 and water. 12.2 Mitochondria and the Citric Acid Cycle: Bacteria are thought to be the evolutionary precursors of mitochondria. The number of mitochondria in a cell are regulated to match the cell’s requirement for ATP. 2 distinct, concentric membranes: the inner and outer membrane. o Outer membrane defines the smooth outer perimeter of the mitochondrion. o Inner membrane lies immediately underneath the outer membrane and has numerous invaginations, called cristae which extend from the perimeter of the inner membrane to the center of the mitochondrion. o Intermembrane space: the space between the outer and inner membranes o Matrix: the central compartment which forms the lumen within the inner membrane. o The cristae increase the surface area of the inner mitochondrial membrane, to increase the ability to synthesize ATP. Charcot-Marie-Tooth subtype 2A: inherited neuromuscular disease in which defects in peripheral nerve function lead to progressive muscle weakness, main in the feet and hands. Human mitochondria 13 proteins are encoded by mitochondrial DNA genes and synthesized inside the mitochondrial matrix space. The remaining proteins are encoed by nuclear genes, synthesized in cytosol, and then imported into mitochondria. Miller syndrome: mitochondrial associated disease which results in multiple anatomical malformations, and connective tissue defects. Porin: the most abundant protein in the outer membrane; a transmembrane channel protein. Ions can pass through these channels when opened. The inner membrane and cristae are the major permeability barriers between the cytosol and the mitochondrial matrix, limiting the rate of mitochondrial oxidation and ATP generation. The inner membrane is 76% protein. o ATP synthase o Proteins responsible for electron transport o A variety of transport proteins that permit the movement of metabolites between the cytosol and the mitochondrial matrix. ADP/ATP carrier: an antiporter that moves newly synthesized ATP out of the matrix and into the inner membrane space in exchange for ADP from the cytosol. In plants, starch is hydrolyzed to glucose. Glycolysis then produces pyruvate that is transported into mitochondria. Mitochondrial oxidation in plants occurs during dark periods and in roots and other nonphotosynthetic tissues at all times. The inner membrane, cristae, and matrix are the sites of most reactions involving oxidation of pyruvate and fatty acids to CO2 and water and ATP from ADP and Pi. Stage 2 Citric acid cycle: o 1. Conversion of pyruvate to acetyl CoA o 2. Oxidation of acetyl CoA to CO2 in the citric acid cycle In the first part of stage II, pyruvate is converte to Acetyl CoA and high energy electrons: o In the matrix, pyruvate reacts with co enzyme A, forming CO2, acetyl CoA, and NADH. o This reaction is catalyzed by pyruvate dehydrogenase and is irreversible o Acetyl CoA: consists of a 2 carbon acetyl group covalently linked to a logner molecule known as coenzyme A (CoA). Plays a central role in the oxidation of pyruvate, FAs and AAs. Is an intermidate in numerous biosynthetic reactions In the mitochondria, the 2 carbon acetyl group is oxidized to CO2 via the citric acid cycle. (The 2 carbons in the acetyl group come from pyruvate; the 3 pyruvate is released as CO2) In the second part of stage II, the citric acid cycle oxidized the acetyl group in acetyl CoA to CO2 and generates high0-energy electrons. o 9 reactions operate in cycle to oxidize the acetyl group of acetyl CoA to CO2. Citric Acid Cycle, TC cycle, Krebs cycle. o The net result: each Acetyl CoA produces 2 molecules of CO2, 3 molecules of NADH, one FADH2, one GTP. NADH and FADH2 are high energy electron carriers that play a role in Stage 3, electron transport. The citric acid cycle begins with the condensation of the two-carbon acetyl group from the acetyl CoA with the four-carbon molecule oxaloacetate to yield the six-carbon citric acid. For each turn of the citric acid cycle, 3 NADH molecules are generated. (2 turns per original glucose molecule) Most citric acid cycle enzymes are water soluble in the mitochondrial matrix. o CoA, acetyl CoA, succinyl CoA, NAD+, NADH, and others. Succinate dehydrogenase, is a membrane bound protein with its active site facing the matrix NET RESULT OF THE GLYCOLYTIC PATHWAY AND THE CITRIC ACID CYCLE Reaction CO2 NAD+ FAD molecules ATP (or GTP) molecules molecules reduce to produced reduced to FADH2 NADH 1 glucose to 2 0 2 0 2 pyruvate (glycolytic pathway) 2 pyruvates to 2 2 0 0 2 acetyl CoA molecules 2 acetyl CoA 4 6 2 2 to 4 CO2 molecules Total 6 10 2 4 NAD+ is required in the cytosol for glycolysis and in the matrix for the citric acid cycle, producing NADH. Oxygen is necessary to oxidize NADH and FADH2 back NAD+ and FAD in the electron transport chain. Although Oxygen is not directly used in the citric acid cycle, the lack of oxygen will stop the citric acid cycle because the amounts of NADH and FADH2 will increase and the availability of NAD+ and FAD to accept protons. In the electron transport chain, oxygen is reduced to water has it oxidizes NADH and FADH to regenerate NAD+ and FAD. There is a small amount of NADH produced in the cytosol during the glycolytic pathway. In order to get the electrons from the cytosolic NADH and regenerate NAD+, electron shuttles are utilized to transport the electrons from cytosol to the matrix. One of the most common is the malate- aspartate shuttle. Fatty acids are also another source of cellular energy. Fatty acids can be taken up from the extracellular space by special transporter proteins. Cells can store glucose as glycogen and fatty acids as triglycerides. In some cells, excess glucose is converted into fatty acids and then triacylglycerols for storage. Animals are unable to convert fatty acids to glucose. When a cell needs to use these storages, they break down glycogen to glucose or hydrolyze triacylglycerols to fatty acids which are then oxidized to generate ATP. Fatty acids are the major energy source for adult heart muscle. In humans, more ATP is generated by the oxidation of fats than the oxidation of glucose. o Oxidation of 1g triacylglycerie to CO2 generates 6 times as much ATP as does the oxidation of 1g of glycogen. o Triglycerides are more efficient than carbohydrates because: They are stored in anhydrous form and can yield more energy when oxidized They are intrinsically more reduced (have more hydrogens) than carbohydrates The primary storage of triacylglycerides is adipose tissue The primary storage site of glucose is muscle and liver The oxidation of fatty acids involves 4 steps just as glucose. o The citric acid cycle, stage 3, and stage 4 are all the same as glucose In stage 1, fatty acids are converted a fatty acyl CoA in the cytosol in a reaction coupled to the hydrolysis of ATP to AMP and PPi. Subsequent hydrolysis of PPi to Pi releases energy that drives this reaction to completion. To transfer the fatty acyl group into the mitochondrial matrix, it is covalently transferred to a molecule called carnitine and moved across the inner mitochondrial membrane by an acylcarnitine transporter protein. o Once transferred into the matrix, the fatty acyl group is dreleased from carnitine and reattached to another CoA molecule. The acitivty of the acylcarnitine transporter is regulated to prevent oxidation of fatty acids when cells have adequate ATP supplies. In the first part of stage 2, each molecule of a fatty acyl CoA is oxidized in a cyclical sequence of four reactions in which all the carbon atoms are converted two at a time to acetyl CoA with the generation of FADH2 and NADH. In the second part of stage 2, these acetyl groups enter the citric acid cycle and are oxidized to CO2. The NADH and FADH2 will be used in stage 3 to generate a proton-motive force that is used in stage 4 to power ATP synthesis. Peroxisomes, small organelles 0.2-1 µm, are capable of oxidizing fatty acids. Peroxisomes are found in all mammalian cells except erythrocytes and are found in plant cells, yeasts, and most other eukaryotic cells. o Oxidize very long chain fatty acids (>C 20which cannot be oxidized by mitochondria. o Peroxisomal oxidation of fatty acids does not produce ATP (as mitochondrial oxidation), instead, energy is released as heat. o Peroxisomes lack an electron transport chain, and electrons from the FADH2 produce during the oxidation of fatty acids are immediately transferred to O2 by oxidases, regenerating FAD and forming hydrogen peroxide. Peroxisomes contain catalase, which quickly decomposes the H2O2, a highly cytotoxic metabolite. o NADH produced during oxidation of FAs is exported and reoxidized in the cytosol. Malate-aspartate shuttle not necessary for peroxisomes o Peroxisomes lack a citric acid cycle, so acetyl CoA generated ruing peroxismal degradation of fatty acids cannot be oxidized further; instead it is transported into the cytosol for use in the synthesis of cholesterol and other metabolites. 12.3 The electron transport chain and generation of the proton-motive force Most of the energy released during the oxidation of glucose and FAs to CO2 is converted into high energy electrons in the reduced coenzymes NADH and FADH2. During stage 3, the energy stored in these coenzymes will be converted into a proton-motive force by the electron transport chain. The conversion of 1 glucose molecule to CO2 yields 10 NADH and 2 FADH2 molecules. Oxidation of these coenzymes has a total ΔG of -613 kcal/mol. Thus the potential free energy present in the chemical bonds of glucose (-686 kcal/mol), about 90% is conserved in the reduced coenzymes. NADH and FADH2 are necessary coenzymes because some reactions involved in glucose and FA oxidation are not energetically sufficient to reduce NAD+. The reactions that are not able to reduce NAD+, are coupled to the reduction of FAD, which requires less energy. The energy of NADH and FADH2 is released by oxidizing them. The mitochondria’s job is to (efficiently) transfer the energy released by this oxidation into the energy in the terminal phosphoanhydride bond in ATP. To efficiently recover the energy, the mitochondria converts the energy of coenzyme oxidation into a proton-motive force using a series of electron carriers, all but one of which are integral components of the inner membrane. The proton-motive force can then be use to efficiently generate ATP. During electron transport, protons are pumped across the inner membrane causing the pH of the mitochondrial matrix to raise when compared to the intermembrane space and cytosol. This also causes the matrix to become more negative than the intermembrane space. o This creates a proton concentration gradient and an electrical gradient across the inner membrane. This is called the proton-motive force. Oxidative phosphorylation: Production of ATP by the transport of electrons from NADH and FADH2 to Oxygen. Oxidative phosphorylation depends on the proton motive force, electron transport, proton pumping, and ATP formation occurring simultaneously. o No ATP is made if a proton motive force cannot be established. o 10 protons are transported out of the matrix for every electron pair transferred from NADH to O2. o The transfer of electrons from FADH2 to O2 is less than that of NADH to O2. Electrons flow downhill through a series of electron carriers Each NADH molecule releases 2 electrons to the electron transport chain. These electrons will ultimately reduce one oxygen atom, creating water. Four large multiprotein complexes compose an electron transport chain in the inner mitochondrial membrane that is responsible for the generation of the proton motive force. Coupled oxidation-reduction reactions are responsible for the transfer of electrons from donor molecules to acceptor molecules through prosthetic groups. o The prosthetic groups are tightly associated with the multiprotein complexes and are organic molecules or metal ions. Heme and Cytochromes: o Heme: iron containing prosthetic group; tightly bound to mitochondrial cytochrome proteins. Each cytochrome is denoted by a letter a, b c, or c1. o Electron flow through the cytochromes occurs by oxidation and reduction of the Fe atom in the center of the heme molecule. o Each cytochrome has a slightly different heme group and surrounding atoms. This generates a different environment for the Fe ion. Each cytochrome has a different reduction potential. Thus, creating the irreversible “downhill” flow of electrons. o All cytochromes, except c, are components of integral membrane multiprotein complexes in the inner mitochondrial membrane. Iron-Sulfur Clusters: o Are nonheme, iron containing prosthetic groups consisting of Fe atoms bonded to inorganic S atoms and to S atoms on cysteine residues. o Iron-sulfur clusters accept and release electrons one at a time. Coenzyme Q (CoQ): o Also called ubiquinone o Only small molecule electron carrier in the chain that is not as essentially irreversible protein-bound prosthetic group. o Carries protons and electrons o Oxidized quinone form can accept a single electron to form a semiquinone (a charged free radical) Addition of a second electron and two protons forms dihydroubiquinone (CoQH2), the fully reduced form. o CoQ and CoQH2 are soluble in phospholipids and diffuse freely in the hydrophobic center of the inner mitochondrial membrane. This is how it participates in the electron transport chain- carrying electrons and protons between the protein complexes of the chain. As electrons flow from one carrier to the next in the electron transport chain, the energy is used to power the pumping of protons to create the proton motive force. o Pumping electrons against the electrochemical gradient across the inner mitochondrial membrane. 4 multiprotein complexes are important for the proton pumping: o NADH-CoQ reductase (complex I >40 subunits) o Succinate-CoQ reductase (complex II 4 subunits) o CoQH2-cytochrome c reductase (complex III 11 subunits) o CoQH2-cytochrome c oxidase (complex IV, 13 subunits) Electrons flow form NADH to complex I (via CoQ/CoQH2) to complex III and then via the soluble protein cytochomre c to complex IV to reduce molecular oxygen. Electrons from FADH2 flow from complex II via CoQ/CoQH2 to complex III and then via cytochrome c to complex IV to reduce molecular oxygen. Protons are simultaneously transported from the matrix to the intermembrane space. o CoQ accepts electrons in the matrix and CoQH2 releases electrons at a site on the intermembrane space of the protein complex, releasing prtons into the intermembrane space. o Transport of each electron pair by CoQ is coupled to movement of 2 protons form the matrix to the intermembrane space. NADH-CoQ Reductase (complex I) Transfers electrons form NADH to CoQ L shaped: with one arm embedded in the membrane with more than 60 transmembrane alpha helices. This arm has four subdomains, three of which have proteins that are members of a family of cation antipoerters. The hydrophilic peripheral arm extends away from the membrane into the cytosolic matrix. The NADH binding site is located at the tip of the peripheral arm. Electrons from NADH flow to FMN (Flavin mononucleotide, a cofactor related to FAD), then down that arm through 7 ion-sulfur clusters then to CoQ (bound at a site partially in the membrane). NAD+ accepts or releases 2 electrons at a time. FMN can accept two electrons but only one at a time. Each pair of electron drops potential energy of -16.6 kcal/mol. The released energy transports 4 protons across the inner membrane per molecule of NADH oxidized by complex I. o 3 cross via the three cation antiporter. The 4 is due to a conformational change of t-helix in the membrane. Succinate-CoQ Reductase (complex II) The citric acid cycle is physically and functionally linked to the electron transport chain. Succinate dehydrogenase (enzyme that oxidizes succinate to fumarate and generates FADH2 in the citric acid cycle) is one of the four subunits of Complex II. The two electrons released in the conversion of succinate to fumarate are transferred to FAD, then to iron-sulfur cluster-regenerating FAD- and finally to CoQ, which bind to a cleft on the matrix side of the transmembrane portions of complex II. The energy released is used to reduce CoQ to CoQH2, but there is not enough energy to pump protons. No proton motive force is generated at this point. Complex II also generates CoQH2 from fatty acyl CoA. o Fatty acyl CoA dehydrogenase (water soluble enzyme) catalyzes the first step of the oxidation of fatty acyl CoA in the mitochondria matrix. There are several fatty acyl CoA dehydrogenase enzymes that mediate the initial step in a 4 step process that removes 2 carbons from the fatty acyl group by oxidizing the carbon in the β position of the fatty acyl chain. o These reactions generate acetyl CoA which enter the citric acid cycle. o FADH2 and NADH are produce. FADH2 remains bound to the enzyme. Electron transfer flavoprotein transfers the electrons from FADH2 in the acyl CoA dehydrogenase to ETF:QO which reduces CoQ to CoQH2 in the inner membrane. CoQH2 intermixes with other CoQH2 molecules generated by complex I and II. CoQH2-cytochrome c Reductase (Complex III) A CoQH2 donates 2 electrons to CoQH2-cytochrome c reductase (complex III), regenerating oxidized CoQ. At the same time, it releases 2 protons that were picked up by CoQ on the matrix face. o This generates part of the proton-motive force Within complex III, the released electrons are first transferred to an iron-sulfur cluster within the complex then to cytochrome c1 or to two b-type cytochromes. Finally, the two electrons are transferred to two molecules of the oxidized form of cytochrome c (a water soluble peripheral protein that diffuses in the intermembrane space). The energy released is enough to translocate 4 protons. (involves proton motive Q cycle) The heme protein cytochrome c and the small lipid-soluble molecule CoQ both serve as mobile electron suhuttles, transferring electrons between the complexes of the electron transport chain. The Q Cycle There are 4 protons translocated from CoQH2 through CoQH2-cytochrome c reductase. These 4 protons are those carried on two CoQH2 molecules, which are converted to wo CoQ molecules during the cycle. Another CoQ molecule receives two other protons from the matrix space and is converted to one CoQH2 molecule. The net overall reaction involves the conversion of only one CoQH2 molecule to CoQ as two electrons are transferred one at a time to two molecules of the acceptor cytochrome c. The Q cycle is responsible for the two-for-one transport of protons and electrons by complex III. The CoQH2 is generated by complex I, complex II, ETF:QO, and complex III itself. In one turn of the Q cycle, 2 molecules of CoQH2 are oxidized to CoQ and release a total fo 4 protons into the intermembrane space. But at another site, CoQH2 is regernated from CoQ and two additional proteins from the matrix space. The Q cycle optimizes the number of protons pumped per pair fo electrons moving through complex III. The Q cycle is found in all plants and animals as well as some bacteria. Electrons are released from CoQH2 by either o the Fe-S, cytochomre c1, then cytochrome c. OR o to cytochrome bL, cytochrome bH, then CoQ. Involves a flexible hinge in the Fe-S containing protein subunit of complex III. Cytochrome c Oxidase (complex IV) CoQH2-cytochrome c reductase (complex III) is reduced by one electron to cytochrome c. Cytochrome c is reoxidized as it transports its electron to cytochomre c oxidase (complex IV). o Mitochondrial complex IV contain 13 different subunits o The catalytic core consists of 3 subunits o Bacterial cytochrome c oxidases only contain the 3 catalytic subunits 4 molecules of reduced cytochrome c bind to the oxidase. An electron is transferred from the heme of each cytochrome c to the pair of copper ions Cu a2+, then to the heme in cytochrome a, and next to CU b2+and the heme in cytochrome a3 that together make up the oxygen reduction center. The 4 electrons are finally passed to O2 yielding four H2O, together with CO2 which are the end products of the overall pathway. Several intermediates may be produced during the oxygen reduction. These intermediates would be harmful to the cell if the escaped from complex IV, but that rarely happens. Complex IV transports only 1 proton per electron transferred. (4 total) o Whereas complex II, using the Q cycle, transports 2 protons per electron transferred. Cyanide binds to heme a3 in mitochondrial cytochrome c oxidase (complex IV), inhibiting electron transport and production of ATP. Cyanide is one of the small molecules that interfere with energy production in the mitochondria. Reduction potentials of electron carriers in the electron transport chain favor electron flow from NADH to O2 The reduction potential for a particular reaction is a mesure of the equilibrium constant of that partial reaction. The reduction potentials of the electron carriers increases steadily from NADH to O2 with the exception of the b cytochromes in CoQH2-cytochomre c reductase complex. The steady increase in reduction potential and decrease in ΔG values of the electron carriers in the ETC favors the flow of electrons from NADH and FADH2 to oxygen. The energy released as the electrons flow down the chain drives the pumping of protons against their concentration gradient from the mitochondrial matrix to the intramembrane space to create the proton motive force. The multiprotein complexes of the electron transport chain assemble into supercomplexes Britton Chance proposed that electron transport complexes might assemble into large supercomplexes. Doing so would bring them all close together and speed up the process. Cardiolipin: unique phospholipid that plays a role in the assembly of these supercomplexes. o Binds to inner membrane proteins (e.g. complex II). o “Glue that holds together the electron transport chain.” Reactive Oxygen Species (ROS) are toxic by-products of electron transport that can damage cells 1-2% of oxygen metabolized by aerobic organisms is reduced to a superoxide anion radical with an unpaired electron. Radicals: atoms that have one or more unpaired electrons in an outer shell, or molecules that contain such an atom. Generally highly chemically reactive. Can alter the structures and properties of those molecules with which they react. Can propogate a chain reaction of radical production. Superoxides and reactive oxygen containing molecules are called reactive oxygen species Can damage lipids, proteins, and DNA; severly interfering with their normal funcitons. ROS contribute to cellular oxidative stress and can be highly toxic. ROS are generated by macrophages and neutrophils to kill pathgoens, but excessive ROS has been implicated in many diverse disease such as heart failure, neurodegenerative diseases, alcohol induced liver disease, diabetes, and aging. The major source of ROS in eukaryotic cells is electron transport in the mitochondria (or chloroplast) ROS are formed when molecular oxygen comes into close contact with the reduced electron carriers. Mitochondria have enzymes that inactivate superoxide by converting it to hydrogen peroxoide and then to water. SOD (Mn-containing superoxide dismutase) found in the mitochondria and catalase are enzymes to rid ROS. Vitamin E helps protect against oxidative stress. In heart muscle cells, peroxisomes are found in the mitochondria, not surprising since the heart is the most oxygen-consuming organ in mammals. Experiments have isolated the multiprotein complexes of the electron transport chain and embedded them in liposomes. When the appropriate electron donor and acceptor are added to the medium, pH changes will occur if the complex transport protons. o For every electron pair that is transferred from NADH to O2, a total of 10 protons are transported from the matrix. o For every electron pair that is transferred from FADH2 to O2, a total of 6 protons are transported across the membrane. The proton-motive force in mitochondria is largely due to a voltage gradient across the inner membrane The main result of the ETC is the generation of the proton motive force. Causes a pH gradient and electrical potential across the mitochondrial inner membrane. The membrane must be poorly permeable to other ions in order to maintain these gradients. Proton pumping by the ETC establishes a large voltage gradient and smaller pH gradient. The electrical potential of the mitochondria is about -160 mV. The pH gradient is about 1 pH unit (tenfold difference in H+ ions) which equals about -60 mV. Total proton motive force is -220 mV, with transmembrane electrical potential responsible for about 73% of that. 12.4 Harnessing the Proton-Motive Force to synthesize ATP Peter Mitchell: 1961, hypothesized that the proton motive force across the inner mitochondrial membrane is the immediate source of energy for ATP synthesis; chemiosmotic hypothesis. Protons actually move through the ATP synthase as they traverse the membrane. ATP synthase is a multiprotein complex that can be subdivided into two complexes called F0 and F1; F0F1 complex o F0 contains the transmembrane portion of the complex o F1 contains the globular portions of the complex that sit above the membrane and point toward the matrix space in the mitochondria The mechanism of ATP synthesis is shared among bacteria, mitochondria, and chloroplasts Although bacteria don’t have membrane bound organelles, the enzymes necessary for the glycolytic pathway and the citric acid cycle are present in the cytosol of bacteria. Enzymes that oxidize NADH to NAD+ and transfer the electrons to O2 reside in the bacterial plasma membrane. The movement of electrons through the membrane carriers is coupled to the pumping of protons out of the cell. The movement of protons back into the cell (down the concentration gradient through ATP synthase) drives ATP synthesis. The bacterial ATP synthase is nearly identical to the mitochondrial/chloroplast ATP synthase, except it is located in the plasma membrane. This makes sense because of the endosymbiotic hypothesis: o The inner mitochondrial membrane would be derived from the bacterial plasma membrane with its cytosolic face pointing toward what became the matrix space of the mitochondria. o In plants, the progenitor’s plasma membrane became the thylakoid membrane and its cytosolic face pointed toward what became the stromal space of the chloroplast. In all cases, ATP synthase is positioned with the F1 domain, which catalyzes ATP synthesis, on the cytosolic face of the membrane. o ATP is always synthesized on the cytosolic face of the membrane. Driven by the proton motive force. The cytosolic face has a negative electric potential relative to the exoplasmic face. The proton motive force across the bacterial plasma membrane is used to power other processes, including the uptake of nutrients such as sugars and the rotation of bacterial flagella. o The membrane potential, the concentration gradients of protons and other ions across a membrane, and the phospoanydride bonds in ATP are equivalent in the interconvertible forms of chemical potential energy. ATP Synthase Comprises F0 and F1 multiprotein complexes Each of the F0 and F1 components are multimeric proteins. The F0 component contains 3 types of integral membrane proteins; a, b, and c o The c subunits form a doughnut-shaped “c-ring” in the plant of the membrane. o The a and b subunits are rigidly linked to one another but not to the c ring. The F1 portion o Water soluble complex of 5 distinct polypeptides with a composition of α3 β3 γδε Normally firmly bound to the F0 subcomplex at the surface of the membrane. The lower end of the rod-like γ subunit is a coiled coil that fits into the center of the c-subunit ring and appears rigidly attached to it. o When the c-ring rotates, the rod like γ subunit moves with it. o The ε subunit is rigidly attached to γ and also forms tight contacts with several of the c subunits of F0 o The α and β subunits are responsible for the overall globular shape of the F1 sub complex and associate in alternating order to form a hexamer, which rests atop the single long γ subunit. o The F1 δ subunit is and the αβ hexamer form a rigid structure anchored to the membrane. The rod like b subunits form a “stator” that prevents the hexamer from moving while it rests on the γ subunit, whose rotation together with the cu subunits of F0 plays an essential role I the ATP synthesis mechanism. ATP synthase is embedded in the membrane. The F1 component forms a knob that protrudes from the cytosolic face. o F1 separated from the membranes is capable of catalyzing ATP hydrolysis (ATP conversion to ADP plus Pi) in the absence of the F0 component, it has been called the F1 ATPase; however, its function in cells is the reverse, to synthesize ATP. o ATP hydrolysis is a spontaneous process; thus energy is required to drive the ATPase “in reverse” to generate ATP. Rotation of the F1 γ subunit, driven by proton movement through F0, powers ATP synthesis Each of the 3 β subunits can bind ADP and Pi and catalyze the endergonic synthesis of ATP when coupled to the flow of protons from the exoplasmic medium to the cytosolic medium. The coupling between proton flow and ATP synthesis must not occur in the same portions of the protein, because the nucleotide-binding sites on the β subunits where ATP synthesis occurs are 9-10 nm from the surface of the mitochondrial membrane. The most widely accepted model for ATP synthesis by the F0F1 complex is: binding-change mechanism posits just such an indirect coupling. o Energy released by the downhill movement of protons through F0 directly powers rotation of the c-subunit ring together with its attached γ and ε subunits. o The γ subunit acts as a cam, or nonsymmetrical rotating shaft, whose rotation within the center of the static αβ hexamer of F1 causes it to push sequentially against each of the β subunits and thus cause cyclical changes in their conformations between three different states. Rotation of the γ subunit relative to the fixed hexamer causes the nucleotide0binding site of each β subunit to cycle through three conformational states in the following order: O: open state that binds ATP very poorly and ADP and Pi weakly. L: loose state that binds ADP and Pi more strongly but cannot bind ATP T: tight state that binds ADP and Pi so tightly that they spontaneously react to form ATP. o In the T state, the ATP is bound so tightly that it cannot readily dissociate from the site. It is trapped until another rotation of the γ subunit returns that β subunit to the O state. o ATP or ADP also binds to regulatory or allosteric sites on the three α subunits; this binding modifies the rate of ATP synthesis according to the level of ATP and ADP in the matrix, but is not directly involved in the synthesis of ATP from ADP and Pi. Multiple protons must pass through ATP synthase to synthesize one ATP The energy requirements show that more than 1 proton must pass to synthesize ATP from ADP and Pi. The downhill movement of 1 mol of protons releases just over 5 kcal of free energy, the passage of at least 2 protons is required for the synthesis of each molecule of ATP from ADP and Pi (5.1 kcal/mol) F0 c ring rotation is driven by protons flowing through the transmembrane channels Each c subunit contains 2 membrane-spanning α helices that form a hairpin like structure. Aspartate residue in the center of one of these helices in each subunit is throught o play a key role in proton meovemnt by binding and releasing protons as they traverse the membrane. The protons traverse the membrane via two staggered, proton half-channels I and II. o Half channels because they only extend halfway across the membrane. o I is open to the exoplasmic surface and II is open to the cytosolic surface. They meet the level of Asp-61. Proton translocation begins when a proton rom the exoplasmic medium moves upwards through half-channel I. As that proton moves into the empty proton-binding site, it displaces Arg-210 side chain, which swings toward the filled proton-binding site of the adjacent c subunit in contact with half-channel II. As a consequence, the positive side chain of Arg-210 displaces the proton bound to Asp-61 of the adjacent c subunit. The displaced proton is now free to travel up half-channel II and out into cytosolic medium When one proton entering form half-channel I binds to the c ring, a different proton is released to the opposite side of the membrane via half-channel II. Rotation of the entire c ring due to thermal/Brownian motion then allows the newly un-protonated c subunit to move into alignment above half-channel I as an adjacent protonated c subunit rotates in to take its place under half- channel II The entire cycle is then repeated, as additional protons move down their electrochemical gradient form the exoplasmic medium to cytosolic medium. The γ subunit is tightly bound to the c subunits. As the c subunits rotate, it takes 120° rotation of the γ subunit to synthesize 1 ATP. Complete rotation of the c ring would generate 3 ATPs. It is estimated that for every 4 protons transported out, 3 are used to synthesize 1 ATP molecule and 1 is used power the export of ATP from the mitochondrion in exchange for ADP and Pi. o Ensure high ratio of ATP to ADP in the cytosol, where hydrolysis of the high energy phosphoanhydride bonds of ATP is utilized to power many energy-requiring reactions. Rate of mitochondrial oxidation normally depends on ADP levels Mitochondria can only oxidize FADH2 and NADH as long as there is a source of ADP and Pi to generate ATP. This is known as respiratory control. o Occurs because oxidation of NADH and FADH2 is obligatorily coupled to proton transport across the inner membrane. If the resulting proton motive force is not dissipated during the synthesis of ATP from ADP and Pi, both the transmembrane proton concentration gradient and membrane electric potential will increase to high levels. At this point, pumping of additional protons across the inner membrane requires so much energy that it eventually cease, blocking the coupled oxidation of NADH and other substrates. Brown fat mitochondria use the proton motive force to generate heat Brown fat gets its name from the color it has from abundant mitochondria. Specialized for heat generation. White fat tissue: specialized for the storage of fat and contains few mitochondria. Brown fat contains thermogenin: a natural uncoupler of oxidative phosphorylation and generation of a proton motive force. Thermogenin dissipates the proton-motive force by rendering the inner mitochondrial membrane permeable to protons. As a consequence the energy released by NADH oxidation in the ETC and used to create a proton gradient is not used to synthesize ATP via ATP synthase. Instead, when protons move back into the matrix through thermogenin, the energy is released as heat. Thermogenin is a proton transporter, not a proton channel, and shuttles protons across the membrane at a rate that is a million fold slower than that of typical ion channels. o Similar to ATP/ADP transporter DNP: lipid soluble chemical that can reversibly bind to and release protons and shuttle them across the inner membrane from the intermembrane space into the matrix. Environmental conditions regulate the amount of thermogenin in brown fat mitochondria. o During the adaptation of rats to cold, the ability of their tissues to generate heat is increased by the induction of thermogenin synthesis. In the newborn human, thermogenesis by brown fat mitochondria is vital to survival.
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