Cell Metabolism (Lecture 17-19) Comparison of passive and active transport In passive transport, a substance diffuses spontaneously down its concentration gradient with no need for the cell to expend energy. Hydrophobic molecules and very small uncharged polar molecules diffuse directly across the membrane. Hydrophilic substances diffuse through transport proteins in a process called facilitated diffusion. In active transport, a transport protein moves substances across the membrane "uphill" against their concentration gradients. Active transport requires an expenditure of energy, usually supplied by ATP. An electrogenic pump. Proton pumps are examples of membrane proteins that store energy by generating voltage (charge separation) across membranes. Using ATP for power, a proton pump translocates positive charge in the form of hydrogen ions. The voltage and H+ gradient represent a dual energy source that can be tapped by the cell to drive other processes, such as the uptake of sugar and other nutrients. Proton pumps are the main electrogenic pumps of plants, fungi, and bacteria. Cotransport. An ATP-driven pump stores energy by concentrating a substance (H1, in this case) on one side of the membrane. As the substance leaks back across the membrane through specific transport proteins, it escorts other substances into the cell. The proton pump of the membrane is indirectly driving sucrose accumulation by a plant cell, with the help of a protein that cotransports the two solutes. Energy flow and chemical recycling in ecosystems The mitochondria of eukaryotes (including plants) use the organic products of photosynthesis as fuel for cellular respiration, which also consumes the oxygen produced by photosynthesis. Respiration harvests the energy stored in organic molecules to generate ATP, which powers most cellular work. The waste products of respiration, carbon dioxide and water, are the very substances that chloroplasts use as raw materials for photosynthesis. Thus, the chemical elements essential to life are recycled. But energy is not: It flows into an ecosystem as sunlight and leaves it as heat. How ATP drives cellular work Phosphate-group transfer is the mechanism responsible for most types of cellular work. Enzymes shift a phosphate group (R) from ATP to some other molecule, and this phosphorylated molecule undergoes a change that performs work.

For example, ATP drives active transport by phosphorylating specialized proteins built into membranes; drives mechanical work by phosphorylating motor proteins, such as the ones that move organelles along cytoskeletal "tracks" in the cell; and drives chemical work by phosphorylating key reactants. The phosphorylated molecules lose the phosphate groups as work is performed, leaving ADP and inorganic phosphate as products. Cellular respiration replenishes the ATP supply by powering the phosphorylation of ADP. Cellular respiration and fermentation are catabolic Fermentation – an ATP-producing catabolic pathway in which both electron donors and acceptors are organic compounds. Can be an anaerobic process Results in partial degradation of sugars Cellular respiration – an ATP-producing catabolic process in which the ultimate electron is an inorganic molecule, such as oxygen. Most prevalent and efficient catabolic pathway Is an exergonic process (ΔG = -2870 kJ/mol or -686 kcal/mol) Can be summarized as: Organic compounds + Oxygen à Carbon dioxide + Water + Energy Carbohydrates, proteins, and fats can all be metabolized as fuel, but cellular respiration is most often described as the oxidation of glucose: C6H12 O6 +6O2 à 6CO2 + 6 H2 O + Energy (ATP + Heat) An introduction to redox reactions Oxidation-reduction reactions – chemical reactions which involve a partial or complete transfer of electrons from one reactant to another; called redox reactions for short. Oxidation – partial or complete loss of electrons Reduction – partial or complete gain of electrons Generalized redox reaction: Electron transfer requires both a donor and acceptor, so when one reactant is oxidized the other is reduced. Xe- + Y à X + YeX = substance being oxidized, acts as reducing agent because it reduces Y Y = substance being reduced; as an oxidizing agent because it oxidizes X.

Methane combustion as an energy-yielding redox reaction During the reaction, covalently shared electrons move away from carbon and hydrogen atoms and closer to oxygen, which is very electronegative. The reaction releases energy to the surroundings, because the electrons lose potential energy as they move closer to electronegative atoms. Electrons “fall” from organic molecules to oxygen during cellular respiration Cellular respiration is a redox process that transfers hydrogen, including electrons with high potential energy, from sugar to oxygen. oxidation C6H12 O6 + 6O2 à 6CO2 + 6H2 O + energy (used to make ATP) reduction Valence electrons of carbon and hydrogen lose potential energy as they shift toward electronegative oxygen.

Released energy is used by cells to produce ATP. Carbohydrates and fats are excellent energy stores because they are rich in C to H bonds.

NAD1 as an electron shuttle. Nicotinamide adenine dinucleotide molecule consists of two nucleotides joined together. The enzymatic transfer of two electrons and one proton from some organic substrate to NAD1 reduces the NAD1 to NADH. Most of the electrons removed from food are transferred initially to NAD1 During the oxidation of glucose, NAD+ functions as an oxidizing agent by trapping energy-rich electrons from glucose or food. These reactions are catalyzed by enzymes called dehydrogenases, which: Remove a pair of hydrogen atoms (two electrons and two protons) from susbstrate Deliver the two electrons and one proton to NAD+ Release the remaining proton into the surrounding solution The high energy electrons transferred from substrate to NAD+ are then passed down the electron transport chain to oxygen, powering ATP synthesis (oxidative phosphorylation). Electrons transport chains convert some of the chemical energy extracted from food to a form that can be used to make ATP. Are composed of electron-carrier molecules built into the inner mitochondrial membrane. Structure of this membrane correlates with its functional role Accept energy-rich electrons from reduced coenzymes (NADH and FADH2); and pass these electrons down the chain to oxygen, the final electron acceptor. The electronegative oxygen accepts these electrons, along with hydrogen nuclei, to form water. Release energy from energy-rich electrons in a controlled stepwise fashion Since electrons lose potential energy when they shift toward a more electronegative atom, this series of redox reactions releases energy.

An introduction to electron transport chains. (a) The exergonic reaction of hydrogen with oxygen to form water releases a large amount of energy in the form of heat and light: an explosion. (b) In cellular respiration, an electron transport chain breaks the "fall" of electrons in this reaction into a series of smaller steps It stores some of the released energy in a form that can be used to make ATP (the rest of the energy is released as heat). Cellular Respiration Respiration is a cumulative function of three metabolic stages: 1. Glycolysis 2. The Krebs cycle 3. The electron transport chain and oxidative phosphorylation Respiration is a cumulative function of three metabolic stages: Glycolysis is a catabolic pathway that: Occurs in the cytosol Partially oxidizes glucose (6C) into two pyruvate (3C) molecules. The Krebs cycle is a catabolic pathway that: Occurs in the mitochondrial matrix Completes glucose oxidation by breaking down a pyruvate derivative (acetyl CoA) into carbon dioxide

Glycolysis and the Krebs cycle produce: A small amount of ATP by substrate-level phosphorylation NADH by transferring electrons from substrate to NAD+ (Krebs cycle also produces FADH2 by transferring electrons to FAD) The electron transport chain: Is located at the inner membrane of the mitochondrion Accepts energized electrons from reduced coenzymes (NADH and FADH2) that are harvested during glycolysis and Krebs cycle. Couples this exergonic slide of electrons to ATP synthesis or oxidative phosphorylation. This process produces most (90%) of the ATP. During glycolysis, each glucose molecule is broken down into two molecules of the compound pyruvate. The pyruvate crosses the double membrane of the mitochondrion to enter the matrix, where the Krebs cycle decomposes it to carbon dioxide.

NADH transfers electrons from glycolysis and the Krebs cycle to electron transport chains, which are built into the membrane of the cristae. The electron transport chain converts the chemical energy to a form that can be used to drive oxidative phosphorylation, which accounts for most of the ATP generated by cellular respiration. A smaller amount of ATP is formed directly during glycolysis and the Krebs cycle by substratelevel phosphorylation. Substrate-level phosphorylation Some ATP is made by direct enzymatic transfer of P group from a substrate to ADP. Phosphoenolpyruvate (PEP) is formed from breakdown of sugar during glycolysis Glycolisis: steps 1-3 The orientation diagram at the right relates glycolysis to the whole process of respiration. Steps 1-5 are the energy-investment phase of glycolysis. Steps 6-10 are the energy-payoff phase of glycolysis. Glycolisis Harvests chemical energy by oxidizing glucose to pyruvate Glycolysis – catabolic pathway during which six-carbon glucose is split into two three-carbon sugars, which are then oxidized and rearranged by a step-wise process that produces two pyruvate molecules. Each reaction is catalyzed by specific enzymes dissolved in the cytosol. No CO2 is released as glucose is oxidized to pyruvate; all carbon in glucose can be accounted for in the two molecules of pyruvate. Occurs whether or not oxygen is present.

Ten reactions, each catalyzed by a specific enzyme, makeup the process we call glycolysis. ALL organisms have glycolysis occurring in their cytoplasm. At steps 1 and 3 ATP is converted into ADP, inputting energy into the reaction as well as attaching a phosphate to the glucose. At steps 7 and 10 ADP is converted into the higher energy ATP.

At step 6 NAD+ is converted into NADH + H+. The process works on glucose, a 6-C, until step 4 splits the 6-C into two 3-C compounds. Glyceraldehyde phosphate (GAP, also known as phosphoglyceraldehyde, PGAL) is the more readily used of the two. Dihydroxyacetone phosphate can be converted into GAP by the enzyme Isomerase. The end of the glycolysis process yields two pyruvic acid (3-C) molecules, and a net gain of 2 ATP and two NADH per glucose. The process is exergonic (ΔG = -140 kcal/mol or -586 kJ/mol); most of the energy harnessed is conserved in the high-energy electrons of NADH and in the phosphate bonds of ATP.

Krebs Cycle (Citric Acid Cycle) Most of the chemical energy originally stored in glucose still resides in the two pyruvate molecules produced by glycolysis. The fate of pyruvate depends upon the presence or absence of oxygen. If oxygen is present, pyruvate enters the mitochondrion where it is completely oxidized by a series of enzymecontrolled reactions. The junction between glycolysis and the Krebs cycle is the oxidation of pyruvate to acetyl CoA. The Krebs cycle reactions oxidize the remaining acetyl fragments of acetyl CoA to CO2. Energy released from this exergonic process is used to reduce coenzyme (NAD+ and FAD) and to phosphorylate ATP (substratelevel phosphorylation). A German-British scientist, Hans Krebs, elucidated this catabolic pathway in the 1930s. The Krebs cycle, which is also known as the citric acid cycle or TCA cycle, has eight enzyme-controlled steps that occur in the mitochondrial matrix.

Anaerobic versus aerobic Anaerobic - lactic acid fermentation; - alcohol fermentation; - cellular (anaerobic) respiration. Aerobic - Krebs cycle; - electron transport. Anaerobic pathways Alcohol fermentation is the formation of alcohol from sugar. Yeast, when under anaerobic conditions, convert glucose to pyruvic acid via the glycolysis pathways, then go one step farther, converting pyruvic acid into ethanol, a C-2 compound. Humans cannot ferment alcohol in their own bodies, we lack the genetic information to do so. Many organisms will also ferment pyruvic acid into, other chemicals, such as lactic acid. Humans ferment lactic acid in muscles where oxygen becomes depleted, resulting in localized anaerobic conditions. This lactic acid causes the muscle stiffness couch-potatoes feel after beginning exercise programs.

The stiffness goes away after a few days since the cessation of strenuous activity allows aerobic conditions to return to the muscle, and the lactic acid can be converted into ATP via the normal aerobic respiration pathways. Aerobic Respiration When oxygen is present (aerobic conditions), most organisms will undergo two more steps, Krebs Cycle, and Electron Transport, to produce their ATP. In eukaryotes, these processes occur in the mitochondria, while in prokaryotes they occur in the cytoplasm. Acetyl Co-A: The Transition Reaction from glycolysis and the Krebs cycle Pyruvic acid is first altered in the transition reaction by removal of a carbon and two oxygens (which form CO2). When the carbon dioxide is removed, energy is given off, and NAD+ is converted into the higher energy form NADH. Coenzyme A attaches to the remaining 2-C (acetyl) unit, forming acetyl Co-A. This process is a prelude to the Krebs Cycle. Krebs Cycle (Citric Acid Cycle) The Acetyl Co-A (2-C) is attached to a 4-C chemical (oxaloacetic acid). The Co-A is released and returns to await another pyruvic acid. The 2-C and 4-C make another chemical known as Citric acid, a 6-C. The process after Citric Acid is essentially removing carbon dioxide, getting out energy in the form of ATP, GTP, NADH and FADH2, and lastly regenerating the cycle. Between Isocitric Acid and a-Ketoglutaric Acid, carbon dioxide is given off and NAD+ is converted into NADH. Between a-Ketoglutaric Acid and Succinic Acid the release of carbon dioxide and reduction of NAD+ into NADH happens again, resulting in a 4-C chemical, succinic acid. GTP (Guanine Triphosphate, which transfers its energy to ATP) is also formed here (GTP is formed by attaching a phosphate to GDP). The remaining energy carrier-generating steps involve the shifting of atomic arrangements within the 4-C molecules. Between Succinic Acid and Fumaric Acid, the molecular shifting releases not enough energy to make ATP or NADH outright, but instead this energy is captured by a new energy carrier, Flavin adenine dinucleotide (FAD). FAD is reduced by the addition of two H's to become FADH2. FADH2 is not as rich an energy carrier as NADH, yielding less ATP than the latter.

The last step, between Malic Acid and Oxaloacetic Acid reforms OA to complete the cycle. Energy is given off and trapped by the reduction of NAD+ to NADH. The carbon dioxide released by cells is generated by the Kreb's Cycle, as are the energy carriers (NADH and FADH2) which play a role in the next step. 1. Pyruvate’s COO- is removed away as CO2; Acetyl CoA adds two-carbon to oxaloacetate. 2. Conversion of citrate to its isomer, isocitrate via adding and removing a molecule of H2O. 3. Loss of CO2 and oxidizing the remaining compound to, reducing NAD+ to NADH. 4. CO2 is lost; remaining 4-C is oxidised by the transfer of e to NAD+ to form NADH and then attached to coenzyme A. 5. Substrate-level phosphorylation CoA is displaced by a P, which turns GDP into GTP. GTP donates P to ADP Converting the latter one to ATP. 6. Two hydrogens are transferred to FAD to form FADH2. 7. Addition of H2O rearranges bonds in the substrate. 8. Last oxidative step converts malate into oxaloacetate and produces NADH. Summary of the Krebs cycle. The cycle functions as a metabolic "furnace" that oxidizes organic fuel derived from pyruvate, the product of glycolysis. The cycle generates 1 ATP per turn by substrate phosphorylation, but most of the chemical energy is transferred during the redox reactions to NAD1 and FAD. The reduced coenzymes, NADH and FADH2, shuttle high-energy electrons to the electron transport chain, which uses the energy to synthesize ATP by oxidative phosphorylation. The inner mitochondrial membrane couples electron transport to ATP synthesis Only few molecules of ATP are produced by substrate-level phosphorylation: 2ATPs per glucose from glycolysis 2ATPs per glucose from the Krebs cycle Most molecules of ATP are produced by oxidative phosphorylation. At the end of the Krebs cycle, most of the energy extracted from glucose is in molecules of NADH and FADH2. These reduced coenzymes link glycolysis and the Krebs cycle to oxidative phosphorylation by passing their electrons transport chain to oxygen. The exergonic transfer of electrons down the ETC to oxygen is coupled to ATP synthesis.

Electron Transport Phosphorylation Krebs Cycle occurs in the matrix of the mitochondrion, the Electron Transport System (ETS) occurs in the membranes known as the cristae. Kreb's cycle completely oxidizes the carbons in the pyruvic acids, producing a small amount of ATP, and reducing NAD and FAD into higher energy forms.

In the ETS those higher energy forms are cashed in, producing ATP. The electron transport chain is made of electron carrier molecules embedded in the inner mitochondrial membrane. Each successive carrier in the chain has a higher electronegativity than the carrier before it, so the electrons are pulled downhill towards oxygen, the final electron acceptor and the molecule with the highest electronegativity. Except for ubiquinone (Q), most of the carrier molecules are proteins and are tightly bound to prosthetic groups (nonprotein cofactors). Prosthetic groups alternative between reduced and oxidized states as they accept and donate electrons. Protein Electron Carriers Flavoproteins Iron-sulfur proteins Cytochromes

Prosthetic Group flavin mononucleotide (FMN) iron and sulfur heme group

Heme group – prosthetic group composed of four organic rings surrounding a single iron atom. Cytochrome – type of protein molecule that contains a heme prosthetic group and functions as an electron carrier in the electron transport chains of mitochondria and chloroplasts There are several cytochromes, each a slightly different protein with heme group. It is the iron of cytochromes that transfers electrons. Each member of the chain oscillates between a reduced state and an oxidized state. A component of the chain becomes reduced when it accepts electrons from its "uphill" neighbor (which has a lower affinity for the electrons). Each member of the chain returns to its oxidized form as it passes electrons to its "downhill" neighbor (which has a greater affinity for the electrons).

At the bottom of the chain is oxygen, which is very electronegative. The overall energy drop for electrons traveling from NADH to oxygen is 53 kcal/mol, but this fall is broken up into a series of smaller steps by the electron transport chain. As molecular oxygen is reduced is also picks up two protons from the medium to form water. For every two NADHs, one O2 is reduced to two H2O molecules. FADH2 also donates electrons to the electron transport chain, but those electrons are added at a lower energy level than NADH. The electron transport chain does not make ATP directly. It generates a proton gradient across the inner mitochondrial membrane, which stores potential energy that can be used to phosphorylate ADP.

Chemiosmosis: the energy-coupling mechanism Cytochromes are molecules that pass the "hot potatoes" (electrons) along the ETS chain. Energy released by the "downhill" passage of electrons. The ADP is reduced by the gain of electrons. ATP formed in this way is made by the process of oxidative phosphorylation. The mechanism for the oxidative phosphorylation process is the gradient of H+ ions discovered across the inner mitochondrial membrane. This mechanism is known as chemiosmotic coupling.

This involves both chemical and transport processes. The mechanism for coupling exergonic electron flow from the oxidation of food to the endergonic process of oxidative phosphorylation is chemiosmosis. Chemiosmosis – the coupling of exergonic electron flow down an electron transport chain to endergonic ATP production by the creation of a protein gradient across membrane. The proton gradient drives ATP synthesis as protons diffuse back across the membrane. The term chemiosmosis emphasizes a coupling between (1) chemical reactions (phosphorylation) and (2) transport processes (proton transport). Process involved in oxidative phosphorylation and photophosphorylation. The site of oxidative phosphorylation is the inner mitochondrial membrane, which has many copies of a protein complex, ATP synthase. This complex: Is an enzyme that makes ATP Uses an existing proton gradient across the inner mitochondrial membrane to power ATP syntheis Cristae, or infoldings of the inner mitochondrial membrane, increase the surface area available for chemiosmosis to occur. Membrane structure correlates with the prominent functional role membranes play in chemiosmosis: Using energy from exergonic electron flow, the electron transport chain creates the proton gradient by pumping H+s from the mitochondrial matrix, across the inner membrane to the intermembrane space. This proton gradient is maintained, because the membrane’s phospholipid bilayer is impermeable to H+s and prevents them from leaking back across the membrane by dffusion.

Drops in the potential energy of electrons moving down the ETS chain occur at three points. These points turn out to be where ADP + P are converted into ATP. Potential energy is captured by ADP and stored in the pyrophosphate bond. NADH enters the ETS chain at the beginning, yielding 3 ATP per NADH. FADH2 enters at Co-Q, producing only 2 ATP per FADH2. This protein complex, which uses the energy of an H+ gradient to drive ATP synthesis, resides in mitochondrial and chloroplast membranes and in the plasma membranes of prokaryotes. ATP synthase has three main parts: a cylindrical component within the membrane, a protruding knob (which, in mitochondria, is in the matrix), and a rod (or "stalk") connecting the other two parts. The cylinder is a rotor that spins clockwise when H1 flows through it down a gradient. The attached rod also spins, activating catalytic sites in the knob, the component that joins inorganic phosphate to ADP to make ATP. The chemiosmosis hypothesis was proposed by Peter Mitchell in 1961, later he would win the Nobel Prize for his work. Fermentation

Pyruvate, the end-product of glycolysis, serves as an electron acceptor for oxidizing NADH back to NAD+. The NAD+ can then be reused to oxidize sugar during glycolysis, which yields two net molecules of ATP by substrate-level phosphorylation. Two of the common waste products formed from fermentation are (a) Ethanol (b) lactate, the ionized form of lactic acid. Food can be oxidized under anaerobic conditions Aerobic – existing in the presence of oxygen Anaerobic – existing in the absence of free oxygen Fermentation – anaerobic catabolism of organic nutrients Glycolysis oxidizes glucose to two pyruvate molecules, and the oxidizing agents for this process is NAD+, not oxygen. Some energy released from the exergonic process of glycolysis drives the production of two net ATPs by substrate-level phosphorylation. Glycolysis produces a net of two ATPs whether conditions are aerobic or anaerobic. Aerobic conditions: pyruvate is oxidized further by substrate-level phosphorylation and by oxidative phosphorylation and more ATP is made as NADH passes electrons to the electron transport chain. NAD+ is generated in the process. Anaerobic conditions: pyruvate is reduced, and NAD+ is regenerated. This prevents the cell from depleting the pool of NAD+, which is the oxidizing agent necessary for glycolysis to continue. No additional ATP is produced. Pyruvate as key juncture in catabolism Glycolysis is common to fermentation and respiration. The end-product of glycolysis, pyruvate, represents a fork in the catabolic pathways of glucose oxidation. In a cell capable of both respiration and fermentation, pyruvate is committed to one of those two pathways, depending on oxygen presence. Alcohol fermentation is the formation of alcohol from sugar. Yeast, when under anaerobic conditions, convert glucose to pyruvic acid via the glycolysis pathways, then go one step farther, converting pyruvic acid into ethanol, a C-2 compound. Humans cannot ferment alcohol in their own bodies, we lack the genetic information to do so. Many organisms will also ferment pyruvic acid into, other chemicals, such as lactic acid. Humans ferment lactic acid in muscles where oxygen becomes depleted, resulting in localized anaerobic conditions. This lactic acid causes the muscle stiffness couch-potatoes feel after beginning exercise programs. The stiffness goes away after a few days since the cessation of strenuous activity allows aerobic conditions to return to the muscle, and the lactic acid can be converted into ATP via the normal aerobic respiration pathways.

The catabolism of various food molecules. Carbohydrates, fats, and proteins can all be used as fuel for cellular respiration. Monomers of these food molecules enter glycolysis or the Krebs cycle at various points. Glycolysis and the Krebs cycle are catabolic funnels through which electrons from all kinds of food molecules flow on their exergonic fall to oxygen. Catabolism can harvest energy stored in fats obtained either from food or from storage cell in the body. Most of the energy of a fat is stored in the fatty acids. A metabolic sequence called beta oxidation breaks the fatty acids down to two-carbon fragments. Readings CH. 9 (160-180)