Metabolism & Bioenergetics PDF Notes - 2022

S.H. Ackerman, PhD Metabolism & Bioenergetics 1 [email protected] 4213 Scott Hall LECTURE TITLE: METABOLISM & BIOENERGETICS LEARNING OBJECTIVES 1. Write the form of the Gibb’s equation that expresses free energy change in term...

S.H. Ackerman, PhD Metabolism & Bioenergetics 1 [email protected] 4213 Scott Hall LECTURE TITLE: METABOLISM & BIOENERGETICS LEARNING OBJECTIVES 1. Write the form of the Gibb’s equation that expresses free energy change in terms of [reactant] and [product] and define the terms/symbols. 2. Explain what chemical equilibrium is and what is conveyed about reactant & product energies if the equilibrium constant (Keq) is less than, equal to, or greater than 1. 3. Describe the conditions under which a chemical reaction, which is defined by a +DGº′ (standard free energy change that is a positive number), will be spontaneous. 4. Calculate the nutritional value of food based on the amount of carbohydrate, fat, and protein. 5. Differentiate between catabolism and anabolism. 6. Explain why the additive nature of free energy changes is important. 7. Explain the term “high energy metabolite” in biochemistry and list all of the possible products from ATP cleavage. 8. Use reduction potentials and the Nernst equation to decide the direction electrons flow; distinguish the electron donor and electron acceptor in redox reaction. OUTLINE I. First law of thermodynamics. II. Free Energy/Gibb’s Equation III. Equilibrium IV. Free energy vs. Q plots V. Food Energetics. VI. Fundamental Features of Metabolism A. Anabolism vs. Catabolism B. Organization of individual chemical reactions in pathways. C. Relationship to Gibbs free energy VII Common Strategies in Metabolism VIII. Nucleotide Triphosphates A. Cleavage of High Energy Metabolite B. Hierarchy of High Energy Metabolites C. NTP Cleavage Products IX. Creation of Reactive Intermediates A. UDP-Glucose B. Fatty Acyl-CoA X. REDOX Energy  Metabolism & Bioenergetics 2 The universe = matter and energy. Matter comprises everything that is physical in nature (has mass, takes up space). Energy is the force that enables matter to do something (change, perform work). First law of thermodynamics Energy in the universe is conserved; it is neither created nor destroyed. Energy can be changed from one form to another and transferred between a system and its surroundings. (Gibbs) Free Energy Chemical reactions proceed with the exchange of free energy between molecules that are directly related to each other through a single chemical transformation. A net free energy change (DG) will be observed if, as individual entities, the free energy of the reactants (Greactants) differs from the free energy of the products (Gproducts). Reversible processes/reactions proceed spontaneously in the direction that yields a final state/product, which contains less free energy relative to the initial state/reactant. Given that DG = Gproducts – Greactants, spontaneous events are defined by a negative value for the free energy change (−DG) In its original form, the Gibbs equation, DG = DH - TDS, relates the driving force behind a spontaneous chemical transformation, DG, to changes in enthalpy and entropy. This version has limited utility in biochemistry b/c enthalpy and entropy are not easily extrapolated biological systems. To explain the effect thermodynamics has on the direction of reversible reactions in vivo, biochemists have found it more DG′ = DGº′ + RT ln Q practical to use an alternate version of the Gibbs equation: The modified expression introduces two new terms, DGº′ and Q, and includes the prime symbol (') in the symbols that define state functions to indicate that it is assumed the reaction is occurring under conditions of physiological pH. Q is equal to the molar ratio of product relative to reactant as it exists in the chemical mixture in real time. DGº′ defines the free energy change under standard state conditions, [R] = 1 M [P] = 1 M [P]/[R] ratio is equal to 1. Can extrapolate the definition of DGº′ to the general concept: DGº′ is the free energy change under all conditions where [P]/[R] = 1, even if the equivalent molar concentrations of R and P are values other than 1 M.  Metabolism & Bioenergetics 3 DGº′ values are not measured directly, but are derived, instead, from the equilibrium constant DGº′ = − RT ln Keq {Keq = e‒(DGº′/RT)} according to the relationship: Equilibrium Equilibrium defines a state of energy balance. This is an extremely important concept b/c the most predominant driving force in nature is to achieve a state of perfect energy balance. In a closed system, once a reversible chemical reaction is initiated, the quantities of the components on either side of the reaction arrows kAB A+B C+D decrease or increase according to the relative distribution of energy. kCD As the energies come into balance, the opposing reaction rates, Rate A+B ➛C+D = kAB ([A][B]) and Rate C+D ➛A+B = kCD ([C][D]) adjust and eventually become equal, and this concept can be expressed as kAB ([A][B]) = kCD ([C][D]), which rearranges to define a mathematical constant, Keq, that is equal to the ratio of products vs. reactants as they exist at equilibrium, when their concentrations define the unique condition where the energy of the reactant(s) is equal to the energy of the product(s). For the reaction proceeding left to right, kAB [C][D] Keq = A+B C+D [A][B] kkAB CD C+D [A][B] For the reaction proceeding right to left, A+ B Keq = kCD [C][D] The state of equilibrium is dynamic, not static; the reactions going L ➛ R and R ➛ L are occurring, but since they are going at the same rate, [P]/[R] remains a constant number equal to the Keq. Keq, like DGº′, is a unique, defining characteristic of every chemical reaction, and it is no surprise that the two parameters are related mathematically (see above). Keq informs about the energy relationship between the reactant(s) and product(s) One observes Keq = 1 for reactions between reactants and products that occupy the same energy level. In fact, as per the equation DGº′ = -RT ln Keq, when Keq = 1, DGº′ is 0. One observes Keq < 1 for reactions in which the free energy of the product(s) exceeds that of the reactant(s). Energy balance is achieved when [R] is in molar excess over [P]. As per the equation DGº′ = -RT ln Keq, when Keq < 1, DGº′ is a positive value. Such reactions are not spontaneous in the standard state because they require energy input from an outside source. One observes Keq > 1 for reactions in which the free energy of the reactant(s) exceeds that of the product(s). Energy balance is achieved when [P] is in molar excess over [R]. As per the equation DGº′ = -RT ln Keq, when Keq > 1, DGº′ is a negative value. Such reactions are spontaneous in the standard state and release the excess energy.  Metabolism & Bioenergetics 4 While the determination of thermodynamic favorability takes DGº′ into consideration, the Gibbs equation DG′ = DGº′ + RT ln Q dictates that the overall free energy change is also a function of the DG′ =isDGº′ actual [P]/[R] ratio in as it exists for the system under observation, which symbolized + RT ln Qby Q. DG′ = DGº′ + RT ln Q In other words, the Gibb’s free energy change is defined, in part, DDG′ D G′ = G′ DDGº′ == − Gº′ RT ++ RT lnRTeq ln+Q K ln Q RT ln Q by the ratio of product(s) to reactant(s) that is known to exist DG′ = − RT ln Keq + RT ln Q when the system is at equilibrium and, in part, by the ratio of DDG′ G′ == −− RT RT ln ln K Keq + RT RT ln ln QQ eq + product(s) to reactant(s) that exists in real time for the system DG′ = − RT ln + RT ln under study. DG′ = − RT ln + RT ln DDG′ G′ == −− RT RT ln ln ++ RT RT ln ln The punchline of the Gibbs equation is best delivered with a G′ vs Q plot, which graphs how free energy changes over the course of a chemical reaction with Q symbolizing the full range of [P]/[R] values in- between the two extremes (100% R, 100% P). To derive DG’ values from this graph, the point-slope formula is used to determine the slope for unique values of Q. The slope is equal to the instantaneous free energy change, DG’, at that Q value. The position in the curve where energy is at the minimum indicates the state of equilibrium. Recall that equilibrium is observed under the unique circumstance when there is perfect energy balance on either side of the reaction arrow and the corresponding ratio, [P]/[R], is Keq. In other words, a reaction is at equilibrium when Q = Keq. What information is obtained from plotting G′ vs Q First, the line tangent to the point that defines equilibrium (the free energy minimum, where Q = Keq) is perfectly horizontal; there is no slope. No slope means no change in free energy at equilibrium, DG’ = 0, which is as expected. The slopes are negative numbers (−DG′) for all values of Q that are smaller than the Keq; the L ➛ R reaction is favored because the [P]/[R] ratio (Q) is moving toward equilibrium. A system that is moving toward equilibrium is favored thermodynamically. The slopes are positive numbers (+DG′) for all values of Q that are larger the Keq; the L ➛ R reaction is not favored ecause the [P]/[R] ratio (Q) is moving away from equilibrium. A system that is moving away from equilibrium is NOT favored thermodynamically. Regardless of the sign (+ or –) of the standard state free energy change (DGº′), a chemical reaction will be spontaneous in a given direction provided that the [P]/[R] ratio that exists in solution is smaller than the equilibrium constant for that particular chemical reaction. When Q < Keq, the overall free energy change is a negative value (–DG′)  Metabolism & Bioenergetics 5 Food Energetics One food calorie is equivalent to 1000 chemical calories, hence if the thermodynamic energy value of food is in units of kcal, the number is equal to the number of nutritional calories in that food. You are expected to know kcal/g Energy value of food for fats, carbs, and proteins and Energy content Metabolic fuel use these values in calculations. (kcal/g) Fats 9 e.g. If given the content of a Carbohydrates 4 person’s diet (grams or gram (6 x 9) percent), calculate total calories. Proteins 4 Very simple arithmetic. Alcohol 7 Organic acids 3 Sugar alcohols 2.4 Soluble Fiber 2 Intermediary metabolism defines pathways of chemical reactions that manage cellular material and energy resources. A metabolic pathway is initiated with a reactant (simple or complex molecule) and ends with a final product. The end product of a metabolic pathway bears little to no resemblance to the starting material. Anabolism & catabolism follow a defined sequence; each reaction is dependent on the preceding step to Anabolic pathway supply the reactant. A given metabolic pathway typically employs a variety of different kinds of chemical reactions. Each reaction in the pathway is catalyzed by a different enzyme. Catabolic pathway The anabolic pathway and the catabolic pathway for a particular complex molecule are different and may even occur in separate cellular compartments.  Metabolism & Bioenergetics 6 Metabolism runs in a particular direction Metabolic pathways are uni-directional and irreversible Circular Spiral Linear (regenerative) (Recursive) Entry to the pathway requires The first and last steps occur that a particular metabolite is once; the intervening already present. This reactions form an internal metabolite is not a net pathway that is repeated as product, it is regenerated needed. with each round. Key differences between catabolic and anabolic pathways anabolism catabolism Catabolism is Energy yielding Anabolism is Energy consuming Convergent Divergent Converging branches multiple pathways feed into pool of common Diverging branches take metabolites metabolites. from a common pool to build a variety of diverse macromolecules.  Metabolism & Bioenergetics 7 Thermodynamics of intermediary metabolism All pathways of intermediary metabolism include at least one individual step that runs in the direction defined by a positive DG'o and would not occur under standard state conditions DG' values are additive. Cells employ two main strategies to deal with chemical reactions that are endergonic in the standard state: 1. Combine a (+) DG'o reaction with a (−) DG'o reaction in a single step. 2. Combine (+) DG'o reactions with (−) DG'o reactions in a single pathway. All that matters is the sign of the net free energy change. The Table shown here lists standard free energy changes for some metabolic reactions that are exergonic (spontaneous, energy yielding) in the standard state. The reverse reactions are all endergonic (not spontaneous, energy consuming). The corresponding DG'o has the same numerical value but is a positive number. The synthesis of glucose-6-phosphate (G6P) is used here to illustrate what is achieved when a (+) DG'o reaction is combined with a (−) DG'o reaction in a single step. The chemical reaction for making G6P from glucose and phosphate is: Glucose + Pi → G6P + HOH DG'o = +3.3 kcal/mol The endergonic reaction shares intermediates in common with the exergonic reaction of ATP hydrolysis: Do not memorize these values ATP + HOH → ADP + Pi DG'o = − 7.3 kcal/mol At some point in time, the two processes were combined in an exergonic kinase reaction, which produces G6P by transferring a phosphoryl group from ATP to glucose and eliminates phosphate and water as reaction components. Glucose + Pi → G6P + HOH DG'o = +3.3 kcal/mol ATP + HOH → ADP + Pi DG'o = − 7.3 kcal/mol Glucose + ATP → G6P + ADP DG'o = − 4.0 kcal/mol  Metabolism & Bioenergetics 8 The TCA cycle is an example of strategy #2. Summing all eight DG'o gives −8.4 kcal/mol as the net standard free D G°' = - 7.7 kcal/mol energy change for the pathway. D G°' = + 3.2 kcal/mol Based on the standard free energy changes, only 2 D G°' = + 7.1 kcal/mol D G°' = - 2.0 kcal/mol reactions are spontaneous in the direction shown, 2 are clearly not spontaneous, and the other 4 are close to D G°' = - 0.9 kcal/mol equilibrium. Yet there is continuous carbon flux through the D G°' = - 8.0 kcal/mol circuit. D G°' = 0 kcal/mol D G°' = - 0.7 kcal/mol Humans and cars both use hydrocarbons for energy Fat (fatty acids) Protein (amino acids) Octane Carbohydrates (sugars) Ethanol The hydrocarbons are oxidized in combustion reactions, which are exothermic and release energy. In combustion engines, the fuel (hydrocarbons) is oxidized directly by molecular oxygen. This reaction is initiated with a spark of heat energy and generates a HUGE burst of heat energy, which causes the gaseous combustion products to expand and move the piston in the cylinder. Probably the most striking feature about intermediary metabolism is the level of complexity. Going back to the car analogy, combustion engines work on the principle of the enormous output of energy from reacting hydrocarbons with molecular oxygen. However, biological life is not capable of handling the amount of energy that would be released by direct oxidation of glucose (656 kcal/mol) or a 16- carbon saturated fatty acid (2338 kcal/mol) in one step. Instead, the energy from nutrients gets released slowly over the course of many chemical steps in small increments (10-15 kcal/mol maximum).  Metabolism & Bioenergetics 9 In humans, fuel (hydrocarbon) oxidation is NOT through direct reaction with molecular oxygen. Dietary nutrients release energy in the form of reducing equivalents (e- and H+) directly to the redox centers of carrier molecules that intermediate between the energy source and the terminal electron acceptor (O2). Carboxylate groups are lost as CO2, and molecular oxygen is reduced to water. ~60% of the energy released initially is lost as heat along the pathway and 40% is conserved in ATP (adenosine triphosphate). CH2 1½O2 Metabolism CO2 ATP Biosynthesis H2O Metabolite and ion transport Movement ADP + Pi Organ function Another reason for complexity is because the molecular components of cells have to be converted to activated intermediates in order to run chemical reactions in vivo. It is not unusual to see that 2-3 chemical steps are needed just to convert an inert molecule into a reactive species.  Metabolism & Bioenergetics 10 How the cell copes with constraints imposed by thermodynamics. ATP hydrolysis is very exergonic DGº′ = -7.3 kcal/mol and correlates with [ADP][Pi] Keq = is 2.3 x 105 M for the ratio [ATP] [ATP] is micromolar (4.3 x 10-6 M) at equilibrium, which is far too low a concentration for the principal energy molecule in the cell. This consideration has selected over time for regulatory mechanisms that maintain [ATP] in the cell far above the equilibrium concentration. Do not memorize these values Common motifs in metabolism A high concentration of circulating ATP (relative to ADP, AMP) or NADH/NADPH (relative to NAD+/NADP+) indicates a high-energy charge. Anabolism is favored under conditions of high energy charge Catabolism is favored under conditions of low energy charge Two high energy metabolites are cleaved to make the creation of one new high energy metabolite an irreversible process. Clever shuttle systems have evolved to transfer metabolites and energy sources across the highly selective inner membrane of mitochondria. Feedback Inhibition: he enzymes for the rate-limiting and/or committed steps in a pathway are often feed-back regulated by molecules that look nothing like the substrates or products of the reactions they catalyze.  Metabolism & Bioenergetics 11 Feedback Inhibition Common regulatory strategy is for the end product of a metabolic pathway to feedback and inhibit an enzyme that catalyzes a reaction at a point much earlier in the pathway. The end products of metabolism typically bear little resemblance to the metabolites that the pathway was initiated with. Allosteric enzymes are perfectly suited for feedback inhibition as in addition to the substrate binding site, these proteins contain allosteric sites to bind effectors that look nothing like the substrate. The end product will target the first step in linear pathway, and the committed step in a branched pathway. This avoids having the cell produce metabolic intermediates that are not going to be used for anything in the cells. Nucleotide Triphosphates Nucleotide triphosphates (NTPs) are composed of a nitrogen base (adenine, guanine, cytosine, uracil), ribose, and three phosphates linked via phosphoanhydride bonds. The vast majority of the ATP used by the human body is synthesized by a combustive process in which reducing equivalents, captured from dietary molecules, are transferred O2. The products released from cleaving a phosphoanhydride bond in NTP’s (see arrows in upper figure), occupy free energy levels significantly below that of the parent molecule (see adjacent figure). Same concept applies to molecules harboring a thioester bond. Hence, the reason why cleaving a phosphoanhydride or thioester bond releases so much energy is a function of the free energies of the molecules involved (products vs. parent), not the bonds. NTP’s and acyl derivatives of Coenzyme A (contain thioester bond) are high energy molecules.  Metabolism & Bioenergetics 12 Hierarchy of high-energy metabolites The hydrolysis of ATP (or another NTP) yields 7.3 kcal/mol and is by far the most commonly employed means for providing the energy needed to drive biochemical reactions that are exergonic in nature. Hierarchy of high-energy metabolites One mightDpredict ATP would be top Although Go of Hydrolysis dog among ATP isthe the high high energy energy metabolites in cells, while, in fact, it occupies often Metabolite D G º (kcal/mol) molecule used most an in cells, it is not the metabolite with the highest intermediate position when -14.8 phosphoenolpyruvate such molecules free are energy of ranked hydrolysis in the cell. according to the DG´º of hydrolysis phosphocreatine -12.0 (see adjacent table). 1,3-bisphosphoglycerate -11.8 The rationale behind metabolites pyrophosphate -8.0 Metabolites with a DG´º of hydrolysis acetyl coenzyme A -7.5 occupying greater athanposition 7.3of higher energy kcal/mol are essential for cases in which the energy b/c potential vs. ATP is they drive the required ATP -7.3 endergonic synthesis of ATP from to glucose-1-phosphate make ATP must be supplied -5.0 by something ADP by theother process than known as molecular oxygen. The following fructose-6-phosphate -3.8 aresubstrate-level four examples of substrate-level phosphorylation in which the phosphorylation. energy for ATP synthesis is -3.3 glucose-6-phosphate provided by cleaving a high energy molecule, independent of O2. Phosphoenolpyruvate + ADP ATP + pyruvate Glycolysis 1,3-bisphosphoglycerate + ADP ATP + 3-phosphoglycerate Succinyl-CoA + GDP +Pi GTP + succinate TCA cycle Phosphocreatine + ADP ATP + creatine Muscle contraction Essential for ATP synthesis without molecular oxygen. All 4 NTPs (ATP, GTP, CTP, UTP) are utilized to provide the energy necessary to drive endergonic reactions. Cleavage occurs by nucleophilic attack at the aP, the bP, or the gP and generates one product that contains a phosphoryl group ( PO3-) and another with phosphate (PO42-). A phosphoryl has 3 oxygens, phosphate has 4. The reactions are essentially identical for all four high energy molecules and are illustrated below for ATP, which is the NTP used most often as the cell’s energy source. Metabolism & Bioenergetics 13 Metabolism & Bioenergetics 14  Metabolism & Bioenergetics 15 NTP reactions involve the transfer of a phosphoryl species to, or from, another molecule. The enzymes are named accordingly: NTP Hydrolase: Phosphoryl transfer between NTP and H2O. NTP kinase: Phosphoryl transfer between NTP and a molecule other than water. Nucleotidyl transferase: Nucleotidyl transfer between NTP and a molecule other than water.  Metabolism & Bioenergetics 16 Nucleotidyl transfer is the most common strategy for activating molecules. The creation of activated intermediates typically involves NTP cleavage at the a phosphate, followed by release of pyrophosphate, and transfer of the nucleotidyl product to the target molecule. Combining the actions of an enzyme that catalyzes nucleotidyl transfer with a pyrophosphatase makes the generation of activated metabolites an irreversible process. An example is shown below for the "activation" of a fatty acid. Fatty Acyl-CoA synthetases catalyze a two- part reaction. First, ATP is cleaved at the a phosphate. Pyrophosphate is released as one product. The other product, the adenylyl group, is transferred to a free fatty acid creating fatty acyl-adenylate. The fatty acyl-adenylate is a high energy metabolite because of the mixed anhydride bond created between the acid carboxylate and adenylyl phosphate. In the second part, CoA-SH enters the active site, the mixed anhydride bond is cleaved (releasing AMP) and a thioester bond is created between CoA and the fatty acid to create a fatty acyl-CoA. Note that the adenylyl group that was generated when ATP is cleaved in step 1 keeps the linking oxygen when the mixed anhydride bond is cleaved in step 2, and is released from the enzyme as adenylate (AMP). Overall, the reaction catalyzed by fatty acid synthetases is: Fatty Acid + ATP + CoA → Fatty Acyl-CoA + AMP + PPi. In essence, going Left to Right, this reaction cleaves one bond in one high energy molecule (ATP) and creates one bond to make a different high energy molecule (fatty acyl-CoA). The energetics equally favor the Right to Left reaction, which if allowed to occur, would eliminate the desired activated metabolite (fatty acyl-CoA). The Right to Left reaction is prevented through the action of pyrophosphatases, which cleave PPi to (2)Pi. The combined actions of the enzyme that does nucleotidyl transfer (fatty acid synthetase) and pyrophosphatase makes the conversion of a low energy metabolite (fatty acid) to a high energy metabolite (fatty acyl-CoA) a uni-directional, irreversible step.  Metabolism & Bioenergetics 17 REDOX Energy Electrons (e-) are a form of energy. Oxidation is the loss of electrons (and H+ sometimes); Energy released. Reduction is the gain of electrons (and H+ sometimes); Energy consumed. The Nernst equation relates the free energy difference to the difference in reduction potentials for a redox pair under standard state conditions (1 M concentrations) and is highly relevant given the importance of redox chemistry in intermediary metabolism. The standard reduction potential (Eo) of an element or a metabolite is the measure (in volts) of the affinity it has for electrons. DEo = Eoelectron acceptor - Eoelectron donor DEo must be a positive number for DGo to be negative (exergonic). Negative Eº correlates with electropositivity, molecule gives up electrons more easily (is a better reducing agent). Positive Eº correlates with electronegativity, molecule gains electrons more easily (is a better oxidizing agent). Electron transfer energetics: e- move to groups with a more positive of Eº because energy flows downhill in this direction. Tables of redox couples typically present the reduction half reaction along with the reduction potential (E'o). A REDOX reaction combines the reduction half reaction of one redox couple with the oxidation half reaction of another. The electron acceptor is identified as the reduced species of the redox couple defined by the more positive E'o. The reduced species of the other redox couple is the electron donor. Provided the reduction potentials for half reactions and the Faraday constant (F = 23 kcal/V mol) are given, the Nernst equation can be used determine the spontaneous direction of a redox reaction. DE'o must be a positive number for DG'o to be a negative value. −DG'o defines a thermodynamically favored reaction in the standard state  Metabolism & Bioenergetics 18 Write balanced redox reaction between the NAD / NADH,H+ couple and the Ferredoxin (+3) / Ferredoxin (+2) couple, and calculate the free energy change. Ferredoxin (+3) + e- g Ferridoxin (+2) Eo = -0.432 V NAD+ + 2e- + 2H+ g NADH + H+ Eo = -0.320 V (2) Ferridoxin (+2) + NAD+ g NADH + H+ + (2) Ferridoxin (+3) DG'o = -2(23 kcal/V)(-0.320 V – (-0.432 V) DG'o = -2(23 kcal/V)(0.112 V) DG'o = -5.15 kcal/mol The reduction potential remains -0.432 V for ferredoxin even though require two ferredoxins to achieve correct stoichiometry fo the reaction. To write a balanced chemical equation, need to multiply ferredoxin by an integer (2) to account for the fact it is a single electron carrier vs. NAD+/NADH, which is a 2 e- carrier. However, one does NOT multiply the reduction potential by the same integer because the reduction potential is an intensive property of a molecule whose value does not depend on the amount of the substance for which it is measured. Khan academy has a nice video that shows why voltage is intensive v. extensive. https://www.khanacademy.org/science/chemistry/oxidation-reduction/cell-potential/v/voltage-as-an- intensive-property#! The problems given below are for practice, and are not to be turned in. I will post the answers on Friday, July 22. Write balanced redox reaction between the NAD+ / NADH,H+ couple and the Lipoic acid / Dihydrolipoic acid couple and calculate the free energy change. Write balanced redox reaction between the cytochrome b (+3) / cytochrome b (+2) couple and the ubiquinone / ubiquinol couple and calculate the free energy change.