• Metabolism is a highly coordinated cellular activity
• By different chemical reactions, we obtain chemical energy in form of ATP • In metabolic processes, convert nutrient molecules into the cell’s own characteristic molecules and degrade biomolecules. Bioenergetics describes the transfer and utilization of energy in biologic systems Examples of energy transfer within the human body Muscle contraction: chemical energy to mechanical energy Vitamin D formation: light energy to chemical energy Photosynthesis: light energy to chemical energy in plants Principles of Bioenergetics • Its can enable to understand why these energy transfers occur. • Biological energy transductions obey the laws of thermodynamics • Energy cannot be created or destroyed, but can be changed from one form to another For any physical or chemical change, the total amount of energy in the universe remains constant; energy may changed from or it may be transported from one region to another • Energy transfer will always proceed in the direction of increased entropy, and the release of “free energy In all natural processes the entropy of the universe increases Free energy changes (ΔG) “Free energy” is referred to as Gibb’s free energy during energy transfers change in Gibb’s free energy is expressed as a ΔG ΔG= ΔH - TΔS ΔG= Change in Gibbs free energy of the reacting system (Gproducts– Greactives) ΔH= Change in enthalpy of the reacting system(Hproducts – Hreactives), change in heat content, heat release or absorbed T= Absolute temperature ΔS= Change in entropy of the reacting system(Sproducts – Sreactives), measure of randomness Sign of ΔG predicts the direction of a reaction (ΔG) provide a measure of the energetic feasibility of a chemical reaction and therefore, allow prediction of whether a reaction can take place. • Under the constant temperature and pressure changes in free energy, determined by the degree to which two factors change during the reaction, enthalpy (ΔH change in heat content of the reactants and products) and entropy (ΔS, a measure of the change in randomness or disorder of reactants and products When a reaction proceeds with the release of free energy, ΔG has a negative value and the reaction is said to be exergonic In endergonic reactions, the system gains free energy and ΔG is positive If ΔG = 0, the reactants are in equilibrium Change in free energy (ΔG) ΔG of the forward and back reactions • free energy of the forward reaction (A → B) is equal in magnitude but opposite in sign to that of the back reaction (B → A). • if ΔG of the forward reaction is −5 kcal/mol, then that of the back reaction is +5 kcal/mol standard free energy change ΔGo • The change in free energy is represented in two ways, ΔG and ΔGo. The • ΔG represents the change in free energy thus, the direction of a reaction at any specified concentration of products and reactants. • standard free energy change, ΔGo energy change when reactants and products are at a concentration of 1 mol/L. pH = 7 • ΔGo can readily be determined from measurement of the equilibrium constant ΔG depends on the concentration of reactants and products ΔG of the reaction A → B depends on the concentration of the reactant and product At constant temperature and pressure, the following relationship can be derived ΔG = ΔGo + RT ln[B]/[A] ΔGo standard free energy change (energy change when reactants and products are at a concentration of 1 mol/L) R is the gas constant (1.987 cal/mol . degree) T is the absolute temperature [A] and [B] are the actual concentrations of the reactant and product ln represents the natural logarithm ΔG0 • ΔG'0 is the difference between the free energy content of the products and the free energy contents of the reactants under standard conditions • ΔG0 = ΔG0 products– ΔG0 reactives • When ΔG0 is negative, the products contain less free energy than the reactants and the reaction will proceed spontaneously under standard conditions • When ΔG0 is positive, the products contain more free energy than the reactants and the reaction will tend to go in the reverse direction under standard conditions ΔG to be negative • consider the reaction: Glucose 6-phosphate →fructose 6-phosphate • concentration of reactant, glucose 6-phosphate, is high compared with the concentration of product, fructose 6-phosphate. • Ratio of the product to reactant is small, and RT ln([fructose 6- phosphate] ⁄ [glucose 6-phosphate]) is large and negative, causing ΔG to be negative. Thus, the reaction can proceed the forward direction. Standard free energy change, ΔGo • Τhe standard free energy change, ΔGo is equal to the free energy change, ΔG, under standard conditions— when reactants and products are at 1 mol/L concentrations • Under these conditions, the natural logarithm of the ratio of products to reactants is zero (ln1 = 0) and, therefore, the equation becomes: ΔG = ΔGo + 0 ΔGo is predictive only under standard conditions: ΔGo can be used to predict the direction a reaction under these conditions, ΔGo is equal to ΔG Relationship between ΔGo and Keq: In a reaction A → B, a point of equilibrium is reached at which no further net chemical change takes place—that is, when A is being converted to B as fast as B is being converted to A. In this state, the ratio of [B] to [A] is constant, regardless of the actual concentrations of the two compounds: Keq =[B]eq/[A]eq Keq is the equilibrium constant ΔGo and Keq • If the reaction is allowed to go to equilibrium at constant temperature and pressure, then at equilibrium the overall free energy change (ΔG) is zero. • ΔG = 0 = ΔGo + RT ln[B]eq/[A]eq
• Actual concentrations of A and B are equal to the equilibrium
concentrations of reactant and product [A]eq and [B]eq, and their ratio as shown above is equal to the Keq. Thus • ΔGo = -RT ln Keq ΔGo of two consecutive reactions are additive • The standard free energy changes (ΔGo) are additive in any sequence of consecutive reactions, as are the free energy changes (ΔG). For example: Glucose + ATP → glucose 6-P + ADP ΔGo = –4,000 cal/mol Glucose 6-P → fructose 6-P ΔGo = +400 cal/mol Glucose + ATP → fructose 6-P + ADP ΔGo = –3,600 cal/mol ΔGs of a pathway are additive • This additive property of free energy changes is very important in biochemical pathways through which substrates must pass in a particular direction (for example, A → B → C → D → ...). • As long as the sum of the ΔGs of the individual reactions is negative, the pathway can potentially proceed , even if some of the individual reactions of the pathway have a positive ΔG. • The actual rate of the reactions does,of course, depend on the lowering of activation energies by the enzymes that catalyze the reactions ATP AS AN ENERGY CARRIER This principle of bioenergetics explains how a thermodynamically unfavorable (endergonic) reaction can be driven in the forward direction by coupling it to a highly exergonic reaction through a common intermediate • Reactions or processes that have a large positive ΔG, such as moving ions against a concentration gradient across a cell membrane, are made possible by coupling the endergonic movement of ions with a second, spontaneous process with a large negative ΔG, such as the exergonic hydrolysis of adenosine triphosphate (ATP). • In the absence of enzymes, ATP is a stable molecule because its hydrolysis has a high activation energy. • Energy coupling in biologic reactions occurs when the energy-requiring and the energy yielding reactions share a common intermediate. Reactions are coupled through common intermediates • Two chemical reactions have a common intermediate when they occur sequentially A+B→C+D D+X→Y+Z • D is the common intermediate and can serve as a carrier of chemical energy between the two reactions. • Many coupled reactions use ATP to generate a common intermediate. These reactions may involve the transfer of a phosphate group from ATP to another molecule. • Other reactions involve the transfer of phosphate from an energy-rich intermediate to adenosine diphosphate (ADP), forming ATP. Energy carried by ATP • ATP consists of a molecule of adenosine (adenine + ribose) to which three phosphate groups • If one phosphate is removed, ADP is produced; if two phosphates are removed, adenosine monophosphate (AMP) results. The standard free energy of hydrolysis of ATP, • ΔGo, is approximately –7.3 kcal/mol for each of the two terminal phosphate groups. Because of this large negative ΔGo, ATP is called a high- energy phosphate compound Compounds have large free energy change • Phosphorylated compounds Thioesters (Acetyl-CoA) • Phosphoenolpyruvate, 1,3-bisphosphoglycerate • Phosphocreatine, ADP, ATP, AMP • PPi, Glucose 1-phosphate, Fructose 6-phosphate Thioesters • In thioesters a sulfur atom is replaced the usual oxygen in the ester bond • Thioesters have large, negative standard free energy change of hydrolysis. • Acetyl coenzyme A is one of many thioesters important in metabolism. The acyl group in these compounds is activated for trans-acylation, condensation or oxidation-reduction reactions. ELECTRON TRANSPORT CHAIN Energy-rich molecules, such as glucose, are metabolized by a series of oxidation reactions ultimately yielding CO2 and water The metabolic intermediates of these reactions donate electrons to specific coenzymes—nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD)—to form the energy-rich reduced coenzymes, NADH and FADH2. These reduced coenzymes donate a pair of electrons to a specialized set of electron carriers called the electron transport chain As electrons are passed down the electron transport chain, they lose much of their free energy. This energy can be stored by the production of ATP from ADP and inorganic phosphate (Pi). This process is called oxidative phosphorylation Mitochondrion • The electron transport chain is present in the inner mitochondrial membrane and is the final common pathway by which electrons derived from different fuels of the body flow to oxygen. • Electron transport and ATP synthesis by oxidative phosphorylation proceed continuously in all tissues that contain mitochondria. • Outer membrane is permeable whereas Inner mitochondrial membrane is impermeable to most ions and metabolites and specialized carriers are required to move ions or molecules across this membrane. • Inner mitochondrial membrane is highly convoluted which increase the surface area of the membrane Matrix of the mitochondrion Gel-like solution in the interior of mitochondria is 50% protein Enzymes responsible for the oxidation of pyruvate, amino acids, fatty acids (by β-oxidation), and involve in TCA cycle. In addition, the matrix contains NAD+ and FAD (the oxidized forms of the two coenzymes that are required as hydrogen acceptors) and ADP and Pi , which are used to produce ATP Organization of electron transport chain The inner mitochondrial membrane have five separate protein complexes, called Complexes I, II, III, IV, and V. Complexes I–IV are part of the electron transport chain Each complex accepts or donates electrons to relatively mobile electron carriers, such as coenzyme Q and cytochrome c. Complex V catalyzes ATP synthesis and so is referred to as ATP synthase Electron transport chain
NADH, produced from a variety of oxidative (catabolic)
processes, is the substrate for Complex I. Succinate, an intermediate of the TCA cycle, is the substrate for Complex II Reactions of the electron transport chain • With the exception of coenzyme Q, all members of this chain are proteins. These may function as enzymes as is the case with the dehydrogenases may contain iron as part of an iron–sulfur center • In the cytochromes may be coordinated with a porphyrin ring • They may contain copper as does the cytochrome a + a3 complex
Formation of NADH: NAD+ is reduced to NADH by dehydrogenases that
remove two hydrogen atoms from their substrate. Both electrons but only one proton (that is, a hydride ion, :H–) are transferred to the NAD+, forming NADH plus a free proton, H+. NADH dehydrogenase The free proton plus the hydride ion carried by NADH are next transferred to NADH dehydrogenase, a protein complex (Complex I) embedded in the inner mitochondrial membrane. Complex I has a tightly bound molecule of FMN, a coenzyme structurally related to FAD FMN accepts the two hydrogen atoms (2e– + 2H+), becoming FMNH2. NADH dehydrogenase also contains iron atoms paired with sulfur atoms to make iron–sulfur centers, necessary for the transfer of the hydrogen atoms to the next member of the chain, coenzyme Q (ubiquinone). Coenzyme Q Coenzyme Q (CoQ) is a quinone derivative with a long, hydrophobic isoprenoid tail CoQ is a mobile carrier and can accept hydrogen atoms both from FMNH2, produced on NADH dehydrogenase (Complex I), and from FADH2, produced on succinate dehydrogenase (Complex II), glycerophosphate dehydrogenase and acyl CoA dehydrogenase CoQ transfers electrons to Complex III. CoQ, then, links the flavoproteins to the cytochromes Cytochromes The remaining members of the electron transport chain are cytochromes. Each contains a heme group (a porphyrin ring plus iron) Cytochrome iron is reversibly converted from its ferric (Fe3+) to its ferrous (Fe2+) form as a normal part of its function as a reversible carrier of electrons Electrons are passed along the chain from CoQ to cytochromes bc1 (Complex III), c, and a + a3 (Complex IV) Cytochrome a + a3 This cytochrome complex is the only electron carrier in which the heme iron has an available coordination site that can react directly with O2, and so also is called cytochrome oxidase. At this site, the transported electrons, O2, and free protons are brought together, and O2 is reduced to water Cytochrome oxidase contains copper atoms that are required for this complex reaction to occur. Site-specific inhibitors Site-specific inhibitors of electron transport have been identified These compounds prevent the passage of electrons by binding to a component of the chain, blocking the oxidation/reduction reaction. Inhibition of electron transport inhibits ATP synthesis because these processes are tightly couple Incomplete reduction of oxygen to water produces reactive oxygen species (ROS), such as superoxide, hydrogen peroxide and hydroxyl radicals. ROS damage DNA and proteins, and cause lipid peroxidation. Enzymes such as superoxide dismutase, catalase, and glutathione peroxidase are cellular defenses against ROS Release of free energy during electron transport • Free energy is released as electrons are transferred along the electron transport chain • The electrons can be transferred as hydride ions (:H–) to NAD+, as hydrogen atoms (•H) to FMN, coenzyme Q, and FAD, or as electrons (e– ) to cytochromes. Redox pairs: Oxidation of one compound is always accompanied by reduction of a second substance oxidation of NADH to NAD+ accompanied by the reduction of FMN to FMNH2 Oxidation-reduction reactions can be written as the sum of two separate half-reactions, one an oxidation reaction and the other a reduction reaction NAD+ and NADH form a redox pair, as do FMN and FMNH2 Redox pairs differ in their tendency to lose electrons. This tendency is a characteristic of a particular redox pair, and can be quantitatively specified by a constant, Eo (the standard reduction potential), with units in volts. Standard reduction potential (Eo) The more negative the Eo of a redox pair, the greater the tendency of the reductant member of that pair to lose electrons. The more positive the Eo, the greater the tendency of the oxidant member of that pair to accept electrons. Therefore, electrons flow from the pair with the more negative Eo to that with the more positive Eo ΔGo is related to ΔEο : The change in free energy is related directly to the magnitude of the change in Eo: ΔGo = – n F ΔEo n = number of electrons transferred (1 for a cytochrome, 2 for NADH, FADH2, and coenzyme Q) F = Faraday constant (23.1 kcal/volt . mol) ΔEo = Eo of the electron-accepting pair minus Eo of the electron-donating pair ΔGo = change in the standard free energy ΔGo of ATP The standard free energy for the phosphorylation of ADP to ATP is +7.3 kcal/mol. The transport of a pair of electrons from NADH to oxygen via the electron transport chain produces 52.58 kcal. Therefore, more than sufficient energy is available to produce three ATP from three ADP and three Pi (3 x 7.3 = 21.9 kcal/mol), The remaining calories are used for ancillary reactions such as Ca2+ transport into mitochondria or released as heat sometimes expressed as a P:O ratio (ATP made per O atom reduced) of 3:1. P:O for FADH2 is 2:1 because Complex I is bypassed. OXIDATIVE PHOSPHORYLATION The transfer of electrons down the electron transport chain is energetically favored because NADH is a strong electron donor and molecular oxygen is an avid electron acceptor. However, the flow of electrons from NADH to oxygen does not directly result in ATP synthesis. Chemiosmotic hypothesis explains how the free energy generated by the transport of electrons by the electron transport chain is used to produce ATP from ADP + Pi Proton pump: Electron transport is coupled to the phosphorylation of ADP by the transport (“pumping”) of protons (H+) across the inner mitochondrial membrane from the matrix to the intermembrane space at Complexes I, III, and IV. Electron transport chain coupled to the transport of protons This process creates an electrical gradient (with more positive charges on the outside of the membrane than on the inside) and a pH gradient (the outside of the membrane is at a lower pH than the inside The energy generated by this proton gradient is sufficient to drive ATP synthesis. Thus, the proton gradient serves as the common intermediate that couples oxidation to phosphorylation. Electron transport chain shown coupled to the transport of protons ATP synthase: The enzyme complex ATP synthase (Complex V) synthesizes ATP using the energy of the proton gradient generated by the electron transport chain. It is also called F1/Fo ATPase because the isolated enzyme can catalyze the hydrolysis of ATP to ADP and Pi The chemiosmotic hypothesis proposes that after protons have been pumped to the cytosolic side of the inner mitochondrial membrane, they reenter the matrix by passing through a channel in the membrane- spanning domain (Fo ) of Complex V, driving rotation of Fo and, at the same time, dissipating the pH and electrical gradients. Fo rotation causes conformational changes in the extra-membranous F1 domain that allow it to bind ADP + Pi, phosphorylate ADP to ATP, and release ATP. Oligomycin: This drug binds to the Fo , domain of ATP synthase, closing the H+ channel, preventing reentry of protons into the mitochondrial matrix, and thus preventing phosphorylation of ADP to ATP.
Uncoupling proteins (UCP)
UCPs occur in the inner mitochondrial membrane These carrier proteins create a “proton leak,” they allow protons to re- enter the mitochondrial matrix without energy being captured as ATP The energy is released as heat, and the process is called nonshivering thermogenesis. UCP1, also called thermogenin, is responsible for the heat production in the brown adipocytes of mammals. c. Synthetic uncouplers: Electron transport and phosphorylation can also be uncoupled by compounds that increase the permeability of the inner mitochondrial membrane to protons. 2,4-dinitrophenol, a lipophilic proton carrier that readily diffuses through the mitochondrial membrane. This uncoupler causes electron transport to proceed at a rapid rate without establishing a proton gradient, much as do the UCPs Again, energy is released as heat rather than being used to synthesize ATP. In high doses, aspirin and other salicylates uncouple oxidative phos - phorylation. This explains the fever that accompanies toxic overdoses of these drugs. Membrane transport systems The inner mitochondrial membrane is impermeable to most charged or hydrophilic substances, numerous transport proteins that permit passage of specific molecules from the cytosol to the mitochondrial matrix. ATP-ADP transport: The inner mitochondrial membrane requires specialized carriers to transport ADP and Pi from the cytosol (where ATP is used and converted to ADP in many energy requiring reactions) into mitochondria, where ATP can be resynthesized. An adenine nucleotide carrier imports one molecule of ADP from the cytosol into mitochondria, while exporting one ATP from the matrix back into the cytosol A phosphate carrier is responsible for transporting Pi from the cytosol into mitochondria Transport of reducing equivalents Inner mitochondrial membrane lacks an NADH transporter, and produced in the cytosol cannot directly enter the mitochondrial matrix. However, two electrons of NADH are transported from the cytosol into the matrix using substrate shuttles. Glycero phosphate shuttle, two electrons are transfer from NADH to dihydroxyacetone phosphate by cytosolic glycerophosphate dehydrogenase. The glycerol 3-phosphate produced is oxidized by the mitochondrial isozyme as FAD is reduced to FADH2. CoQ of the ETC oxidizes FADH2. The glycero phosphate shuttle, therefore, results in the synthesis of two ATPs for each cytosolic NADH oxidized. Malate-aspartate shuttle produces NADH in the mitochondrial matrix and, therefore, yields three ATPs for each cytosolic NADH oxidized by malate dehydrogenase as oxaloacetate is reduced to malate. A transport protein carries malate into the matrix