Bioenergetics and Oxidative Phosphorylation PDF

 Bioenergetics and oxidative Phosphorylation Objectives ▪ To understand the general concepts of Bioenergetics; Enthalpy, entropy and free energy change and standard free energy and the mathematical relation between them ▪ To understand the concepts of high energy compounds and know the...

 Bioenergetics and oxidative Phosphorylation Objectives ▪ To understand the general concepts of Bioenergetics; Enthalpy, entropy and free energy change and standard free energy and the mathematical relation between them ▪ To understand the concepts of high energy compounds and know the most important examples, like ATP ▪ To know the general concept of oxidative phosphorylation and the general mechanism of ATP production. 1  Bioenergetics ▪ Enthalpy, entropy and free energy. ▪ Free energy change and standard free energy change and the relationship between them and the equilibrium constant ▪ ATP is the universal energy carrier in the biological systems ▪ Structural basis of the high phosphate group transfer potential ▪ Phosphorylated compound with high phosphate group transfer potential, PEP, phosphocreatine ▪ ATP has an intermediate group-transfer potential 2  Oxidative Phosphorylation ▪ Electron carriers, NADH and FADH2 ▪ Mitochondria are the respiratory organelles in the cell ▪ NADH dehydrogenase has two prosthetic groups FMN and iron-sulfur cluster ▪ QH2 is the entry for electrons from FADH2 ▪ Cytochrome Reductase ▪ Cytochrome oxidase catalyses the transfer of electrons from CytC to O2 ▪ Chemiosmaotic hypothesis in the phosphorylation of ADP 3  Bioenergetics & Thermodynamics ▪ Bioenergetics: is the quantitative study of energy transduction in living cells and nature and the chemical process underlying these transductions. ▪ Bioenergetics is concerned only with the initial and final energy states of reaction components: ▪ NOT the mechanism of the reaction, ▪ NOT, the time needed for the reaction to occur. ▪ It allows for predicting the spontaneousity of the reaction and whether a reaction will take place or not. 4  Bioenergetics & Thermodynamics Systems tend to go forward to a lowest-energy state; e.g: fall goes downhill, oxidation of fatty acids produces a lot of energy. 5  Bioenergetics & Thermodynamics Factors that determine the direction of a reaction The direction and extent to which a chemical reaction proceeds is determined by these factors: 1. Enthalpy 2. Entropy 3. Temp 6  Bioenergetics & Thermodynamics Enthalpy: ΔH, a measure of the change in heat reaction content of the reactant and product. ΔH= H2(product)- H1(reactants) ΔH +ve ➔ Endothermic ΔH -ve ➔ Exothermic 7  Bioenergetics & Thermodynamics Oxidation of fatty acids produce a lot of energy and heat. This is a spontaneous reaction and ∆H is negative. For a reaction to be spontaneous usually it produces heat (∆H is negative). ▪ But the melting of Ice is a spontaneous reaction even it is endothermic reaction and ∆H is positive. ➔ ∆H alone is not sufficient to predict the direction of a reaction 8  Bioenergetics & Thermodynamics Entropy (∆S): a measure of randomness or disorder of the reactants and products. Systems have a natural tendency to randomize and the degree of randomness of a system is defined as S which is the entropy. ∆S= S2 (product)-S1 (reactant) ∆S +ve → Increased entropy ∆S -ve → Decreased entropy 9 Entropy (∆S): a measure of randomness or disorder of the reactants and products. 10  Entropy Systems tend to increase the entropy (∆S +ve), e.g. Homogenization of sucrose solution with water is spontaneous. Entropy of ordered state is lower than that of the disordered state of the same system. But organisms create ordered structures from less organized forms of energy and matter. Neither the entropy nor the enthalpy alone can predict the 11 direction of the reaction. Neither the entropy nor the enthalpy alone can predict the direction of the reaction 12 How can we determine if a reaction occurs spontaneously and which require input of energy? 13  Gibbs free energy Free Energy: Gibbs free energy that correlates the entropy and the enthalpy mathematically which allow to predict in which direction a reaction proceeds spontaneously. Free Energy change, ΔG, Predicts the change in the free energy and thus direction of reaction at any specified concentration of products and reactants ΔG = ΔH - T Δ S T is the absolute temperature in Kelvin (K): K = ºC + 273 If ΔG is –ve, the reaction proceeds spontaneously. 14  Gibbs free energy ΔG = ΔH - T Δ S T is the absolute temperature in Kelvin (K): K = ºC + 273 ▪ The sign of the ΔG predicts the direction of the reaction. ΔG is –ve ➔ exergonic reaction. ΔG is +ve ➔ endergonic reaction. ΔG is zero ➔ equilibrium. ΔG of the forward and back reactions A→ B ΔG = -500 cal/mol, spontaneous in this direction ▪ The back reaction B→A ΔG= 500 cal/mol, non-spontaneous at this direction 15  Free Energy change profile G is –ve ➔ exergonic reaction ➔ spontaneous from A to B ΔG = -500 cal/mol, spontaneous in this direction 16  Free Energy change profile G is +ve ➔ endergonic reaction➔ non- spontaneous from B to A ΔG = 500 cal/mol non- spontaneous at this direction 17 18 19  Standard free energy change Standard free energy change (ΔGº): Free energy change under standard conditions; that when reactant and product concentration are kept at 1M conc. 20 G depends on the concentration of both reactants and products. For a reaction A ↔ B A: the reactants B: the product [B] ΔG  ΔG  RTlno [A] ∆Go is the standard free energy change R is the gas constant (1.987 cal/mol K) T is the absolute temperature (Kelvin) [A] and [B] are the actual concentrations of the reactant and product ln represents the natural logarithm The sign of G and Gº can be different 21 G depends on the concentration of both reactants and products. For a reaction A ↔ B A: the reactants B: the product [B] ΔG  ΔG  RTln o [A] The sign of G and Gº can be different Gº gives prediction of the direction of the reaction only at the standard conditions: At standard conditions the [A]=[B]=1 G = Gº + RTln1 G = Gº at standard conditions 22 Relation between equilibrium constant (Keq) and ΔGº A ↔ B at equilibrium [B] eq K  eq [A]eq [B] eq ΔG  ΔG  RTln o [A]eq at equilibrium G=0 [B]eq 0  ΔG o  RTln [A]eq ΔG o   RTlnk eq 23 Relation between equilibrium constant (K eq) and ∆Gº ΔG  RTlnk eq o ➔If Keq=1 → Gº=0 ➔If Keq>1 → Gº < 0 (-ve) ➔If Keq 0 (+ve) 24  Equilibrium of transamination reactions Most of the transamination reactions have an equilibrium constant near 1, allowing the reaction to proceed in both a.a degradation and biosynthesis depending on the relative concentrations. 25  The reaction of Glucose 6-PO4 into Fructose 6-PO4 under three different conditions Glucose 6-PO4 ↔ Fructose 6-PO4 First: At nonequilibrium conditions: Glucose 6-PO4 ➔ Fructose 6-PO4 ΔG = -0.96 kcal/mol 26  The reaction of Glucose 6-PO4 into Fructose 6-PO4 under three different conditions Glucose 6-PO4 ↔ Fructose 6-PO4 Second: At standard conditions: Fructose 6-PO4 ➔ Glucose 6-PO4 ΔG = +0.4 kcal/mol 27  The reaction of Glucose 6-PO4 into Fructose 6-PO4 under three different conditions Glucose 6-PO4 ↔ Fructose 6-PO4 Third: At equilibrium conditions: Glucose 6-PO4 ↔ Fructose 6-PO4 ΔG = 0 kcal/mol 28  What do you think is the right answer? Under standard conditions, the reaction of isomerization of glucose-6-phosphate to fructose-6 phosphate is endergonic (+0.4 kcal/mol), this is the second step in glycolysis. Which of the following will make the reaction favorable: a. Keep high concentration of fructose-6-phosphate. b. Maintaining a high concentration of glucose-6-phosphate. c. Using fructose-6-phosphate immediately in the next step. d. Maintaining a high concentration of glucose-6-phosphate and Using fructose-6-phosphate immediately in the next step. 29 30  What do you think is the right answer? Under standard conditions, the reaction of isomerization of glucose-6-phosphate to fructose-6 phosphate is endergonic (+0.4 kcal/mol), this is the second step in glycolysis. Which of the following will make the reaction favorable: a. Keep high concentration of fructose-6-phosphate. b. Maintaining a high concentration of glucose-6-phosphate. c. Using fructose-6-phosphate immediately in the next step. d. Maintaining a high concentration of glucose-6- phosphate and Using fructose-6-phosphate immediately in the next step. 31  Gibbs free energy ΔG = ΔH - T Δ S T is the absolute temperature in Kelvin (K): K = ºC + 273 ▪ The sign of the ΔG predicts the direction of the reaction. ΔG is –ve ➔ exergonic reaction. ΔG is +ve ➔ endergonic reaction. ΔG is zero ➔ equilibrium. 32 33 34 35 ΔGº of two consecutive reactions are additives and also ΔG of pathways are additives Reactions or processes with a large +ve ΔGº as moving against electrochemical gradient are made possible by coupling the endergonic process with a large –ve process as hydrolysis of ATP Favorable and unfavorable reactions are coupled through common intermediates A+ B → C + F ΔGº1 (non-spontaneous) F→E ΔGº2 (spontaneous) A + B + F→C+ F+ E A+ B → C + E ΔGº3= ΔGº1 + ΔGº2 (spontaneous) F is a common intermediate and can serve as energy carriers for this reaction 36  Standard Free-Energy Changes Are Additive Glucose + Pi → glucose 6-phosphate+ H2O (ΔGº =13.8 kJ/mol ) ATP + H2O → ADP+ Pi (ΔGº =-30.5 kJ/mol) Sum: Glucose + ATP → glucose 6-phosphate+ ADP ΔGº =13.8 kJ/mol + (-30.5 kJ/mol) = -16.7 kJ/mol 37  ATP is the universal energy carrier in biological systems ATP: nucleotide consists of adenine, ribose and triphosphate unit, the active form of ATP is complex with Mg+2 or Mn+2 ATP is energy rich molecule because of its triphosphate unit that contain 2 phosphanhydrid bonds, large free energy is released when ATP is hydrolyzed to ADP + Pi or to AMP and PPi ATP + H2O ↔ ADP + Pi + H+ ΔGº= -7.3kcal/mol ATP + H2O ↔ AMP + PPi + H+ ΔGº= -7.3kcal/mol 38  The free energy liberated in the hydrolysis of ATP is used to drive reactions that requires an input of free energy ATP is formed from ADP and Pi when fuel molecules are oxidized ATP-ADP cycle: the energy exchange in biological system Motion Biosynthesis Active transport ATP is Signal amplification continuously formed and consumed ATP ADP Photosynthesis Oxidation of Fuel molecules 39  Some biosynthesis reactions are driven by nucleotide analogous to ATP and these are: Gaunosine triphosphate: GTP Cytidine triphosphate: CTP Uridine triphosphate: UTP ATP + GDP → ADP + GTP Nucleoside diphosphate kinase, found in all cells, catalyzes the reaction. 40 Structural basis of the high P group transfer potential of ATP ATP + H2O↔ ADP + Pi + H+ ΔGº = -7.3kcal/mol Glycerol 3-phosphate + H2O ↔ Glycerol + Pi ΔGº= -2.2kcal/mol ➔ATP has a stronger tendency to transfer its terminal phosphoryl group to water than dose the glycerol 3-phosphate ➔ ATP has high phosphate group transfer potentials. Why? 1- Electrostatic repulsion 2- Resonance 41 stabilization Other compounds have high phosphate group transfer potential 1. Phosphoenol pyruvate (PEP) has a higher group transfer potential than dose ATP. PEP can donate P to ADP to produce ATP PEP ↔ pyruvate + Pi ΔGº = -62 kJ/mol ADP + Pi ↔ ATP ΔGº = +31 kJ/mol PEP + ADP ↔ Pyruvate +ATP ΔGº = -31 kJ/mol ?????? 42 Other compounds have high phosphate group transfer potential 2. Phosphocreatine have a higher group transfer potential than dose ATP. ADP + Pi ↔ ATP ΔGº = +31 kJ/mol 43 44  ATP is the currency of energy It is significant that ATP has a group-transfer potential that is intermediate among the biological important phosphorylated molecules. This intermediate position enable ATP to function efficiently as a carrier of phosphoryl groups. There is NO enzyme in cells that transfer P from high-P donor to low energy acceptor. It should first transfer first to ATP to form ADP 45  ATP is continuously formed and consumed ATP is the intermediate donor of free energy in biological systems rather than as a long-term storage form of energy. ATP molecule is consumed after 1 min of its formation and the turnover of ATP is high, human consumes about 40 kgs of ATPs in 24 hr. ATP hydrolysis is coupled to the reaction to shift the reaction toward the product A→ B ΔGº = +4 kcal/mol non-spontaneous ATP + H2O → ADP + Pi ΔGº = -7.4 kcal/mol A + ATP + H2O → B + ADP + Pi + H+ ΔGº = -3.4 kcal/mol Spontaneous 46 47  Oxidative Phosphorylation 48 49 50 51 52  Electron Transport Chains a) Uncontrolled reaction b) Cellular respiration  Substrate-level phosphorylation The process that generates almost 90% of the ATP is called oxidative phosphorylation because it is powered by redox reactions A smaller amount of ATP is formed in glycolysis and the citric acid cycle by substrate-level phosphorylation 54 PEP ↔ pyruvate + Pi ΔGº = -62 kJ/mol ADP + Pi ↔ ATP ΔGº = +31 kJ/mol PEP + ADP ↔ Pyruvate +ATP ΔGº = -31 kJ/mol 55 Electron Carriers NAD + FAD 56 * NAD+ is the oxidized form of nicotinamide adenine NADH: generation of ATP dinucleotide, NADH is the reduced form NADPH: reductive biosynthesis * NAD+ is the major e- acceptor in oxidation of fuel molecules. Oxidizing agent reducing agent NAD+ + 2e- + H+ → NADH Oxidized form reduced form NAD+ is strong oxidizing agent that can oxidize secondary alcohol into keton * electron donor= reducing agent (reductant) electron acceptor= oxidizing agent (oxidant) 57 * Flavin adenine dinucleotide (electron carrier molecule) FAD: Oxidized Form FADH2: Reduced Form FAD + 2e- + 2H+ → FADH2 Oxidizing agent reducing agent Oxidant reductant FAD is strong oxidizing agent it can oxidize the alkaline into alkene 58  Oxidative Phosphorylation Oxidative phosphorylation: is the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH2 to O2 by a series of electron carriers. NADH, FADH2 formed glycolysis, fatty acid oxidation and citric acid cycle. They have a pair of electrons with high transfer potential when these electrons are transferred to O2, a large free energy is liberated. O2 H2O ATP ADP + Pi -- - - - ++++ H+ 59 H+  Oxidative Phosphorylation The flow of electrons from NADH or FADH2 to O2 through protein complexes in the inner membrane of the mitochondria leads to the pumping of protons out of the mitochondrial matrix, this makes pH and transmembrane electrical gradient. ATP is synthesized when the proton flows to the mitochondrial matrix. O2 H2O ATP ADP + Pi -- - - - ++++ H+ 60 H+ Chemiosmosis Couples the Electron Transport Chain to ATP Synthesis 61 Chemiosmotic Theory 62 63 64 65 ▪ The Respiratory chain consists of three proton pumps, linked by two mobile electron carriers. ▪ Electrons are transferred from NADH to O2 through a chain of three large protein complexes Complex I: NADH dehydrogenase, NADH-Q reductase. Complex III: Cytochrome reductase. Complex IV: Cytochrome oxidase. The above protein complexes are pump protons Ubiquinone (Q): carries electrons from NADH dehydrogenase (I) to cytochrome reductase (III) Cytochrome C: carries electrons from cyt-reductase (III) to cytochrome oxidase (IV). Complex II: succinate dehydrogenase, (succinate- Q reductase), doesn't pump protons, production of FADH2 from succinate. 66  NADH Complex I NADH dehydrogenase Complex II Q FADH2 FAD Ubiquinone (Q), Cytochrome C are mobile e-carriers Cytochrome reductase. Complex III Cytochrome C Cytochrome oxidase. Complex IV 67 O2 Oxidative Phosphorylation 72  NADH dehydrogenase (Complex I) The electrons of NADH enter the chain at the NADH dehydrogenase, the initial step is the binding of NADH and then the transfer the two electrons to the flavin mono nucleotide (FMN) prosthetic group of this protein to give the reduced form FMNH2 NADH + H+ + FMN FMNH2 +NAD+ Electrons transfer from the Fe-S cluster of complex I are shuttled to Coenzyme Q NADH FMN reduced Fe-S Q NAD+ FMNH2 oxidized Fe-S QH2 The flow of two electrons from NADH to QH2 leads to pumping of four H+ from the matrix to intermembrane space 73 QH2 shuttle electrons from Oxidized form of Q complex I to cytochrome reductase (complex III) It is hydrophobic Intermediate quinone diffuse rapidly within the inner membrane of mitochondria Reduced form 74 Electrons flow from Ubiqinol to cyto. C through Cytochrome reductase Cytochrome is an electron-transferring proteins that contain a heme prosthetic group. Their iron atoms alternate between a educed ferrous (+2) state and an oxidized ferric (+3) state during electron transport. Cyt. Reductase catalyzes the transfer of 2 e- from QH2 to Cyt. C (water-soluble protein) and this is coupled to the pumping of 4 H+ to the inter-membrane space * Cyt. Reductase has two types of cytochromes; b and C1 Cyt. C1 and Cyt. C have iron- protoporphyrine1X the same as heme of myoglobin, hemoglobin and these hemes are covalently linked to protein 75  Cytochrome reductase Complex III QH2 transfer one of its electrons to Fe-S cluster in the reductase. Then this electron is shuttled to Cyt. C1 then to Cyt. C which carries it away from the complex 76  Complex IV: Cytochrome Oxidase. Cytochrome Oxidase catalyses the transfer of electrons from Cyt C to O2 In this reaction 2Cyt C(+2) + 2H+ +1/2 O2 2Cyt C(+3) +H2O This process accomplished by pumping 2 protons from matrix to intermembrane space Cytochrome Oxidase contains two heme A groups called heme a and heme a3 , they are different because they differ in their location in the location. Cyt. Oxidase contains also two copper ions called CuA and CuB as prosthetic group O2 is reduced into water 77  Complex II: succinate dehydrogenase (Succinate-Q oxireductase) QH2 is the entry for electrons from FADH2 of Flavoproteins FADH2 is formed in citric acid cycle by the oxidation of the succinate to fumarate by succinate dehydrogenase (complex II) which is integral protein in the mitochondrial inner membrane, FADH2 doesn't leave the complex, but its electrons are transferred to Fe-S cluster then to Q for the entry to the electron transport chain, the same thing for the FADH2 moieties of glycerol dehydrogenase, and Fatty acyl Co dehydrogenase transfer their high potential electrons to Q to from QH2, these enzymes are not proton pumps 78 * Oxidation and Phosphorylation are coupled by a proton-motive force NADH + ½ O2 + H+ → H2O + NAD+ ΔG0= -52.6 kcal/mol ADP + Pi + H+ →ATP + H2O ΔG0= +7.3 kcal/mol * ATP synthesis is mediated by mitochondrial ATPase (ATP synthase in the inner membrane of mitochondria) * Oxidation of NADH is coupled to Phosphorylation of ADP into ATP The chemiosmotic hypothesis 79  Oxidation of NADH is coupled to Phosphorylation of ADP into ATP (The chemiosmotic hypothesis) The transfer of electrons through the respiratory chain leads to the pumping of protons from the matrix to the other side of the inner mitochondrial membrane. The H+ concentration becomes higher on the systolic side and the electrical potential is generated and this proton motive force drives the synthesis of ATP by the ATP-synthase complex. Oxidation of 1 NADH ➔ 3ATP Oxidation of 1 FADH2 ➔ 2ATP 80 Oxidative Phosphorylation 81  Electrons transfer in the respiratory chain can be blocked by specific inhibitors ▪ Oligomycin: drug bind to ATP synthase that prevents the reentry of H+ ➔ prevents ATP synthesis ➔ prevent electron transport so electron transport and phosphorylation are coupled 82 The End 83