Phosphorylation - Binding-Change Model

OUTLINE: Introduction and review Transport Glycogenolysis Glycolysis Other sugars Pasteur: Anaerobic vs Aerobic Exam-1 material Fermentations Pyruvate Exam-2 material Chemiosmotic theory: Phosphorylation ATPase pyruvate dehydrogenase (ox-decarbox; S-ester) Mitchell Hypothesis Krebs’ Cycle Binding-Change Model How did he figure it out? Overview Connection to the proton motive force 8 Steps Citrate Synthase (C–C) Net ATP production Aconitase (=, -OH) Regulation Isocitrate dehydrogenase (ox-decarbox; =O) Energetics Regulation Summary Ketoglutarate dehydrogenase (ox-decarbox; S-ester) Succinyl-CoA synthetase (sub-level phos) Succinate dehydrogenase (=) Fumarase ( -OH) Malate dehydrogenase (=O) Oxidative Phosphorylation Energetics (–0.16 V needed for making ATP) Mitochondria Transport (2.4 kcal/mol needed to transport H+ out) Electron transport Discovery Four Complexes Complex Complex Complex Complex I: NADH à CoQH2 II: Succinate à CoQH2 III: CoQH2 à Cytochrome C (Fe2+) IV: Cytochrome C (Fe2+) à H2O Phosphorylation Mechanism Binding-Change Model WHAT drives the g-subunits motion? John E. W alker Nobel Prize in Chemistry 1997 ATP cannot be released from one site unless and until ADP and Pi are bound at the other…………without substrates IT ALL GRINDS TO A HALT! 1 Phosphorylation Experimental evidence for the rotary/binding-change mechanism The g-subunit is tightly bound to the C10 ring with the help of the e-subunit ATP Synthase moving a bead Phosphorylation Mechanism • Protons in excess outside the matrix forces them on a specific Asp residue in the C-subunit. • But, it must enter through the a-subunit. • Once bound, it causes a conformational change to rotate the c-ring and make available another proton binding site. • Protons can’t get access to the matrix until a complete revolution and contact with the a-subunit again, which has a tunnel to the matrix. • Proton translocation causes a rotation of the F0 subunit and the central shaft g. 2 Phosphorylation Mechanism Matrix (N-side) 61 E. coli [12] H. sapiens IMS (P-side) [8] The number of c-subunits in the F0 ring varies from 8-17. Bovine & S. cerevisiae [10] Phosphorylation Mechanism Translocation of three Coupling Proton Translocation protons fuels synthesis of one ATP. L to ATP Synthesis T (Lets assume there are 9 c-subunits in F0) •Proton translocation causes a rotation of the F0 subunit and the central shaft g. •This causes a conformational change within all the three ab pairs. •The conformational change in one of the three pairs promotes condensation of ADP and Pi into T ATP. •The conformational change in another drives the release of ATP. •This conformational change opens the binding site for ADP and Pi. O O L O T L 3 Phosphorylation The Respiratory Chain and ATP Synthase Produce ATP by a Chemiosmotic Mechanism Animation of ATP Synthase Phosphorylation Transport of ADP and Pi into the Matrix also uses proton-motive force 3 3 Translocation of a fourth proton per ATP is required to facilitate cotransport of substrates into and products out of the mitochondria. 4 Oxidative Phosphorylation Net Production of ATP by Oxidation of NADH • 10 protons pumped to P-side. • For an F0 with 9 c-subunits, 9 protons are allowed down their concentration gradient and one 360° rotation of the F1 subunit produces 3 ATP molecules; or 3 protons per ATP. • But, one proton is needed to get substrate Pi into matrix, so the total is 4 protons per ATP. • So, for each 2e– from NADH, the 10 protons will yield 2.5 ATP molecules • For 2e– from FADH2, the 6 protons will yield 1.5 ATP molecules. • Different organisms have different numbers of c-subunits, so this number varies species to species. Humans have 8. Oxidative Phosphorylation Net Production of ATP by Oxidation of Glucose • In prokaryotic systems, organelles do not segregate machinery, so all electron carriers can easily feed directly into the electron-transport chain. • In eukaryotic systems, organellar segregation prevents NADH from the cytosol from directly entering the electrontransport chain at Complex I. • NAD+ pools are kept segregated and cannot directly cross the mitochondrial inner membrane. • Two methods are used to feed the electrons from NADH from the cytosol into the mitochondria: – glycerol-3-phosphate shuttle – malate-aspartate shuttle 5 Oxidative Phosphorylation Converting Cytosolic Electron Carriers (NADH) to the Mitochondria Glycerol-3Phosphate Shuttle Malate-Aspartate Shuttle • This more complicated shuttle is mostly present in liver, heart, and kidney. • Will be discussed when we do amino-acid degradation • It moves NADH equivalents form cytosol to NADH equivalents to the mitochondria. Oxidative Phosphorylation Net Production of ATP via Catabolic Pathways TABLE 19-5 ATP Yield from Complete Oxidation of Glucose * Process Direct product Final ATP Glycolysis 2 NADH (cytosolic) 2 ATP 3 or 5a 2 Pyruvate oxidation (two per glucose) 2 NADH (mitochondrial matrix) 5 Acetyl-CoA oxidation in citric acid 6 NADH (mitochondrial matrix) cycle (two per glucose) 2 FADH2 2 GTP Total yield per glucose 15 3 2 30 or 32 If the malate/aspartate shuttle is used to transfer reducing equivalents into the mitochondrion, yield is 5 ATP. If the glycerol 3-phosphate shuttle is used, the yield is 3 ATP. a • • • Every F0 turn uses 8-17 H+ Every turn gets 3 ATP Additional 3 H+ to transport 3 Pi What is the yield for c17? * • This Table assumes F0 is c9 and uses 9 H+ per turn • Additional 3 H+ to transport Pi needs 12 H+ per 3 ATP • This is 4 H+ per ATP • NADH pumps10 H+, so 10/4 = 2.5 ATP/NADH oxidized 6 Oxidative Phosphorylation • Primarily regulated by substrate Regulation availability – NAD+ and ADP/Pi • Local feedback inhib. – Succ-CoA, Citrate, Ac-CoA, and Glc6P. • Inhibition of OxPhos leads to accumulation of NADH. – causes feedback inhibition cascade up to Pyruvate Kinase in Glycolysis – lack of NAD+ in TCA causes accumulation of citrate, which feeds back to inhibit PFK-1 • Inhibitor of F1 (IF1) – prevents hydrolysis of ATP during low oxygen – IF1 only active at lower pH in matrix, encountered when electron transport is stalled (i.e., low oxygen) Oxidative Phosphorylation UNCOUPLING Chemically uncoupling ET and ATP biosynthesis: Recall: • In addition to chemical uncouplers (DNP & FCCP), there are times when uncoupling is needed physiologically • Uncoupling protein 1 (UCP-1) in babies • Hibernating animals 7 Reactive Oxygen Species Can Damage Biological Macromolecules • Ubiquinone is naturally “leaky” and facilitates partial reduction of nonComplex III targets. • Single electron tranfers result in free radicals. • One method by which the cell can correct freeradical production of reduced glutathione, which fuels the glutathione shuttle Summary:Oxidative Phosphorylation We learned that: • the reduced cofactors pass electrons into the electron- transport chain in mitochondria • stepwise electron transport is accompanied by the directional transport of protons across the membrane against their concentration gradient • the energy in the electrochemical proton gradient drives synthesis of ATP by coupling the flow of protons via ATP synthase to conformational changes that favor formation of ATP in the active site 8