TCA Cycle & OXPHOS PDF

TCA Cycle and Oxidative Phosphorylation Jeff Powers, PhD. Department of Molecular Biology [email protected]  Learning Objectives Explain how cytosolic pyruvate is oxidized to acetyl-CoA and CO2 in the mitochondria - Describe t...

TCA Cycle and Oxidative Phosphorylation Jeff Powers, PhD. Department of Molecular Biology [email protected]  Learning Objectives Explain how cytosolic pyruvate is oxidized to acetyl-CoA and CO2 in the mitochondria - Describe the roles of pyruvate dehydrogenase complex (PDHc; E1, E2, E3), four water-soluble vitamins, and lipoate in pyruvate oxidation - Discuss the regulation of PDH by allosteric effectors and covalent modification - Discuss in brief the clinical significance of PDHc deficiency / dysregulation Explain how acetyl-CoA is oxidized in the TCA cycle to generate CO2, reducing equivalents, and GTP - Describe the eight successive reaction steps, and exergonic and endergonic reactions (e.g., reactions catalyzed by citrate synthase and malate dehydrogenase) in the TCA cycle - Identify the key TCA cycle enzymes that catalyze the formation of CO2, reducing equivalents (NADH and FADH2; energy conservation), and GTP/ATP (substrate-level phosphorylation) - Discuss the regulation of TCA cycle by substrate availability and allosteric effectors - Discuss in brief the clinical significance of dysregulated TCA cycle enzymes (including SDH)  Learning Objectives Describe the four protein complexes (I, II, III, and IV) and mobile electron carriers (Q and cytochrome c) involved in electron transfer through the respiratory chain Explain how the respiratory chain oxidizes reducing equivalents (NADH or FADH2) and acts as a proton pump Discuss the chemiosmotic theory of oxidative phosphorylation (ATP synthesis) Identify the sites of action of key inhibitors of respiratory chain or oxidative phosphorylation Describe how glycerophosphate or malate shuttle transfers reducing equivalents to the mitochondria Describe in brief the significance of superoxide dismutase Many figures for this lecture were created by Lakshman Segar, Ph.D., and are being included with his permission  % -Carbons) Aotyl-Cotfrom Where can go we G 13 carb) 2 Carbons Making our way here 12a Glycolysis-Cytosol b toward many Mitochond  The outer mitochondrial membrane is permeable to most small ions and molecules because of the channel protein - porin. (VDAC – voltage dependent anion channel) Allows passage of small molecules and ions. The inner membrane, which is folded into ridges called cristae, is impermeable to most molecules. ↳ wher ETC The inner membrane is the site of electron transport and ATP synthesis. Transporters shuttle metabolites across the inner membrane The citric acid cycle and fatty acid oxidation occur in t outer = Mem Space blur inner the matrix.  Nutrients Carbohydrate Lipid Protein Digestion & Absorption Glucose Fatty acids Amino acids Blood All roads lead to Aatyl-Cod Moving into Matrix Pyruvate Acetyl-CoA Acetyl-CoA formation Nucleus Mitochondria in the mitochondria Lakshman Segar, Ph.D.  Glucose gluc Transporter. Plasma membrane Glucose (C6) & RBCs? reaced ↳ Pyruvate is carbon A gainarban Mentor Glycolysis 2 NADH ︖ Lactate b ↳ Alinated 2 Lose Carbon * b Alanine ATP Acetyle Cot ︖ Pyruvate (C3) & (2 molecules) not a take Most tissues (bk no Mitochondrial direction Modified from Medical Biochemistry 2nd Ed Baynes & Dominiczak Lakshman Segar, How is pyruvate transported to the mitochondrial matrix? Pyruvate Pyruvate carrier Located on IMM - Pyruvate Lakshman Segar,  How is pyruvate (C3) oxidized to acetyl-CoA (C2)? decarboxylizedproduce more NADH Haan's Kreb's PDHC Pyruvate Acetyl-CoA Hans Krebs Why do we begin the TCA cycle with pyruvate and not acetyl-CoA? 1937 Lakshman Segar, Pyruvate undergoes oxidative decarboxylation of to form acetyl-CoA & W/ Z Starting Out Pyruvate Acetyl-CoA − + PDHC CH3-C-COO + NAD + CoA CH3-C S-CoA + NADH + H+ + CO2 ॥ ॥ 1st fully Oxidied O O Carbon Pyruvate (C3) Lipoic (2 molecules) acid B Thiamin , B2 Riboflavin Pyruvate 2 CO2 As B3 dehydrogenase Niacin complex (PDHC) prosthetic BS Pantothenic groups or acid 2 NADH coenzymes Acetyl-CoA (C2) (2 molecules) Lakshman Segar,  Inner Lipoic Pyruvate dehydrogenase complex membrane acid -- multienzyme complex -- Thiamin Riboflavin (E1 + E2 + E3) Niacin Pantothenic acid Er E2 E3 Pyruvate Dihydrolipoyl Dihydrolipoyl dehydrogenase transacetylase dehydrogenase /required 4 Froen them) Prosthetic - Made > Thiamin-PPi Lipoic acid FAD groups Coenzyme A Made from & NAD+ Bi MadeFrom Bs - (CoA-SH) Niacin Essential Nutrient Lakshman Segar,  Just A Reference Catalytic coenzymes: Thiamine pyrophosphate, lipoic acid, FAD – not permanently altered Stoichiometric coenzymes: Coenzyme A and NAD – function as substrates  Coenzyme A Examples of acyl groups: CoASH Acetyl CoA Succinyl Bs CH3-COS-CoA Acetyl-CoA: ॥ O High band Energy Aaty) IMPORTANT Gr 2 C acetyl group is activated (“TURBO-charged”) =. ↓ shuttled off toTCA Hydrolysis of acetyl-CoA (exergonic reaction) Acetyl-CoA + H2O acetate− + CoA + H+ (ΔG°ˊ = −31.4 kJ/mol) thermodynamically favorable Lehninger, Principles of Biochemistry 6th Ed Marks’ Basic Medical Biochemistry: A Clinical Approach, 5e Lakshman Segar,  Pyruvate undergoes oxidative decarboxylation of to form acetyl-CoA Pyruvate Acetyl-CoA − + PDHC CH3-C-COO + NAD + CoA CH3-C S-CoA + NADH + H+ + CO2 ॥ ॥ O O Regulation & : Lipoic acid Thiamin Riboflavin Niacin Pantothenic acid Harper’s Illustrated Biochemistry, 32e Lakshman Segar, Regulation of PDH by allosteric effectors and covalent modification (phosphorylation/dephosphorylation) to slow These Act it all hasaccumulation O it down ATP-stimulate Pyruvate => by Activity sinhibitor dehydrogends its to stow down - 6 Active form if p on it = deactivated direction op ↳ Aatyl-Cot Energy depleated was direct gluconeogie slows down State High Energy Mansalin Harper’s Illustrated Biochemistry, 32e seen Harper’s Illustrated Biochemistry, 32eMusde in Contraction Lakshman Segar, directiont o Cot ↓TCA Muscle Contraction Ca2+ Release  PDH Kinase PDH Phosphatase high in Activated Activated Energy Abundanc factors  Clinical significance of PDHc Pyruvate E1 dehydrogenase #XX Deficiency of α subunits (rare) Congenital Neurodegeneration lactic acidosis Muscle spasticity EC 1.2.4.1 Dietary deficiency is * Thiamin-PPi d o s or ↓ absorption aci (as in alcoholics) ac tic Seen Chronic Alcoholies n dl ca in - Inhibit Abscription of u v i pyr Dihydrolipoyl ta l fa i al ly en t E2 transacetylase p o t 33 EC 2.3.1.12 As Arson Poisony Arsenic Arsenite or Lipoic acid Mercuric ions reacts with 80 Coenzyme A −SH groups Inhibit BorEs Hg (CoA-SH) of lipoic acid Mercury Lakshman Segar,  CH3-C S-CoA ॥ O TCA cycle enzymes b > how to going Mitochondrial > TCA matrix electrons extracted Ch NAD-FAD Oxidation of NADH acetyl moiety of CO2 acetyl-CoA to CO2 NADH CO2 TCA cycle Reduction of NADH coenzymes FADH2 NAD+ and FAD 8 reaction steps Electron Transport Chain Oxidative Phosphorylation ATP Lakshman Segar, The citric acid cycle oxidizes the acetyl fragment of acetyl CoA to CO2. In the process of oxidation, high-energy electrons are captured in the form of NADH and FADH2. A key function of the citric acid cycle is to harvest high-energy electrons from carbon fuels. Acetyl Cot Oxidation bl take Idearboxylated e- (decarboxylated)  (breathe ofp) Metabolic H20 by product Mnemonic for TCA cycle intermediates - Citrate is a sour substrate for metabolic oxidation CH3-C S-CoA ॥ O Oxaloacetate > Citrate Malate > Fumarate Isocitrate Succinate Succinyl-CoA α-ketoglutarate Medical Biochemistry 2nd Ed Baynes & Dominiczak Lakshman Segar,  TCA cycle 8 reaction steps oA c c i nyl-C Su etase h synt Harper’s Illustrated Biochemistry, 32e Lakshman Segar, most are exerganic Spontaneousy Happen - Formation of Citrate Synthesis Citrao as Squiggly Citroyl CoA (transient intermediate) hydrolysis highly exergonic Modified from Harper’s Illustrated Biochemistry, 32e Citrate + free CoA (ΔG°ˊ = −32.2 kJ/mol) thermodynamically favorable Lehninger, Principles of Biochemistry 6th Ed Oxidation of Malate to Oxaloacetate Reversible reaction Modified from Harper’s Illustrated Biochemistry, 32e (ΔG°ˊ = +29.7 kJ/mol) appears to be thermodynamically unfavorable In intact cells: Reaction proceeds in the forward direction – oxaloacetate is continually removed by highly exergonic citrate synthase reaction Lehninger, Principles of Biochemistry 6th Ed Lakshman Segar,  α-ketoglutarate undergoes oxidative decarboxylation to form succinyl-CoA α-KGDH complex α-ketoglutarate + NAD+ + CoA Succinyl S-CoA + NADH + H+ + CO2 Lipoic acid Thiamin Riboflavin Niacin Pantothenic acid Mechanism of oxidative decarboxylation → similar to PDHc  Come Can · back Citrate (2 molecules) (4) Around37 min. Highly Endergonic (2) Mark ICDH 2 NADH (6) Hydroy & Moving - α-KGDH 2 NADH produced SuccTK ~ 2 ATP (2 GTP) SDH 2 FADH2 MDH 2 NADH > - c c i Su etase nyl-C oA (6) h synt Oxaloacetate (2 molecules) X O How many ATPs are formed in the Oxidative givg CO2 TCA cycle (one turn)? decarboxy > - O Harper’s Illustrated Biochemistry, 32e Lakshman Segar, (S)  Amphibolic nature of TCA cycle – energy production and biosynthetic pathways (gluconeogenesis, amino acid synthesis, and fatty acid synthesis) / Anaplerosis (“filling up”) – anaplerotic reactions / substrates What TCA intersects Harper’s Illustrated Biochemistry, 32e Lakshman Segar,  CH3-C-S-CoA TCA cycle ॥ O > > ETC Oxidation of NADH acetyl moiety of CO2 acetyl-CoA to CO2 NADH CO2 Reduction of NADH coenzymes ETC in IMM FADH2 NAD+ and FAD Electron Transport Chain Oxidative Phosphorylation ATP Lakshman Segar, Controlled primarily by energy charge of the cell (ATP abundance vs Deplation) Transported to cytoplasm to control PFK (inhibit) Look backD Sonin & Q asked  Summa Anaplerotic Reactions  Oxidative Summay &54 00 : min Phosphorylation Intermembrane Space v kind left · pow e- the Lots o (3TC) creatin  Oxidation of H+ H+ H + H+ reducing equivalents gradient NADH and FADH2 out of Matrix pumped * to Membran Space ATP synthesis drives ↳ gradiend ETC (e → e → e → e → O2) - - - - Oxidative (Respiratory chain) ATP Phosphorylation Glucose oxidation to carbon dioxide and water C6H12O6 + 6O2 6CO2 + 6H2O Lakshman Segar, Direction of electron flow & flow from Lower-Hight e-potential  need Carrier) (carrier) 1+ 344 2 - 344 never 1, 2 , 3-4 - Comple whe we get ATP from Carrier makes ↓  Components of the respiratory chain (Electron transport chain) Flow of electrons from reducing equivalents (NADH) to Oxygen know ETC In men detail J know both the names Modified from Harper’s Illustrated Biochemistry, 32e Components of the respiratory chain (Electron transport chain) Flow of electrons from reducing equivalents (FADH2) to Oxygen Modified from Harper’s Illustrated Biochemistry, 32e NAD+ accepts two electrons as FAD accepts two electrons as hydride ion to form NADH two hydrogen atoms to form FADH2 Marks’ Basic Medical Biochemistry: A Clinical Approach, 5e  Mobile electron carriers used interchangeably [Q, coenzyme Q, CoQ or ubiquinoneJ → diffuses rapidly within the membrane accepts from either Complex → carries two electrons to ↓ drops of + Harper’s Illustrated Biochemistry, 32e Cytochrome c, Cyt c → soluble protein → carries one electron  Iron-sulfur proteins (Nonheme iron proteins; Fe-S) in Complexes I, II, and III good dcpa Harper’s Illustrated Biochemistry, 32e Fe-S → participate in single electron transfer reactions (one Fe atom → Fe2+ to Fe3+ and vice versa Heme A in cytochromes a and a3 Fe anbada Grun · Capabl of Oxidative stress-so usually exclosed in stupp Marks’ Basic Medical Biochemistry: A Clinical Approach, 5e Components of the respiratory chain - Electron flow Modified from Harper’s Illustrated Biochemistry, 32e Complex I: NAD-Q Oxidoreductase https://www.youtube.com/watch?v=rdF3mnyS1p 0  Two electron carrier to a one INTER-MEMBRANE SPACE electron SIDE carrier MATRIX SIDE https://www.youtube.com/watch?v=rOv-X7VFbp 0 Complex IV skipped to her, Respiratory chain oxidizes reducing equivalents (NADH or FADH2) and acts as a proton pump 7Creata NADH-total (14) Harper’s Illustrated Biochemistry, 32e FADH2-110)  Chemiosmotic Hypothesis Electron transport and ATP synthesis are coupled by a proton gradient across the inner mitochondrial membrane NADH + H+/FADH2 NAD+/FAD + e- + H+  Proton gradient drives ATP synthesis – Chemiosmotic theory of oxidative phosphorylation every 4 protons >- 1 ATP ↳ explains FADH doesn't produce why as much ATP synthase functions as a rotary motor From TCA Cycles /Every ↑ compose turn of TCA Cycle) a 3 NADH (2 5). = %S. small gradin 1 FADH (1 S) 1S :·. = ATP ↓ Not FOATP * Total ATP Lange Gradient flow down Canc Gradient Harper’s Illustrated Biochemistry, 32e. Marks’ Basic Medical Biochemistry: A Clinical Approach, 5e  form Roles of phosphate transporter and adenine nucleotide transporter NAD" Oxidized = in ATP synthesis (within mitochondria) and ATP exit (from mitochondria) NADH = 2.5 ATP it doesn't Ib/c FADH = 1 5 ATP. Istill need to more aut of Mitochondria X to cytosol S [ Harper’s Illustrated Biochemistry, 32e  Inhibitors of respiratory chain / oxidative phosphorylation Inhibit z Ibinds to bemoglobin Garbas better than Moroida ② ② - How cydride Work Inhibits Complex 4 Barbiturate Compl. I Inhibits oxidation and phosphorylation by blocking proton flow through ATP synthase Harper’s Illustrated Biochemistry, 32e  Glycerol 3-phosphate & lyo colysis lyard phosphate shuttle High in muscle 5 ATP (NADH) > it - go togoAp insteadop.. produced DHA - SATPINADACarboxylation ↑ e at in Glycolysis [ J + 20 (from TCA) 32 ATP/o30 when glucose is fully Oxidized mather Glycerophosphate shuttle transfers reducing equivalents from cytosol to mitochondrial matrix (utilizes cytosolic and mitochondrial isoforms of Glycerol-3-P DH) Harper’s Illustrated Biochemistry, 32e  Malate shuttle transfers reducing equivalents from cytosol to mitochondrial matrix Started zony opp e 29 min mark-an Transcript and (utilizes cytosolic and mitochondrial isoforms of Malate DH) Same Enzyme but dif Locations Glutamate-oxaloacetate Glutamate-oxaloacetate transaminase (or) transaminase (or) Aspartate aminotransferase Aspartate aminotransferase Harper’s Illustrated Biochemistry, 32e  brown Adipose Tissue(babes possess lots of this I they red to generate Heat /yellow Powde 2 4DNP = ↳ cause weight loss by uncomply , > - creates a more b pearmedba membrane death via Hypothermia  > - disrupts t , Superoxide dismutase o about 1 % of e-spit upp-t combined / 2 happens Allth time > - reactive moty O Species (Superoxide) I break H82 neutralize somethy Marks’ Basic Medical Biochemistry: A Clinical Approach, 5e