Developmentof ComplexCurriculaforMolecularBionicsand InfobionicsProgramswithina consortial* framework** Consortiumleader PETER PAZMANY UNIVERSITY Consortiummembers SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER The Project has beenrealisedwiththesupportof theEuropean Union and has beenco-financedbytheEuropean SocialFund*** 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 1 **Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben ***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg. PETER PAZMANY UNIVERSITY SEMMELWEIS UNIVERSITY sote_logo.jpg dk_fejlec.gif INFOBLOKK 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 2 Semmelweis University BIOCHEMISTRY THE ROLE OF MITOCHONDRIA IN BIOENERGETICS (BIOKÉMIA) (A MITOKONDRIUMOK SZEREPE A BIOENERGETIKÁBAN) TRETTER LÁSZLÓ http://semmelweis-egyetem.hu/ Biochemistry: The role of mitochondria in bioenergetics 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 3 http://semmelweis-egyetem.hu/ Energy Relationships between Energy Production (Catabolism) and Energy Utilization (Anabolism) CATABOLISM Energy is released From: Carbohydrates M Lipids M Proteins M ANABOLISM Energy is used For: Motion, locomotion M Transport processes, M Thermogenesis M Synthesis of macromolecules M ATP NADH NADPH ADP+Pi NAD+ NADP+ “M” indicates mitochondrial involvement Biochemistry: The role of mitochondria in bioenergetics 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 4 Introduction to mitochondria The citric acid cycle The electron transport chain, components, function Mitochondrial ATP production, the chemiosmotic mechanism Communication between mitochondria and the extramitochondrial environment Table of contents http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsIntroduction to mitochondria 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 5 http://semmelweis-egyetem.hu/ Electron microscopic picture of a mitochondrion MitochondrionDEF:Intracellular organelle responsible for the aerobic ATP production. http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsIntroduction to mitochondria 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 6 Pre 1900 First descriptions of mitochondria in cells; speculations about them as bacteria 1930's 1930 Formulation of urea and TCA cycle by H. Krebs 1950's 1950 Identification of mitochondria by electron microscopy (Palade, Sjostrand) 1950s Mitochondria as site of fatty acid oxidation (Lehninger, Kennedy) Mitochondria as site of respiration and oxi. phosphorylation 1953 Discovery of cytoplasmic inheritance in yeast (Ephrussi, Slonimski) 1955 Definitiverespiratorychainanalysis(Chanceand Williams, Ernster) 1960's & 70's 1960CharacterizationofthemaincomponentsofrespiratorychainandATPsynthaser.Descriptionofmobileelectroncarriers 1961Discovery of mtDNA (M.M.K. and S. Nass) 1962-70's Elaborationof chemiosmotictheoryP. Mitchell 1980's 1980sAmtDNAsequenchingDescriptionofmtproteinsynthesis.InvitromitochondrialproteintransportmtDNAreplicationcharacterization. 1981 Complete sequence of mammalian DNA 1988 Firstmitochondrialdiseasedescription. 1990's 1990s First sequences of plantmtDNA 1990s Transformation of mtDNA in yeast (introduction of DNA by microprojectiles) 1997 Mitochondriumok és apoptósis: bcl2, cytochrome c; a PTPty 1997 Direct demonstration of rotation of ATP synthase 2000 1995-2000 Crystal structures of complex III, complex IV, F1 ATPsynthase 2000 The MitochondriaResearch Societyand theMitochondrionjournal Mitochondrial chronology http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsIntroduction to mitochondria 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 7 ENZYMES IN MITOCHONDRIAL COMPARTMENTS OUTER MEMBRANEINTERMEMBRANE SPACE MAO (monoamine oxidase)Adenylate kinase, Kinurenin hidroxilaseNukleosidediphospho kinase NADH cyt c reductase Carnitine acyl transferase I Porin INNER MEMBRANEMATRIX Electron transport chainCitric acid cycle enzymes ATP synthaseEnzymes of beta oxidation TransportersPyruvate dehydrogenase Glutamate dehydrogenase Ornithine transcarbamoylase http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsIntroduction to mitochondria 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 8 PARTICIPATION OF MITOCHONDRIA IN METABOLISM MAJOR PATHWAYS TCA cycle-universal Beta oxidation–most of the energy is derived from fatty acid oxidation Urea cycle–essential for the Nitrogen metabolism .-ALA synthesis, porphyrins, cytochromes Pyrimidin synthesis –dihydroorotate dehydrogenase Cyt P450 –OH C20, C22 -side chain cleavage -11ß, 18ßOH Kidney1.24 hidroxylation -cholecalciferol steroid hormone synthesis http://semmelweis-egyetem.hu/ } Biochemistry: Mitochondria in bioenergetics/The citric acid cycle 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 9 Learning objectives: At the end of the presentation students could be able: • To understand the cyclic character of the tricarboxylic acid cycle • To understand the bioenergetic importance of the cycle • To understand the meaning of the regulation of the cycle http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/The citric acid cycle 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 10 Citric acid cycleDEF: is the final common pathway for the oxidation of carbohydrates, lipids and proteins. Citric acid cycle generates reducing equivalents for the terminal oxidation. Acetyl-CoA C2 CoA Oxaloacetate C4 Citrate C6 CO2 CO2 Citric acid cycle: the catalytic role of oxaloacetate Synonyms of citric acid cycle: Krebs cycle Tricarboxilic acid cycle (TCA cycle) Szent Györgyi-Krebs cycle http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/The citric acid cycle 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 11 Characteristics of citric acid cycle Acetyl-CoA is combined with oxaloacetate 2 molecules of CO2are formed Oxaloacetate is regenerated During oxidation reducing equivalents are formed Reducing equivalents enter into the respiratory chain, ATP is generated in the oxidative phosphorylation Hypoxia inhibits the cycle because oxygen is the final oxidant of reducing equivalents Enzymes of the cycle are located in the mitochondrial matrix; freely or attached to the mitochondrial inner membrane Reducing equivalentsDEF: A general term for an electron or an electron equivalent in the form of hydrogen atom or hydride ion http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/The citric acid cycle 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 12 Reactions of the citric acid cycle Acetyl-CoA Compounds with red: inhibitors with green: activators http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/The citric acid cycle 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 13 Chemical reactions in the citric acid cycle (enzyme, type of reaction) Citrate synthase: condensation Aconitase: dehydration, hydration Isocitrate dehydrogenase: oxidative decarboxylation Alpha ketoglutarate dehydrogenase: oxidative decarboxylation Succinyl-CoA synthase: substrate level phosphorylation Succinate dehydrogenase: dehydrogenation Fumarase:hydration Malate dehydrogenase: dehydrogenation http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/The citric acid cycle 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 14 Regulation of the citric acid cycle Enzyme Inhibitor Activator Citrate synthase NADH, succinyl-CoA, citrate, ATP ADP Isocitrate dehydrogenase ATP, NADH Ca2+, ADP Alpha ketoglutarate dehydrogenase ATP, NADH, succinyl-CoA Ca2+ Succinate dehydrogenase oxaloacetate There is no phosphorylation/dephosphorylation in the cycle Local intermediates concentrations regulate the flux. http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/The citric acid cycle 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 15 Entry of glucose-derived carbons into the cycle Connection between glycolysis and the TCA cycle: pyruvate dehydrogenase complex Oxidative decarboxylation of pyruvate Pyruvate Acetyl lipoamide Hydroxyethyl TPP Coenzymes: TPP HS-CoA lipoamide FAD NAD http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/The citric acid cycle 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 16 + + + - - - - - Regulation of PDH complex Phosphorylated form: inactive Dephosphorylated form: active Activators: Ca2+, pyruvate, ADP, NAD+ Inhibitors:, acetyl-CoA, ATP, NADH http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/The citric acid cycle 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 17 Summary of oxidation of pyruvate on PDH complex + citric acid cycle NADH produced4 FADH produced1 Subst. level phosph.1 CO2produced3 http://www.youtube.com/watch?v=A1DjTM1qnPM&feature=related Click in the “show” mode for the video about the citric acid cycle and the respiratory chain http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 18 Overview of the electron transport chain http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 19 Learning objectives At the end of the presentation students could be able: To understand and to reproduce the flow of electrons through the respiratory chain To combine the knowledge about the respiratory chain with Mitchell’s postulates To understand the effect of respiratory chain inhibitors on the oxidoreduction state of the individual chain components http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 20 Electron transport chainDEF: a sequence of electron-carrying proteins that transfers electrons from respiratory substrates to molecular oxygen in aerobic cells. Synonyms: respiratory chain, electron transfer chain Components: small molecular mass components -NAD/NADH, -FAD/FADH2 -FMN/FMNH2 -iron-sulphur centers -heme groups -Coenzyme Q macromolecules (proteins) http://semmelweis-egyetem.hu/ } prosthetic groups of proteins Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 21 Respiratory Chain Components Localization Prostheticgroups Function NADPH / NADP(almost 100% reduced) matrix space (separate pool in the cytosol) - mobile carrier energy-linked transhydrogenaseNADPH+NAD+=>NADH+NADP+ Integrant membrane protein none proton pump 2H+/2e-1 NADH / NAD(less than 30% reducedt) matrix space (separate cytosolic pool) - mobile carrier NADH dehydrogenase(complex 1) membrane spanning multi-subunit protein non-heme iron& FMN proton pump 4H+/2e-1 succinate dehydrogenase(complex 2) membrane spanning multi-subunit protein non-heme iron& FAD no proton pumping ubiquinol / ubiquinone lipidsoluble - mobile carrier ubiquinol:cytochrome c reductase (complex 3) membrane spanning multi-subunit protein non-heme iron, heme b & hemec1 proton pump 4H+/2e-1 cytochrome c(ferrous / ferric) inter-membrane space heme c mobile carrier cytochrome c oxidase(complex 4) membrane spanning multi-subunit protein copper, heme a& heme a3 proton pump 2H+/2e-1 [ F0 / F1 ATPase(ATP synthetase)(complex V.) membrane spanning multi-subunit protein none proton pump3H+ / ATP Components of the respiratory chain and oxidative phosphorylation http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. nadh TÁMOP –4.1.2-08/2/A/KMR-2009-0006 22 nad A NADH, the most important reducing equivalent .max=250 nm Oxidized form (NAD+) Reduced form (NADH) .max=340 nm wavelength http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 23 Isoalloxazine Flavin mononucleotide FMN Flavin adenine dinucleotide FAD Riboflavin Flavines: FAD and FMN Flavoproteins in the respiratory chain Complex IFMN Complex IIFAD Glycerophosphate FAD dehydrogenase http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 24 The oxidoreduction of Coenzyme Q Coenzyme Q: antioxidant Semiquinone: prooxidant Function: mobile lipophylic electron carrier in the membrane http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 25 Structure of iron-sulfur centers Localization of iron sulfur proteins Complex I., complex II, complex III, http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 26 / hemea hemec Heme a Heme c hemeb Heme b Prosthetic groups of cytochromes CytochromesDEF:heme proteins, electron carriers in respiration and in other oxido-reduction reactions http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 27 Complexes of the electron transport chain, Complex I Membrane spanning Proton pumping activity Oxidation of NADH of the TCA cycle detailed structure of complex I with inhibitors http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 28 Complexes of the respiratory chain: complex II No proton pumping activity http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 29 CoQcycle The flow of electrons in complex III and the coenzyme Q cycle Dimeric form Membrane spanning Proton pumping activity http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 30 The flow of electrons in the complex IV Membrane spanning Proton pumping, but less proton can be pumped than in complex I or III. Water formation on the matrix side http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 31 Respiratory complexes are organized according to their redox potentials http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/Electron transport chain 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 32 Summary http://www.youtube.com/watch?v=KXsxJNXaT7w&feature=related In “show” mode click to the link below: respiratory chain movie Electrons flow in the respiratory chain from NADH to oxygen Respiratory complex I, III, and IV are able to pump protons to the inner membrane space 10 protons are pumped out/NADH oxidation The stoichiometry of proton pumping 4; 4; 2; per pair of electrons passed through the complex Respiratory complexes are organized accoding to their redox potentials http://semmelweis-egyetem.hu/ Biochemistry:Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 33 Introduction In mitochondria ATP is synthesized via oxidative phosphorylation. Oxidative phosphorylationDEF: oxidation (oxygen consumption, or oxidation of reducing equivalents) is coupled to phosphorylation i.e. to ATP synthesis. The oxygen consumption thus coupled to ATP synthesis http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 34 http://semmelweis-egyetem.hu/ 1)The membrane-located ATPase systems of mitochondria and chloroplasts are hydro-dehydration systems with terminal specificities for water and ATP; and their normal function is to couple reversibly the translocation of protons across the membraneto the flow of anhydro-bond equivalents between water and the couple ATP/(ADP + Pi). 2)The membrane-located oxido-reduction chain systems of mitochondria and chloroplasts catalyse the flow of reducing equivalents, such as hydrogen groups and electron pairs, between substrates of different oxido-reduction potential; and their normal function is to couple reversibly the translocation of protons across the membrane to the flow of reducing equivalentsduring oxido-reduction. 3)There are present in the membrane of mitochondria and chloroplasts substrate-specific exchange-diffusion carrier systemsthat permit the effective reversible transmembrane exchange of anions against OH-and of cations against H+; and the normal normal function of these systems is to regulate the pH and osmotic differential across the membrane, and to permit entry and exit of essential metabolites (e.g., substrates and phosphate acceptor) without collapse of the membrane potential. 4)The systems of postulates 1, 2, and 3 are located in a specialized coupling membranewhich has a low permeability to protons and to anions and cations generally. MITCHELL, P. Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic type of mechanism. Nature 191:144–148, 1961. Nobel Prize in Chemistry 1978 Mitchell’s postulates H+ ADP+Pi ATP reducing equivalents H2O H+ Ions, metabolites H+ http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 35 nfgz030 Oxidation–Reduction Potentials of the Mitochondrial Electron Transport Chain Carriers http://semmelweis-egyetem.hu/ Biochemistry:Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 36 Energy derived from NADH oxidation NADH + H++ 1 O2 .Go’=-NF.Eo’n= number of electrons, F=Faraday number =2(96.5 kJ/V*mol)(1.14 V).Eo’=st. redox potential difference =220 kJ/molbetween NAD/NADH and oxygen/water pairs As a consequence of proton pumping activity membrane potential (matrix negative) and pH difference is formed across the two sides of the membrane. Proton motive force (pmf) .µH+=..m-60.pH (mV) Energeticsof ATP synthesis http://semmelweis-egyetem.hu/ Biochemistry:Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 37 How much energy is released when one proton moves from the intermembranespace to the matrix?l In energized mitochondria...pH~0.75; ..~150-200 mV Proton motive force: .G=2.3RT.pH + F..=5.70kJ/mol*.pH + (96.5kJ/V*mol)...20 kJ/mol Energy required to pump out one proton cca20 kJ/mol 10 protons would cost about .G~200 kJ However, the oxidation of NADH releases 220 kJ. The efficacy of proton pumping about 200/220 (Energy derived from NADH oxidation NADH + H+ + 1 O2 .Go’= -NF.Eo’ =2(96.5 kJ/V*mol)(1.14 V) =220 kJ/mol) Energeticsof ATP synthesis http://semmelweis-egyetem.hu/ Biochemistry:Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 38 F1FoATP-ase produces ATP from the energy of proton gradient http://www.youtube.com/watch?v=uOoHKCMAUMc Click to the link below to play the movie about the ATP synthesis http://semmelweis-egyetem.hu/ Biochemistry:Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 39 Functional characteristics of the ATP synthase molecule http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 40 The molecular mechanism of ATP synthesis http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 41 CytosolInner membraneMatrix ADP3- ATP4- H3PO4- H+ Phosphate carrier Adenine Nucleotide translocase ANT is electrogenic: there is a net movement of one negative charge from the matrix to the intermembrane space ANT can be reversed. In that case ATP from the cytosol enters into the mitochondria Electrogenic transportDEF: A transport is defined as electrogenic if during the transport process there is a net charge movement. As a consequence of it a current is generated Transports essential for the oxidative phosphorylation Phosphate carrier is electroneutral http://semmelweis-egyetem.hu/ Biochemistry:Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 42 How much energy is required to synthesize one mol ATP? How much energy is liberated during the hydrolyzis of 1 mol ATP? .G= .Go’ + RTln[ADP][Pi]/[ATP] .G=-30.5kJ/mol + 8.315*298 =-54.7 kJ/mol muscle cell .G=-51.8 kJ/mol Red Blood Cell 2 protons are not enough(2x20=40 kJ less than 50-55kJ) 3 proton is more than enough but: Because of the ATP/ADP exchanger one extra proton is required Total cost: 4 protons/ATP 10 protons/NADH could form 2.5 mol ATP/NADH oxidation http://semmelweis-egyetem.hu/ Biochemistry:Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 43 P/O ratioDEF: P/O or ATP/O ratio (where O refers to an oxygen atom) is the number of ATP molecules formed when one oxygen atom is reduced to water P/O ratio for NADH oxidation: 10 protons are pumped during oxidation 1 NADH will reduce 1 oxygen atom 10 protons are enough to synthesize 2.5 mol ATP-t How much is the P/O ratio during the oxidation of succinate? Succinate oxidation does not involve proton pumping at complex I. Complex II is unable to pump protons. The number of proton pumped 4(complex III) +2(complex IV)=6 6 protons produce 1.5 mol ATP P/Osuccinate=6/4=1.5 http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 44 Energy balance of glycolysis+citric acid cycle Site ATP substrate level phosphorylation ATP from oxidation of reducing equivalent sum Glycolysis P-glycerate kinase Pyruvate kinase 1 1 2 Gliceraldehyde-P dehydrogenase 2x2.5 5 Pyruvate dehydrogenase 2x2.5 5 Citric acid cycle ICDH 2x2.5 5 .-KGDH 2x2.5 5 Succinate thiokinase 2 2 Succinate dehydrogenase 2x1.5 3 Malate dehydrogenase 2x2.5 5 Summary 4 28 32 http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 45 Uncoupling of oxidative phosphorylation Normal Uncoupled ADP+Pi High energy protons Low energy protons X X uncoupler Protons cross the ATP synthase and drive ATP synthesis Protons are carried by the protonophor (uncoupler).Protons cross the membrane without using ATP synthase. Proton gradient is dissipated without ATP synthesis http://semmelweis-egyetem.hu/ UncouplingDEF: Oxidation (oxygen consumption) is not coupled to phosphorylation (ATP) synthesis. Oxidation is fast, ATP synthesis is slow or non-existent. P/O ratio is low, even could be zero. Biochemistry: Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 46 Experiments proving the chemiosmotic ATP generation INNER MEMBRANE ULTRASOUND INSIDE OUT SUBMITOCHONDRIAL PARTICLES MECHANICAL SHAKING MEMBRANE VESICLES ELECTRON TRANSPORT + ATP SYNTHESIS - F1PARTICLES ELEKTRON TRANSPORT – ATP SYNTHEZIS - ATP-ASE ACTIVITY + RECONSTRUCTION EL. TRPORT + ATP SYNTHESIS + F1 Fo NADH+O2 ADP+Pi NAD+H2O ATP http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 47 .[H+] Time (sec) pH electrode O2 injector mitochondria O2injection Experiments proving the chemiosmotic ATP generation Mitochondria respire (oxygen consumption and proton pumping activity) OXYGEN DECREASES, PROTON PUMPING ACTIVITY DECREASES http://semmelweis-egyetem.hu/ Biochemistry:Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 48 Experiments proving the chemiosmotic ATP generation The role of proton motive force in ATP synthesis 1961 Peter Mitchell pH:7.5 pH 4.0 pH 4.0 pH:7.5 pH 4.0 pH 8.0 Equilibrationwith pH 4.0 buffer Buffer pH 8.0 +ADP + Pi ADP+Pi ADP+Pi ATP ATP ATP synthesis Artifitially constructed pH gradient http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 49 Liposomes H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ Bakteriorhodopsin ADP+Pi ATP F0 F1 H+ Artifitial vesicles From purified phospholipids Protons pumped by bacteriorhodopsin Can be utilized to synthesize ATP The role of proton motive force in ATP synthesis http://semmelweis-egyetem.hu/ Biochemistry: 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 50 The essence of oxidative phosphorylation MEMBRANE ------ + + + + + + + ATP ADP+PI H+ O2H2O http://semmelweis-egyetem.hu/ Biochemistry:Mitochondria in bioenergetics/ Mitochondrial ATP production 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 51 Summary The energy of substrate oxidation forms proton gradient High energy protons are used for ATP synthesis 3 protons are necessary to cover the actual expenses of ATP synthesis However, besides the respiratory chain and the ATP synthase ANT and Pi carrier are also essential for the oxidative phosphorylation ANT is an electrogenic carrier, every catalytic cycle decreases membrane potential The electrogenic property of ANT adds a further proton to the costs of ATP synthesis, so 4H+s are needed to synthesize 1 ATP molecule The P/O ratio for NADH is 2.5 Uncoupling decreases P/O ratio http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 52 Learning objectives At the end of the presentation students could be able: To understand the essential role of mitochondrion-cytosol communication: -in the metabolite transport (with special emphasis to the oxidation of glycolytic NADH) -in the calcium homeostasis. Students can understand the importance of various Ca2+uptake and Ca2+release pathways The importance of mPTP opening can not be overestimated in pathology. Students could get an impression about the nonphysiological conditions associated with mitochondria. The unique properties of mtDNA and the flow of information between the cytosol, nucleus and the mitochondrion make this chapter especially attractive. http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 53 Phosphate Malate Pyruvate OH- Malate Citrate Glutamate Aspartate Aspartate –glutamate carrier Malate Alpha-ketoglutarate malate –.-KG carrier Dicarboxylate carrier Tricarboxylate carrier Monocarboxylate carrier Mitochondrial substrate carriers http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 54 Shuttle of reducing equivalents Glycolysis produces NADH. NADH could reduce pyruvate to lactate, however lactate formation prevents entry of pyruvate into the matrix, thus oxidative phosphorylation is inhibited. Cytosolic NADH accordingly should be oxidized in the mitochondria, but mitochondrial inner membrane is impermeable to NAD+and NADH. Those mechanisms which help the oxidation of cytosolic NADH in the mitochondria are called “shuttles”. Glycerophosphate dehydrogenase localization: inner membrane, outer surface. Redox potential between complex I and III (like complex II) thus 6H+/glycerophosphate oxidation are pumped out. http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 55 Malate Glutamate Aspartate –glutamate carrier Malate malate –.-KG carrier The malate-aspartate shuttle cytosol matrix IM Inner Membrane glyceraldehyde3-P GLYCOLYSIS glucose pyruvate 1,3 bisphosphoglycerate Pi NAD NADH .-Ketoglutarate OA Aspartate OA .-KG Aspartate Glutamate NAD NADH NADH NAD GOT GOT RESPIRATORY CHAIN http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 56 glyceraldehyde3-P GLYCOLYSIS glucose pyruvate 1,3 bisphosphoglycerate Pi NAD NADH O || CH |HCOH | CH2O P CH2OH | C=O | CH2O P CH2OH |HCOH | CH2O P O || COP |HCOH | CH2O P Glycerol 3-P Dihyroxyacetone P FAD FADH2 The glycerophosphate shuttle Glycerol-P dehydrogenase Cytosol Inner membrane Matrix http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 57 Mitochondrial Ca2+homeostasis UNIPORTER http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 58 Mitochondrial Uptake of Ca2+ Na+/Ca2+-exchange is important ..mdependent slower kinetics ATP: purinergicagonist Gq->PLC->IP3 FCCP: uncoupler CGP-37157: Na-Ca exch inhib. Cell Calcium 2001; 30.5: 31 http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 59 Mitochondrial Ca2+transporters http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 60 Ca2+uniporter – Ca2+elecrtophoretic uptake (charge carried: 2) – Voltage dept, Ca2+activated – Km~10µM – InhibitorsRuthenium red, Ru360 (-) – Kompetetive inhibitors bound and transported in the channels: Sr2+,Mn2+,Ba2+ – Allosteric inhibitors: Mg2+,Mn2+, – Spermin (+) – ATP>ADP>AMP (-) – Unknown molecular identity Rapid uptake mode – Ca2+uptake [Ca2+]o~400nM Transient uptake, – Reset – Inhibitor: Ruthenium red, Ru360 (-but at higher conc.) – ATPactivated – Mg2+does not inhibit – Unknown molecular identity http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 61 Na+-indept Ca2+-efflux (NICE) – nH+-Ca2+antiporter, n>2 ..mrequired – Ruthenium Red insensitive – Slow, early saturation Na+-dept Ca2+-efflux (NCE) – 3Na+-Ca2+antiporter – ..mrequired – Mg2+,Sr2+,Ba2+,Mn2+ (-) – Amilorid, trifluoperazin, diltiazem, CGP-37157 (-) – Mátrix pH dept (opt.= pH 7.6) Ca2+-cycling – Could contribute to the fine tuning of[Ca2+]matrix(e.g.: cardiomyocyte) http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 62 ch14f14 pyruvate Ca2+activates TCA cycle [Ca2+]matrix Pyruvate dehydrogenase complex .-ketoglutarate dehydrogenase Isocytrate dehydrogenase + + + [Ca2+]matrix Pyruvate dehydrogenase complex .-ketoglutarate dehydrogenase Isocytrate dehydrogenase + + + http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 63 Hypothetical scheme of mitochondrial Permeability Transition Pore (mPTP) http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 64 Mitochondrial permeability transitionDEF:The mitochondrial permeability transition involves a sudden (and initiallyreversible) increase in permeability of the IMM to solutes up to 1.5 kDa. It is commonly definedby its inhibition by cyclosporin A. http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 65 Regulationof mPTP The most important mPTP opener is high matrix [Ca2+] ! – Voltage sensitive: IMM depolarization opens• gating potential is shifted by agonist (more negative ..m) • or antagonists (less negative ..m) – Divalent cations:• [Ca2+]Matrix(+) • [Mg2+]Matrix, [Mn2+]Matrix, divalent cations outside (-) – Matrix pH: Alkalization is permissive (>= pH 7.3); OH-,Pi(+) – Thiol oxidation: Oxidation (disulphide formation) of ANT (+)• redox status is in equilibrium with matrix glutathione. – Oxidation of pyridine nucleotides favours permeability transition. http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 66 Regulationof mPTP – ANT ligands: • ADP, bongkrekate (-) • Atractyloside (+) – Metabolites: • Glucose and creatine inhibit (-) (action on hexokinase and creatine kinase) • Coenzyme Q (-) • Long-chain fatty acids, ceramide and ganglioside GD3 (+) – Anti-and pro-apoptotic members of the Bcl-2 family. Multiple conductance state – Low conductance state for selective permeation of H+or Ca2+ http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 67 Metabolic consequences of mPTP opening Uncoupling of the respiratory chain with collapse of the proton gradient Cessation of ATP synthesis Matrix Ca2+outflow Depletion of reduced glutathione Depletion of NADPH Hypergeneration of O2 .– Release of intermembrane proteins Self-amplifying process (on neighborhooding mitochondria) http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 68 Physiological function of mPTP Periodic reversible opening of the permeability transition pore allows for – release of Ca2+from the mitochondrial matrix, thereby participating in Ca2+homeostasis and/or the generation of Ca2+waves – The ANT/VDAC couple (and its interacting proteins hexokinase and creatine kinase) may also participate in regulating ATP/ADP transport/synthesis. Irreversible permeability transition – triggers mitochondrial autophagy (a process by which cells digest parts of their cytoplasm), apoptosis or necrosis. http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 69 Physiological uncoupling: brown adipose tissue cold adaptation UCP-1 protein uncoupling Heat generation http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 70 The mitochondrial genom 16,569base pair 7 of43 subunits ofcomplexI (ND1, 2, 3, 4, 4L, 5, and 6), 1of 11 subunits of complex III (cytochromeb, cyt b), 3of 13 subunits of complex IV (COI, II, and III), 2of 16 subunits of ATP synthase(ATPase 6 and 8), + smalland large rRNAs, 22 tRNAs More than 98% of mitochondrial proteins are imported! http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergeticsMitochondrion-cytosol communication 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 71 Summary There is an intensive communication between the mitochondria and the other intracellular compartments. Metabolite trafficking is essential, because metabolic fuels should enter into the mitochondria, ATP, and waste products have to leave it. The NADH shuttle systems are particularly important in the oxidation of cytosol-generated NADH. Mitochondria play an important role in the Ca2+homeostasis. Ca2+can be an important signal for mitochondria to increase energy production, but Ca2+overload could kill mitochondria opening the mPTP. Although probably the most important mitochondrial task is to supply the cell with energy, during cold adaptation mitochondria participate in the thermogenesis using the uncoupling proteins. http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 72 Recommended literature Orvosi Biokémia (Ed. Ádám Veronika) Textbook of Biochemistry Ed. Thomas Devlin 5th-7th edition http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 73 Questions Calculate P/O ratio if only complex IV is in the position to pump protons Which enzymes in the mitochondria are activated by calcium? How many ATP is produced in the citric acid cycle if NADH is oxidized in the respiratory chain? What is the effect of uncoupling on the transmembrane pH difference? What are the transporters for the mitochondrial calcium efflux What are the well-known functions of the hypothetical constituents of the mPTP? http://semmelweis-egyetem.hu/ Biochemistry: Mitochondria in bioenergetics 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 74 Questions Which statements are true for the thermodinamics of the respiratory chain? A: The change of redoxpotential during the oxidation of a NADH is 1.14V B: The energy of a single proton in energized mitochondria is about 20 KJ/mol C: In order to synthesize 1 ATP molecule we need the energy of 4 protons D: The standard free energy of ATP hydrolysis differs from the actual one E:All of the statements are true Which of the following enzymes produce NADH in the citroc acid cycle A: Aconitase B: Glutamate dehydrogenase C: Succinate dehydrogenase D: Malate dehydrogenase E: Citrate synthase http://semmelweis-egyetem.hu/