Pyruvate fates depend on O 2 conditions of the cell Where does O 2 come into play? O 2 not required O 2 not required for O 2 is terminal acceptor of How are reducing agents ( ) used to make ATP? 1
Production of ATP from reducing agents, NADH/FADH 2, from glycolysis and TCA cycle Electron Transport - e - from NADH/FADH 2 are passed along chain of - aerobic process, O 2 acts as - energy in e - transport used to pump Oxidative Phosphorylation - use of the DG in H + 2
Electron Transport - NADH/FADH 2 are oxidized to NAD + and FAD - e - are transferred through a chain of, - main enzymes are called - e - are ultimately accepted by O 2 - completes process for complete - NAD + and FAD can be reused 3
Electron Transport - production of H + gradient - in e - transport chain use energy of to pump H + across inner membrane to intermembrane space A.K.A.: H + gradient, gradient, gradient 4
Oxidative phosphorylation - - energy stored in H + gradient - what other mechanism used energy stored in H + gradient? 5
Enzyme complexes of e - transfer 4 enzyme complexes - - Complexes I, III, IV - Complex II takes e- from FADH2 and donates to CoQ Complex II 6
Enzyme complexes of e - transfer Complex I - - carries out first step of - transfer of e - to Coenzyme Q (CoQ, ubiquinone) - more than e - transfer - NADH to flavin (FMN) NADH oxidized to NAD +, FMN reduced to FMNH 2 - FMNH 2 to FMNH 2 oxidized to FMN, Fe-S oxidized to Fe-S reduced - Fe-S reduced to CoQ CoQ reduced to CoQH 2 Fe-S reduced to Fe-S oxidized 7
Enzyme complexes of e - transfer Complex I - NADH-CoQ oxidoreductase - some H + moved to intermembrane space, - e - carries can only transfer, - pumped to intermembrane space 8
Enzyme complexes of e - transfer Coenzyme Q (ubiquinone) - membrane - 9
Enzyme complexes of e - transfer Complex II - - second entry point - carries out e - transfer from - subunits e - transfer - FADH 2 to Fe-S protein FADH 2 oxidized to FAD, Fe-S oxidized to Fe-S reduced - Fe-S reduced to CoQ Fe-S reduced to Fe-S oxidized, CoQ to CoQH 2 FADH 2 FAD + Complex II 10
Enzyme complexes of e - transfer Complex II - Succinate-CoQ oxidoreductase - FADH 2 comes from - complex II reaction weakly FADH 2 FAD + - not enough DG to transport Complex II 11
Enzyme complexes of e - transfer Complex III - CoQH 2 -cytochrome c oxidoreductase, - transfers e- from CoQH 2 through - cytochrome: containing protein - dimer of subunit complexes 12
Enzyme complexes of e - transfer Cytochrome - a heme binding protein - heme similar to O 2 binding heme in hemoglobin and myoglobin - Cyt heme binds - reduction of for e - transfer 13
Enzyme complexes of e - transfer Complex III e - transfer: - CoQH 2 releases two e - - Cyt c can only accept/transfer - first e - will be passed to Fe-S protein - second e - is passed to Cyt b and cycled back to CoQ - second e - is then passed on to Cyt c - energy from reactions transports 14
Enzyme complexes of e - transfer Complex IV - Cytochrome c oxidase - catalyzes last step of - e - transferred through - Cu ions act as e- transfer intermediates to - subunits - energy from reactions transports H + to intermembrane space 15
Enzyme complexes of e - transfer Complex IV - Cytochrome c oxidase - Cty c is loosely bound to outer - can freely move from complex III to IV, transferring e - to complex IV - O 2 acts a final - this is link between O 2 and aerobic metabolism 16
Enzyme complexes of e - transfer - e - will only flow - CoQ will not donate - e - move from high energy to low energy 17
Proton gradient formation 1. H + from NADH in Complex I 2. - proteins of complexes take up H + from - these H + are released into intermembrane space 18
Oxidative phosphorylation How is the H+ gradient used to produce ATP? How is chemical energy of H+ gradient converted into chemical energy of ATP? ATP synthase - energy in H + gradient - complex enzyme that - portions of enzyme found on of innermembrane 19
ATP synthase Oxidative phosphorylation 20
Oxidative phosphorylation Chemiosmosis - generation of ATP by across a membrane by ATP synthase - a.k.a oxidative phosphorylation F 0 = subunit of ATP synthase that acts as an F 1 = subunit of ATP synthase ATP synthase links the to the phosphorylation reaction 21
Oxidative phosphorylation Function of F 1 subunit in ATP synthesis - three sites for - exist is 3 states: O - open, low binding affinity 1 L - loose-binding of substrate, T - tight-binding of substrate, 2 - each binding site can be in one of the states - movement of H + through F 0 causes conformational change 1. ATP bound to T, 3 2. H + movement, T will change to L changes to T 3. T forms 22
Oxidative phosphorylation How is conformational change in F 0 accomplished? - c, g, e subunits of F 0 and F 1 - H + movement - rotor causes conformational change in ATP Synthase http://www.youtube.com/watch?v=pjdpty1whdq Electron transort and ATP synthase http://www.youtube.com/watch?v=xbj0nbzt5kw&feature=related 23
Glycerol-phosphate shuttle NADH can not cross the mitochondrial membranes NADH e - from glycolysis must be carried Into - DHAP reduced to glycerol phosphate - moved to matrix - oxidized to DHAP, reducing - FADH 2 can be used for H + gradient formation - 1.5 ATP from FADH 2 - occurs in 24
Malate-Aspartate shuttle More complex but more efficient, 2.5 ATP from NADH - Oxaloacetate reduced to malate in cytosol, - transport to matrix - oxidation to, produces NADH - conversion to aspartate - transport to cytoplasm - conversion oxaloacetate - occurs in 25
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Evolution of mitochondria in Eukaryotic cells Mitochondria have many similarities to prokaryotic cells (bacteria) - their own - many of the same - divide separately from rest of eukaryotic cell, direct their own division - have their own Endosymbiosis- early eukaryotic cell form symbiosis with bacteria that could carry out aerobic metabolism (Krebs cycle, e - transport, oxidative phos) Mitochondria were at one time a bacteria that has Chloroplasts in plant cells also 28
History Herman Moritz Kalckar (1908-1991) - Dutch born biochemist - worked at University of Copenhagen - in early 1940 s established link between sugar oxidation and ATP production - British biochemist Peter D. Mitchell (1920-1992) - worked at Edinburgh University - 1n 1961 discovered chemiosmosis as mechanism for ATP production - 1978 Nobel Prize for Chemistry 29
History Paul D. Boyer (1918 - ) - American born biochemist - worked at UCLA - in 1973 discovered conformation binding change in ATP synthase - in 1982 proposed rotational catalysis of ATP synthase - British born chemist John E. Walker (1941 - ) - worked at Laboratory of Molecular Biology of the Medical Research Council, Cambridge, UK - determined structure of enzyme in oxidative phosphorylation Both awarded Nobel Prize in Chemistry, 1997 30