Chapt. 21 oxidative phosphorylation

1. Chapt. 21 oxidative phosphorylation • Ch. 21 oxidative phosphorylation • Student Learning Outcomes: • Explain process of generation • of ATP by oxidative phosphorylation: • NADH + FAD(2H) donate e- to O2 -> H2O • ATP synthase makes ATP (~3/NADH, ~2/FAD(2H) • Describe chemiosmotic model, H+ gradient • Describe complications of deficiency of ETC – • anemia, cyanide, OXPHOS diseases • Describe transport through mitochondrial membranes

2. I. Oxidative phosphorylation summary • Oxidative phosphorylation overview: • Multisubunit complexes I, coQ, III, IV pass e- to O2 • H+ are pumped out -> electrochemical gradient • H+ back in through ATP synthase makes ATP Fig. 1

3. Proton Motive Force • Proton motive force: • Electrochemical potential gradient • Membrane is impermeable to H+ • pH gradient ~ 0.75 pH units Fig. 2

4. ATP synthase Figs. 3,4 • ATP synthase (F0F1 ATPase): • F0 inner membrane (12 C) • F1 matrix has stalk, headpiece • H+ go through a-c channel • 12 protons/turn -> 3 ATP • Binding change mechanism: • Turning releases ATP

5. B. Components of Electron Transport Chain • Components of Electron Transport chain: • Series of transfers of e- down energy gradient • Series of oxidation reduction reactions • e- finally to O2 -> H2O • H+ pushed across membrane Fig. 5

6. Components of Electron Transport Chain • NADH dehydrogenase: 42 subunits, • FMN binding proteins • Fe-S binding proteins (transfer single e-) • binding site for CoQ • pass e- to CoQ; transfers 4 H+ Fig. 5,6

7. Components of Electron Transport Chain • Complex II: succinate dehydrogenase (from TCA) • FAD bound e- from TCA, • Other FAD from other paths • Not sufficient energy to transfer H+ when pass e- to CoQ Fig. 5

8. Coenzyme Q • Coenzyme Q is not protein bound. 50-C chain inserts in membrane, diffuses in lipid layer • Also called ubiquionone (ubiquitous in species) • Transfer of single e- makes it site for generation of toxic oxygen free radicals in body Fig. 7

9. Cytochromes have heme groups • Cytochromes have heme groups: • Proteins with hemes • Fe3+ -> Fe2+ as gain e- • Transfer e- to lower potential Figs. 5,8; Heme A is in Cyt a, Cyt a3

10. C. Pumping of protons not well understood • Cytochrome C oxidase: Cyt a, Cyt a3, O2 binding: • Receives e- from Cyt c (takes 4 to make 2 H2O) • Transfers to O2; • Pumping of H+ not well understood; must couple to e- transport and ATP; otherwise backup Fig. 5

11. D. Energy yield • Energy yield from oxidation by O2: • NADH: Dg0’ ~ -53 kcal; FAD(2H) ~ -41 kcal • Each NADH 2e- -> ~ 10 H+ pumped; • Takes ~ 4 H+/ATP -> 2.5 ATP/ NADH; 1.5/ FAD(2H) • or if ~ 3H+/ATP -> 3 ATP/ NADH; 2/ FAD(2H) Fig. 5

12. E. Inhibition of chain, sequential transfer • Once start ETC, must complete transfer of e- • In absence of O2, backup since carriers full of e- • Inhibitors like cyanide (binds Cyt c oxidase) • mimics anoxia: prevents proton pumping • Cyanide binds Fe3+ in heme of Cyt a a3 • CN in soil, air, foods (almonds, apricots) Fig. 5

13. OXPHOS diseases from mutated Mitochondrial DNA OXPHOS diseases from mutated mitochondrial DNA Human mt DNA is 16.569 kb: 13 subunits of ETC: 7 of 42 of Complex I 1 of 11 Complex III 2 of ATPsynthase 22 tRNA, 2 rRNA

14. Table 21.1 examples OXPHOS diseases from mt DNA • Point mutations in tRNA or ribosomal RNA genes: • MERFF (myoclonic epilepsy and ragged red fiber): • tRNAlys progressive myoclonic epilepsy, mitochondrial myopathy with raged red fibers, slowly progressive dementia • Severity of disease correlated with proportion mutant mtDNA • LHON (Leber’s hereditary optic neuropathy): • 90% of cases from mutation in NADH dehydrogenase • Late onset, acute optic atrophy

15. Nuclear genes can cause OXPHOS • Mutated nuclear genes can cause OXPHOS: • About 1000 proteins needed for Oxidation phosphorylation are encoded by nuclear DNA. • Electron transport chain, translocators • Need coordinate regulation of expression of genes, import of proteins into mitochondria, regulation of mitochondrial fission • Nuclear regulatory factors • for transcription in nucleus, mt • Often recessive autosomal

16. III. Coupling of electron transport and ATP synthesis • Concentration of ADP controls O2 consumption: • Or phosphate potential ( [ATP]/[ADP][Pi]) • ADP used to form ATP • Release ATP requires H+ flow • H+ decreases proton gradient • ETC pumps more H+, uses O2 • NADH donates e-, makes NAD+ to return to TCA cycle or other Fig. 10

17. Uncoupling agents dissipate H+ gradient without ATP • Uncoupling agents decrease H+ gradient without generating ATP: • Ex. DNP is a chemical uncoupler: • lipid soluble, carries H+ across membrane Fig. 11

18. Uncoupling proteins form channels, thermogenesis • Uncoupling proteins form channels for protons: • Ex. UCP1 (thermogenin) makes heat in brown adipose tissue (nonshivering thermogenesis); many mitochondria; • Infants have lots of brown adipose tissue, not adults Fig. 12

19. IV. Transport through mitochondrial membranes • Transport across inner mitochondrial membranes uses channels, translocases: • Form of active transport using proton gradient : • ANT exchanges ATP: ADP • Symport H+ with Pi • Symport H+, pyruvate Fig. 13

20. Transport across outer membrane: • Transport across outer membrane: • Rather nonspecific pores: • VDAC voltage-dependent anion channels • Often kinases on cytosolic side Fig. 13

21. Mitochondrial permeability transition pore • Mitochondrial permeability transition pore: • Large nonspecific pore: • Will lead to apoptosis (cell death) • Highly regulated process • Hypoxia can trigger • Pore opens, lets H+ flood in, • Anions, cations enter • Mitochondria swell and • Irreversible damage Fig. 14

22. Key concepts • Reduced cofactors NADH, FAD(2H) donate e- to electron transport chain • ETC transfers e- to O2 -> H2O • As e- transferred, H+ pushed across membrane; • H+ gradient used by ATP synthase to make ATP • O2 consumption tightly coupled to ATP synthesis • Uncouplers disrupt process – poisons • OXPHOS diseases from mutations in mt DNA or in nuclear DNA • Compounds transported across mt membranes

23. Review question • 5. Which of the following would be expected for a patient with an OXPHOS disease? • A high ATP:ADP ratio in the mitochondria • A high NADH:NAD+ ratio in the mitochondria • A deletion on the X chromosome • A high activity of complex II of the electron-transport chain • A defect in the integrity of the inner mitochondrial membrane

24. Review question p. 392 • Decreased activity of the electron transport chain can result from inhibitors as well as from mutations in DAN. Why does impairment of the ETC result in lactic acidosis? • Inhibit ETC -> Impaired oxidation of pyruvate, fatty acids and other fuels; therefore more lactate and pyruvate in blood. • NADH oxidation requires complete transfer of e- to O2, so defect in chain increase NADH:NAD+ and inhibit pyruvate dehydrogenase.