1Chapt. 21 oxidative phosphorylationCh. 21 oxidative phosphorylationStudent Learning Outcomes:• Explain process of generationof 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 I. Oxidative phosphorylation summaryOxidative 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 ATPFig. 1Proton Motive ForceProton motive force: • Electrochemical potential gradient• Membrane is impermeable to H+• pH gradient ~ 0.75 pH unitsFig. 2ATP synthaseFigs. 3,4ATP synthase (F0F1ATPase):• F0inner membrane (12 C)• F1matrix has stalk, headpiece• H+ go through a-c channel• 12 protons/turn -> 3 ATP• Binding change mechanism:• Turning releases ATP2B. Components of Electron Transport ChainComponents 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 membraneFig. 5Components of Electron Transport ChainNADH 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. 5,6Components of Electron Transport ChainComplex 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 CoQFig. 5Coenzyme QCoenzyme 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 bodyFig. 73Cytochromes have heme groupsCytochromes have heme groups:• Proteins with hemes• Fe3+ -> Fe2+ as gain e-• Transfer e- to lower potentialFigs. 5,8; Heme A is in Cyt a, Cyt a3C. Pumping of protons not well understoodCytochrome C oxidase: Cyt a, Cyt a3, O2binding:• 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 backupFig. 5D. Energy yieldEnergy yield from oxidation by O2:NADH: Dg0’ ~ -53 kcal; FAD(2H) ~ -41 kcalEach 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. 5E. Inhibition of chain, sequential transferOnce 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. 54OXPHOS diseases from mutated mitochondrial DNAHuman mt DNA is 16.569 kb:13 subunits of ETC:7 of 42 of Complex I1 of 11 Complex III2 of ATPsynthase22 tRNA, 2 rRNAOXPHOS diseases from mutated Mitochondrial DNA Table 21.1 examples OXPHOS diseases from mt DNAPoint mutations in tRNA or ribosomal RNA genes:MERFF (myoclonic epilepsy and ragged red fiber):• tRNAlysprogressive myoclonic epilepsy, mitochondrial myopathy with raged red fibers, slowly progressive dementia• Severity of disease correlated with proportion mutant mtDNALHON (Leber’s hereditary optic neuropathy):• 90% of cases from mutation in NADH dehydrogenase• Late onset, acute optic atrophyNuclear genes can cause OXPHOSMutated 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 autosomalIII. Coupling of electron transport and ATP synthesisFig. 10Concentration of ADP controls O2 consumption:• Or phosphate potential ( [ATP]/[ADP][Pi])1. ADP used to form ATP2. Release ATP requires H+flow3. H+decreases proton gradient4. ETC pumps more H+, uses O25. NADH donates e-, makes NAD+to return to TCA cycle or other5Uncoupling agents dissipate H+ gradient without ATPUncoupling agents decrease H+ gradient without generating ATP:Ex. DNP is a chemical uncoupler:• lipid soluble, carries H+across membraneFig. 11Uncoupling proteins form channels, thermogenesisUncoupling 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 adultsFig. 12IV. Transport through mitochondrial membranesTransport across inner mitochondrial membranes uses channels, translocases:• Form of active transport using proton gradient :• ANT exchanges ATP: ADP • Symport H+with Pi• Symport H+, pyruvateFig. 13Transport across outer membrane:Fig. 13Transport across outer membrane:Rather nonspecific pores:• VDAC voltage-dependent anion channels• Often kinases on cytosolic side6Mitochondrial permeability transition poreFig. 14Mitochondrial 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 damageKey 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 membranesReview question5. Which of the following would be expected for a patient with an OXPHOS disease?A. A high ATP:ADP ratio in the mitochondriaB. A high NADH:NAD+ ratio in the mitochondriaC. A deletion on the X chromosomeD. A high activity of complex II of the electron-transport chainE. A defect in the integrity of the inner mitochondrial membraneReview question p. 392Decreased 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