ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION
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ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION

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ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION Presentation Transcript

  • Electron Transport and Oxidative Phosphorylation M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
  • Electron Transport and Oxidative Phosphorylation *Introduction* stage 3 of respiration NADH & FADH oxidized, electrons are “carried” (ETS) energy in form of ATP (Ox/Phos)  aerobic acceptor = oxygen
  • Mitochondrion -- A. football shaped (1-2μ), 1-1000s in each cell B. electron transport and oxidative phosphorylation Cytosol
  • C. Outer membrane- permeable to small molecules D. Inner membrane- electron transport enzymes embedded;  also ATP synthase  Cristae increase area  Impermeable to small molecules Integrity required for coupling ETS to ATP synthesis Cytosol
  • E. Matrix TCA enzymes, other enzymes; also ATP, ADP, NAD+, NADH, Mg2+, etc.
  •  The Electron Transport System is the mechanism the cell uses to convert the energy in NADH and FADH2 into ATP.  Electrons flow along an energy gradient via carriers in one direction from a higher reducing potential (greater tendency to donate electrons) to a lower reducing potential (greater tendency to accept electrons).  The ultimate acceptor is molecular oxygen.
  • -- The overall voltage drop from NADH E = -(-0.32 V) to O Eº = +0.82 V is Eº = 1.14 V
  • -- This corresponds to a large free energy change of G = - nFE = -220 kJ/mole (n =2) -- Since ATP requires 30.5 kJ/mole to form from ADP, more than enough energy is available to synthesize 3 ATPs from the oxidation of NADH.
  • NADH Dehydrogenase- Complex I NADH-CoQ oxidoreductase Contains FMN/FMNH2 and an Iron Sulfur Center as Electron Carriers NADH is substrate Coenzyme Q is second substrate
  • NAD+/NADH NADP+/NADPH Never covalently bound- freely diffusible  Nicotinamide 
  • Flavin mononucleotide = FMN Flavin adenine dinucleotide = FAD Riboflavin = ring + ribitol isoalloxazine ring ribitol
  • 2H++2e Coenzyme Q Coenzyme Q Coenzyme Q = Ubiquinone a lipid in inner membrane  carries electrons  polyisoprene tail  moves freely within membrane CoQ CoQH2 (reduced form)
  • For NADH, one of two entry points into the electron transport chain: -- So the oxidation of one NADH results in the reduction of one CoQ -- Another important function of the enzyme will be mentioned later.
  • Succinate Dehydrogenase- Complex II Succinate:CoQ oxidoreductase Similar reaction can be written yielding CoQH2 Second entry into electron transport Substrate is succinate Contains Iron Sulfur Center  FAD is reduced, not FMN CoQH2 carries electrons to cytochrome b
  • Cytochromes - proteins in ETS Carry electrons Contain heme or heme-like group  carries electrons only: Fe(III) + e-  Fe(II)
  • -- Cytochromes in respiration are on inner mitochondrial membrane  cytochromes b, c1, c, a, a3 , relay electrons,one at a time, in this order 
  •  COMPLEX III = b, an Fe-S and c1.  Cytochrome c is mobile.  COMPLEX IV = a+a3 = cytochrome a-a3 = cytochrome c oxidase -- large protein. -- both a and a3 contain heme A and Cu -- a3 Cu binds to oxygen and donates electrons to oxygen cytochrome a3 - only component of ETS that can interact with O2
  • Cytochrome c oxidase Heme A and Cu act together to transfer electrons to oxygen Cu(II)  Cu(I)e- from cyt c to a
  • Sequence of Respiratory Electron Carriers Inhibitors in green
  • How is amount ATP synthesized measured? Quantify P/O ratio Definition: # Pi taken up in phosphorylating ADP per atom oxygen (½O2), in other words per 2e-. NADH 3 FADH2 2
  • Experimental, we know As electrons are passed through: NADH oxidized by CoQ Cytochrome b oxidized by cytochrome c1 Cytochrome a oxidized by O2 Each yields enough energy to synthesis about one ATP So oxidation of NADH yields about 3 ATPs Oxidation of FADH2 gives only 2 ATPs (succinate dehydrogenase & others)
  • What about energy and ATP stoichiometry? -- measured -- 220 kJ/mole from NADH oxidation -- Each ATP produced: ADP + Pi  ATP G = +30.5 kJ/mole [3 (30.5)/220] 100 = 41% efficiency
  • Oxidative Phosphorylation -- (ox-phos) Definition: Production of ATP using transfer of electrons for energy = coupled --for NADH, we know cyt b O2 NADHFMN-FeS  CoQ  FeScyt c  cyt aa3  cyt c1  ATP ATP ATP Complex I Complex III Complex IV Note: Several small energy steps
  • What are the requirements for coupling? -- Lehninger in the 50's and 60's  Intact mitochondria = intact inner membrane, respiratory chain  Pi  ADP  NADH or other reductant no other metabolites needed!
  • Acceptor Control Suspend intact mitochondria with NADH and Pi Add ADP add ADP O2 taken up time add ADP Requires ADP for oxygen uptake = coupling
  • How is this coupling accomplished? -- It was originally thought that ATP generation was somehow directly done at Complexes I, III and IV. -- We now know that the coupling is indirect in that a proton gradient is generated across the inner mitochondrial membrane which drives ATP synthesis.
  • ATP Synthetic Machinery = FoF1 ATP synthase Complex -- in inner mitochondrial membrane Matrix
  • -- knob-like projections on the matrix side called F1 spheres. -- responsible for ATP production since when removed by trypsin treatment, the resulting membranes still transport electrons but do not make ATP.
  • FoF1 ATP synthase -- ATP synthesized on matrix side. -- electron transport complexes and FoF1 ATP synthase arranged on the inner membrane of the mitochondrion facing in and lining the membranes bordering the cristae. *********************************************************
  • Chemiosmotic Theory --Peter Mitchell -- A proton gradient is generated using energy from electron transport. --The vectorial transport of protons (proton pumping) is done by Complexes I, III, IV from the matrix to intermembrane space of the mitochondrion.
  • -- The protons have a thermodynamic tendency to return to the matrix = Proton-motive force The proton move back into the matrix through the FoF1ATP synthase driving ATP synthesis.
  • The proton pumps are Complexes I, III and IV. Protons return thru ATP synthase
  • The return of protons “downhill” through Fo rotates Fo relative to F1, driving ATP synthesis. Note: Subunit  rotates through F1.
  • ATP synthesis at F1 results from repetitive comformational changes as  rotates  rotates 1/3 turn- energy for ATP release animation
  • Experimental corroboration  Uncoupling. The compound 2,4 dinitrophenol (DNP) allows proton through the membrane and uncouples. Blocking. The antibiotic oligomycin blocks the flow of H+ through the Fo, directly inhibiting ox-phos.
  • Respiratory Control -- Most mitochondria are said to be tightly coupled. That is there is no electron flow without phosphorylation and no phosphorylation without electron flow. -- Reduced substrate, ADP, Pi and O2 are all necessary for oxidative phosphorylation.
  • For example, in the absence of ADP or O2 electron flow stops, reduced substrate is not consumed and no ATP is made = acceptor control. Under certain conditions, coupling can be lost. -- A toxic, nonphysiological uncoupler, DNP, was described previously.
  • -- Brown adipose (fat) cells contain natural uncouplers to warm animals - cold adaptation and hibernation.
  • Shuttling Reducing Equivalents from Cytosolic NADH -- Electrons from NADH are shuttled across the mitochondrial membrane by carriers since NADH cannot cross inner membrane. -- reoxidation of cytosolic NADH leads to different energy yields depending on mechanism the cell uses to shuttle the reducing equivalents.
  • -- The dihydoxyacetone phosphate shuttle yields 2 ATP/NADH -- The malate shuttle yields 3 ATP/NADH
  • cytosolic malate dehydrogenase mitochondrial malate dehydrogenase
  • Review of the Energy Yield from Glycolysis, Pyruvate Dehydrogenase and the TCA Cycle Glycolysis: glucose 2pyruvate + 2NADH+2ATP 6-8 ATPs Pyruvate Dehydrogenase: pyruvate  acetyl CoA + NADH 6 ATPs TCA cycle: acetyl CoA 2CO2+3NADH+FADH2+GTP 2x12ATPs OVERALL yield from glucose 36-38 ATPs