Respiratory Chain & Oxidative Phosphorylation PDF

 Harper’s Illustrated Biochemistry  Lippincotts  Explain how aerobic organisms capture the available free energy from substrates metabolism.  Occur in mitochondria.  Respiration is coupled to oxidative phosphorylation ▪ Generate ATP  Gib...

 Harper’s Illustrated Biochemistry  Lippincotts  Explain how aerobic organisms capture the available free energy from substrates metabolism.  Occur in mitochondria.  Respiration is coupled to oxidative phosphorylation ▪ Generate ATP  Gibbs free energy  Reactions with large G are made possible by coupling endergonic process with exergonic process. absorbs energy from surround release energy  Eg: endergonic movement of ions against concentration gradient across membrane. ▪ Coupled with exergonic hydrolysis of ATP  Outer membrane – permeable to most metabolites  Inner membrane - selectively permeable  A matrix within.  Inner mitochondrial membrane.  Electrons are passed down.  Lose much of free energy.  Some are captured-stored by ATP production.  energy released  Energy liberated during the oxidation of carbohydrate, fatty acids, and amino acids ▪ as reducing equivalents (H or electrons).  Reducing equivalents, produce most by ▪ β-oxidation ▪ citric acid cycle  Mitochondria contain the respiratory chain ▪ collects and transports reducing equivalents ▪ directing them to their final reaction with oxygen to form water ▪ the machinery for trapping the liberated free energy as high-energy phosphate  The reduced NADH oxidized by a metalloflavoprotein enzyme—NADH dehydrogenase.  Passes reducing equivalents on to Q.  Electrons flow from Q through the series of cytochromes to molecular oxygen.  The terminal cytochrome aa3 (cytochrome oxidase) ▪ the final combination of reducing equivalents with molecular oxygen ▪ has a very high affinity for oxygen ▪ allowing the respiratory chain to function at maximum rate until the tissue has become depleted of O2.  Protein complexes ▪ I, II, III, IV and V. ▪ Carriers are integral protein ▪ Prostetic groups that accept/donate electron.  Complex I-IV : contain part of ETC. ▪ Each complex accepts/donates electron to mobile electron carriers. ▪ Coenzyme Q, cytochrome c ▪ Electron eventually combine with oxygen & protons – water  Complex V ▪ Spans the inner mitochondria membrane. ▪ Catalyzes ATP – ATP synthase.  The iron-sulfur protein (FeS; nonheme iron).  Associated with flavoproteins, cytochrome b.  Are thought to take part in the oxidoreduction mechanism between flavin and Q ▪ involves only a single e− change ▪ the iron atom undergoing oxidoreduction between Fe2+ and Fe3+.  Flow of electrons spontaneous ▪ next carrier has greater affinity for electrons compared to previous.  In addition to NADH, FADH2, types of redox centers in the complexes ▪ FMN (flavin mononucleotide) ▪ Fe-S (iron-sulfur clusters) ▪ CoQ (ubiquinone) ▪ Cytochromes  NADH dehydrogenase/NADH:ubiquinone oxidoreductase.  Embedded in the inner membrane of mitochondria.  Has FMN (flavin mononucleotide) molecule ▪ coenzyme structurally related to FAD. ▪ accept the two H (:H-, free H+) ▪ FMNH2  Electron transfers to Fe-S cluster → ubiquinone (CoQ). ▪ form ubiquinol, QH2 (freely diffuses within the membrane).  4 H+ translocated from the mitochondrial matrix to intermembrane space.  Mobile carriers.  Quinone derivative.  The structure very similar to vitamin K & E, plastoquinone (found in chloroplasts).  Oxidized-quinone, reduced-quinol, under aerobic or anaerobic conditions, respectively.  Collects reducing equivalents from fixed flavoprotein complexes ▪ passes them on to the cytochromes.  2 forms exist ▪ Succinate dehydrogenase (TCA) ▪ Acyl-CoA dehydrogenase (-oxidation).  Associated with FAD - flavoprotein.  Has Fe-S clusters as prostetic groups.  Direct the transfer of electrons from their substrate molecule (succinate, acyl CoA) to CoQ via FADH2.  The remaining members of ETC.  Contain heme group (a porphyrin ring & iron).  Iron: reversibly converted from ferric (Fe3+) to ferrous (Fe2+) form –reversible carrier of electron.  Electrons → CoQ → cytochromes bc1 (complex III) → c → a + a3 (complex IV).  Cytochrome c ▪ Mobile carrier with the intermembrane space.  Cytochrome bc1 complex.  Electrons transfer from ubiquinol.  Fe-S clusters.  Cytochromes ▪ Electron-transfering proteins. ▪ Contains heme prosthetic group. ▪ Accept & donate ONE electron per redox cycle.  Electrons transfer translocate more H+ (4).  Cytochrome a + a3.  Combined - cytochrome c oxidase.  4 electrons from 4 molecules of cytochrome c transferred to O2 - two molecules of water. O2 + 4e- + 4H+ → 2H2O  2 protons are translocated across the membrane.  Reduced products must be recycled: ▪ Oxidation of NADH & FADH2 by electron acceptor, O2. ADP + Pi ATP  NADH + H+ ½ O2 → NAD+ + H2O ADP + Pi ATP  FADH2 + ½ O2 → FAD + H2O  Oxidized coenzymes reformed.  Energy released is coupled to ATP synthesis.  Complex I (NADH dehydrogenase)  Complex II (succinate dehydrogenase)  Complex III (cytochrome bc1 complex)  Complex IV (cytochrome c oxidase)  Reactions by dehydrogenase (eg: TCA cycle)  Remove two H from substrate.  Transferred two electrons & one proton (hydride ions, :H-) to NAD+.  Form NADH + H+.  NADH carries ▪ :H-, free H+  Pyruvate and α-ketoglutarate dehydrogenase have complex systems involving lipoate and FAD prior to the passage of electrons to NAD.  Electron transfers from other dehydrogenases, eg, L(+)-3- hydroxyacyl- CoA dehydrogenase, couple directly with NAD.  Preparatory step.  Mediated by Substrate Shuttles.  NADH cannot penetrate the mitochondrial membrane, but it is produced continuously in the cytosol (glycolysis).  Under aerobic conditions - extramitochondrial NADH does not accumulate ▪ presumed to be oxidized by the respiratory chain.  The transfer of reducing equivalents through the mitochondrial membrane requires substrate pairs ▪ linked by suitable dehydrogenases on each side of the mitochondrial membrane.  The mechanism of transfer using the glycerophosphate shuttle.  Present in some tissues (eg, brain, white muscle), in others (eg, heart muscle) it is deficient.  Combination of 2 activities: ▪ The flow of electrons to O2 via ETC ▪ NADH + H+ + ½ O2 → H2O + NAD+ ▪ Down hill flow of electrons release large amount of E. ▪ Drive another process. ▪ Phosphorylation of ADP by Pi synthesizing ATP ▪ ADP + Pi  ATP + H2O ▪ Catalyzed by ATP synthase.  Mitchell hypothesis ▪ ETC and oxidative phosphorylation are coupled by a proton gradient across the inner mitochondria membrane ▪ The proton motive force caused by the electrochemical potential difference (negative on the matrix side) drives the mechanism of ATP synthesis.  Each of the respiratory chain complexes I, III, and IV acts as a proton pump.  Inner mitochondria membrane is permeable to protons: ▪ Accumulate in intermembrane space: creates a PROTON GRADIENT. ▪ Electrochemical potential difference across the membrane.  Electrochemical potential ▪ Consists of: ▪ a chemical potential (difference in pH). ▪ an electrical potential.  Chemiosmotic hypothesis ▪ Protons re-enter matrix. ▪ Dissipating the pH & electrical gradients.  Once established, inhibits further transport of reducing equivalents through the respiratory chain.  Discharged by back translocation of protons through ATP synthase ▪ depends on availability of ADP and Pi.  Synthesizes ATP in the presence of Pi + ADP.  Consist of several protein subunits: ▪ F1 ▪ projects into the matrix ▪ contain the phosphorylation mechanism. ▪ three α- and three β-subunits ▪ F0 ▪ consists of several protein subunits ▪ spans the membrane ▪ forms the proton channel. ▪ γ-subunit. ▪ Protons flow through F0 –F1 ▪ Cause the F0 causes to rotate ▪ drive ATP production in the F1 complex ▪ ADP and Pi are taken up sequentially by the β-subunits to form ATP.  The respiratory chain provides most of the energy captured during catabolism  Approximately 68% of the free energy from the oxidation of foodstuffs captured.  The remaining free energy - liberated as heat. Availability of ADP  Oxidation and phosphorylation are tightly coupled ▪ oxidation cannot proceed via the respiratory chain without phosphorylation of ADP. ▪ Energy metabolism-maintain constant ATP level. ▪ Electrons flow down ETC to O2 in the presence of ADP for phosphorylation. ▪ [ADP] regulate the electrons flow. ▪ Level of ADP triggers increase in rate of catabolic reactions. Availability of ADP  When work is performed, ATP is converted to ADP, allowing more respiration to occur - replenishes the store of ATP.  Concentration of inorganic phosphate.  Rate of respiration increases ▪ If increase - as in exercise ▪ the capacity of the respiratory chain becomes saturated ▪ or the PO2 decreases below the Km for cytochrome a3.  ADP/ATP transporter ▪ facilitates entry of cytosolic ADP into and ATP out of the mitochondrion might also becomes ratelimiting. Transporter systems in the inner mitochondrial membrane. 1 , phosphate transporter; 2 , pyruvate symport; 3 , dicarboxylate transporter; 4 , tricarboxylate transporter; 5 , α-ketoglutarate transporter; 6 , adenine nucleotide transporter. Also present (but not shown) are transporter systems for glutamate/aspartate , glutamine, ornithine, neutral amino acids, and carnitine.  Uncoupling by ▪ uncoupling protein/thermogenin – animals ▪ uncoupling agent-2,4-dinitrophenol (dieting aid).  Provides a channel to return pumped protons from intermembrane space to matrix.  Proton flow bypass ATP synthase ▪ Energy release as heat instead of ATP.  Site specific inhibitors of electron transport.