21a-Biochemistry-Lecture21a.pdf

ENERGY FROM REDUCED FUELS IS USED TO SYNTHESIZE ATP IN ANIMALS BY OXIDATIVE PHOSPHORYLATION Carbohydrates, lipids, and amino acids are the main reduced fuels for the cell (that undergo oxidation) Electrons from reduced fuels are transferred to oxidized cofactors NAD+ or FAD (that become reduced). El...

ENERGY FROM REDUCED FUELS IS USED TO SYNTHESIZE ATP IN ANIMALS BY OXIDATIVE PHOSPHORYLATION Carbohydrates, lipids, and amino acids are the main reduced fuels for the cell (that undergo oxidation) Electrons from reduced fuels are transferred to oxidized cofactors NAD+ or FAD (that become reduced). Electrons from NADH and FADH2 are passed to proteins in the respiratory chain. The ultimate electron acceptor for these electrons is oxygen in eukaryotes. The energy from NADH and FADH2 is used to phosphorylate ADP and make ATP (oxidative phosphorylation). ENERGY FLOW IN CELLULAR RESPIRATION Catabolism of proteins, fats, and carbohydrates in the three stages of cellular respiration. - Stage 1: oxidation of fatty acids, glucose, and some amino acids yields acetyl-CoA. - Stage 2: oxidation of acetyl groups in the citric acid cycle includes four steps in which electrons are abstracted. - Stage 3: electrons carried by NADH and FADH2 are funneled into a chain of mitochondrial (or, in bacteria, plasma membrane–bound) electron carriers—the respiratory chain—ultimately reducing O2 to H2O. This electron flow drives the production of ATP. STAGE 3 OF CELLULAR RESPIRATION: ELECTRON TRANSFER AND OXIDATIVE PHOSPHORYLATION In the third stage (stage 1 is acetyl-CoA production, stage 2 is acetyl-CoA oxidation), the reduced coenzymes (NADH and FADH2) are reoxidized giving up protons and electrons. The latter are transferred to O2, the final electron acceptor, via a chain of electron carrying molecules (the respiratory chain). In the course of these transfers, a large amount of energy is captured in the form of ATP, a process known as oxidative phosphorylation. Electrons carried by NADH and FADH2 are funneled into a chain of mitochondrial (or, in bacteria, plasma membrane-bound) electron carriers—the respiratory chain—ultimately reducing O2 to H2O. This electron flow drives the production of ATP. Basic principles: 1. Flow of electrons through membrane bound carriers 2. Coupling of exergonic electron flow to proton transport across membrane 3. Transmembrane flow of protons down the concentration gradient through ATP synthase to form ATP CHEMIOSMOTIC THEORY Ø ATP formation from ADP and Pi (ADP + Pi à ATP) is highly thermodynamically unfavorable How do we make it possible? 1/ The energy released by electron transport is used to transport protons against the electrochemical gradient of the mitochondrial membrane 2/ The flow of protons down the electrochemical gradient provides the energy needed to phosphorylate ADP In this case, phosphorylation of ADP is not a result of a direct reaction between ADP and some high-energy phosphate carrier (like in substrate level phosphorylation) CHEMIOSMOTIC THEORY Chemiosmotic theory, (Peter Mitchel,1961): “The electrochemical energy due to proton concentration differences between inter membrane space and mitochondrial matrix acts as proton-motive force to drive protons through proton channel with ATP synthase activity”. § The energy yielding “downhill” flow of electrons through a number of electron carriers is coupled to the “uphill” transport of protons out of the matrix, across a protonimpermeable membrane (inner membrane), conserving the free energy of fuel oxidation as a transmembrane electrochemical potential. § The transmembrane flow of protons down their concentration gradient, back into the matrix, through a transmembrane complex (ATP synthase) provides the free energy for the synthesis of ATP (coupling of proton flow to the synthesis of ATP). CHEMIOSMOTIC ENERGY COUPLING REQUIRES MEMBRANES The proton gradient needed for ATP synthesis can be stably established across a membrane that is impermeable to ions – Plasma membrane in bacteria – Inner membrane in mitochondria Membrane must contain proteins that couple the “downhill” flow of electrons in the electron-transfer chain with the “uphill” flow of protons across the membrane Membrane must contain a protein that couples the “downhill” flow of protons to the phosphorylation of ADP STRUCTURE OF A MITOCHONDRION Double membrane leads to four distinct compartments: 1.Outer Membrane (OM): – Relatively porous membrane allows passage of metabolite and ions (up to about 5000 molecular weight) through membrane channels called, porins). 2.Inter Membrane Space (IMS): – similar environment to cytosol – higher proton concentration (lower pH) 3.Inner Membrane (IM): – Relatively impermeable, with proton gradient across it (the only molecules that can cross this membrane are those for which there are specific transporters). – Location of electron transport chain complexes – Convolutions called Cristae serve to increase the surface area (the inner membrane of a single liver mitochondrion may have more than 10,000 sets of electron-transfer systems (respiratory chains) and ATP synthase molecules, distributed over the membrane surface) 4.Matrix (M): – Location of the citric acid cycle and parts of lipid and amino acid metabolism – Lower proton concentration (higher pH) THE CHEMIOSMOTIC MECHANISM FOR ATP SYNTHESIS In mitochondria: u electrons move through a chain of membrane-bound electron carriers (the respiratory chain) spontaneously, driven by the high reduction potential of oxygen and the relatively low reduction potentials of the various reduced substrates (fuels) that undergo oxidation in the mitochondrion. u Electron carriers pump protons out of the matrix, thus creating an electrochemical potential that stores the energy u This energy drives ATP synthesis by ATP synthase THE ELECTRON TRANSPORT CHAIN (ETC) IN MITOCHONDRIA ELECTRON-TRANSPORT CHAIN PROTEIN COMPLEXES CONTAIN A SERIES OF ELECTRON CARRIERS § In addition to NAD and FAD coenzymes (see previous lectures), three electron carriers function in the electron transport chain: - Ubiquinone or coenzyme Q or Q - Cytochromes - Iron-sulfur cluster § Each protein complex* contains multiple redox centers consisting of: - Flavin Mononucleotide (FMN) or Flavin Adenine Dinucleotide (FAD) that serves as the initial electron acceptors (for Complex I and Complex II) and can carry two electrons by transferring one at a time - Cytochromes a, b or c - Iron-sulfur cluster * most of the protein complexes are integral membrane proteins with prosthetic groups capable of accepting and donating one or two electrons. UBIQUINONE OR COENZYME Q, OR Q § Ubiquinone or Coenzyme Q is a small conjugated dicarbonyl compound, soluble in the lipid layer and can therefore diffuse readily within the inner layer of the membrane. § Can accepts either one or two electrons and can therefore act as a junction between a oneelectron and a two-electron donor. Complete reduction of ubiquinone requires two electrons and two protons, and occurs in two steps through the semiquinone radical intermediate. § Upon accepting two electrons, it picks up two protons to give an alcohol, ubiquinol. Ubiquinol can freely diffuse in the membrane, carrying electrons with protons from one side of the membrane to another side. § Thus, Q couples electron flow to protons movements § Q is a mobile electron carrier transporting electrons from Complexes I and II to Complex III CYTOCHROMES Cytochromes a and b and some of c are integral membrane proteins containing Hemes. Prosthetic groups of Cytochromes are iron coordinating porphyrin ring derivatives (a, b or c differ by ring addition and can be distinguished by their absorption bands in the visible range). Each cytochrome has, when the iron is in the reduced Fe2+ state, three absorption bands. Prosthetic groups of cytochromes. (a) Each group consists of four five-membered, nitrogen-containing rings in a cyclic structure called a porphyrin. The four nitrogen atoms are coordinated with a central Fe ion, either Fe2+ or Fe3+. Iron protoporphyrin IX is found in b-type cytochromes and in hemoglobin and myoglobin. Heme c is covalently bound to the protein of cytochrome c through thioether bonds to two Cys residues. Heme a, found in a-type cytochromes, has a long isoprenoid tail attached to one of the five-membered rings. The conjugated doublebond system (shaded light red) of the porphyrin ring has delocalized π electrons that are relatively easily excited by photons with the wavelengths of visible light, which accounts for the strong absorption by hemes (and related compounds) in the visible region of the spectrum. Cytochrome c of mitochondria is a soluble protein associated with the outer surface of the inner membrane through electrostatic forces. It is a one electron carrier (transports one electron at a time) It is a mobile carrier for a single electron from the cytochrome bc1 (complex III) to cytochrome oxidase (complex IV). IRON-SULFUR CLUSTERS One electron carriers Coordination of iron and sulfur by cysteines in the protein - There are at least eight iron-sulfur proteins active in the electron transport chain (Found in Complex I, II and III). Iron-sulfur proteins contain iron coordinated to an inorganic sulfur atom or to a sulfur atom from a side chain of cysteine. These iron-sulfur centers range from simple structures where a single iron is coordinated to four cys-SH groups (a) to more complex iron-sulfur centers with two (b) or four irons (c) coordinated to sulfur. - The reduction potential of these proteins varies from -0.65 V to + 0.45 V depending on the microenvironment within the protein. STANDARD REDUCTION POTENTIAL OF ELECTRON CARRIERS OF THE RESPITORY CHAIN Fumarate + 2H+ + 2e– à succinate 0.031 The sequence of electron carriers in the chain follows the order of increasing reduction potentials, and this can be measured experimentally (see next slide). FREE ENERGY OF ELECTRON TRANSPORT Electrons are transferred from lower (more negative) to higher (more positive) reduction potential (E). The Reduction Potential difference ∆E for two redox couples is: ∆Eo′ = Eo′(e- acceptor) – Eo′(e- donor) u And the free energy difference is: ∆Go′ = –nF∆Eo′ For a negative DG, a positive DE is needed Thus, E(acceptor) > E(donor) u Free Energy released is used to pump proton, storing this energy as the electrochemical gradient Electron transfer from NADH to O2 Sum DG’0 = ~220 KJ/mol Electron transfer from Succinate to O2 Sum DG’0 = ~150 KJ/mol OVERVIEW OF THE FLOW OF ELECTRONS AND PROTONS IN THE THE RESPITORY CHAIN Glycerol-3-P (cytosolic) Fatty-acyl-CoA Complex I and II catalyze electron transfer to ubiquinone from the two main electron sources arising from the operation of the citric acid cycle; NADH (from three separate oxidation reactions (isocitrate, α-ketoglutarate and malate) and FADH2 arising from the oxidation of succinate Complex III carries electrons from reduced ubiquinone to cytochrome c. Complex IV completes the sequence by transferring electron to O2. PATH OF ELECTRONS FROM NADH, SUCCINATE, FATTY ACYL–COA, AND GLYCEROL 3-PHOSPHATE TO UBIQUINONE. NADH from glycolysis (mitochondrial) Cytosolic G3PDH Ubiquinone (Q) is the point of entry for electrons derived from reactions in the cytosol, from fatty acid oxidation, and from succinate oxidation (in the citric acid cycle). Electrons from NADH pass through a flavoprotein with the cofactor FMN to a series of Fe-S centers (in Complex I) and then to Q. Electrons from succinate pass through a flavoprotein with the cofactor FAD and several Fe-S centers (in Complex II) on the way to Q. Glycerol 3-phosphate donates electrons to a flavoprotein (glycerol 3-phosphate dehydrogenase) on the outer face of the inner mitochondrial membrane, from which they pass to Q. Acyl-CoA dehydrogenase (the first enzyme of β oxidation) transfers electrons to electron-transferring flavoprotein (ETF), from which they pass to Q via ETF: ubiquinone oxidoreductase. COMPLEX I: NADH:UBIQUINONE OXIDOREDUCTASE One of the largest macro-molecular assemblies in the mammalian cell Over 40 different polypeptide chains, encoded by both nuclear and mitochondrial genes NADH binding site in the matrix side Noncovalently bound flavin mononucleotide (FMN), accepts two electrons from NADH These two electrons pass through a series of Fe-S centers (one electron at a time), then to ubiquinone Q to form QH2, which diffuses into the lipid bilayer. This electron transfer also drives the expulsion from the matrix of four protons per pair of electrons. COMPLEX II: SUCCINATE DEHYDROGENASE In the conversion of succinate to fumarate, FAD accepts two electrons from succinate* Electrons are passed, one at a time, via iron-sulfur centers to ubiquinone, which becomes reduced QH2 Complex II does NOT transport protons * Note: This reaction is step 6 of the CAC (see the CAC cycle) COMPLEX III: UBIQUINONE:CYTOCHROME C OXIDOREDUCTASE Uses two electrons from QH2 to reduce two molecules of cytochrome c Additionally contains iron-sulfur clusters, cytochrome bs, and cytochrome cs Four protons are transported (through the Q cycle) to the Inter Membrane Space: CYTOCHROME C The second mobile electron carrier (the first one being Q) A soluble heme-containing protein in the Inter Membrane Space (IMS) Heme iron can be either ferrous (Fe3+, oxidized) or ferric (Fe2+, reduced) Cytochrome c carries a single electron from the cytochrome bc1 (complex III) to cytochrome oxidase (complex IV). Thus, two Cyt c are needed to carry 2 electrons. COMPLEX IV (CYTOCHROME OXIDASE) PASSES ELECTRONS TO O2 Two electrons are used to reduce one half oxygen molecule into one water molecule (or four electrons reduce one oxygen into two water molecules) two protons are picked up from the matrix (as substrate) per two electrons (or four protons per four electrons) and are consumed in this process Two protons (per two electrons) are passed from the matrix to the inter-membrane space (or four protons per four electrons) Reduction of O2 to H2O requires 4 electrons (or 2 pairs) SUMMARY OF THE ELECTRON FLOW AND PROTON MOVEMENTS IN THE RESPIRATORY CHAIN - Two electrons reach Q through Complexes I or II. Four protons are pumped through complex I but no protons are pumped from complex II. The reduced Q (QH2) serves as a mobile carrier of two electrons and passes them to Complex III, which in turn passes them to another mobile mobile carrier, cytochrome c (two cytochromes per two electrons). Four protons are transferred to the IMS in the process. Complex IV then transfers the two electrons from reduced cytochrome c to half O2, transferring two protons in the process. Thus, a pair of electron flow through Complexes I, III, and IV is accompanied by a flow of 10 protons from the matrix to the intermembrane space, while a pair of electron flow through Complexes II, III, and IV (bypassing complex I in this case) is accompanied by a flow of only 6 protons from the matrix to the intermembrane space only. THE TRANSFER OF ELECTRON THROUGH THE ETC IS HIGHLY EXERGONIC Transfer of electrons from NADH to O2 : 2 NADH + 2 H+ à 2 NAD+ 2 H20 The ΔE’o for the redox pair NAD+/NADH is -0.320 V and for the redox pair O2/H2O is 0.816 V. The ΔE’o (∆Eo′ = Eo′(e- acceptor) – Eo′(e- donor)) for the combined reaction is therefore 1.14 V, and the free-energy change for the reaction is given by ΔG’o = - nFΔE’o = - 2(96.5 kJ/Vxmol)(1.14 V) = - 220 kJ/mol of NADH Transfer of electrons from succinate to O2: (gives a smaller, but still substantial, negative value of ΔG’o of – 151 kJ/mol) The ΔE’o for the redox pair Fumarate/Suucinate = 0.031 V and for the redox pair O2/H2O = 0.816 V. The ΔE’o (∆Eo′ = Eo′(e- acceptor) – Eo′(e- donor)) for the combined reaction is therefore 0.785.V, and the free-energy change for the reaction is given by: ΔG’o = - nFΔE’o = - 2(96.5 kJ/Vxmol)(0.785 V) = - 151.5 kJ/mol of succinate) In respiring mitochondria, the ratio of NADH/NAD+ is well above unity, which implies that the actual-free energy change is even more negative than that calculated from the ΔE’o value. ENERGY OF ELECTRON TRANSFER IS USED TO PUMP PROTONS FROM THE MATRIX INTO THE INTER-MEMBRANE SPACE. u For each electron pair transferred from NADH to O2, four protons are pumped out by Complex I, four by Complex 3, and 2 by Complex IV (10 in total). Complex I à Complex IV 1NADH + 11H+(N) + ½O2 ——> NAD+ + 10H+(P) + H2O u For each electron pair transferred from FADH2 to O2, four protons are pumped out by Complex 3, and 2 by Complex IV (6 in total). Complex II à Complex IV FADH2 + 6H+(N) + ½O2 ——> FAD + 6H+(P) + H2O The difference in number of protons transported by NAD and FAD reflects differences in ATP synthesized. REACTIVE OXYGEN SPECIES CAN DAMAGE BIOLOGICAL MACROMOLECULES : ROLE OF ENZYMES, NADPH AND GLUTATHIONE IN PROTECTION - When the rate of electron entry into the respiratory chain and the rate of electron transfer through the chain are mismatched, superoxide radical ( O2-) production increases at Complexes I and III as the partially reduced ubiquinone radical ( Q-) donates an electron to O2. - Superoxide acts on aconitase, a 4Fe-4S protein, to release Fe2+. In the presence of Fe2+, the Fenton reaction leads to formation of the highly reactive hydroxyl free radical ( OH). - The reactions shown in blue defend the cell against the damaging effects of superoxide. Reduced glutathione (GSH) donates electrons for the reduction of H2O2 and of the oxidized Cys residues (—S—S—) of enzymes and other proteins, and GSH is regenerated from the oxidized form (GSSG) by reduction with NADPH.