Exam 3 Review Metabolism and Energy Production

CH 462 2022 Exam 3 Review Metabolism and Energy Production Review these enough that you know context for Electron Transport Chain A. Glycolysis (V&V Chapter 17) B. Fates of pyruvate (V&V Sections 17-3 and 21-2) C. Citric Acid Cycle (V&V Chapter 21) 1 New material: Electron Transport Chain/Oxidative Phosphorylation (V&V Chapter 22) D. Respiratory electron transport realizes most of the energy from glucose These are significant facts, but will be mainly needed for context: 1. Glycolysis and the TCA yield very little ATP (4 per glucose) 2. Glycolysis/TCA produce a lot of reduced cofactors (10 NADH and 2 FADH2) a) Energy from oxidation of these provides ~2500 kJ/mol b) C6H12O6 + 6 O2 → 6 CO2 + 6 H2O ∆Go = -2870 kJ/mol 3. Purpose of mitochondrial e– transport a) Oxidize NADH and FADH2 b) O2 is ultimate e– acceptor c) Free energy used to establish H+ gradient formed across inner mitochondrial membrane d) ATP synthesis is coupled to release of H+ gradient 4. Mitochondria a) Bacterium-sized organelle b) Contains own genome, but many genes have relocated in nucleus c) Outer membrane (highly permeable) d) Inner membrane (highly impermeable, even to small, charged species like H+) e) Surface area of inner membrane increased by folding (cristae) f) Inner membrane encloses matrix 5. Cofactors You need to know how each of these cofactors participates in electron transport (1 or 2 electron carrier, proton carrier, etc). You also need to know the basic structures of these. a) Redox Cofactors i) Nicotinamide (2 e- carrier) NAD+ + 2 e- + H+ → NADH ii) Flavin (1 or 2 e- carrier) FAD + e- + H+ → FADH + e- + H+ → FADH2 b) Ubiquinone (Q) (lipid soluble, 1 or 2 e- carrier) i) Benzoquinone nucleus 2 ii) a. 1 e– reduction yields semiquinone radical (•QH) b. 2nd e– reduction yields quinol (QH2) c. can accept 1 or 2 e– at a time isoprenoid tail is hydrophobic, keeping Q in lipid bilayer c) Fe porphyrins (1 e- carriers) i) a-type cytochromes contain Heme A ii) b-type cytochromes contain Fe protoporphyrin IX iii) c-type cytochromes contain covalently linked Heme C d) Fe-S clusters (1 e- carriers) i) Fe-S cluster (Fe coordinated by four Cys sulfurs ii) Fe2-S2 cluster (planar Fe2S2 assembly coordinated by four Cys sulfurs) iii) Rieske cluster (Fe2-S2 assembly coordinated by 2 Cys and 2 His) iv) Fe4-S4 cluster (cubic Fe4S4 assembly coordinated by four Cys sulfurs) v) Redox potential varies greatly depending upon environment 6. Redox potentials and e– transfer You should recognize that the electrons are moving downhill in energy, but you do not need to know specific potentials. a) Reaction is favorable for transfer of e– from donors with low potential (NADH, E° = -320 mV) to acceptors of high potential (O2, E° = +820 mV) b) Respiratory chain involves series of e– transfers 7. Four protein complexes convert energy of reduced compounds to H+ gradient For each of these complexes, you need to know function and a general outline of the process. You should also appreciate important structural features. a) Complex I (NADH dehydrogenase) i) Transfers 2 e– from NADH to ubiquinone (NADH + Q + H+ → NAD+ + QH2) ii) Four protons are concomitantly pumped from matrix to intermembrane space Conformational changes from reduced (but unprotonated) Q2drive proton pumping—know how this works b) Complex II (succinate dehydrogenase) 3 i) ii) iii) Succinate from TCA is oxidized by FAD cofactor which transfers e– for Q reduction succinate + Q → fumarate + QH2 no protons are pumped (other than 2 Hs carried by QH2) why not? c) Complex III (cytochrome c/ubiquinone oxidoreductase) i) QH2 (#1) transfers 2 e– to complex and releases 2 H+ to intermembrane space a. Rieske iron-sulfur center (Fe2S2 cluster) accepts 1 e– which is then transferred to Cyt c b. Heme bL accepts other e– ii) Q cycle Know how Q-cycle works! a. e– is transferred from heme bL to Q (via heme bH) Q + H+ + e– → •QH b. QH2 (#2) transfers electrons to cyt c and Heme bL and 2H+ to intermembrane space as above c. e– is transferred from heme bL to •QH (via heme bH) • QH + H+ + e– → QH2 d. QH2 repeats reactions with Complex III e. Net reaction is: QH2 + 2cyt cox + 2H+(matrix)→Q + 2cyt cred + 4H+(intermembrane space) d) Complex IV (cytochrome oxidase) i) 4 cyt cred transfer 4 e– to O2 a. 2 cyt cred sequentially donate 2 e– to CuA center b. e–s are transferred from CuA center to Fe-Cu center (Cu + heme a) c. O2 binds heme a and is reduced to O22– (peroxy anion) d. Transfer of 2 e– is repeated to reduce O22– to 2 O2– (oxide) e. Protonation of oxides (via proton channels) to produce H2O removes total of 4 H+ from matrix side f. An additional 4 H+ are concomitantly pumped to intermembrane space via proton channels g. Note graphics in text vary as to whether they refer to the reduction of ½ O2 (2 e–) or a whole O2 (4 e–) e) Net for each NADH (2e–) is 10 H+ pumped 4 8. Mitochondrial metabolism produces ROS a) ROS (reactive oxygen species include superoxide (•O2–), hydrogen peroxide (H2O2), and hydroxyl radical (•OH). b) These species can damage biomolecules (DNA, proteins, lipids, etc) c) Numerous systems are present to safely eliminate these species before damage can occur (catalylase, superoxide dismutase, etc) 9. ATP synthase You need to understand process by which the proton gradient is translated into chemical energy (ATP). a) Excess protons make the intermembrane space more acidic while matrix becomes more alkaline b) ATP synthase uses transport of protons from high [H+] → low [H+] to drive phosphorylation of ADP c) F0 domain i) H+ permeable transmembrane rotor (c10 subunit) ii) Other subunits form stator iii) Rotation is coupled to rotation of central γ-subunit of F1 a. Asp-61 near center of transmembrane c unit accepts proton from intermembrane space b. c10 complex rotates protonated Asp away from channel and Arg sidechain in a-subunit c. Proton released through channel into matrix d. Electrostatic attraction between deprotonated Asp and Arg in a-subunit helps turn complex e. Conformational change in c protein α-helix also promotes turning (pushing off of a-subunit) d) F1 domain i) Contains active site for condensation of ADP and Pi a. Active site (stabilizes ADP binding b. Mg2+ and movement of Ala activates loss of H2O from Pi c. Unfavorable reaction is driven by tighter binding of F1 to ATP product than to ADP d. Further conformational change ejects ATP from active site ii) F1 structure a. Heterohexamer (α3β3) surrounding γ-subunit (axle) 5 b. δ- and ε-subunits contact membrane and α3β3 assembly c. the γ-subunit can turn but α3β3δε assembly cannot iii) Each of three β-subunits has different structure due to interactions with asymmetric γ-subunit a. One β-site binds ATP b. One β-site binds ADP + Pi c. One β-site is empty e) ATP synthesis/rotational catalysis i) Movement of 3 H+ from P-side to N-side rotates γ 120° a. Rotation causes ejection of ATP from its β-site b. Change in conformation of ADP site pushes condensation reaction (ADP + Pi → ATP + H2O) c. ADP binding to empty β-site ii) Repetition causes synthesis of another ATP at cost of 3 H+ f) “Uncoupling” relieves proton gradient without ATP synthesis i) Protein mediated (thermogenin) used to produce heat ii) Chemicals such as 2,4-dinitrophenol can perform same process E. Bacterial respiratory chain (aerobic bacteria) Be able to compare and contrast the bacterial and mitochondrial systems. 1. Bacteria have inner membrane similar to mitochondria 2. Bacterial periplasmic space is similar to mitochondrial intermembrane space of 3. A functional analog of Complex I/II a) Transfers e– from NADH, succinate, or glycerol to a quinone b) Bacteria use menaquinone (a naphthoquinone) 4. Analog of Complex IV a) Transfers e– from reduced menaquinone (QH2) to oxygen b) Proton gradient is created by pumping H+ to periplasmic space 5. Proton gradient can be used to synthesize ATP F. Bacteriorhodopsin 6 Background for proton pumping by conformational changes. a) Primitive system to capture light energy b) Found in archeabacteria such as halophiles c) Light-driven conformational change drives pumping of protons G. Photosynthesis (V&V Chapter 24 1. Reverse of glycolysis/TCA cycle/oxidative phosphorylation 2. 6 CO2 + 6 H2O → C6H12O6 + 6 O2 3. requires 686 kcal/mol of energy 4. photosynthetic cells use sunlight to provide energy (ATP and NADPH) for reactions H. Chloroplasts 1. Bacterium-sized organelle 2. Contains own genome, but many genes have relocated in nucleus 3. Outer membrane (highly permeable) 4. Inner membrane (highly impermeable, even to small, charged species like H+) 5. Inner membrane encloses third compartment (thylakoids) which is highly folded to increase surface area (encloses lumen) 6. Inner membrane encloses stroma I. Cofactors You need to know how each of these cofactors participates in photophosphorylation. You also need to know the basic structures of these, but will not be asked to draw them. 1. Chlorophylls a) Mg2+ tetrapyrroles with hydrophobic phytol side chains b) Chlorophyll a c) Chlorophyll b has some minor modifications of tetrapyrrole ring d) Bacteriochlorophyll a has other modifications including reduction of a C=C in tetrapyrrole e) Strongly absorb red and blue light (hence green color) f) Pheophytin similar to chlorophyll but without Mg2+ 7 2. Other pigments a) Phycobilins i) Ring-opened tetrapyrroles (no Mg2+) ii) Absorbs orange-to-green light b) Carotenoids i) Conjugated -system absorbs light in high efficiency ii) Includes -carotene and lutein J. Light Reactions 1. Involves use of photons (energy) to oxidize H2O to O2 2. Light-harvesting complex You need to know how light energy is “harvested” and funneled into the reaction center. a) Contains numerous chromophores (chlorophylls, luteins, etc) b) Serves as antenna (absorbs light) c) Energy (excitons) is transferred to reaction center by numerous energy transfers between chromophores 3. Reaction centers a) All contain a “special pair” of chlorophylls know definition i) Close arrangement of chlorophylls causes overlap of electronic states ii) Energy is transferred to RC chlorophyll by antenna complex iii) Excited state of chlorophylls can donate e– to acceptor (quinone, chlorophyll or pheophytin) iv) Transfer of e– from donor creates charge separation b) Bacteria reaction centers Simpler versions of process in chloroplasts… i) Type II RC (purple bacteria) a. RC uses P870 (special pair) chromophore b. Excited state (P870*) transfers e– to pheophytin c. e– is transferred from pheo to quinone A (QA) then to QB i. QA is tighlty bound by protein complex 8 ii. QB is mobile d. e is transferred to Cyt bc1 complex i. similar to mitochondrial complex III ii. transfers e– to mobile Cyt c2 and creates proton gradient iii. H+ gradient drive ATP synthase e. Cyt c2 donates e– back to P870 (P870•+) – ii) Type I RC (green sulfur bacteria) a. RC uses P840 (special pair) chromophore b. Excited state (P840*) can transfer e– to ferridoxin (Fd, an FeS protein) or to quinone i. e– transferred to Fd used for NADH synthesis 1. Fd-NAD oxidoreductase 2. Reduction of H2S can provide e– to reduce P840•+ ii. e– is transferred from reduced quinone (QH2) to Cyt bc1 complex 1. similar to mitochondrial complex III 2. transfers e– to mobile Cyt c553 and creates proton gradient 3. H+ gradient drive ATP synthase 4. Cyt c553 donates e– back to P840 (P840•+) 4. Oxygenic photosynthesis (noncyclic photophosphorylation) You will need a detailed knowledge of the Z-scheme for the light reactions of oxygenic photosynthesis. Focus on functions of PSII, cyt b6f and PSI a) Occurs in green plants and cyanobacteria b) Type I and II RCs are coupled c) Oxygen evolving center provides e– to re-reduce chromophores d) Photosystem II (PS II) i) oxygen evolving complex: produces electrons to reduce oxidized RC a. oxidizes water 2H2O → O2 + 4H+ + 4e– b. manganese cluster i. contains 4 Mn ions + Ca2+ (structure unknown) 9 ii) iii) ii. serves as charge accumulation site c. four e– are transferred sequentially to P680•+s with concomitant transfer of 4H+ to lumen side d. 4 e- from water oxidation reduces Mn4 cluster Analog of type II RC Light (excitons) excite special pair chromophore (P680) a. Excited state (P680*) transfers e– to pheophytin b. e– is transferred from pheophytin to plastoquinone A (PQA) then to PQB i. PQA is tightly bound by protein complex ii. PQB is mobile electron carrier e) Cyt b6f complex i) 2e– are transferred to Cyt b6f complex from reduced PQB a. similar to mitochondrial complex III b. creates proton gradient (Q cycle) + transfers e– to mobile plastocyanin i. H+ gradient drive ATP synthase ii. plastocyanin is 1e– carrier ii) plastocyanin donates e– to P700•+ from photosystem I f) Photosystem I (PS I) i) Analog of type I RC ii) RC uses P700 (special pair) chromophore a. Excited state (P700*) can transfer e– to the A0 chlorophyll b. A0 chlorophyll passes e– to A1 phylloquinone, which transfers e– through Fe-S clusters to ferridoxin (Fd, an FeS protein) c. e– transferred to Fd used for NADPH synthesis by Fd-NADP+ oxidoreductase 5. Cyclic Photophosphorylation This pathway is used to produce additional ATP without NADPH a) Uses PSI and Cyt b6f to produce proton gradient without oxidation of water or production of NADPH b) Reduced ferridoxin is shunted to transfer electrons to Cyt b6f Know how this is energetically possible. 10 11