Biochemistry 301 Week 8 Notes
Biochemistry 301 Week 8 Notes BBMB 301
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This 11 page Class Notes was uploaded by Emily on Sunday February 28, 2016. The Class Notes belongs to BBMB 301 at Iowa State University taught by Robert Thornburg in Spring 2016. Since its upload, it has received 14 views. For similar materials see Survey of Biochemistry in General Science at Iowa State University.
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Date Created: 02/28/16
Biochemistry 301 – Survey of Biochemistry Professor Robert Thornburg LECTURE 19 – CHAPTER 19 – CITRIC ACID CYCLE AND GLYOXYLATE CYCLE By: Emily Settle Central Metabolism o When O2 is available pyruvate from glycolysis can be further oxidized Far more ATP generated than occurs in glycolysis only Lots more NADH, FADH2 generated as carbon is oxidized NADH oxidized back to NAD+ as O2 is reduced to H2O o Acetyl-CoA – central metabolite for many pathways Enters citric acid cycle for complete oxidation to CO2 Pyruvate Oxidation o Stage 2 Pyruvate + CoASH acetyl-CoA One CO2 released Fully oxidized carbon One NADH produced Five coenzymes used Catalyzed by the pyruvate dehydrogenase complex o Stage 3 Citric acid cycle Acetyl-CoA 2CO2 + CoASH Three NADH produced One FADH produced One GTP produced 8 enzymes, cyclic reaction pathway used Citric Acid Cycle o Step 1 - addition Acetyl group condenses with oxaloacetate (OAA) Catalyzed by citrate synthase Hydrolysis of CoA thioester provides energy Lyase Reaction Addition across a double bond Formation of new C-C bond, ATP not involved Hydrolysis to release Coenzyme A o Citrate Synthase Mechanism Enzyme can hydrolyze thioester of citryl-CoA intermediate but not acetyl-CoA substrate Latter would be useless and wasteful Active site for hydrolysis of thioester does not form until after citryl-CoA intermediate present OAA must bind before acetyl-CoA binding site is created – induced fit Catalytic residues for thioester hydrolysis not in position until after lyase reaction is complete – after citryl-CoA has formed as the intermediate o Step 2 – rearrangement - Citrate to isocitrate Catalyzed by aconitase Two lyase reactions Elimination to form a double bond – dehydration Addition to a double bond – hydration Looks like an isomerization reaction but its NOT These two steps are both lyase reactions Rearrangement enables subsequent oxidation reactions o Step 3 - Isocitrate to α-ketoglutarate Catalyzed by isocitrate dehydrogenase Hydroxyl oxidized to ketone, forming a “β-keto acid” Decarboxylation Products are a α-ketoglutarate and CO2 Β ketone group necessary to stabilize transition state Oxidation-reduction reaction, then lyase reaction o Step 4 - α-ketoglutarate to succinyl-CoA Catalyzed by α-ketoglutarate dehydrogenase complex Same reaction as pyruvate dehydrogenase complex Different acyl group: succinyl- rather than acetyl- Lyase (bond cleavage), then oxidation-reduction o Step 5 – Succinyl-CoA to succinate Catalyzed by succinyl-CoA synthase Free energy released Thioester converted to carboxylic acid Captured in synthesis of GTP from GDP + Pi This is a substrate level phosphorylation Similar to glycolysis reactions, different than aerobic ATP production o Steps 6-8: regeneration of oxaloacetate to complete the cycle 6) Succinate to fumarate – 2e- transfer Catalyzed by succinate dehydrogenase (redox reaction) FAD reduced to FADH2, does not leave the enzyme 7) Fumarate to malate Catalyzed by fumarase (lyase reaction – addition to double bond) Hydration 8) Malate to oxaloacetate – 2e- transfer Catalyzed to malate dehydrogenase (redox reaction) NAD+ reduced to NADH Citric Acid Cycle is Multifunctional o Moves carbon atoms between biomolecule types Acetyl-CoA from lipid or amino acids enters gluconeogenesis Involves the glyoxylate cycle Glucose changed into amino acids, lipids, nucleotides, and organic cofactors Ex α-ketoglutarate glutamic acid o This the cycle participates both in catabolism and anabolism The term for such metabolic pathways is amphibolic Anaplerotic Reactions o Intermediates must be at constant concentrations Necessary for energy capture when required Intermediates drawn off to other pathways must be replenished Replenishing reactions are called anaplerotic o Ex pyruvate + CO2 OAA (step one of gluconeogenesis) Catalyzed by pyruvate carboxylase Carbon from glucose enters citric acid cycle without oxidation without passing through pyruvate decarboxylate complex ATP required Glyoxylate Cycle o Converts two acetyl-CoA (added independently) to one oxaloacetate (OAA) OAA then used to make glucose via gluconeogenesis Or to make amino acids o Glycolate pathway starts exactly the same as the citric acid cycle Oxaloacetate Citrate Isocitrate o Isocitrate is cleaved in two different ways o Loss of CO2 = citric acid cycle o Loss of succinate = glycolate cycle Glyoxylate Cycle o Acetyl-CoA+ OAA to isocitrate Citric acid cycle reactions 1 and 2 o Isocitrate lyase (glycolate cycle) Instead of isocitrate dehydrogenase (citric acid cycle) o Glyoxylate is coupled with another acetyl-CoA Produces malate o Malate is oxidized to make oxaloacetate Regular citric acid cycle o Summary Input: two acetyl-CoA and one OAA Output: two OAA Net: two acetyl-CoA converted to one OAA o Cycle enzymes present in plants and some microbes Not in mammals: cannot survive on a lipid-only diet Allows plants to survive on lipids only Germinating oil seeds Later we learn how acetyl-CoA is produced from lipids Biochemistry 301 – Survey of Biochemistry Professor Robert Thornburg LECTURE 20 – CHAPTER 20 – ELECTRON TRANSPORT By: Emily Settle Aerobic Metabolism o Acetyl-CoA From pyruvate via glycolysis From amino acids or lipids o Complete oxidation to CO2 o High energy electron carriers FADH2, NADH o Electron Transport Chain Electron transfer to O2 forms H2O Energy released used to make ATP o O2 required Terminal electron acceptor Mitochondrial Structure o Double membrane system Two soluble compartments Intermembrane space Matrix o Location of PDH complex and citric acid cycle Inner membrane, folded into cristae o ATP is the ultimate consumer of the proton gradient How to measure electron flow o Consider mixing Cu+, Cu2+, Fe2+, and Fe3+, all at 1M concentration Electrons will flow from one ion to another but… Which way will the electrons flow between these ions? Cu+ + Fe3+ Cu2+ + Fe2+ net flow from copper to iron or iron to copper? Electrons will move spontaneously to the lowest energy state o Half-reactions Cu+ Cu2+ + e- Fe2+ Fe3+ + e- Measure the redox potential using a voltage cell o Voltage cells indicates direction of electron flow In this example the direction of flow is from copper to iron If electrons were moving the other direction, the voltage would have the opposite sign Standard Reduction Potential (ii) o Oxygen is lowest energy electron acceptor o Reduced carbon is high in energy Quantification of Redox Energy o Greater difference in E^degree’ between two members of the redox pair means more energy is released when the electrons are transferred o NADH to O2 (for example) Half reactions: NAD+ + 2H+ + 2e- NADH + H+ (reverse for e- donor) E = +-.32 V ½ O2 + 2H+ + 2e- H2O E = .82 V NADH + H+ + ½ O2 H2O + NAD+ (full reaction) E = 1.14 V o Equate ΔG to ΔE ΔG = -nF ΔE F = 9.648 x 10^4 C*mol^-1 -54 kcal/mol (-222 kJ/mol) for NADH to O2 Sufficient to make 7 ATP at -7.4 kcal/mol (-30.5 kJ/mol) each Formation of High Energy Electron Carriers o Steps in glycolysis, PDH, and the citric acid cycle all produce either NADH or FADH2 Electron source is carbon originating in glucose or other food molecules Electron Transport Chain o ETS – protein complexes embedded in mitochondrial inner membrane o Electrons from NADH or FADH2 transported through these protein complexes o Hydrogen ions (protons) are transferred across membrane along with the electrons o Transfer of protons establishes a proton gradient across the membrane o Protein complexes Complex I – NADH dehydrogenase Complex II – Succinate dehydrogenase Complex III – Cytochrome b-c1 complex Complex IV – Cytochrome c oxidase o Electrons move from NADH or FADH2 to O2 NADH electrons enter Complex I FADH2 electrons enter Complex II o Redox reactions with numerous donor/acceptor pairs Organized into four multisubunit complexes Located in mitochondrial inner membrane o Energy released used to move (pump) protons from matrix to intermembrane space Electron Carriers o Five types of electron carrier are used in the electron transport chain NAD+/NADH NAD+ + 2e- + 2H+ NADH + H+ FAD/FADH2 FAD + 2e- + 2H+ FADH2 And the related compound FMN/FMNH2 o Same functionality as FAD/FADH2 Iron-sulfur proteins o Fe+++ + e- Fe++ Coenzyme Q o CoQ + 2e- + 2H+ CoQH2 Cytochromes o Contain heme, in which the iron atom is the electron acceptor/donor o Fe+++ + e- Fe++ Cytochromes o Heme cofactors, Fe atoms are the e- carrier o Three types of cytochrome, called a, b, or c o Differences in the specific structure of the heme group o Distinguished by absorbance maximal wavelengths o Sevel different cytochromes (1 iron centers) 3 b types: b560, b562, b566 2 c types: c, c1 2 a types: a, a3 Coenzyme Q (mobile carrier) o Isoprenoid (lipid) Soluble in phospholipid bilayer of mitochondrial inner membrane Can move within the membrane from one electron transfer complex to another Binds and releases protons when reduced or oxidized, respectively Respiratory Complexes o Four large multisubunit respiratory complexes can be isolated from the inner membrane Embedded in the mitochondrial membrane, detergent needed to release them o Several electron transfers occur within each complex The last acceptor of each complex diffuses in the membrane, acts as the electron donor for the next complex The mobile electron carriers as CoQH2 and Cyt c Complex I: NADH-Q Oxidoreductase o NADH oxidized, CoQ reduced o Electron carriers: FMN, 3 Fe-S clusters, CoQ o When CoQ accepts electrons, it also accepts protons Those protons come from the matrix Oxidized CoQ enters complex from membrane, returns to membrane as CoQH2 Protons on CoQH2 are released into the intermembrane space at a later step Complex III o Energy supplied by redox reaction NADH + H+ + CoQ NAD+ + CoQH2 Drives active transport of protons from matrix to intermembrane space Complex II: Succinate-Q Reductase o FADH2 formed in citric acid cycle at the succinate dehydrogenase step Succinate dehydrogenase is part of complex II Membrane bound FADH2 never leaves the complex (different than NADH) o Electrons from FADH2 transferred to Fe-S centers, then to CoQ No proton pumping into intermembrane space at this step Complex III: Q-Cytochrome c Oxidoreductase o CoQH2 oxidized Electrons donated successively to 3 heme Fe, 1 Fe-S center, finally to Cyt c o Four protons move from matrix side to intermembrane Not strictly a “pump” Protons absorbed to CoQ on one side, then the reduced from CoQH2 moves in the membrane, then protons released on the other side and CoQ regenerated In contrast, proton pumping actually carries individual protons from one side of the membrane to the other Complex I and Complex IV are active proton pumps o Overall reaction: CoQH2 + 2Cyt c (Fe2+) + 2H+ (matrix) CoQ + 2Cyt c (Fe2+) + 4H+ (intermembrane space) o The Q cycle proton pump First CoQH2 enters complex 2H+ released to intermembrane space, CoQ released One e- passed through carriers to Cyt c, Fe3+ reduced to Fe2+ The other electron passed to CoQ to form CoQ- Second CoQH2 binds complex 2H+ released to intermembrane space One electron passed through carriers to Cyt c, Fe3+ reduced to Fe2+ The other electron passed to CoQ to form CoQ- Two protons absorbed by CoQ2- to form CoQH2 which then leaves the complex Complex IV: Cytochrome c Oxidase o 4Cyt c (Fe2+) + 4H+ (matrix) + O2 4Cyt c (Fe3+) + H2O o During this process 4H+ from the matrix are moved to the intermembrane space o Electron acceptors are Cu2+, then two cytochrome heme Fe3+, then another Cu2+, then O2 o Protons absorbed from matrix onto reduced oxygen, forming H2O Aside: Oxygen Starvation Results from Nutrient Runoff o Nutrient runoff from Iowa farms into Mississippi River, to Gulf of Mexico Rich marine environment, algae species thrive to excess of natural ecosystem Cells die, are consumed by aerobic bacteria These consume oxygen in the water for respiration, again in excess of what the ecosystem can tolerate Aquatic life (fish and crustaceans) cannot survive due to lack of oxygen Oxidative Stress: Reactive Oxygen Species (ROS) o ROS are free radicals Free radicals contain unpaired electrons These unpaired electrons make ROS highly reactive Free radicals participate in a wide variety of disease states from cancer to asthma to stroke, and atherosclerosis o Enzyme systems eliminate ROS o Superoxide dismutase Removes electron from one superoxide Donates it to a second superoxide to form hydrogen peroxide HO-OH o Catalase 2HOOH 2H2O + O2 o Exercise increases the levels of superoxide dismutase and catalase Lots of respiration, lots of ROS made by Complex IV SOD, catalase needed Long term benefits to keep damage level from ROS low Biochemistry 301 – Survey of Biochemistry Professor Robert Thornburg LECTURE 21 – CHAPTER 21 – OXIDATIVE PHOSPHORYLATION By: Emily Settle Energy of Electron Transport o During ET, NADH and FADH2 are oxidized, and O2 is reduced Full reaction: NADH + H+ + ½O2 H2O + NAD+ ΔE = 1.14V o There is a large free energy change when NADH is oxidized by O2 ΔG = -nfΔE = -222kJ/mole o Sufficient for 7 reactions of ADP + Pi ADP ΔG = 7.4kcal/mol (30kJ/mole each) o No ATP is generated in the ET reactions (chapter 19) o Energy released by oxidation of NADH, FADH2 has been stored in the proton gradient Proton-Motive Force o Electrochemical gradient across mitochondrial inner membrane Lower pH in intermediate space than matrix Chemical concentration gradient stores energy Concentration of charge also stores energy Positive charges are concentrated in regions of higher H+ concentration o Force is exerted when barrier to proton movement is released This force is used to synthesize ATP Assay for e- transfer and ATP synthesis o Lyse cells, collect mitochondria by centrifugation o Incubate mitochondria in solution with succinate, ADP and Pi O2 will be present in the solution, but no ATP is present when the incubation is started o At selected times measure [O2] and [ATP] Loss of O2 (it is converted to H2O) shows that electron transfer is occurring Increase in ATP shows that ATP synthesis is occurring Uncoupling of ATP Synthesis and e- Transfer o Coupling ATP synthesis does not occur in mitochondria unless electron transfer also is occurring If proton gradient becomes too extreme, then no longer enough energy in electron transfer to continue pumping protons ATP synthase activity releases the proton gradient Likewise, e- transfer does not occur unless ATP synthesis also is occurring Electron transfer needed to provide energy for ATP synthesis o Uncoupling Uncouplers artificially create a pore that allows passage of H+ Eliminate the proton gradient across the mitochondrial inner membrane In these conditions electron transfer can continue without production of ATP o This proves that the pH gradient causes ATP synthesis when the potential energy stored in that gradient is released Chemiosmotic Hypothesis o Proton gradient coupled to activity of ATP synthase, to catalyze the reaction: ADP + Pi ATP + H2O o Proof Bacteriorhodopsin is a light-dependent proton pump Reconstitute in artificial membrane vesicles along with ATP synthase from mitochondria Add ADP + Pi, in the dark No ATP generated Expose to light ATP produced Thus, ATP synthase activity requires a proton gradient The ATP Synthase Complex o The enzyme that makes ATP is located in the mitochondrial inner membrane F1F0 ATP synthase o F1 component 5 polypeptide types, 9 total polypeptides: α3, β3, Υ, δ, ԑ Hydrophilic Matrix side of mitochondrial inner membrane Attached to F0 o F0 component Integral membrane complex 10-14 c subunits (depending on species), provide the proton pore 1 a subunit, 2 b subunits o F0 a, b, and F1 δ connect the two components Mechanism of ATP Synthase o The pH gradient powers rotation of a mechanical motor C subunit ring of F0 can rotate within the lipid bilayer A subunit of F0 is adjacent to the c ring, fixed in place in the membrane o A subunit provides protons to each c subunit, one at a time H+ enters a subunit in a channel in a exposed to intermembrane space Low pH, high [H+] H+ transferred to Asp residue of c, protonates carboxylate group The charged aspartate (-COO-) residue has now been neutralized (-COOH) o Neutralized c can rotate to interact with lipid in inner membrane Electrostatic effects drives movement Entire c ring rotates o C subunit that accepted the proton now in contact with a different channel in a That channel exposed to matric, high pH low [H+] environment Proton dissociates, again according to pKa The next c subunit in the ring has moved into place to accept a proton Mechanism of ATP Synthase o Υԑ subunits of F1 are fixed onto the c ring, rotate with it Remainder of F1 is fixed in place Υ subunit rotates through the fixed structure of the α and β subunits o α is the catalytic site for ATP synthesis ADP + Pi ATP + H2O Energy of physical motion of Υ as it interacts with α is coupled to the chemical energy change The total free energy change is negative, allowing synthesis of ATP Observation of Rotational Motor o Αβ core of F1 fixed to substrate, Υ free to move Rigid, fluorescent filament attached to Υ so that rotation can ve observed in microscope Single molecules visualized Add ATP ATP synthase reaction is running in reverse: ATP + H2O ADP + Pi o Υ subunit can be observed to spin 120 degree rotation per each ATP hydrolyzed Opposite direction to what happens in ATP synthesis ATP/ADP Exchange o ATP made in the mitochondrial matrix must be transported to the cytoplasm where it can be used for metabolism o ATP/ADP translocator exchanges matrix ATP for ADP in cytoplasm Intermembrane space is net positively charged owing to proton pump Exchanging ATP for ADP increases negative charge in intermembrane space 4 negative charges on ATP, 3 negative charges on ADP Thus, the proton gradient (powered by oxidation of carbon) is used in part to move ATP out of the matrix into the cytoplasm Uncoupling of ATP synthase and ET o Energy from proton gradient can be released without making ATP o Heat energy released Energy that is no used in ATP synthase is converted into heat energy o Hibernation (and human infants) Fats converted to acetyl-CoA (chapter 26) Acetyl-CoA to CO2 NADH, FADH2 generated ET in presence of uncoupled protein (UCP = thermogenin) generates heat Use of Respiration to Reoxidize Cytoplasmic NADH o Recall that NADH generated in glycolysis in the cytoplasm must be oxidized to regenerate NAD+ o How does this NADH get into mitochondria to be oxidized, then back out to cytoplasm to continue glycolysis? IT DOESN’T. o Electron shuttles are used Glycerol-3-phosphate shuttle NADH electrons from glycolysis enter mitochondria as FADH2 Some energy lost o NADH FADH2 CoQH2
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