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Biochem Final Exam Study Guide

by: Kiara Lynch

Biochem Final Exam Study Guide BIO 412-01

Marketplace > La Salle University > Biology > BIO 412-01 > Biochem Final Exam Study Guide
Kiara Lynch
La Salle

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These notes cover the material on the final exam. Ch 13, 15 (metabolism), 16 (glycolysis), 17 (citric acid cycle), and 18 (oxidative phosphorylation).
Stefan Samulewicz
Study Guide
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This 16 page Study Guide was uploaded by Kiara Lynch on Tuesday February 2, 2016. The Study Guide belongs to BIO 412-01 at La Salle University taught by Stefan Samulewicz in Summer 2015. Since its upload, it has received 46 views. For similar materials see Biochemistry in Biology at La Salle University.


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Date Created: 02/02/16
EXAM 4 STUDY GUIDE CHAPTER 13  Free energy change o A gradient is a form of energy o Increased concentration on the outside vs inside  becomes form of energy that can be released and captured by cell and used to do work  Pumps o Use ATP o Na+ out, K+ in o Reticulocyte ghost  Adjust concentration of materials outside to create a cell that contains whatever you want  Isolated RBCs are put in a hypotonic solution (fresh water)  Highly concentrated cell  water into membrane and cell swells  Gaps appear in membrane  Needs to be controlled before cell bursts  Drop cell into solution of appropriate concentration and cell shrinks back to normal  Flushes out hemoglobin but still have proteins and membrane pumps o Pumping molecules across membranes  Integral transmembrane proteins; allosteric  2 conformation states  Binding site to each side of membrane  Changes in affinity cause binding and release  Key to functioning of pumps  Center of membrane  Aspartic acid residue- capable of being phosphorylated which leads to a conformational change o NaK pump  4 subunits  Alpha- pump; binding sites  Conformational change affected by cardiotonic steroids from fox glove o Binds to extracellular portion of NaK pump o Freezes it in E2 P conformation o Na K gradient not getting restored o Antiporter can’t remove Ca from cytoplasm  Ca binds to tropomyosin in heart muscles and allows them to keep contracting  Increased [calcium]  greater muscle contraction  Side effect- closes gap junctions  Natural electrical signals don’t permeate  Selectivity filter has 4 binding sites +  Hydrated K ions enter these sites 1 at a time, losing hydration shells  2 ions occupying adjacent sites repel each other  pushed out  1. Binding site to inside  High affinity for Na+  E1- 3 Na bind  Dephosphorylation  ATP binds  2. E1 phosphorylation  Conformational change  Binding site to outside  Eversion- changes shape and releases Na  3. E2 phosporylation  High affinity for K  2 K bind  Triggers dephosphorylation  E2 dephosphorylated  Reverse conformation 2+  Eversion back to E1 conformation o Ca pump structure  2 Ca 2+ions bind in center of transmembrane domain  Asp- binds phosphoryl group in P domain  2 binding sites in center of membrane  Muscle contracts  release Ca  P domain contains Asp acid residues  A domain  Ca from cytoplasm binds to sites  conformational change  Ca released to lumen of SR  conformational change back  Ca 2+ATPase transports Ca through mem 2+  Ca binding from cytoplasm  ATP binding  ATP cleavage, transfer of P to Asp 351  ADP release and eversion of enzyme to release Ca on opposite side of membrane  Hydrolysis of Asp 2+  Eversion to prepare for binding of Ca from cytoplasm o Secondary transporters- pumps that don’t use ATP  Use energy of existing gradient to create a different gradient  Antiporter- can transport 2 substrates in opposite directions  Pump that allows A to flow with gradient (high to low concentration) and pumps B against gradient (low to high concentration)  Symporter- can transport 2 substrates in the same direction  A moves with gradient- high to low  B moves against gradient- low to high  Uniporter- can transport 1 substrate in either direction o Lactose permease  Bacterial cells use proton gradients for flagellum and to pump lactose against gradient into cell  Structure of lactose permease with bound lactose analog  Amino terminal and carboxyl terminal  2 halves that surround sugar and are linked to one another by only a single stretch of polypeptide  Mechanism  Lactose exposed to outside  protonate amino acid residue (binds H+ from outside)  binding of lactose  eversion (change conformation, release lactose, also releases proton to inside with gradient)  Use proton gradient to move lactose against gradient CHAPTER 15- Metabolism  Creatine phosphate- high energy phosphate bond energy storage o Energy used for motion, active transport, biosynthesis, signal amplification, oxidation of fuel molecules, or photosynthesis  Anaerobic metabolism- upregulated, ATP generated at higher rate, forms lactic acid  Glucose metabolism o Glucose10 steps pyruvate o Needs electron carriers o Pyruvate forms either lactate (anaerobic) or acetyl coA (aerobic) o Adenosine triphosphate (ATP)  Universal currency for energy exchange  High phosphoryl-transfer potential (3 phosphates)  Gamma phosphate is the one that is broken  In the middle of the standard free energy chart  have to be able to make ATP (need molecules with higher phosphoryl energy transfer) and can give energy  Resonance stabilization- ADP + P is more stable than ATP; ATP pushes for phosphoryl release  Improbable resonance structures- repulsion hydration o Contributes little to terminal part of ATP because 2 + chargets are adjacent to each other o More water can fit around P in ADP than ATP (higher degree of hydration)  Proton Gradient o Capture electrons  oxidation of fuel molecules (little ATP produced) o Electrons  electron carriers  shuttle through electron transport chain (in inner mitochondrial membrane)  produces proton gradient (no ATP) o Proton gradient moves through proton complex  spins and produces ATP (looks like prokaryotic flagella motion)  Electron Carriers o Nicatinamide adenine dinucleotide (NAD)  Reactive site (Hydrogen) – accepts 2 e- and a proton  Reduce NAD+ to NADH  other proton released to solution o Flavin adenine dinucleotide (FAD)  Oxidized form- reactive sites N=-=N in ring  Electron carrying unit and AMP unit  Reduced form – FADH2  Hydrogens attached to nitrogens electrons and H+ carried by isoalloxazine ring CHAPTER 16- Glycolysis  ATP  myosin  actin o Low O2 (last seconds of a sprint)  lactic acid buildup o Normal O2 (long slow run)  O2 incorporated into pathways  Some fates of glucose o Glucose  pyruvate  fermentation (ethanol) or lactate formation or complete oxidation  Energy in the form of sugar o Glucose- aldehyde; molecules in active site- 6C ring structure broken  becomes linear o Fructose- ketone, 5C ring  Sugars into cell- glucose transporter protein o 12 transmembrane alpha helices  Isoforms - Glut 1-5 in family  Transport glucose in downhill fashion (low [] compared to outside)  No energy used  eversion  Glycolytic pathway  Know intermediates and enzymes; where ATP is used or generated; where e- are captured or donated; where molecules enter or leave pathway  Prime system with 2 ATP, create molecules with high phosphoryl transfer potential o Hexokinase  Traps glucose in cell and begins glycolysis  Want glucose to stay in  add charges so it can’t move through membrane  becomes glucose 6-phosphate  Packaged to make glycogen  Induced fit model- lobes separate in absence of glucose, lobes come together and surround glucose when present (substrate- glucose and ATP)  creates environment for catalysis  Enzyme hydrolysis  ATP can bind with H2O in active site (no hydrolysis)  conformational change when glucose is in active site  functioning enzyme  P can only be added to glucose (no hydrolysis) o Phosphoglucose isomerase  Convert glucose 6-phosphate so it can accept a second phosphate  Fructose 6-phosphate formed – 5 member ring, opens up a C o Phosphofructokinase  Committed step- highly unlikely to be reversed  Isomerization- aldose  ketose (6C to 5C ring)  Converts to fructose 1,6-biophosphate by adding P to #1 C  Allosteric sets pace of glycolysis o Aldolase  Breaks into two 3C molecules each with a phosphate  Readily reversible aldol condensation  Products are immediately shuttled through rest of pathway  Forms Glyceraldehyde 3-phosphate (GAP) and Dihydroxyacetone phosphate (DHAP) which is converted into 2 GAPs o Triose phosphate isomerase  Causes isomerization  Converts DHAP to 2 GAPs  Central core- 8 parallel beta strands surrounded by 8 alpha helices  Alpha beta barrel (also present in aldolase, enolase, and pyruvate kinase)  His and Glu- at active site of barrel responsible for catalysis  Loop- 10 to 12 amino acids; ribbon of alpha helices  Lid to close off active site on substrate binding  Traps substrate and potential intermediate and prevents release and side reactions  Enediol intermediate o Highly reactive  loop is important o Traps in active site  no reaction or loss of P o Glyceraldehyde 3-phosphate dehydrogenase (GAP dehydrogenase)  Converts GAP to 1,3-Biophophoglycerate (1,3-BPG)  3C with 1 P  3C with 2 P  Needs energy to occur  Coupled reaction  NAD+ reactive site accepts H:-  Favorable oxidation of GAP drives unfavorable acyl phosphate formation (dehydration)  Forms temporary covalent bond with intermediate  Cofactor NAD+  Active site o Cys and his residues; adjacent to bound NAD+ o Thioester bond o Sulfur atoms of cys links with substrate to form transitory thioester intermediate  Carbonyl C- remove hydride, has partial positive charge  Creates nucleophile and forms thioester bond allows hydride to be transferred to NAD+ oxidized NADH  Energy trapped  powers reaction  Break bond with cysteine residue  Cys reacts with aldehyde of substrate (hemiacetyl) oxidation with transfer of hydride to NAD+ (thioester) reduced NADH exchanged for NAD+  orthophosphate attacks thioester forming 1,3-BPG o Phosphogylcerate kinase  Converts 1,3-BPG to 3-Phosphoglycerate  ADP into active site  kinase removes P from 1 substrate and moves it to the other  generates ATP o Phosphoglycerate mutase  Converts 3-phosphoglycerate to 2-Phosphoglycerate  Position of phosphate group shifts  Mutase- enzyme that catalyzes an intramolecular shift of a chemical group o Enolase  Converts 2-phosphoglycerate to phosphenolpyruvate o Pyruvate kinase  Converts phosphenolpyruvate to pyruvate  2 molecules of pyruvate formed  2ATP per glucose + potential for more energy production (P on NADH)  Oxygen is not required for cycle but only 2 ATP would be produced  pyruvate has energy oxygen can use to produce more ATP  Pyruvate o No O2 fermentation – ethanol and lactic acid formation (need NADH- limiting factor; is recycled)  Remove 1 C (CO2)  2C acetaldehyde reduction + NADH ethanol (NADH can participate in glycolysis again) o O2 further oxidation o Pyruvate decarboxylase  Converts pyruvate to acetaldehyde  H+ in, CO2 out o Alcohol dehydrogenase  Converts acetaldehyde to ethanol  NADH in NAD+ out (can participate in glycolysis again)  Active site  Contains Zn ion bound to 2 Cys and 1 His  Zinc binds acetaldehyde (hydride acceptor) substrate through its O2 atom polarizing substrate so it more easily accepts hydride from NADH (Hydride donor) o Maintaining Redox Balance  NADH produced by glyceraldehyde 3-phosphate dehydrogenase reaction must be reoxidized to NAD+ for glycolytic pathway to continue  In alcoholic fermentation, lactate dehydrogenase oxidizes NADH while generating lactic acid  No net redox reaction o Lactate dehydrogenase  Converts pyruvate to lactate  Regeneration of NAD+ in reduction of pyruvate to lactate orethanol sustains continued process of glycolysis under anaerobic conditions  Absence of O2- in muscles- 1 step reaction  Lactate dehydrogenase has a feedback loop  Lactic acid buildup changes pH and affects O2  harms cells o Obligate anaerobes  O2 is toxic  Clostridium tentani tentanus; clostridium botulinum botulism; clostridium perfringens gas gangrene; bartonella hensela cat scratch fever; bacterioides fragilis abdominal, pelvic, pulmonary, and blood infections o Fermentation products  Glucose lactate  Lactate acetate  Glucose ethanol  Ethanol acetate  Arginine CO2  Pyrimidines CO2  Purines formate  Ethylene glycol acetate  Threonine propionate  Leucine 2-alkylacetate  Phenylalanine propionate o Binding sites for NAD+ in dehydrogenases  Nicotinamide-binding half  Adenine-binding half  Together form a motif Rossman fold  NAD+ binds in an extended conformation o Sucrose= glucose + fructose  Fructose enters glycolysis as fructose 6-phosphate o Lactose= glucose + galactose  Galactose enters glycolysis as glucose 6-phosphate o Fructose Mechanism  Enters glycolytic pathway in liver through fructose 1-phosphate pathway  Fructose- 6C prime with ATP  adds P  Splits into 2 3C molecules  Glyceraldehyde+ ATP  into pathway  Dihydroxyacetone phosphate  isomerized into glycolytic pathway o Galactokinase  Interconversion pathway  Phosphorylation of galactose to galactose 1-phosphate o Lactase  Microbes can’t use it symptoms of intolerance o Galactokinase  Galactose to Galactose 1-phosphate  Galactose buildup  galactitol  precipitates and forms cataracts  Galactitol- osmotically inactive, water will diffuse into lens  cataract  High incidence of cataract formation with age in population that consume substantial amounts of milk CHAPTER 17- THE CITRIC ACID CYCLE  Captures electrons to generate ATP  Oxidizing carbon fuels (acetyl CoA); source of precursors for biosynthesis; intermediates can be used as building blocks  Most fuel molecules enter the cycle as acetyl CoA  Glycolysis pyruvate in cytoplasm  Gets into matrix, outer membrane pores, inner membrane needs transporters pyruvate translocase- proton symporter o Inner mitochondrial membrane  Cristae- folds that increase and maximize surface area so more proteins and enzymes can be present  Proteins of electron transport chain that make up ATP o Outer membrane has pores that are size discrimination  Lose 2C as (CO2), lose 2NADH, lose 1 FADH2, 1 ATP  6C molecule  oxidative decarboxylation  4C molecule  generate high energy phosphate bond GTP  Cellular respiration o Capture high energy electrons for electron transport chain reduce O2 to generate H+ gradient o Citric acid cycle is the meeting point for different sources of energy (fatty acids, glucose, amino acids)  Pyruvate (3C) to Acetyl CoA (2C) is the link between glycolysis and the citric acid cycle o Shuttle 2C molecule from glycolysis into Krebs cycle o Needs enzyme o Carrier of acetyl group captures 2e- NADH, release 3 C as CO2  Pyruvate dehydrogenase complex o Dozens of subunits together to take in pyruvate and pump out acetyl coA o Intermediate- side reactions o Pyruvate + CoA + NAD+  acetyl-CoA + CO2 + NADH o E1- pyruvate dehydrogenase component  24 subunits  Oxidative decarboxylation of pyruvate; removes CO2  Negative charge on carbonyl carbon of TPP, ring of lipoamide forms bond with carbonyl carbon  + charge attracts electron of CO2 we want to get rid of  pulls e- toward ring and makes cleavage of the bond releasing the CO2 easier  TPP is acidic; can lose H+ and form bond to attract e- for CO2 removal o E2- Dihydrolipoyl transacetlyase  24 subunits  Acetyl group transferred to CoA  Highly reactive disulfide bond at end of ring that can be reduced and reoxidized  A is attached to long hydrocarbon chain (long and flexible)  Prosthetic group attached to end of Lysine (long flexible side chain)  Highly reactive group at end of “rope”  move intermediates from one active site to the other o E3- dihydrolipoyl dehydrogenase  12 subunits  Captures e-; transfers to NAD+  NADH  E- removed  Oxidation and reformation of disulfide bond  Tightly bound FAD (e- and H+)– never leaving active site  NAD+ in takes e- from FADH2 and carries it to the e- transport chain  Flip and enter E1 active site  disulfide bond broken and 2C acetyl group transferred to form thioester bond with cofactor o Coenzymes- catalytic factors TPP (thiamine pyrophosphate), lipoic acid, and FAD o 1. Pyruvate decarboxylated at E1 active site forms hydroxyethyl TPP intermediat, CO2 leaves; active site deep within E2 complex; connected to enzyme by 20 angstrom long hydrophobic channel o 2. E2 into lipoamide arm of domain into deep channel of E1 o 3. E2 transfers acetyl group to lipoamide; acetylated arm leaves E1 and enters E2 cube to active site active site deep in cube at subunit interface o 4. Acetyl moiety transferred to CoA; acetyl CoA leaves cube; reduced lipoamide arm swings to active site of E3 o 5. At E3 active site, lipoamide oxidized by coenzyme FAD; reactivated lipoamide is ready to begin another cycle o 6. NADH produced with reoxidation of FADH2 to FAD  Pyruvate  acetyl CoA (3steps); steps are coupled to preserve free energy derived from decarboxylation step to drive formation of NADH and acetyl CoA o Decarboxylation o Oxidation o Transfer to CoA  Krebs Cycle o Oxidizes acetyl groups from carbohydrates, fatty acids, and amino acids o Cycle  just like one big enzyme o All reactions in the mitochondria o 2C in, 2C out each cycle o Intermediates are building blocks o Acetyl > CO2, transfer 4 pairs of e-, get 1 GTP  Synthase- enzyme catalyzing a synthetic reaction o 2 units jointed without direct precipitation of ATP o Citrate synthase (oxaloacetate 4C + acetyl CoA 2C  citrate 6C  Induced fit model- ensures carbons are transferred only to correct substrate  Dimer- closure of hinge around active site  acetyl CoA binds  His and Asp  Charged, can accept and donate H+  Aldol condensation between substrates via enol intermediate  conformational change holds in place (degrades if it gets out) o Aconitase  Dehydrates and rehydrates citrate and cis-aconitate  Moves location of hydroxyl  Non- heme Fe-S protein  4 Fe bound to 4 inorganic S and 3 Cys S atoms; 1 Fe available for binding  Iron cluster  Isocitrate dehydrogenase o 1 oxidative decarboxylation (isocitrate 6C  alpha ketoglutarate 5C) releases CO2  Dehydrogenase oxidative decarboxylation; dihydrolipoyl trans-  transfer; dihydrolipoyl dehydrogenase regeneration of oxidized form of lipoamide  Succinyl-CoA synthetase o Link inorganic phosphate to GDP; GTP released  used for microtubules, actin fiber formation, or converted to ATP without loss of energy o 2 active sites- P to succinyl and P to mobile His His transports to 2d active site  GTP released o 2 subunits  His residue between CoA and ADP- beta subunit; picks up phosphoryl group near CoA & swings over to transfer it to nucleotide bound in ATP grasp domain  Rossmann fold- in alpha subunit binds enzyme CoA, ATP/GDP grasp  Succinate dehydrogenase o Only enzyme anchored in inner mitochondrial membrane  direct link to e- transport chain; capture more e-  hydration  Intermediates are building blockes o 32 succinyl CoA + gly  porphyrin rings (heme) o α ketoglutarate  glutamate  glutamine, proline, arginine o oxaloacetate  Asp  Asn, Met, Thr, Ile, Lys o intermediates drawn off for biosynthesis when the energy needs of the cell are met; replenished by formation of oxaloacetate from pyruvate  Pathway integration o Pyruvate (3C) carboxylase + C  oxaloacetate (4C) o Fatty acids  acetyl CoA o Rate of cycle increases during exercise- requires replenishment of oxaloacetate (formation of pyruvate) and acetyl CoA (metabolism of pyruvate and fatty acids)  Beri Beri o Neurological and cardiovascular disorder o Anorexia, enlargement of heart, paresthesia (itching), muscle weakness o Tremor: penetrates motion and sensation of hands, feet, and sometimes whole body o Deficiency in vitamin B1 (thymine)  Vit B1 in pork, red meat, whole grain, nuts, peas, spinach o No TTP  can’t convert pyruvate to CoA  can’t make enough ATP  Arsenite poisoning o Need a disulfide to chelate o Arsenite inhibits pyruvate dehydrogenase complex by inactivating dihydrolipoamide o Sulfhydryl reagents relieve inhibition  form complex with arsenite o If mercury and arsenite get into skin high affinity for disulfide bonds; interrupts flow of molecules (pyruvate dehydrogenase complex)  causes neurological problems because glucose cannot be formed (glucose is energy for the brain) CHAPTER 18- OXIDATIVE PHOSPHORLYATION  Fuel and oxygen delivered to inner mitochondrial matrix by blood flow; Krebs cycle occurs in matrix; glycolysis occurs in cytoplasm o Mitochondria form network inside fibroblast cell oxidize carbon fuels to form cellular energy (ATP) o Mitochondria have cristae to increase surface area to fit more proteins; 2 membranes  ATP formed in matrix  Oxygen and ATP synthesis are coupled by transmembrane proton fluxes o Electrons from NADH and FADH2 through 4 complexes to reduce oxygen to water o 3 complexes pump protons from matrix to exterior of mitochondria o Protons return by flowing through 4 complex, ATP synthase which powers syn. of ATP  Similar appearance of mitochondrial genomes o Symbiotic relationship o Mitochondria look like single cell bacteria; have their own genome (most circular); code their own RNAs  Overlapping gene complements of mitochondria o Protozoan are the most closely related bacterial strain to the one that invaded eukaryotic cells and developed a symbiotic relationship  7.3 kcal/mol needed to make 1 ATP  Redox potentials o Electron transfer potential- ability of substance to give electrons up to one another o Phosphoryl transfer potential- free energy to make ATP  Voltmeter o Measures willingness to give up electrons (e- transfer potential) o Electrons on molecular O2 o 2 aqueous chambers- oxidant and reductant at 1M o 1M protons and H2 gas o Are electrons flowing to H2 gas or are they staying  Electron transport chain o 3 enzyme complexes  lose free energy  O2 to water o Pumps protons across inner mitochondrial membrane  protons drive motor that makes ATP o Electron flow down energy gradient from NADH to O2 o Gradient formed o Fe is a component of 1, 3, 4, and cytochrome C  Prosthetic groups associated with protein complexes o Hemes  Fe-Sulfur Iron Clusters- Fe bound by 4 cysteines  Types: Fe-S, 2Fe-2S, 4Fe-4S o Copper o Flavin  Oxidation states: FMN- oxidized; FMNH2- reduced o Quinone  Oxidation states: quinone- oxidized; quinol-reduced  Complex 1: NADH-Q-oxidoreductase (NADH-Q) o Q- hydrophobic molecule in inner mitochondrial membrane (Q pool) o NADH + Q (oxidized) + 5H+ (from matrix)  NAD+ + QH2 (reduced) + 4H+ (to cytoplasm) o In from mitochondria, out to cytoplasm o E- into complex from NADH through FMN and a series of Fe clusters to Q o Pumping 4H+ and taking up 2H+ from matrix  Complex 2: Succinate-Q reductase o Part of Krebs cycle- succinate deyhydrogenase o FAD  FADH2 o No electrons pumped o Lower energy than e- of NAD  not as much ATP made  Complex 3: Q-cytochrome C oxidoreductase (Q) o Transmembrane alpha helices o Inner mitochondrial matrix o Homodimer- parallel distinct polypeptide chains o 3 hemes and 2Fe-2S clusters near cytoplasmic edge  positioned to mediate electron transfer reactions between quinones in membrane and cytochrome C in inner mitochondrial membrane o Coenzyme Q can carry 2 e- and Cytochrome C can carry only 1 e-  Potential for loss of energy  prevented by Q cycle  2 halves o Docking site for Q  Carrying electrons  1 e- travels up via complex 1 to cytochrome C  drifts to next complex  1 e- stays associated with complex  different Q (oxidized)  lands on other partially reduced Q  becomes fully reduced and leaves binding site to enter pool  Complex 4: Cytochrome C oxidase o 13 polypeptide chains o Most of complex and 2 hemes are embedded in membrane o Heme α3-Cu inBsite of reduction of O2 to H2O o Covalent bond between His and Tyr o Cu ACu Broup near intermembrane space  better accepts electrons from Cyt. C o CO backbone o Variety of prosthetic groups for e- to proceed through  Hemes (with His); Cu Cys; Cu, His, and Tyr  Heme A- redox potential  in different environment within Cytochrome C oxidase o e- from Cytochrome C to molecular oxygen  requires 4e-; 2 steps of 2  cytochrome C on docking site  release e- to heme  reduce iron and copper  can bind O2  cycle begins and ends with molecule in oxidized form  4 Cyt C donate 4e-  binding and cleavage of O2  import of 4H+  2H2O  Water released from enzyme to regenerate initial state o Peroxide bridge  Fe 3+and Cu 2+  Designed to keep O2 in active site until water forms  Oxygen bound to heme is reduced to peroxide by presence of CuB  Break bond between oxygens and get enough energy to form water o Proton transport  H+ from mitochondrial matrix  Break bond between hydroxyl and Fe&Cu to form water  Pumps 4 protons from 1 side of membrane to the other  -4 outside (gradient)  4H+ to form water (chemical H+) o e- flow through respiratory chain  powers proton pumping and results in O2 reduction o Conservation of 3-D structure of Cytochrome C  Changed little evolutionarily  Can interact with protein complexes efficiently o Used as a tool for molecular trees- shows changes in amino acids  Oxygen free radicals contribute to intensity of diseases o Oxygen free radicals may escape complex 4 o Extra 2 e- o Highly reactive o Want to react with proteins o Ex: DNA  damage  Superoxide dismutase o Scavenges oxygen free radicals  High binding affinity o Enzyme accepts e-  reduced form  molecular O2 o Enzyme finds another oxygen free radical and converts it to H2O2 o Oxidized form reacts with 1 superoxide ion to form O2ndnd generate reduced form of enzyme. Reduced form reacts with 2 superoxide and 2H+ to form H2O2 and regenerate oxidized form of enzyme o Catalase  Eliminates H2O2 from cells; H2O2 H2O + O2  Distance dependence of electron transfer rate o Optimal distance of an e- carrier in complex  optimizes diffusion of e- o Rate of e- transfer decreases as e- donor and acceptor move apart o In vacuum- rate decreases more gradually because variations in structure of intervening protein medium affects rate  Chemiosmotic hypothesis o e- transfer through respiratory chain leads to pumping of protons from matrix to cytoplasm side of inner mitochondrial membrane o pH gradient and membrane potential constitute a proton motive force used to derive ATP synthase  Synthetic system- bacteriorhodopsin in synthetic vesicle o ATP made when reconstructed membrane vesicle containing bacteriorhodopsin an ATP synthase are illuminated o Orientation reverse of mitochondrion  Proton pump run by light  conformational changes (normally pumps H+ out)  proves point that proton gradients are used as energy  Protons escape produce ADP and P  ATP outside of cell (backwards)  No H+ gradient  no ATP  ATP synthase o Transmembrane  Stationary subunit A  Rotating C ring- 2 α helices, hollow ring o P loop, NTPase domains of α and β subunits o Part of enzyme embedded in inner mitochondrial membrane; remainder in matrix o Asymmetrical- different faces exposed to α and β subunits  Interactions change as it spins o F0  γ subunit, € subunit  Attached to C ring; as C ring spins, they spin o F1  Stationary α and β helices  Anchors and attaches to α and β hexamer to create ATP  Stationary B subunit  ATP synthesis mechanism o ADP attacks P aid releases H2O and ATP o ATP forms on its own without a proton motive force  ATP synthase is present but just sits there  Just needs ADP and P toibe present  ATP wouldn’t release just forms and breaks o Get ATP out of active site with γ subunit o Radioactive water  Incorporated into P i  In active site (no H+ gradient)  Gives ADP and P i  ATP forms and comes apart  ATP synthase nucleotide o Binding sites are not equivalent o γ in center of hexamer o α- contains ATP but does not participate in reaction o 3 conformations of β subunits  O- open; low affinity for substrate  L- loose; binds substrate loosely, not very catalytically active  T- tight; ATP forms  ATP synthase binding mechanism o γ rotation changes subunits o T- ADP and P, iTP does not release ATP  120° rotation CCW; T becomes O o O- ATP release; ADP and P biid  120° rotation CCW; O becomes L o L- traps substrate  120° rotation CCW; L becomes T o T- substrate in, forms ATP o 1 rotation- 3 ATP formed  3 subunits go through all 3 conformations o Proof- ATP observation of ATP driven rotation in ATP synthesis  ATP hydrolyze  γ subunit spun  Fix hexamer to surface  Fluorescently labeled actin filament is seen spinning  Hydrolyze or form ATP based on direction of rotation  Proton conducting unit of ATP synthase o Subunit c- 2 α helices span membrane  Aspartic acid in 1 helices lies at center of membrane  Neutralizes charge  C ring rotates  Asp out into hydrophobic part of membrane o Subunit a- 2 half channels that allow H+ to pass partially through membrane  Proton motion across C ring drives rotation of C ring Proton in from inner membrane space into cytoplasm ½ channel to neutralize charge on Asp in C subunit C ring rotates clockwise by 1 c subunit moving Asp out of membrane into matrix ½ channel Proton into matrix, resets initial state o H+ gradient  spins C ring  spins γ  conformational changes of β subunits  once around  3 ATP- depends on number of protons outside  Glycerol 3-phophate shuttle o Glycolysis e- from cytoplasm into e- transport chain o 2 forms of glycerol phosphate dehydration  Free floating and tightly bound  Converts dihydroxyacetone phosphate to glycerol 3-phosphate  oxidized  E- into e- transport chain  Glycerol 3-phosphate back into dihydroxyacetone phosphate o e- from NADH can enter into mitochondrial e- transport chain by being used to reduce dihydroxyacetone phosphate to glycerol 3-phosphate  reoxidized by e0 transfer to an FAD in membrane bound glycerol 3- phosphate dehydrogenase  subsequent e- transfer to Q  QH2 allows e- to enter chain o NADH (cytoplasmic) + H + E-FAD (mitochondrial)  NAD (cyt) + E- FADH 2mit)  ATP-ADP translocase o More protons on outside (+)  attracts negative charges of ATP  Gradient provides energy for process o ATP in mitochondrial matrix o Transmembrane protein binding site for ATP and ADP o Eversion  Exposed to cytoplasm- ADP binds o Eversion  Opens to inside of matrix  ADP in; ATP binds o Eversion  ATP release to cytoplasm o Structure- 3 similar subunits o Transporters (carriers)  Transmembrane proteins that carry specific ions and charged metabolites across inner mitochondrial membrane  #ATP/NADH = 2.5 ATP/pair of e- on NADH  #ATP/FADH = 2.5 ATP/pair of e- on FADH (FA2H doesn2t flow through complex)


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