Biochemistry MCDB 310 Final Exam Review
Biochemistry MCDB 310 Final Exam Review 310
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BIOCHEMISTRY EXAM 4: FINAL EXAM REVIEW Chapter 17 and 18: Fatty Acid and Amino Acid Oxidation Triacylglycerols (Triglycerides, Fats, Neutral Fat) Three fatty acids Ester linkages to glycerol Nonpolar Relatively inert, chemically. Fatty Acids: Principle Form of Stored Energy In Many Organisms 1. Carbons are almost completely reduced ● Oxidation of fatty acids results in more energy per carbon than oxidation of carbs ○ 9 cal/g vs. 4 kal//g for glucose. 2. Triacylglycerols require less solvation than fatty acids & polysaccharides (less H bonds needed) ● Fatty acids can be more compactly stored 3. Triacylglycerols are relatively inert (chemically) ● Can be stored long term in large quantities. Stored Fuel in A 70 KG 154 lb Person Start off with glucose, all available glucose in person can get oxidized. Then break down glycogen in muscle + liver. Use feeder pathways (mannose, trehalose) and bring them to glycolysis. After using up available carbs go to storage fats. Extract energy from ketone bodies Then oxidize proteins (enzymes, skeletal, muscle proteins) Glucose → Fats → Proteins Digestion of Fat 1. Some fatty acids are released by lipasesin acidic environment of stomach 2. Fatty acids are released mainly induodenum ● Secretion ofalkaline pancreatic juice ● Alkaline pH is optimum forpancreatic lipases and nonspecific esterase to liberate fatty acids ● Alkaline bile salts such as taurocholic acid from gall bladder ● Bile salt: amphipathic (polar and nonpolar ends) detergents form micelles. Processing of Fatty Acids Di and monoacylglycerols, fatty acids, glycerol ● Diffuse acrossintestinal epitheliu (intestinal mucosa) ● Convert BACK to triacylglycerides: packaged with dietary cholesterol and lipoproteins intohylomicrons . ● Chylomicrons move through lymphatic system and bloodstream to tissues ○ Target muscles and adipose tissues ● Lipoprotein lipase, activated by apoCIIapolipoproteins) in capillary, hydrolyzes triglycerides and converts to fatty acids + glycerol. ● Fatty acids oxidized as fuel or reesterified for storage. (FA +glycerol enter tissues) Chylomicron remnants are transported to liver ● Excess triglycerides in liver ○ Energy for liver ○ Conversion to ketone bodies ○ Conversion into VLDL → Transported to adipose ○ Fatty Acids oxidized to CO2 (Betaoxidation) Chylomicrons Phospholipid micelles Triglycerides (80%) inside = nonpolar Apolipoproteins on surface Combinations of apolipoproteins and lipids produce differedensity particle: LDL, VDL Signaling: Mobilization of Storage Fat Epinephrine and Glucagon (signals for low blood glucose) → activate adenylyl cyclase in adipose membrane → High levels of cAMP → PKA → Activation of lipases. Fatty acid and glycerol can be used for fuels. Triglycerides converted to FA+ glycerol → glycerol make new triglycerides or membrane lipids. Glycerol gets phosphorylated by glycerol kinase by ATP → Glycerol 3P. Glycerol 3 Kinase phosphorylates glycerol Glycerol 3P → Dihydroxyacetone P (this conversion makes NADH) Then converted to Glyceraldehyde 3P → Glycolysis. SUMMARY: Oxidation of Fatty Acids Stage 1: βOxidation successive removal of C2 units (acetyl coA + NADH + FADH2) Stage 2: Citric Acid cycle: Oxidation of acetyl groups to CO2 + H2O Stage 3: Oxidative Phosphorylation : uses reducing equivalents from other stages Fatty Acid Oxidation Takes place in mitochondria Oxidation starts with carbon that is β to the carboxyl carbon (βoxidation) Oxidation of fatty acids produce: acetyl coA, FADH2, NADH. Activation of fatty acids and transport into mitochondria (addition of coenzyme A) Overall reaction: FA + CoASH + ATP → FACoA + AMP + 2Pi. Acyl Carnitine/Carnitine Transporter Rate limiting step of acyl chain oxidation Facilitated diffusion/antiport. βOxidation Saturated fatty acids, even numbers of carbons: 4 steps Step 1: Reduction (Dehydrogenation) ● 3 isozymes: ○ LACD (1218) ○ MACD (414) ○ SACD (48) ● FAD is prosthetic group ● Electrons immediately donated to ETF (electron transferring flavoprotein) of the respiratory chain. Step 2: Hydration Step 3: Reduction (Dehydrogenation) (This makes NADH) ● NADH makes 2.5 ATP Step 4: Acetyl transferase reaction + Add Coenzyme A Citric Acid cycle and βoxidation follow similar steps because it’s efficient and saves energy! Use 1 ATP to put on Coenzyme A Use 1 ATP for transport system So 108 ATP 2 = 106 ATP total. If acyl coA already in mitochondrial matrix = 108 ATP, no transport involved. 106108 depending on locations of substrates. Methods for calculating potential ATP Yields Assume even numbers of carbons, saturated Divide number of carbons in half ● This is the total acetyl coA molecules ● How many ATP from 1 Acetyl coA via CTA = 10 ATP molecules. Total acetylcoA minus 1 = total NADH and total FADH Ex: Myristate = 14C so 7 AcetylcoA, so 6 NADH and 6 FADH2. Total NADH (2.5) = ATP molecules Total FADH2 (1.5) = ATP molecules Add all ATP molecules. Subtract ATP for “activation” depending on where substrate located. βOxidation of Unsaturated Fatty Acids EnoylcoA hydratase cannot work on cis double bonds 1. Convert cis to trans 2. Moves bond one carbon 3. One enzyme required 4. Enters second step of beta oxidation βOxidation of Polyunsaturated Fatty Acids Cis to trans (isomerize) Reduce Isoemerize Same enzyme for monounsaturated Another enzyme for polyunsaturated Enters second step of beta oxidation Oxidation of Odd Carbon Number Fatty Acids Oxidize as usual Last step: 1 acetyl coA → Citric acid cycle And 1 PropionylcoA For odd number, it’s beta oxidation all the way until the end. Propionyl coA is carboxylated with bicarbonate and biotin and requires ATP → Form methylmalonyl coA. MethylmalonylcoA Mutase Reaction Reversal of alkyl group with H on adjacent carbon WITHOUT adding H to solvent. Coenzyme B12: Deoxyadenosylcobalamin Key feature: relatively weak covalent bond (110 kJ/mol) between cobalt and 5’ deoxyadenosine Homolytic cleavage of bond produces a radical, the basis for the mutase reaction. Other Ways to Use Fatty Acid Oxidation Products Beta oxidation in peroxisome/glyoxysome A difference in the first step 3 differences from mitochondria Other Ways to Oxidize Fatty Acids woxidation in endoplasmic reticulum ● Oxidation starts at the wcarbon ● The carbon most distant from carboxyl group aoxidation: branched fatty acids ● Phytol is a product from chlorophyll degradation. Acetyl coA can be converted into ketone bodies or enter citric acid cycle. Liver makes lots of acetyl coA and converts to acetoacetate and is broken down into other compounds which move to brain. Brain uses betahydroxybutyrate and makes more acetyl coA. Ketone Body Formation Very little normally Present in starvation, fasting, and diabetic patients. Starvation ● Gluconeogenesis depletes citric acid cycle intermediates and that leads to high acetyl coA → ketone body formation. Untreated Diabetes Low [insulin] = no fatty acid synthesis Fatty acids enter mitochondria to be degraded Citric acid cycle intermediates low in concentration. Acetyl coA accumulates Increased ketone body formation from acetyl coA in liver, release into body ● Leads to ketosis, lowering blood pH (acidosis) ● Can cause coma + death ● Normally, secrete ketone bodies of less than 125 in urine, but with ketosis = 5,000 ketone bodies. We have to tear up any protein we have when we’re really low on energy. Use whatever we can find through Kreb’s cycle to make energy Glucogenic amino acids = amino acids that can be used to make glucose, take amine group off and use carbon skeleton to make glucose. Oxidize these amino acids to make glucose Ketogenic amino acids make ketone bodies = make intermediates like OA for citric acid cycle. Glucogenic Amino Acids : Arginine, Glutamine, Histidine, Proline make Glutamate. ● Asparagine and Aspartate make OA Ketogenic Amino Acids: ● Isoleucine, Leucine , Tryptophan make AcetylcoA. Amino Acid Oxidation 1. Protein rich diet in carnivores (90% of energy comes from protein) 2. Endogenous proteins are used as fuel during starvation or uncontrolled diabetes ● Enzymes, cytoskeletal, membrane protein 3. Amino acids are NOT stored to a significant degree. Peptidases: break peptide bonds in amino acids. Turn amine groups into ammonia → urea cycle for nitrogen removal. Turn carbon skeletons into aketo acids and enter citric acid cycle for energy production. Nitrogen Rich Molecules Dispose of Excess Nitrogen Nitrogen rich molecules like dispose excess nitrogen through: ammonia, urea, and uric acid. ● Ammonia : make ammonia and get rid of ammonia = ammonotelic animals: fish + amphibia ● Urea: ureotelic animals: Humans + terrestrial vertebrates + sharks ● Uric acid: uricotelic animals. Birds + reptiles Carbon in these molecules is highly oxidized Most of the energy has been extracted. Digestion of Proteins to Amino Acids Low pH activates zymogens Neutralization High pH activates other zymogens Pepsinogen turns intopepsin and breaks peptide bonds. First part of small intestine = breaking peptide bonds under acid environment Then neutralization Then at high pH = alkalization = more peptide bonds broken. Have lots of amino acids that come across microvilli on intestinal wall Parietal cell secrete HCl Chief cell = Pepsinogen Amino Group Catabolism Protein metabolism produces lots of ammonia Amine/ammonia is toxic and metabolically expensive ● Lots of energy needed to acquire new nitrogen ● Lots of energy needed to dispose old nitrogen Strategy ● Transfer amines to carrier molecules for safe transport via blood ● Transfers amines into mitochondria. ● 3 Amino group carriers: Glutamine, Alanine, Glutamate ● Source of most amino groups: amino acids from ingested protein ● Alanine from muscles + Glutamine from muscle + tissues. Aminotransferases All aminotransferases have same prosthetic group: PYRIDOXALPHOSPHATE (PLP) VIT B6 Covalently bound to ALL aminotransferases via a highly conserved Lysine. Glucose: Alanine Cycle Cori Cycle Step 1: liver Transamination = formation of Glutamate Step 2: Liver cytosol = Glutamate transport into mitochondria Step 3: Liver mitochondria: Oxidative Deamination releases ammonia. Step 1: Formation of Carbamoyl Phosphate in Mitochondria Phosphoryl ON Phosphate OFF Bicarbonate is from respiration Enzyme: carbamoylphosphate synthetase I → Urea cycle Urea Cycle Liver disposal of NH4+ Both cytosol and mitochondria Urea is transferred into Bloodstream → kidney → Urine Carbamoyl phosphate + Ornithine to citrulline. ATP transfers AMP = adenylation. This makes citrullyl AMP intermediate. Makes argininosuccinate by using asparatate Aspartate is product of aminotransferase reaction. Argininosuccinate cleaved + releases fumarate and arginine Arginine then releases urea and forms ornithine. AspartateArgininosuccinate Shunt: Use of Fumarate Urea cycle makes fumarate. When citrulline turns into argininosuccinate it releases fumarate. Fumarate turns into Malate Malate goes through antiport transporter and enters mitochondria Malate goes in, alphaketoglutarate comes out. (malatealpha ketoglutarate antiporter) Malate hits the citric acid cycle. Chapter 19: Oxidative Phosphorylation and Photophosphorylation Overview These 2 processes provide most ATP for most biological reactions for most organisms. Oxidative Phosphorylation ● Catabolism of carbs, lipids, amino acids converge on cellular respiration ● Involved reduction. (Electrons donated by NADH and FADH2) ● Happens in mitochondria and bacterial membranes. Photophosphorylation ● This process by which sunlight is captured and used to drive ATP synthesis ● ABSOLUTELY REQUIRES LIGHT. ● Involves oxidation of water to O2 (NADP+ is final e acceptor) ● Happens in chloroplasts Oxidative Phosphorylation and Photophosphorylation Similar Mechanisms 1. Both promote electron flow through a chain of membrane bound carriers 2. Both utilize e flow to produce uphill proton transport across a membrane impermeable to H ● Coupling conserves free energy of oxidation as an electrochemical potential. 3. The flow of protons across a membrane down the concentration gradient provides free energy for ATP synthesis ● Couples proton flow to ADP phosphorylation. Brief Review of Universal Electron Carriers Oxidative phosphorylation begins with electron transfers ● Dehydrogenases collect electrons from catabolic pathways and transfer them to ○ Nicotinamide nucleotides (NAD+ and NADP+) ○ Flavin nucleotides (FAD and FMN) ○ Electron Transferring Flavoprotein ● NADH and NADPH DO NOT cross the INNER mitochondrial membrane. The Universal Electron Carriers NAD and FAD/FMN Ubiquinone (Coenzyme Q) ● Lipid solublelong isoprenoid side chain ○ Remains in the lipid bilayer ○ Very mobile ○ Shuttles electrons between carriers ● Acceptors one electron (semiquinone radical) or two (ubiquinol) Cytochromes Contain ironheme groups Tightly, but noncovalently, associated with the protein Most are integral proteins of the inner mitochondrial membrane 3 classes: ● Iron protoporphyrin IX (b type cytochromes) ● Heme C (c type cytochromes) ● Heme A (a type cytochromes) The Universal Electron Carriers IronSulfur Proteins ● Iron associated with inorganic sulfur or Cys sulfur (not heme sulfur) ● Participate in one electron transfers ● RieskeIron Sulfur proteins: iron coordinates with 2 His instead of 2 Cys. Side Note on the Universal Electron Carriers The respiratory chain: electrons move from NADH, Succinate, or other donors to: ● Flavoproteins ● Ironsulfur proteins ● Ubiquinone (coQ) ● Cytochromes (and copper centers) ● Molecular oxygen (final e acceptor) Summary of Electron Transport Chain I: NADH dehydrogenase ● Prosthetic groups: FMN, FeS II: Succinate dehydrogenase ● Prosthetic groups: FAD, FeS III: Ubiquinone:cytochrome c oxidoreductase cytochrome c ● Cytochrome c Prosthetic groups:= Heme ○ Cytochrome c is not part of enzyme complex, it moves between complexes III and IV as freely soluble protein. ● Ubiquinone: cyt c oxidoreductase Prosthetic groups:= hemes, FeS IV: Cytochrome oxidase ● Prosthetic groups: Hemes; CuA and CuB. 4 MultiEnzyme Complexes Complex I: NADH: ubiquinone oxidoreductase ● Carrier from NADH to ubiquinone ● Uses one flavoprotein FMN ● 42 polypeptides ● 6 different iron centers. ● Embedded in the membrane ○ One domain extends into matrix to interact with soluble NADH (arm) ● Catalyzes two coupled reactions ○ 1. Electron transfer drives ○ 2. Proton movement ○ 1. Transfers a hydride (from NADH) and a matrix H to ubiquinone (exergonic) ○ 2. Transfers 4 protons through the inner membrane per pair of electrons ○ Invest 2 electrons, move 4 protons. ● Two electrons go through complex I, 4 protons go to matrix. Complex II: Succinate Dehydrogenase ● Carrier from succinate to ubiquinone ● REVIEW: Only membrane bound enzyme in the Kreb’s cycle. ● 4 proteins + 2 different prosthetic groups ○ One proteon has FAD (covalent) ○ One protein has 1 FeS center. ● Other entry points pass electrons to ubiquinone (not through complexes I or II) ○ (Enoyl coA) First step of betaoxidation passes electrons from the substrate (acylcoA) to ETF ○ Completed electron transfer to coenzyme Q ■ FAD → Electron transfer flavoprotein → ETF: ubiquinone oxidoreductase → Ubiquinone Q ○ Glycerol3 Phosphatefrom triglyceride metabolism or reduction of dihydroxyacetone phosphate (glycolysis) ■ Oxidized by GAPDH (outer surface of inner membrane) ■ Dehydrogenase channels electrons to ubiquinone (Q) via FAD. Complex III: Cytochrome bc1 (or ubiquinone:cytochrome c oxidoreductase) ● Carrier from ubiquinone to cyt c ● Transports 2 protons through inner membrane ● One FeS center ● 4 total hemes. ● Specific passage of electrons through complex III is known ○ Electron path in other complexes is not known. ○ Switch from 2 electron carrier (NADH, FADH2, Q) to 1 electron carriers (cytochromes, Cu) ○ Function: ■ 4 H+ move through the membrane for every pair of electrons passed to cytochrome c (2 needed) ■ Cytochrome c passes the electrons to complex IV Complex IV: Cytochrome Oxidase ● Carrier from cytochrome c to O2. ● Oxygen is final e acceptor ● Reduces 1 O2 to 2H2O using 4 H+ from matrix and 4 e ● 2 complete Q cycles ● Composed of functional subunits ○ FeS centers ○ Cu centers ○ Hemes Balance Sheet Passing two electrons from NADH through the respiratory chain NADH + H + ½ O2 = NAD+ + H2O 2 e from NADH 1 H2O from ½ O2. Balance Sheet Net reaction using NADH is highly exergonic RexOx pairs: ● NAD+/NADH ΔE = 0.32 V ● O2/H2O ΔE = 0.816 V ● Total E = 1.14 V ● ΔG’º =n F ΔE’º ● = (2) (96.5 kJ/V x Mol) (1.14V) = 220 kJ/mol of NADH Represents standard free energy changes, not transformed standard free energy changes, which are higher. The net reaction using the succinate/fumarate transfer yields ● ΔE’º= 0.031 V and ΔG’º = 150 kJ/mol The net energy from both reactions is mostly used to pump H+ from the matrix. ● For each electron pair ○ 4 H + pumped out by Complex I ○ 4H + pumped out by Complex III ○ 2H+ pumped by Complex IV ○ 10H+ total using NADH Review: Plant Mitochondria Alternative NADH Oxidation Mechanism In dark/low illumination plants supply ATP from mitochondria In light: plan: Gly to Ser to obtain NADH in mitochondria 2 glycine + NAD+ = serine + NADH + H + CO2 + NH4 Counterproductive: reaction is not controlled by need ● Plants produce NADH even they do not need to produce ATP ● Excess NADH must be oxidized to produce a pool of NAD+ ○ Electrons are transferred from NADH to ubiquinone and then directly to O2. ○ Produces NAD+ ○ Complexes III and IV are not used. ATP Synthesis Proton Motive Force provides energy to drive ATP synthesis The force from the H+ gradient moving downhill ADP + Pi → ATP required 50 kJ of free energy ATP synthesis results from coupling H+ flux to phosphorylation. Some inhibitors block the passage of electrons to O2. ● Blocks ATP synthesis ● Known as: Uncoupling Oxidative Phosphorylation Opposite is also true: blocking ATP synthesis blocks electron transfer ● Oligomycin nhibits ATP synthase and blocks electron transfer to O2. Uncoupling of Phosphorylation from Electron Transfer ● FCCP, DNP, Valinomycin, Thermogenin ● Thermogenin: in brown adipose tissue, forms proton conducting pores in inner mitochondrial membrane. ● Value of uncoupling movement of protons to ATP synthesis: You need ATP all the time, but you don’t need the same level of ATP all the time. So you can afford to uncouple a few electrons and avoid making tons of ATP. Brown Fat Found in many animals, including humans High concentration of lipids and mitochondria Normally expresses thermogenin ● Uncouples oxidative phosphorylation ● Provides heat in addition to that from normal metabolism Thermogenin Signaling ● Several receptors: UCP1, UCP2 ● UCP1 expressed in brown fat. ● Low temperatures induce signaling through thermogenin. ● Normal human infants have significant brown fat ● High concentration of mitochondria may provide more energy under conditions of high metabolic activity ● Brown fat in human babies and adults may provide heat, but only under low temperature conditions. ● Normal human baby temperature: 97100.4F ● Fever accepted threshold: 100.4F The Two Functional Domains of ATP Synthase Fo: The Respiratory Chain ● Integral membrane protein ● Oligomycinsensitive (“o” in Fo) ● Isolated from mitochondrial membranes (ATP synthase) using detergent treatment ● Stripped membranes contain intact respiratory chain ● Contains a passive proton pore. F1: ATP Synthesis F Type ATPase ● Peripheral membrane protein ● First oxidative factor identified (1 in F1 = first) ● Can be purified away from Fo ● Isolated F1 can catalyze ATP synthesis The ATP Synthase Paradox Catalytic formation of ATP is reversible The Puzzle ● ΔG’º for the reaction approaches 0 kJ/mol ● ΔG’º is expected to be ~ 30.5 kJ/mol The Solution ● Affinity changes, and the terminal phosphoryl oxygen associates and dissociates several times before ATP is released from the enzyme. Reaction favors ATP synthesis because F1/Fo affinity for ATP (10M) is much greater 5 than for ADP (10 M) ● The difference in Kd alone is about 40 kJ/mol ● ATP synthesis requires 3050 kJ/mol Continued synthesis of ATP depends upon ● 1. Cyclic high ATP affinity ● 2. Low ADP affinity Structure of the F1/Fo Complex F1 contains 9 subunits ● 5 different onesα3 3 δε ● Alternating alpha and beta subunits ● Beta subunits have the catalytic site. Functional differences are dictated by β and γ subunit structures γ subunit binds only 1 β subunit at a time β subunits are all in different conformations ● Each one binds different things on a cyclic basis ● One β subunit doesn’t bind anythin β empty ● One β subunit binds ADP = β ADP ● One β subunit binds ATP = βATP Rotational Catalysis Model ATP Synthesis Csubunits of Fo rotate in the plane of the membrane ● Due to passive H transport Movement of protons causes ● Association of γ subunit with one of the 3 β subunits ● ATP is made One rotation = all 3 β subunits through all 3 conformations One rotation = synthesis of 3 ATP If there are 12 csubunits, then every 4H transfers = 1 ATP. What is the relationship between electrons transported and ATP molecules synthesized? A history lesson Assumptions were: number of electrons moved AND number of ATP molecules formed were whole numbers (integers) Values of these numbers are derived from P/O ratios P/O ratio: Phosphates transferred (P) to Oxygens oxidized (O) P/O ratios for NADH and Succinate were 3 and 2. Since Chemiosmotic theory has been accepted ● Key values are: ○ Protons pumped out per pair of electrons transferred ○ NADH = 10 Succinate = 6. ● Number of protons required to drive the synthesis of 1 ATP = 4: Two get ADP, ATP and Pi through the membrane. ● New protonbased P/O ratios: ○ NADH (2.5) (old was 3) 10/4 ○ Succinate (1.5) (old was 2) 6/4 ProtonMotive Force: Transfer of Phosphorylation Reactants/Products Transport mechanisms move and ADP/Pi into the matrix and ATP out of the matrix. 1. Adenine Nucleotide Translocase (Antiport) 3 4 ● Exchanges ADP for ATP in the intermembrane space ● A charge difference: one more negative charge is transported out than in ● Proton motive force drives ATP/ADP exchange. 2. Phosphate Translocase (Symport) ● Moves 1 H and 1 H2PO4 into the matrix: A symport ● This proton decreases the electrochemical gradient NADH Shuttles: NADH & Oxaloacetate Moved Where They Are Needed Problem ● NADH and OA are produced in the cytosol ● They are required in the mitochondrial matrix in high concentration ● NADH dehydrogenase is in the mitochondrial matrix ● Mitochondrial membranes are impermeable to NADH, NAD+, and OA Solution ● Move reducing equivalents and carbon skeletons through mitochondrial membranes. NADH Shuttles: MalateAspartate Shuttle 1. Reducing equivalents of NAD are transferred to OA producing malate (via cytosolic malate dehydrogenase) 2. Malate moves through inner mitochondrial membrane via the malateaketoglutarate transporter (an antiport) 3. Reducing equivalents are passed from malate to NAD+ in the matrix (via matrix malate dehydrogenase) (2 pools of same enzyme) 4. OA and NADH are regenerated NADH Shuttles: Glycerol 3Phosphate Shuttle Delivers NADH reducing equivalents through glycerol 3P Cytosolic ● Glycerol 3P dehydrogenase ● Glycerol 3P passes the electrons directly from NADH to FAD Mitochondrial ● Glycerol 3P dehydrogenase ● Moves electrons from FADH2 to Q 2 pools of same enzyme Reduced FADH2 passes the electrons to Complex III (not to Complex I) Summary of Metabolic ATP Synthesis → Regulation of Oxidative Phosphorylation: Regulation According to Cellular Energy Needs 1. Complete oxidation of 1 glucose produced 3032 ATP ● Anaerobic glycolysis produces 2 ATP ● Complete oxidation of one palmitoylcoA produces 108 ATP 2. Aerobic oxidation and transfer of electrons to O2 produces the vast majority of ATP at a rapid rate ● Production of ATP is regulated to fit fluctuating metabolism needs 3. Cellular respiration (O2 oxidation) is limited by [ADP] called ACCEPTOR CONTROL. Cellular needs can be estimated by using the mass action ratio ● Defined as [ATP]/[ADP] = product/reactant ● In resting conditions the ratio is very high (Most of the AxP is maximally phosphorylated ● Under high energy consumption = ATP levels drop; ratio drops proportionately ● BIG PICTURE: The ratio actually changes very little, indicating the high sensitivity and the rapid response even under extreme conditions. Review: ATP Producing Pathways The ATP/ADP ratio controls ● Oxidative Phosphorylation ● Citric Acid Cycle ● Glycolysis ● Pyruvate Oxidation ● Most control is FEEDBACK INHIBITION As ATP hydrolysis increases, so does ● Electron transfer ● Pyruvate oxidation ● ATP synthesis ● These events increase glycolysis and [pyruvate] All increase rapidly and proportionately . Summary of regulation mechanism Pathways are not single sets of reactant and products Pathways obtain reactants from any source ATP synthesis is primarily controlled by: ● ATP/ADP ratio ● NADH/NAD+ ratio ● Feedback inhibition ● Multiple levels of control Chapter 19: Photophosphorylation Photophosphorylation Photophosphorylation: process by which sunlight drives electron transfers, powering ATP synthesis using a H+ gradient. Involvesoxidation of H2O to O2 ● NADP+ is the final electron acceptor. The general reaction: CO2+ H2O = O2 + Carbohydrate Absolutely requires light. H2O is NOT a good electron donor (like NADH) E = +0.82V (large positive E = more likely to be reduced = gain electrons not donating electrons) ● Sunlight assists in creating a good electron donor ● Electrons flow through a series of membrane associated electron carriers ● Protons are pumped across a membrane to create an electrochemical gradient (potential) ○ Similar to Complex III of Oxidative Phosphorylation ● The electrochemical potential drives ATP synthesis ○ ATP Synthase Complex catalyzes the phosphorylation. Photosynthesis has Two Phases Light dependent: Photophosphorylation ● Energy from light is absorbed chlorophyll and other pigments ● Energy is eventually conserved as NADPH and ATP ● O2 is produced. Carbon Assimilation/Carbon Fixation (Not Dark) ● Driven by the products of the light reactions ● NADPH and ATP reduce CO2 to a new triose ● “New” carbon + existing C coupled to production of starch, triose phosphates, carbs. Electron Flow in Chloroplasts Chloroplasts reduce electron receptors in the presence of light according to Hill Reaction ● 2H2 + 2A → 2AH 2+ O2 ● A is an electron acceptoHill reagen) ● This implies that water is the electron donor ● Final electron acceptor (A) in chloroplast = NADP+ + ● 2H2 + 2NADP → 2NADPH + O 2 Light Absorption Visible light electromagnetic radiation ● Wavelengths: 400 700 nm (violet to red) ● One mole of photons of visible light = 170300 kJ ○ ADP + Pi → ATP requires 3050 kJ ● Conclusion: light has enough energy to do the job. Chromophores: Light absorbing molecules with different energy states Absorbing Light Energy ● Absorbing a photon of light raises the chromophore to the next level ● Photon absorption requires exactly enough energyexcito) to raise the energy state (an lectronic transition st) ○ Molecule is in aexcited stat ○ Relatively unstab. Chromophores: Light Absorbing Molecules with Different Energy States Releasing the energy ● A chromophore (pigment) returns to ground state when energy is released as heat, light, or biological work. Emitted lightfluorescence) is longer wavelength (less energy) than that which was absorbed. Photosynthetic Pigments Chlorophylls (Primary Pigments) ● Green pigments in the thylakoid membranes ● Contain polycyclic, planar rings resembling hemes + phytol side chains. ○ Mg2+ is at the center of the ring in coordination bonds ● Strongly absorb light in the visible spectrum, due to alternating singledouble bond ring structure. ● Different chlorophylls (a and b) absorb nonoverlapping different wavelengths of light. Light Harvesting Complexes Chlorophylls are always associated with binding proteins Chlorophyll + binding protein = light harvesting complexes (LHCs) ● Proteinsstabilizthe chlorophyll in 3D space Energy transfer requires contact between pigments, binding proteins, and membrane components ● Example: LHCII ○ 7hlorophyll a(purple) ○ 4hlorophyll b(green) ○ 2Lutein (another pigment) Phycobilins In cyanobacteria and red algae Utilize a ring (not cyclic) structure (no heme, no central Mg2+) (chlorophylls use rings + cyclic structure, unlike phycobilins) Extended polyene chains are similar to chlorophylls. Covalently linked to specific binding proteinsphycobiliproteins Phycobiliproteinsrganize in complexesphycobilisomes ● Serve as light gathering structures ● Light energy is used by the reaction center ● What is this reaction? Accessory (Secondary) Pigments Carotenoids ● Present in thylakoid membranes ● Yellow, red, or purple Examples: ● Βcarotene: redyellow ● Lutein : yellow Absorb light outside the range of chlorophylls ● Carotenoids and chlorophylls are complementary Chlorophylls Channel Energy From Sunlight to Reaction Centers Photosystems: functionallyarranged pigments ● All pigments can absorb light ● Only a few chlorophylls transduce light energy into chemical energy ● Associated withphotochemical reaction centers. ● Rest of pigment molecules transmit light energy to reaction centers. ● Light harvesting (antenna)molecules. Exiton Transfer: The Photophosphorylation OxidationReduction Reaction Chlorophyll in solution absorbs sunlight energy ● Loses it rapidly as heat or fluorescence Light harvesting chlorophyll moleculesin reaction center) efficiently transfer energy (very little loss) 1. One molecule absorbs a photon of light (exciton) and is excited 2. Energy is randomly transferred to a neighboring light harvesting molecule, exciting it ● First one returns to the ground state Process continues asexciton transfe until exciting a specialized pair of chlorophyll a at the reaction center. The Oxidation/Reduction Reaction Energy passed to chlorophyll a in reaction center causes 1 e to be promoted to next orbital. This electron is passed to electron acceptor molecule ● Part of the plaelectron transport chain ● Leaves an electron hole in donor chlorophyll a ● Electron acceptor gains a negative charge Electron is replaced through transfer from a neighboring electron donor. Donor becomes positively charged. Conclusion: reaction centers are the source of electrons passed down the transport chain. Bacterial Photochemical Reaction Centers PheophytinQuinone Reaction Centers (Type II) In purple bacteria Electrons pass through heophytin: ● Chlorophyll without central Mg2+ Contains 3 molecules 1. One reaction center P870) 2. Cytochrome bc1 electron transfer complex (similar to mitochondrial complex III) 3. ATP synthase. Mechanism ● Light → electron transfer to pheophytin → (semiquinone intermediatelike mitochondria) → Electron transfer to a quinone ● Product isQB 2similar tQH2f Complex III) ● Quinone passes electrons to cytochrome bc1 complex ● Electrons flow back to the reaction ce =yclic pathway ○ Ready to start another round PheophytinQuinone Reaction Centers (Type II) Structure of the complex indicates rapid exchange in channelfashion. Electrons provide energy for proton movement via cytochrome bc1 complex Resulting proton gradient powers ATP production by the ATP synthase. Bacterial Photochemical Reaction Centers Iron Sulfur Reaction Centers (Type I) Found in green sulfur bacteria Electron transfer similar to Type II = Result is ATP synthesis Reaction center’s lost electrons are replaced by oxidati2 f H Mechanistic Differences from Type II ● Some electrons can pass from the reaction centferredoxin (ironsulfur protein) ● Ferridoxin passes electrons ferridoxin NAD reductas = produces NADH ● Iron sulfur complex contains2 ○ Oxidized to HSO2 4 ○ This reaction is characteristic of these bacteria. Thermodynamics of Bacterial Reaction Centers Key element of reaction centers ● Electron transfer is carefully controlled by precise structural arrangements of interacting molecules. ● Little energy is lost as heat as a result of structural control ○ Heat is produced, but not from inefficiency. ● Reactions proceed with extreme speed ○ Essentially no energy is stored in intermediates. ● The resulting energy gain is significant ○ ΔE = 0.95V and ΔG = 180 kJ/mol ● Overall energy yield is 30% ● Most of the rest is lost as heat ● Kinetics indicate these reactionnearly irreversib. Higher Plant Reaction Centers Plant thylakoid two distinct reaction center types ● They areomplementary ● They resemble the two bacterial systems Each chloroplast has hundreds of each system in thylakoid membranes. Photosystem II (PSII) ● Pheophytinquinone type ● P680 reaction center ● Moves protons across the thylakoid membrane. Photosystem I (PSI) ● Ferridoxin Type ● P700 reaction center ● Reaction center passes electrons to ferridoxin (FeS protein) ● Electrons from ferridoxin reduce NADP+ Reaction Centers Act in Tandem to Move Electrons From H2O to NADP+ Using Sunlight Plastocyanin oves electrons between PSII and PSI (one electron at a time) H2O is oxidized to O2 ● Replaces electrons moving between photosystem Calledxygenic photosynthesis Bacteria:anoxygenic photosynthesis. Higher Plant Reaction Centers Electrons flow from H2O to NADP+ ● 2H2O + 2NADP+ + 8 photons = O2 + 2NADPH + 2H+ ● For every 2 photons absorbed, one electron is transferred to NADP+ PSI and PSII are Physically Separated PSII inrana stacks PSI and the ATP Synthase are in unstacked stromal thylakoids Associations of PSII and PSI is regulated by ● Sunlight ● Protein phosphorylation Both have access to stroma (NADP+, ADP, Pi, water) Cytochrome b6f is uniformly distributed. PSII = grana PSI/ATP Synthase = unstacked thylakoid membranes Cytochrome b6f = uniformly distributed. Second Link Between PSII and PSI: Cytochrome b6f Complex and the Q Cycle 3 components: 1. Cytochrome b 2. FeS protein 3. Cytochrome f Another Q cycle (as in mitochondria) Electrons pass one at a time from quinone to cytochrome b6. Result: proton pumping as electrons are transported. Cytochrome b6f Complex The proton pump ● Protons move from stroma to the thylakoid lumen ● 4 net H move per pair of electrons ● Develops an electrochemical gradient The volume of the space in chloroplasts is small ● Moving a small number of protons has a large effect ● Result: 3 unit pH difference = 1000 fold difference in [H+] Charge and concentration differences drive ATP synthesis = electrochemical gradient. Final Piece of the Model: Splitting Water by the OxygenEvolving Complex of PSII How is the electron donated by P680 replaced? Water is the source of the electrons that end up in NADPH ● Bacteria use acetate, succinate, malate, and sulfide as alternative electron donors. ● Overall reaction: 2H2O = 4H + 4 e + O2. ● 4 photons of light required to split 2 waters. Electrons pass from water to P680 one at a time from the oxygen evolving complex . ● 4 Mn2+ ions become more oxidized with each of the 4 electrons passed. Mn2+ complex takes 4 e from 2H2O ● This releases 4H and one oxygen radical ● The 4H are released into the thylakoid lumen ● Proton pump is driven by e transfer. Lumen: site of high [H] ATP Synthesis in Photophosphorylation PSII and PSI combine to transfer electrons from water to NADP+ ● Energy is conserved in NADPH These reaction are coordinated with ● Production of an electrochemical potential ● Proton pump in the thylakoid membrane This electrochemical gradient drives ATP synthesis. An Electrochemical Gradient is Coupled to ADP Phosphorylation Mechanism for ATP synthesis is analogous to that in mitochondria ● ATP synthesis is catalyzed by CF1/CFo complexes ● Outer surface of thylakoid membranes ● ATP is produced by rotational catalysis. Balance Sheet for Photophosphorylation: We have a model but all is not known About 12H move from the stroma to the thylakoid lumen per 4 electrons passed (per O2 formed) ● 4 H by the Oxygen Evolving Complex ● Up to 8 H by the cytochrome b6f complex Electrochemical potential is derived from ● 3 pH unit difference across the thylakoid membrane ● Most of the the electrical potential is lost due to counterion movement ○ Unlike mitochondria = little is lost. Energy stored in the proton gradient per mole of protons: ΔG = 17 kJ/mol ● 12 moles of protons translate into 200 kJ ● Enough to drive the synthesis of several ATP ● ATP yield is about 3 per mole of O2 (experimental value) Lipid Synthesis Synthesis of Fatty Acids Lipid synthesis is unlike carbohydrate synthesis 1. Lipid synthesis versus lipid oxidation ● Different enzymes ● Different pathways ● Different parts of the cell 2. A 3 carbon intermediate malonylcoA) is involved in synthesis but not in oxidation. ● NOTE :MethylmalonylcoA is produced by oxidation of acyl chains with odd numbers of carbons. Fatty Acid Synthesis: Fundamental Strategy Acetyl coA + MalonylcoA ● 2C + 3C = 4C ● Decarboxylation removes 1 C as CO2. ● Continue by adding 2C at a time. ○ RequiresMalonyl CoA “Activated” ○ Requiresfatty acid syntha, a large complex. Synthesis of MalonylCoA Acetyl CoA + Bicarbonate → Malonyl CoA ● 2C + 1C → 3C Catalyzed by AcetylCoA carboxylase ● Biotin s a prosthetic group ● Covalently attached toLysine ● COO is transferred to biotin (requires ATP) ● Then to Acetyl coA. Overview of the 4 Repeated Steps That Add 2C 3C + 2C = Step 1 Step 2 = Reduction Step 3 = Dehydration (loss of water Step 4 = Reduction Fatty Acid Synthesis: Overview Each round adds 2C (net) and requires 4H+ and 4 e ● Product is palmitate (16:0) ● 4 e and 4H added per step ● Release requires one water. Fatty Acid Synthase: A Complex With 7 Different Active Sites 7 polypeptides and 3 accessory proteins All intermediates remain covalently associated with complex ● Channeling ● Covalent attachment to 2 thiol groups 1. Ketoacyl Synthase (KS) 2. Acyl Carrier Protein (ACP) ● May serve as a flexible arm ● Thioester hydrolysis is highly exergonic ○ Drives thecondensation reaction Fatty Acid Synthase: Charging Both thiols are bound with the correct groups 1. Acetyl group (of acetylcoA) is transferred to Cys of KS 2. Malonyl group (of MalonylcoA) is transferred to the SH of ACP The 4 Steps of Chain Lengthening Step 1: Condensation of Acetyl + Malonyl Groups → AcetoacetylACP ● Acetoacetyl bound to SH o CP pantothenate. ● Actually aβketobutyrylACP ● CO2 produced: originally from HCO3 ● Same CO2 is attached and removed = Makes overall reaction favored. Step 2: Reduction of the Carbonyl Group ● Carbonyl of Acetoacetyl coA is reduced → Dβhydroxybutaryl ACP ● Electron/proton donor is NADPH ● ANABOLIC Step 3: Dehydration ● H2O is produced from groups removed from C2 and C3 ● Produces a double bond: : transbutenoylACP (4carbons) Step 4: Reduction of Double Bond ● Product: a butyrylACP ● NADPH is electron/proton donor (reducing agent) Formation of Palmitate By Repeating the 4 Reaction Butyryl (4C) group is then transferred From the SH group of ACP to the SH group of KS Formation of Palmitate: Recharging and Repetition Another malonyl group is added to ACP (recharging) The four reactions repeat, using the existing butyryl group ● Result: a 6C product 7 total rounds of condensation yield: palmitoylACP Palmitate is released (16:0) ● Mechanism not known ● A hydrolysis reaction using 1 H2O releases palmitate from ACP Balance Sheet for Palmitate Synthesis The overall reaction is costly: 8 AcetylcoA + 7 ATP + 14 NADPH + 14H → Palmitate + 8 CoA + 6H2O + 7 ADP + 7 Pi + 14 NADP+ ATP used for adding CO2 to acetyl coA NADPH used for reducing double bonds. One water is used to release palmitate. Cellular Location of Fatty Acid Synthesis Occurs in cytosol of many organisms ● Same as amino acid, glucose, and nucleotide synthesis ● Segregates these anabolic processes from the catabolism in mitochondria Occurs in stroma of chloroplasts ● The key reactant NADPH is
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