Intro Bio Lecture 9 1 Characteristics of metabolic pathways Aside from its role in energy metabolism: Glycolysis is a good example of a metabolic pathway. Two common characteristics of a metabolic pathway, in general: 1) Each step = a small chemically reasonable change 2) The overall DGo is substantial and negative. 2 dihydroxyacetone phosphate fructose-1,6- glucose-6- fructose-6- diphosphate phosphate phosphate glyceraldehyde-3-phosphate Glycolytic pathway, Glycolysis glucose 1,3-diphosphoglyceric acid phosphoenol-pyruvic 2-phosphoglyceric 3-phosphoglyceric acid acid acid yeast lactic acetaldehyde ethanol acid pyruvic acid oxidative pathways Handout 8A 3 Fermentation goes all the way to the right Efficiency: glucose--> 2 lactates, without considering the couplings for the formation of ATP's (no energy harnessing): Δ Go = -45 kcal/mole kcal/mole Out of this comes 2 ATPs, worth 14 kcal/mol. 31 kcal/mole output as heat. So the efficiency is about 14/45 = ~30% Not bad at all. Since 2 ATPs ARE produced, taking them into account, for the reaction: Glucose + 2 ADP + 2 Pi → 2 lactate + 2 ATP ΔGo = -31 kcal/mole (45-14) Very favorable. All the way to the right. Keep bringing in glucose, keep spewing out lactate, Make all the ATP you want. 4 Energy yield But all this spewing of lactate turns out to be wasteful. Using oxygen as an oxidizing agent glucose could be completely oxidized, to: … CO2 That is, burned. How much energy released then? Glucose + 6 O2 → 6 CO2 + 6 H2O DGo = -686 kcal/mole ! Compared to -45 for glucose → 2 lactates (both w/o ATP production considered) Complete oxidation of glucose, Much more ATP But nature’s solution is a bit complicated. The fate of pyruvate is now different 5 From glycolysis: acetyl-CoA 6 pyruvic acid Scores: per glucose 2 NADH 2 ATP pyruvic acid 2 NADH 2 CO2 citric acid oxaloacetic acid Krebs cycle Citric acid cycle isocitric acid malic acid Tricarboxylic acid cycle TCA cycle fumaric acid a keto glutaric acid succinic Handout 9A acid 6 Acetyl-OCoA O|| CH3 –C –OH + co-enzyme A → acetyl~CoA acetic acid (acetate) coA acetate group pantothenic acid (vitamin B5) 7 Per glucose FromB glycolysis: acetyl-coA pyruvate Input 2 oxaloacetates 2 NADH 2 ATP pyruvic acid 2 NADH 2 NADH 2 CO 2 NADH 2 2 CO2 citric acid 2 CO2 oxaloacetic acid 6 CO2 Krebs Cycle isocitric acid malic acid fumaric acid a keto glutaric acid succinic acid 8 GTP is energetically equivalent to ATP GTP + ADP → GDP + ATP ΔGo = ~0 G= guanine (instead of adenine in ATP) 9 acetyl-CoA B Per 10glucose 2 oxaloacetate 2 NADH 2 ATP pyruvic acid 2 NADH 2 NADH 2 ATP 2 NADH 2 FADH2 citric acid 2 NADH oxaloacetic acid 2 CO2 2 CO2 Krebs cycle 2 CO2 isocitric acid 6 CO malic acid 2 fumaric acid a keto glutaric acid succinic acid 10 FAD = flavin adenine dinucleotide Business end (flavin) ~ Vitamin B2 ribose adenine - ribose FAD + 2H. → FADH 2 11 D acetyl-CoA Per 12glucose oxaloacetate pyruvic acid 2 NADH 2 ATP 2 NADH 2 ATP 2 NADH 2 NADH citric acid 2 FADH2 oxaloacetic acid 2 CO2 2 CO2 Krebs cycle 2 CO isocitric acid 2 malic acid Note label is in OA after one turn of cycle, half the time fumaric acid on top, half on bottom. So a keto glutaric acid no CO2 from Ac-CoA after succinic just one turn. (CO2 in first acid turn is from OA). Succinic dehydrogenase 12 E Per glucose13 2 NADH 2 ATP pyruvic acid 2 NADH 2ATP 2 NADH 2 NADH 2 FADH2 citric acid 2 NADH oxaloacetic acid 2 CO2 2 CO2 Krebs Cycle 2 CO isocitric acid 2 malic acid fumaric acid a keto glutaric acid succinic acid 13 Glucose + 6 O2 → 6 CO2 + 6 H2O : By glycolysis plus one turn of the Krebs Cycle: 1 glucose (6C) → 2 pyruvate (3C) → 6 CO2 √ 2 X 5 NADH2 and 2 X 1 FADH2 produced per glucose 4 ATPs per glucose (2 from GLYCOL., 2 from KC as GTP) NADH2 and FADH2 still must be reoxidized …. No oxygen yet to be consumed No water produced yet Paltry increase in ATP so far Hans Krebs 14 Oxidation of NADH by O2 NADH2 + 1/2 O2 --> NAD + H2O ΔGo = -53 kcal/mole If directly coupled to ADP → ATP (7 kcal cost), 46 kcal/mole waste, and heat So the electrons on NADH (and FADH2) are not passed directly to oxygen, but to intermediate carriers, Each transfer step involves a smaller packet of free negative energy change (release) 15 The electron transport chain (ETC) Iron-sulfur protein NADH2 NAD FMNH2 CoQ FADH2 The electron transport chain NADH Fe+3 FMN CoQH FAD 2 Net: NADH2 + ½ O2 → NAD + H2O Fe+2 Cytb-Fe+3 CoQ +2 +2 Fe Cytb-Fe Cytc1-Fe+3 Fe+3 +2 Cytc-Fe energy ~ Free Cytc1-Fe+2 Cyta-Fe+3 Cytc-Fe+3 +2 Cyta-Fe+2 Cyta3-Fe + Cyta3-Fe+3 O2 + 4H heme 2H2O Ubiquinone, or Coenzyme Q Cytochromes are proteins Up to 50 C’s long 16 H FMN FMNH2 2H+, 2e- R R H Ubiquinone, or heme Coenzyme Q heme Up to 50 C’s long a cytochrome protein with a heme in a pocket 17 Mitochondria and the chemiosmotic theory of oxidative phosphorylation Electron transport chain (ETC) is in the inner membrane. Oxidative phosphorylation is in the lollipops (separate from ETC). The Krebs cycle enzymes are inside the mitochondria, in the matrix. The enzymes of glycolysis are outside the mitochondria, in the cytoplasm. 18 The electron transport chain H+ ions (protons) are pumped out as the electrons are transferred (outside) FADH2→ FAD Complex: I II III IV Nelson and Cox, Principles of Biochemistry 19 Schematic idea of H+ being pumped out Inter-membrane Compartment + + e- + e- + Conformational Relaxation change back (absorbs energy) 20 Outline of Energy Metabolism OXPHOS: 1 NADH from glycolysis Substrate level phosphorylation 1 NADH from Krebs pre-entry (SLP): 2 ATP total: 3 NADH from Krebs 1 ATP from 1 FADH2 from Krebs Glycolysis we recover NAD and FAD by 1 ATP (GTP) ETC (4), but from Krebs WHAT ABOUT ATP (5)? 21 FoF1 Complex: Oxidative phosphorylation (ATP formation) 22 Close-up of crista, showing proton flow the inside 1 pair of NADH electrons going down ATP the ETC → ~10 H+’s pumped out synthase crista + ADP + + + ATP + + + + + + + + + the outside 23 Close-up of crista, showing proton flow ATP synthase the inside crista + ADP + + Pi + + + + ATP + + + + the + + outside ~10 H+’s ~10 H+’s 3 ATP 3 ADP + 3 Pi 1 NAD + 1 H2O 1 NADH2 + ½ O2 24 Close-up of crista, showing proton flow ATP synthase ? the inside ? crista + ADP + + + + + ATP + + What about E. coli? Its cell membrane houses all components 25 Chemiosmotic theory Proton motive force (pmf) Chemical gradient Electrical gradient Electrochemical gradient Water-pump-dam analogy Peter Mitchell 1961 (without knowing mechanism) “The Mitchell Hypothesis” Nobel Prize 1978 26 A test of the chemiosmotic theory Artificial phospholipid membrane H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ ETC Complex I’s +NADH H+ H+ H+ Add NADH H+H+ H+ H+ H+ H+ H+ H+ H+ pH drops 27 A second test of the chemiosmotic theory H+ H+ + + + H+ H H H + + + H+ H+ H H H H+ H+ H+ H+ + H+ + + H H+ H H + + + H H+ H H + H H+ + H + H+ ADP+Pi H H+ + H + H H+ H+ H+ H+ + + H H H+ + + H H H+ 28 H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ ADP + Pi H+ H+ H+ ATP H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ Artificially produced mitochondrial membrane vesicle with ADP and Pi trapped inside 29 ATP is formed from ADP + Pi A third test: Dinitrophenol (DNP): an uncoupler of oxidative phosphorylation - + H+ DNP’s -OH is weakly acidic in this environment. DNP can easily permeate the mitochondrial inner membrane. Outside the mitochondrion, where the H+ concentration is high, DNP picks up a proton. After diffusing inside, where the H+ concentration low, it gives up the proton. So it ferries protons from regions of high concentration to regions of low concentration, thus destroying the proton gradient. Electron transport chain goes merrily on and on, but no gradient is formed and no ATP is produced. 30 Next: How is the ATP actually made (from ADP + Pi) by simply re-entering the mitochondrion? 31 ATP synthase (or the F0F1 complex) MW = ~500,000 the inside H+ outside a c F0 e g b b a F d 1 the outside inside Gamma subunit acts as a cam Gamma subunit is inserted inside the αβs 32 Cryo electron microscopy Math, computational algorithms Joachim Frank 2017 Nobel Prize in Chemistry* Rubinstein JL, Walker JE, Henderson R. Structure of the mitochondrial ATP synthase by *Dept. of Biological Sciences electron cryomicroscopy. The EMBO Journal. Columbia University ☺ 2003;22(23):6182-6192. 33 34 not top view ATP synthase fixed b fixed inside a a b b a http://employees.csbsju.edu/hjakubowski/classe s/ch331/oxphos/olcouplingoxphos.html a subunit c subunit fixed not outside fixed Flow of protons turns the C-subunit wheel. C-subunits turn the gamma cam. 35 Movie link 36 ATP synthase action Start here (top view) alpha+beta ADP Pi + + +H +H +H+ gamma Three conformational states of the α-β subunit: L, T, and O 37 ATP synthase action 38 Outside Mitochondria Inside 39 Is it really a motor? Actin molecules Attach a big arm Detach the C-subunits 10 nm total length =~1 micron10 40 Testing the ATP synthase motor model by running it in reverse (no H+ gradient, add ATP) actin filament Actin labeled Actin is a muscle by tagging it with protein polymer fluorescent molecules Attached to the gamma subunit Add lots of ATP His-tag Junge et al.