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 1 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 2 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 3 Close-up of crista, showing proton flow ATP synthase ? the inside ? crista + ADP + + + + + ATP + + What about E. coli? Its cell membrane houses all components 4 ATP synthase (or the F0F1 complex) MW = ~500,000 the inside H+ outside a c F0 ε γ b α β F δ 1 the outside inside Gamma subunit acts as a cam Gamma subunit is inserted inside the αβs 5 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. 6 7 not top view ATP synthase β fixed fixed α α inside β β α 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. 8 Movie link 9 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 10 Outside Mitochondria Inside 11 Is it really a motor? Actin molecules Attach a big arm Detach the C-subunits 10 nm 10 total length =~1 micron 12 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. (1997) TIBS 22, 420-423 Hiroyuki Noji, Ryohei Yasuda, Masasuke Yoshida & Kazuhiko Kinosita Jr. (1997) Direct observation of the rotation of F1-ATPase. Nature 386, 299 - 302. 13 Run reaction in reverse: add ATP drive counter-clockwise rotation of cam Here the driving motor (c) has been cut away from the cam (γ) 4 3 2 1 5 ATP hydrolysis Start here counter-clockwise Blue subunit= gamma subunit, cam rotation driven by αβ subunits. Notice the cam is driven to rotate counterclockwise 14 The arm can be seen! Extrapolate to zero load: 6000 rpm! 100 rps so ~300 ATP/sec http://www.colum bia.edu/cu/biology/ courses/c2005/mo vies/fof1_rot_2700 nm.mpg 15 More numbers: ATP accounting For each pair of electrons given up by NADH: • Each of the 3 ETC complex (I, III, IV) pumps enough H+ ions to allow the formation of 1 ATP. • So 3 ATPs per pair of electrons passing through the full ETC. • So 3 ATPs per 1/2 O2 • So 3 ATPs per NADH2 • But only 2 ATPs per FADH2 (skips complex I) 16 Nelson and Cox, Principles of Biochemistry 17 More exergonic ∆Go for no fumarate+NADH2 succinate + NAD fumarate formation succinate + FAD with FADH2 than with NADH yes H2 fumarate + FADH2 ) 2 ATP ) O (relative to ATP mol ATP generated by the ’ (kcal/ ° G Free energy change energy Free ATP synthetase is ATP ∆ called is oxidative phosphorylation, or oxphos. 18 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 Total: 17 ATP 1 ATP (GTP) 5 NADH = 15 ATP from Krebs 1 FADH2 = 2 ATP Grand total (E. coli): 17 + 2 = 19 per ½ glucose or 38 per 1 glucose 19 Cellular location: eukaryotes CYTOPLASM MITOCHONDRIA In bacteria, the ETC is in the plasma membrane. 20 ATP accounting • 38 ATP/ glucose in E. coli • 36 ATP/glucose in eukaryotes • Cost of bringing in the electrons from NADH from glycolysis into the mitochondrion = 1 ATP per electron pair • So costs 2 ATPs per glucose, subtract from 38 to get 36 net. 21 Efficiency • 36 ATP/ glucose, worth -7 X 36 = -252 kcal/mole of glucose o • ∆G for the overall reaction glucose + 6 O2→ 6 CO2 + 6 H2O: -686 kcal/ mole • Efficiency = 252/686 = 37% • Once again, better than most gasoline engines. • and Energy yield: 36 ATP/ glucose vs. 2 ATP/glucose in fermentation (yet fermentation works) • So with or without oxygen, get energy from glucose 22 Alternative sources of carbon and energy Shake = milk: milk sugar = lactose = disaccharide = glucose – galactose beta-galactosidase +HOH → glucose + galactose glucose → glycolysis, etc. galactose (3 enzymatic steps) glucose 23 Alternative sources of carbon and energy Bun = starch = poly-alpha-glucose G-1-P → G-6-P glycolysis 24 Alternative sources of carbon and energy Lettuce = cellulose = polysaccharide Poly-beta glucose →| (stays as the polysaccharide) We have no enzyme for catabolizing cellulose 25 Alternative sources of carbon and energy French fries = fat (oil) = triglyceride 26 (Triglyceride) Lipases (hydrolysis) Glycolysis (at DHAP) 27 Glycerol as an alternative sole carbon and energy source for E. coli ATP +NAD + NADH2 DHAP glycerol glycerol phosphate (dihydroxy acetone phosphate) - O glycolysis 2 +O ? 2 CO2 + H2O 28 and ADP + Pi ATP Glycerol + ATP → glycerol phosphate → DHAP NAD → NADH2 29 ATP +NAD + NADH2 DHAP glycerol glycerol phosphate (dihydroxy acetone phosphate) glycolysis - O2 +O2 Glycerol cannot be fermented. CO2 + H2O E. coli CANNOT grow on glycerol in the absence of air These pathways are real, and they set the rules. 30 Stoichiometry of chemical reactions must be obeyed. No magic is involved. Fatty acid catabolism (oxidation) O || CH3-(CH2)n-CH2-CH2-C-OH ATP + Coenzyme A-SH O || CH3-(CH2)n-CH2-CH2-C-CoA + HOH FAD FADH2 FAD FADH2 O || CH3-(CH2)n-CH=CH-C-CoA etc. +HOH OH O | || CH3-(CH2)n-CH-CH2-C-CoA NAD NADH2 O O || || CH3-(CH2)n-C-CH2-C-CoA + CoA Krebs Cycle O O || || CH3-(CH2)n-C-CoA + CH3-C-CoA 31 Fatty acido -2 Acetyl-CoA Alternative sources of carbon and energy Hamburger = protein Proteases (e.g., trypsin) → → 20 AAs Stomach acid (pH1) also helps by denaturing protein making it accessable to proteolytic attack Each of the 20 AA’s has its own catabolic pathway, and ends up in the glycolytic or Krebs cycle pathways But first, the N must be removed: 32 Handout 9-2 Deamination and transamination of amino acids Transamination Alanine alpha-keto-glutaric acid Pyruvate Glutamic acid Oxidative de-amination HOH NAD NADH 2 Glutamic acid alpha-keto-glutaric acid 33 E.g., degradation of phenylalanine (6 steps) Phe builds up and gets metabolized to an injurious product (phenyl pyruvate) PKU (phenylketonuria) transaminase Products = Fumaric acid → Krebs and Acetoacetate → 2 Acetyl-CoA → Krebs 34 You are what you eat Catabolism Anabolism ATP STARCH glucose GLYCOGEN GLYCOLYSIS ATP pyruvate ATP FATS acetyl-CoA FATS KREBS O.A. -K.G. AMINO ACIDS ATP ATP AMINO ACIDS E.T.C. NADH 2 NAD PROTEINS ATP PROTEINS O2 H2O 35 Handout 9-2 monomers Flow of glucose in E. coli Macromolecules Polysaccharides Lipids Nucleic Acids Proteins intermediates Each arrow = a specific chemical reaction glucose biosynthetic pathway 36 Handout 9-2 You are what you eat Catabolism Anabolism ATP STARCH glucose GLYCOGEN GLYCOLYSIS ATP pyruvate ATP FATS acetyl-CoA FATS KREBS O.A. -K. G. AMINO ACIDS ATP ATP AMINO ACIDS E.T.C. NADH2 NAD PROTEINS ATP PROTEINS O 2 H2O 37 Handout 9-2 Biosynthesis of monomers E.g., • Fatty acids (acetyl CoA from Krebs cycle) • Amino acids (Serine: 3-phospho-glyceric acid from glycolysis) 38 Fatty acid biosynthesis O || CH3-(CH2)n-CH2-CH2-C-OH Repeated addition of 2-carbon units (acetyl-CoA molecules via this same set of reactions) O || Handout 9-1 CH3-CH22 -CH -C-CoA NADPH NADP Start at bottom 2 O || CH3-CH= CH-C-CoA -HOH OH O | || CH3-C-CH -C-CoA H 2 NADPH2 NADP O O || || + CoA CH3-C-CH2-C-CoA Krebs Cycle O O || || CH -C-CoA + CH -C-CoA 3 3 39 Acetyl-CoA Acetyl-CoA Handout 9-1 right Handout 9-3 Phosphoester group (Glycolytic intermediate) Glutamate is the amino donor hydrolysis Handout 9-3 40 41 Biosynthesis of macromolecules monomers Flow of glucose in E. coli Macromolecules Polysaccharides Lipids Nucleic Acids Proteins intermediates Each arrow = a specific chemical reaction glucose biosynthetic pathway 42 Biosynthesis of macromolecules 1) Lipids 2) Polysaccharides 3) Proteins 43 Triglyceride (fat) biosynthesis DHAP NADPH (dihydroxy- Glycerol phosphate acetone phosphate) O + Fatty acid 1 || -C-(CH ) -CH3 O 2 x || -C-(CH ) -CH3 O 2 x || -C-(CH ) -CH3 + Fatty acid 2 2 y Phosphatidic acid O + Fatty acid 3 || -C-(CH ) -CH3 2 x O || -C-(CH2)y-CH3 Phospholipid O || -C-(CH ) -CH3 2 z Triglyceride 44 Handout 9-3 45 Polysaccharide: Hyaluronic acid n 46 Polysaccharide synthesis Hyaluronic acid (joint lubricant) N-acetyl-glucosamine Glucuronic acid COO- Enz.1 Enz. 2 Enz.1 + Enz. 2 Enz.1 Enz. 2 Enz.1 Enz. 2 Enz.1 Enz. 2 Enz.1 Hyaluronic acid (polysaccharide) via ~2 enzymes 47 Biosynthesis of proteins e.g., an enzyme like hexokinase: met-val-his-leu-gly ….. If this done like lipids and polysaccharides, we need an enzyme for each linkage First an enzyme that will condense val to met to make met-val.