AP Biology AP Biology John D. O’Bryant School of Mathematics and Science October 24, 2012

AP Biology Agenda  Do Now: HW Review  Cellular Respiration: Lecture/Discussion, Lab  Quiz

AP Biology Cellular Respiration Stage 1: Glycolysis

AP Biology What’s the point? The point is to make ATP ! ATP

AP Biology Glycolysis glucose      pyruvate 2x2x 6C3C In the cytosol? Why does that make evolutionary sense? That’s not enough ATP for me!  Breaking down glucose  “glyco – lysis” (splitting sugar)  ancient pathway which harvests energy  where energy transfer first evolved  transfer energy from organic molecules to ATP  still is starting point for ALL cellular respiration  but it’s inefficient  generate only 2 ATP for every 1 glucose  occurs in cytosol

AP Biology Evolutionary perspective  Prokaryotes  first cells had no organelles  Anaerobic atmosphere  life on Earth first evolved without free oxygen (O 2 ) in atmosphere  energy had to be captured from organic molecules in absence of O 2  Prokaryotes that evolved glycolysis are ancestors of all modern life  ALL cells still utilize glycolysis You mean we’re related? Do I have to invite them over for the holidays? Enzymes of glycolysis are “well-conserved”

AP Biology 10 reactions  convert glucose (6C) to 2 pyruvate (3C)  produces: 4 ATP & 2 NADH  consumes: 2 ATP  net yield: 2 ATP & 2 NADH glucose C-C-C-C-C-C fructose-1,6bP P-C-C-C-C-C-C-P DHAP P-C-C-C G3P C-C-C-P pyruvate C-C-C Overview DHAP = dihydroxyacetone phosphate G3P = glyceraldehyde-3-phosphate ATP 2 ADP 2 ATP 4 ADP 4 NAD + 2 2Pi2Pi enzyme 2Pi2Pi 2H2H 2

AP Biology Glycolysis summary endergonic invest some ATP exergonic harvest a little ATP & a little NADH net yield 2 ATP 2 NADH 4 ATP ENERGY INVESTMENT ENERGY PAYOFF G3P C-C-C-P NET YIELD like $$ in the bank -2 ATP

AP Biology PiPi 3 6 4,5 ADP NAD + Glucose hexokinase phosphoglucose isomerase phosphofructokinase Glyceraldehyde 3 -phosphate (G3P) Dihydroxyacetone phosphate Glucose 6-phosphate Fructose 6-phosphate Fructose 1,6-bisphosphate isomerase glyceraldehyde 3-phosphate dehydrogenase aldolase 1,3-Bisphosphoglycerate (BPG) 1,3-Bisphosphoglycerate (BPG) 1 2 ATP ADP ATP NADH NAD + NADH PiPi CH 2 CO CH 2 OH PO CH 2 OP O CHOH C CH 2 OP O CHOH CH 2 OP O OP O P O H CH 2 OH O CH 2 P O O CH 2 OH P O 1st half of glycolysis (5 reactions) Glucose “priming”  get glucose ready to split  phosphorylate glucose  molecular rearrangement  split destabilized glucose

AP Biology 2nd half of glycolysis (5 reactions) Payola! Finally some ATP! 7 8 H2OH2O 9 10 ADP ATP 3-Phosphoglycerate (3PG) 3-Phosphoglycerate (3PG) 2-Phosphoglycerate (2PG) 2-Phosphoglycerate (2PG) Phosphoenolpyruvate (PEP) Phosphoenolpyruvate (PEP) Pyruvate phosphoglycerate kinase phosphoglycero- mutase enolase pyruvate kinase ADP ATP ADP ATP ADP ATP H2OH2O CH 2 OH CH 3 CH 2 O-O- O C PH CHOH O-O- O-O- O-O- C C C C C C P P O O O O O O CH 2 NAD + NADH NAD + NADH Energy Harvest G3P C-C-C-P PiPi PiPi 6 DHAP P-C-C-C  NADH production  G3P donates H  oxidizes the sugar  reduces NAD +  NAD +  NADH  ATP production  G3P    pyruvate  PEP sugar donates P  “substrate level phosphorylation”  ADP  ATP

AP Biology Substrate-level Phosphorylation P is transferred from PEP to ADP kinase enzyme ADP  ATP I get it! The P i came directly from the substrate! H2OH2O 9 10 Phosphoenolpyruvate (PEP) Phosphoenolpyruvate (PEP) Pyruvate enolase pyruvate kinase ADP ATP ADP ATP H2OH2O CH 3 O-O- O C O-O- C C C P O O O CH 2  In the last steps of glycolysis, where did the P come from to make ATP?  the sugar substrate (PEP) ATP

AP Biology Energy accounting of glycolysis  Net gain = 2 ATP + 2 NADH  some energy investment (-2 ATP)  small energy return (4 ATP + 2 NADH)  1 6C sugar  2 3C sugars 2 ATP2 ADP 4 ADP glucose      pyruvate 2x2x 6C3C All that work! And that’s all I get? ATP 4 2 NAD + 2 But glucose has so much more to give!

AP Biology Is that all there is?  Not a lot of energy…  for 1 billon years + this is how life on Earth survived  no O 2 = slow growth, slow reproduction  only harvest 3.5% of energy stored in glucose  more carbons to strip off = more energy to harvest Hard way to make a living! O2O2 O2O2 O2O2 O2O2 O2O2 glucose     pyruvate 6C 2x2x 3C

AP Biology 7 8 H2OH2O 9 10 ADP ATP 3-Phosphoglycerate (3PG) 3-Phosphoglycerate (3PG) 2-Phosphoglycerate (2PG) 2-Phosphoglycerate (2PG) Phosphoenolpyruvate (PEP) Phosphoenolpyruvate (PEP) Pyruvate ADP ATP ADP ATP ADP ATP H2OH2O NAD + NADH NAD + NADH PiPi PiPi 6 Glycolysis glucose + 2ADP + 2P i + 2 NAD +  2 pyruvate + 2ATP + 2NADH But can’t stop there!  Going to run out of NAD +  without regenerating NAD +, energy production would stop!  another molecule must accept H from NADH  so NAD + is freed up for another round PiPi NAD + G3P 1,3-BPG NADH NAD + NADH PiPi DHAP raw materials  products

AP Biology NADH pyruvate acetyl-CoA lactate ethanol NAD + NADH NAD + NADH CO 2 acetaldehyde H2OH2O Krebs cycle O2O2 lactic acid fermentation with oxygen aerobic respiration without oxygen anaerobic respiration “fermentation” How is NADH recycled to NAD + ? Another molecule must accept H from NADH recycle NADH which path you use depends on who you are… alcohol fermentation

AP Biology Fermentation (anaerobic)  Bacteria, yeast 1C 3C2C pyruvate  ethanol + CO 2  Animals, some fungi pyruvate  lactic acid 3C  beer, wine, bread  cheese, anaerobic exercise (no O 2 ) NADHNAD + NADHNAD + back to glycolysis 

AP Biology recycle NADH Alcohol Fermentation 1C 3C2C pyruvate  ethanol + CO 2 NADHNAD + Count the carbons!  Dead end process  at ~12% ethanol, kills yeast  can’t reverse the reaction bacteria yeast back to glycolysis 

AP Biology recycle NADH  Reversible process  once O 2 is available, lactate is converted back to pyruvate by the liver Lactic Acid Fermentation pyruvate  lactic acid 3C NADHNAD +  Count the carbons! O2O2 animals some fungi back to glycolysis 

AP Biology Pyruvate is a branching point Pyruvate O2O2 O2O2 mitochondria Krebs cycle aerobic respiration fermentation anaerobic respiration

AP Biology What’s the point? The point is to make ATP ! ATP

AP Biology H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ And how do we do that? ATP But… Have we done that yet? ADP P +  ATP synthase  set up a H + gradient  allow H + to flow through ATP synthase  powers bonding of P i to ADP ADP + P i  ATP

AP Biology NO! There’s still more to my story! Any Questions?

AP Biology 10 reactions  convert glucose (6C) to 2 pyruvate (3C)  produces: 4 ATP & 2 NADH  consumes: 2 ATP  net: 2 ATP & 2 NADH glucose C-C-C-C-C-C fructose-1,6bP P-C-C-C-C-C-C-P DHAP P-C-C-C G3P C-C-C-P pyruvate C-C-C Overview ATP 2 ADP 2 ATP 4 ADP 4 NAD Pi2Pi 2Pi2Pi 2H2H

AP Biology Cellular Respiration Stage 2 & 3: Oxidation of Pyruvate Krebs Cycle

AP Biology pyruvate       CO 2 Glycolysis is only the start  Glycolysis  Pyruvate has more energy to yield  3 more C to strip off (to oxidize)  if O 2 is available, pyruvate enters mitochondria  enzymes of Krebs cycle complete the full oxidation of sugar to CO 2 2x2x 6C3C glucose      pyruvate 3C1C

AP Biology Cellular respiration

AP Biology intermembrane space inner membrane outer membrane matrix cristae Mitochondria — Structure  Double membrane energy harvesting organelle  smooth outer membrane  highly folded inner membrane  cristae  intermembrane space  fluid-filled space between membranes  matrix  inner fluid-filled space  DNA, ribosomes  enzymes  free in matrix & membrane-bound mitochondrial DNA What cells would have a lot of mitochondria?

AP Biology Mitochondria – Function What does this tell us about the evolution of eukaryotes? Endosymbiosis! Dividing mitochondria Who else divides like that? Advantage of highly folded inner membrane? More surface area for membrane- bound enzymes & permeases Membrane-bound proteins Enzymes & permeases Oooooh! Form fits function! bacteria!

AP Biology pyruvate    acetyl CoA + CO 2 Oxidation of pyruvate NAD 3C2C 1C [ 2x ]  Pyruvate enters mitochondrial matrix  3 step oxidation process  releases 2 CO 2 (count the carbons!)  reduces 2 NAD  2 NADH (moves e - )  produces 2 acetyl CoA  Acetyl CoA enters Krebs cycle Where does the CO 2 go? Exhale!

AP Biology Pyruvate oxidized to Acetyl CoA Yield = 2C sugar + NADH + CO 2 reduction oxidation Coenzyme A Pyruvate Acetyl CoA C-C-C C-C CO 2 NAD + 2 x []

AP Biology Krebs cycle  aka Citric Acid Cycle  in mitochondrial matrix  8 step pathway  each catalyzed by specific enzyme  step-wise catabolism of 6C citrate molecule  Evolved later than glycolysis  does that make evolutionary sense?  bacteria  3.5 billion years ago (glycolysis)  free O 2  2.7 billion years ago (photosynthesis)  eukaryotes  1.5 billion years ago (aerobic respiration = organelles  mitochondria) 1937 | 1953 Hans Krebs

AP Biology 4C6C4C 2C6C5C4C CO 2 citrate acetyl CoA Count the carbons! 3C pyruvate x2x2 oxidation of sugars This happens twice for each glucose molecule

AP Biology 4C6C4C 2C6C5C4C CO 2 citrate acetyl CoA Count the electron carriers! 3C pyruvate reduction of electron carriers This happens twice for each glucose molecule x2x2 CO 2 NADH FADH 2 ATP

AP Biology So we fully oxidized glucose C 6 H 12 O 6  CO 2 & ended up with 4 ATP! Whassup? What’s the point?

AP Biology  Krebs cycle produces large quantities of electron carriers  NADH  FADH 2  go to Electron Transport Chain! Electron Carriers = Hydrogen Carriers What’s so important about electron carriers? H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ ATP ADP + P i

AP Biology Energy accounting of Krebs cycle Net gain=2 ATP =8 NADH + 2 FADH 2 1 ADP1 ATP ATP 2x 4 NAD + 1 FAD4 NADH + 1 FADH 2 pyruvate          CO 2 3C 3x3x 1C

AP Biology Value of Krebs cycle?  If the yield is only 2 ATP then how was the Krebs cycle an adaptation?  value of NADH & FADH 2  electron carriers & H carriers  reduced molecules move electrons  reduced molecules move H + ions  to be used in the Electron Transport Chain like $$ in the bank

AP Biology What’s the point? The point is to make ATP ! ATP

AP Biology H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ And how do we do that? ATP But… Have we done that yet? ADP P +  ATP synthase  set up a H + gradient  allow H + to flow through ATP synthase  powers bonding of P i to ADP ADP + P i  ATP

AP Biology NO! The final chapter to my story is next! Any Questions?

AP Biology Cellular Respiration Stage 4: Electron Transport Chain

AP Biology Cellular respiration

AP Biology What’s the point? The point is to make ATP ! ATP

AP Biology ATP accounting so far…  Glycolysis  2 ATP  Kreb’s cycle  2 ATP  Life takes a lot of energy to run, need to extract more energy than 4 ATP! A working muscle recycles over 10 million ATPs per second There’s got to be a better way! I need a lot more ATP!

AP Biology There is a better way!  Electron Transport Chain  series of proteins built into inner mitochondrial membrane  along cristae  transport proteins & enzymes  transport of electrons down ETC linked to pumping of H + to create H + gradient  yields ~36 ATP from 1 glucose!  only in presence of O 2 (aerobic respiration) O2O2 That sounds more like it!

AP Biology Mitochondria  Double membrane  outer membrane  inner membrane  highly folded cristae  enzymes & transport proteins  intermembrane space  fluid-filled space between membranes Oooooh! Form fits function!

AP Biology Electron Transport Chain Intermembrane space Mitochondrial matrix Q C NADH dehydrogenase cytochrome bc complex cytochrome c oxidase complex Inner mitochondrial membrane

AP Biology G3P Glycolysis Krebs cycle 8 NADH 2 FADH 2 Remember the Electron Carriers? 2 NADH Time to break open the piggybank! glucose

AP Biology Electron Transport Chain intermembrane space mitochondrial matrix inner mitochondrial membrane NAD + Q C NADH H 2 O H+H+ e–e– 2H + +O2O2 H+H+ H+H+ e–e– FADH NADH dehydrogenase cytochrome bc complex cytochrome c oxidase complex FAD e–e– H H  e- + H + NADH  NAD + + H H p e Building proton gradient! What powers the proton (H + ) pumps?…

AP Biology H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ ATP NAD + Q C NADH H 2 O H+H+ e–e– 2H + +O2O2 H+H+ H+H+ e–e– FADH NADH dehydrogenase cytochrome bc complex cytochrome c oxidase complex FAD e–e– Stripping H from Electron Carriers  Electron carriers pass electrons & H + to ETC  H cleaved off NADH & FADH 2  electrons stripped from H atoms  H + (protons)  electrons passed from one electron carrier to next in mitochondrial membrane (ETC)  flowing electrons = energy to do work  transport proteins in membrane pump H + (protons) across inner membrane to intermembrane space ADP + P i TA-DA!! Moving electrons do the work! H+H+ H+H+ H+H+

AP Biology But what “pulls” the electrons down the ETC? electrons flow downhill to O 2 oxidative phosphorylation O2O2 H2OH2O

AP Biology Electrons flow downhill  Electrons move in steps from carrier to carrier downhill to oxygen  each carrier more electronegative  controlled oxidation  controlled release of energy make ATP instead of fire!

AP Biology H+H+ ADP + P i H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ We did it! ATP  Set up a H + gradient  Allow the protons to flow through ATP synthase  Synthesizes ATP ADP + P i  ATP Are we there yet? “proton-motive” force

AP Biology  The diffusion of ions across a membrane  build up of proton gradient just so H+ could flow through ATP synthase enzyme to build ATP Chemiosmosis Chemiosmosis links the Electron Transport Chain to ATP synthesis So that’s the point!

AP Biology Peter Mitchell  Proposed chemiosmotic hypothesis  revolutionary idea at the time 1961 | proton motive force

AP Biology H+H+ H+H+ O2O2 + Q C ATP Pyruvate from cytoplasm Electron transport system ATP synthase H2OH2O CO 2 Krebs cycle Intermembrane space Inner mitochondrial membrane 1. Electrons are harvested and carried to the transport system. 2. Electrons provide energy to pump protons across the membrane. 3. Oxygen joins with protons to form water. 2H + NADH Acetyl-CoA FADH 2 ATP 4. Protons diffuse back in down their concentration gradient, driving the synthesis of ATP. Mitochondrial matrix 2 1 H+H+ H+H+ O2O2 H+H+ e-e- e-e- e-e- e-e- ATP

AP Biology Cellular respiration 2 ATP ~36 ATP ++ ~40 ATP

AP Biology Summary of cellular respiration  Where did the glucose come from?  Where did the O 2 come from?  Where did the CO 2 come from?  Where did the CO 2 go?  Where did the H 2 O come from?  Where did the ATP come from?  What else is produced that is not listed in this equation?  Why do we breathe? C 6 H 12 O 6 6O 2 6CO 2 6H 2 O~40 ATP  +++

AP Biology  ETC backs up  nothing to pull electrons down chain  NADH & FADH 2 can’t unload H  ATP production ceases  cells run out of energy  and you die! Taking it beyond…  What is the final electron acceptor in Electron Transport Chain? O2O2  So what happens if O 2 unavailable? NAD + Q C NADH H 2 O H+H+ e–e– 2H + +O2O2 H+H+ H+H+ e–e– FADH NADH dehydrogenase cytochrome bc complex cytochrome c oxidase complex FAD e–e–

AP Biology What’s the point? The point is to make ATP ! ATP

AP Biology Do Now (Quiz) 5. During which of the following phases of cellular respiration does substrate-level phosphorylation take place?  A) glycolysis  B) the citric acid cycle  C) "grooming" of pyruvate  D) oxidative phosphorylation  E) glycolysis and the citric acid cycle

AP Biology Do Now (Quiz)  1. Which of the following metabolic pathways is common in aerobic and anaerobic metabolism?  A) the citric acid cycle  B) oxidative phosphorylation  C) chemiosmosis  D) glycolysis  E) electron transport chain

AP Biology Do Now (Quiz)  2. As a result of glycolysis there is a net gain of ________ ATPs.  A) 0  B) 1  C) 2  D) 4  E) 36

AP Biology Do Now (Quiz)  3. Which of the following is a result of glycolysis?  A) conversion of FAD to FADH2  B) production of CO2  C) conversion of glucose to two three-carbon compounds  D) a net loss of two ATPs per glucose molecule  E) conversion of NADH to NAD+

AP Biology Do Now (Quiz)  4. A culture of bacteria growing aerobically is fed glucose containing radioactive carbon and is then examined. During the citric acid cycle, radioactivity would first appear in  A) NADH.  B) citrate.  C) FADH2.  D) oxaloacetic acid.  E) CoA. 

AP Biology Do Now (Quiz)  5. At the end of the citric acid cycle, most of the energy remaining from the original glucose is stored in  A) CO2.  B) pyruvate.  C) ATP.  D) NADH.  E) FADH2. 

AP Biology Do Now (Quiz)  6. Which kind of metabolic poison would most directly interfere with glycolysis?  A) an agent that reacts with oxygen and depletes its concentration in the cell B) an agent that binds to pyruvate and inactivates it C) an agent that closely mimics the structure of glucose but is not metabolized D) an agent that reacts with NADH and oxidizes it to NAD+ E) an agent that blocks the passage of electrons along the electron transport chain

AP Biology Do Now (Quiz)  7. How does pyruvate enter the mitochondrion?  A) active transport B) diffusion C) facilitated diffusion D) through a channel E) through a pore

AP Biology Lab: Alcoholic Fermentation in Yeast

AP Biology Lorenzo’s Oil