The Krebs cycle adds a 2-carbon molecule to a 4-carbon molecule to produce a 6-carbon molecule. If we wanted to create an alternative cycle that uses 10-carbon input molecules, what could the rest of the Krebs cycle intermediates be?
Lab 7: Understanding Cellular Respiration
LAB GOALS:
- To understand the logic of the process of cellular respiration.
- To conquer the fear and loathing of navigating these biochemical processes & pathways!
OUTLINE:
- Overview and Introduction
- Glycolysis
- Krebs Cycle
- Electron Transport and Oxidative Phosphorylation
- Putting it all together!
INTRODUCTION:
Today we are going to use simple problem-solving techniques to figure out how our cells can dismantle the glucose molecule to provide energy in the form of ATP. Although it can look daunting at first, these processes actually follow a logical progression that can be deciphered without too much agony.
STOW AWAY ALL YOUR NOTES & DEVICES! USE LOGIC ONLY FOR THIS ACTIVITY!
PART 1: Glycolysis
Obtain a GLYCOLYSIS packet for your group. This packet should contain:
- 9 Green Cards
- 4 Yellow ATP Cards
- 1 Yellow NAD Card
(It’s a good idea to check before you start that you have all the necessary cards.)
On the mitochondrion mat, order the green cards to show the 9 steps of glycolysis. This is a problem-solving exercise, not a memorization test. You should be able to order the green cards without any prior knowledge of glycolysis solely by elimination and chemistry logic. The final solution will make sense with the arrows on the laminated map. You can start adding the yellow cards where they make sense.
NOTE: Biological chemistry is driven by carbon and phosphate. If you try to keep track of hydrogen or oxygen, these reactions will drive you crazy! Why? Because we are working in an aqueous environment and parts of water molecules are enveloping all the reactants almost all of the time. Focus on carbon!
When you arrive at a Glycolysis pathway that you think is correct, ask your instructor to check your progress. If you have not yet arrived at the correct pathway order, keep working until you do! When you arrive at a correct pathway, do a ‘speed round’ by mixing up the cards and trying to recreate the correct solution as quickly as possible. Less than 20 seconds is good. (The reason we do this is not to encourage speed-memorization, but to practice logic at high speeds, which is a different method for challenging our brain to make sense of a complicated pathway).
When you have finished, discuss the following questions briefly with your group. Answers are located on the last page of this guide.
Glycolysis Discussion Questions:
- In step 3, there is an arrow between reactant and product. Does this arrow signify an enzyme?
- There are two ATP-spending cards in this process, and two ATP-creating cards. Is this process ‘ATP neutral’?
- [A really hard thought question] Which of the enzymes in Glycolysis do you think evolved earliest?
After you have finished with the Glycolysis packet, replace all of the cards in the packet and exchange it for a Linking Step & Krebs Cycle Packet.
PART 2: Arranging the Krebs Cycle and Linking Step
Check to make sure your Linking Step & Krebs Cycle Packet contains:
- 12 Red cards of varying sizes
- 6 Yellow cards (1 ATP, 4 NADH and 1 FADH2)
Now that you’ve practiced some problem-solving with the Glycolysis Packet, you are ready for an even more difficult challenge. Starting with pyruvate (which you might recognize as the 3-carbon molecule that was the end result of glycolysis) you should attempt to place the cards in order through the Linking Step and the Krebs Cycle.
You’ll need to create a Krebs Cycle that can be repeatedly loaded with input molecules. Notice that there is a circular pathway that is ‘fed’ by the linking step. The molecules you chose to be the reactants in the step that starts this circular pathway need to correspond to the larger molecule that starts the cycle. In other words, what you put in should correctly start the series of reactions. When you find the correct solution, this will make sense.
At some points in this cycle, redox reactions will take place. Occasionally, we need to determine whether a redox reaction has occurred between organic molecules that have the same number of carbons. We have a specific algorithm for this:
- Count the C-H bonds in the reactant.
- Count the C-C bonds in the reactant. Double bonds count as 2 C-C bonds.
- Add these numbers together to get the ‘Reactant High Energy Bond Number.’
- Count the C-H bonds in the product.
- Count the C-C bonds in the product. Double bonds count as 2 C-C bonds.
- Add these numbers together to get the ‘Product High Energy Bond Number.’
- If the Reactant High Energy Bond Number does not equal the Product High Energy Bond Number, then a redox reaction has occurred.
Does this algorithm make sense to you? This is a formal way of deciding whether the energy state of a molecule has changed simply by counting the bonds that tend to have more energy in them (C-C and C-H). You may need this algorithm to determine where to place certain yellow cards in this packet.
NOTE: The electron-carriers FADH2 and NADH can be tricky. Each carries a hydrogen and 2 electrons. Each is reduced to their energy-carrying form by oxidizing carbon. The energy levels of these two carriers are slightly different (as we will see in the Electron Transport Chain). However, there is no logical reason that you should be able to determine which redox step is FADH2 rather than NADH. Your instructor will help you with this detail. This is due simply to a difference in the enzyme used to catalyze that step.
Use of the lower-output FADH2 may be a way to protect against poisons or mutations that might damage NADH usage (an internal redundant system). It may also be vestigial; we may simply be in the process of evolving towards use only of higher-energy NADH and this is the last enzyme that has yet to make the evolutionary switchover. Like many evolutionary questions, we don’t know for sure (and the scope of this question is a bit beyond this class).
CoA-containing intermediates have a high-energy S-C bond. In one place, this high-energy bond is used to create an important C-C bond. In another place, CoA-C energy is released in a catalyzed reaction that produces GTP. GTP is of similar energy to ATP, and is converted quickly to that more common energy currency.
When you arrive at a Krebs Cycle that you think is correct, ask your instructor to check your progress. If you have not yet arrived at the correct pathway order, keep working until you do! When you arrive at a correct pathway, do a ‘speed round’ with the Krebs Cycle. Less than 25 seconds is good.
When you have finished, discuss the following questions briefly with your group. Answers are located on the last page of this guide.
Linking Step and Krebs Cycle Discussion Questions:
- Give a one-sentence basic summary of the purpose of the Krebs Cycle.
- The Krebs cycle adds a 2-carbon molecule to a 4-carbon molecule to produce a 6-carbon molecule. If we wanted to create an alternative cycle that uses 10-carbon input molecules, what could the rest of the Krebs cycle intermediates be? (There are many possible answers.)
- What is the major difference in the linking step between prokaryotes and eukaryotes? In which type of cell is the linking step probably more difficult to carry out?
After you have finished with the Linking Step & Krebs Cycle Packet, replace all the cards and exchange it for an Electron Transport & Oxidative Phosphorylation Packet.
PART 3: Re-Enacting the Electron Transport Chain
Make sure your Electron Transport & Oxidative Phosphorylation Packet contains:
- 8 Blue or Purple cards of varying sizes
- 3 Yellow Cards
- A handful of small H+ and e- cards
You’ve already recreated the processes that have led to the production of reducing equivalents in the form of NADH and FADH2. In this third packet, you’ll demonstrate the use of those electrons to produce a proton gradient which will be used to create ATP.
Begin by placing the complexes in the mitochondrial membrane of your laminated eukaryotic cell. NADH drops off two electrons and a proton at Complex I, while FADH2 electrons and protons are added to Complex II. Both Complex I and II can then transfer electrons to a slightly lower energy state on Coenzyme Q. Electrons move from higher energy states to lower energy states from this mobile protein Q to Complex III to mobile protein Cytochrome C to Complex IV. At Complex IV, electrons are transferred to the extremely low energy electron acceptor O2.
During this process, the energy of the electrons is used to transport protons to the inner mitochondrial membrane at Complexes I, III and IV.
NOTE: In prokaryotes, this process occurs in the cellular membrane. While essentially the same, the key difference is that prokaryotes are pumping protons into the outer environment, ending up with a net gradient inside the cell that is relatively low in protons, pulling protons back into the cell through ATP Synthase.
Demonstrate the flows of electrons, protons and the changing ATP/ADP molecules as the process continues first for NADH, and then repeat the demonstration for FADH2. You should be able to watch the membrane gradient build up and then recover. All group members should feel comfortable leading this demonstration and talking through the process. When you feel comfortable with your understanding of this process, demonstrate it for your instructor with input from all group members. When your instructor is confident in your understanding, move on to the following discussion questions.
Electron Transport & Oxidative Phosphorylation Discussion Questions:
- Why are electrons delivered via NADH more valuable than those delivered to the ETC via FADH2?
- What is the role of oxygen in cellular respiration?
- How are the ETC complexes arranged in the inner mitochondrial membrane?
After you have finished with the Electron Transport & Oxidative Phosphorylation Packet, replace all the cards and put it back in the piles with the other packets.
