LAB TOPIC 8

Cellular Respiration

Laboratory Objectives: 

After completing this exercise, you will be able to 

1. Discuss the concept of oxidation and reduction in cellular metabolism and the  mechanism of action of the coenzymes NAD+, NADP+ and FAD. 
2. Describe the anaerobic catabolism of glucose using the following terms: glucose,  PGAL, pyruvic acid, ethanol, CO₂, lactic acid, NAD+, NADH·H+ (also known as 

NADH or NADH₂ or NAD2H), ADP, ATP 

3. Describe the part of aerobic catabolism of glucose called the Krebs cycle using the terms: acetyl CoA, citric acid, ketoglutaric acid, succinic acid, malic acid, oxaloacetic acid, FAD, FAD2H, GDP, GTP 
4. Describe electron transport and oxidative phosphorylation using the terms: FMN,  Co Q, cytochromes, cytochrome oxidase and oxygen. 
5. Compare substrate phosphorylation and oxidative phosphorylation. 

* Prior to coming to lab, you should read the corresponding chapter in your textbook and this laboratory exercise. 

Introduction

Anaerobic Metabolism

When we think of respiration most of us picture the act of breathing. Respiration, however, occurs at several levels; cellular respiration is performed by the cells of all organisms (bacteria to elephants) and it involves the stepwise, enzymatic catabolism of glucose with the simultaneous release of energy. Some of the energy is "trapped" in the universal biological form -- ATP. The reactions are many and some are quite complex. We will attempt to simplify the process and cull out the information needed to understand cellular respiration at its most basic level. 

The process of cellular respiration can be divided into two groups of reactions; one group requires oxygen and is therefore called aerobic while the second group does not require oxygen and is called anaerobic. The anaerobic reaction produces ethanol and occurs in yeasts.  Let us look briefly at the anaerobic group first. 

The anaerobic reactions involve a stepwise series of chemical interactions in which a glucose molecule is split into two molecules called PGAL (phosphoglyceraldehyde) and the two PGALs are oxidized to pyruvic acid. Note that oxidation can occur without oxygen. Whenever electrons or hydrogens are removed from a molecule, that molecule is oxidized. The molecule that receives the electrons or hydrogens is reduced. Electrons and hydrogen cannot be removed from a molecule without another molecule there to receive them - when something is oxidized, something else must be simultaneously reduced. The cell is equipped with special molecules designed to be hydrogen acceptors and donors and others that are designed to be electron acceptors and donors. NAD+ (nicotinamide adenine dinucleotide - a derivative of the vitamin niacin) is a hydrogen carrier. NAD+ can be reduced to NADH·H+ when it accepts two hydrogens: for simplicity's sake, we will label NAD+ and NADH·H+ as NAD and NAD2H. NAD2H can be oxidized back to NAD when it donates the two hydrogens.

In the reaction above (Figure 8.1) ethanol is oxidized to become acetaldehyde while NAD is reduced to become NAD2H.

In the reaction above (Figure 8.2), pyruvic acid is reduced to become lactic acid, while NAD2H is oxidized to become NAD.

Overall,the anaerobic reactions can be written as below (Figure 8.3).

The reaction above is read: glucose (six carbons) with the expenditure of 2ATPs is split into 2 PGAL molecules (three carbons) - each PGAL, in a series of reactions, is lowered in energy and oxidized into a pyruvic acid. In that series of reactions, 4 ATP molecules are generated (2 for each PGAL) thereby trapping some of the energy released from the glucose molecule. At the same time, each PGAL that is oxidized causes the reduction of an NAD to an NAD2H. You can see that 2 ATPs are used to start the reactions and 4 ATP's are made so there is a net gain of 2 ATP molecules for each glucose molecule metabolized in this way. The 2 ATPs represent less than 5% of the energy available in the glucose molecule. The ATP molecules are quickly used [broken down to ADP and inorganic phosphate (Pi) with the release of the stored energy] to drive all the energy-requiring reactions in the cell: therefore, there is always a supply of ADP and phosphate to keep the anaerobic reactions going. 

As cells metabolize glucose and accumulate pyruvic acid, they also accumulate NAD2H and soon the cell's supply of NAD is exhausted. NAD is available in very limited quantities within the cell. Without NAD, no further oxidations can take place because there is no NAD to be reduced to NAD2H to match with the PGAL and derivatives that need to be oxidized, and the anaerobic reactions would cease. ATP would no longer be generated and the energy-requiring reactions necessary for life would stop and the cell would die. There must be a way of regenerating. NAD from NAD2H. 

Cells have an additional step in which pyruvic acid is reduced by NAD2H. In animal muscle and some microorganisms, pyruvic acid is reduced to lactic acid (known as  lactic acid fermentation) while in some other microorganisms, such as yeast, it is reduced to ethanol and carbon dioxide (alcohol fermentation). 

The anaerobic metabolism of glucose to pyruvic acid is known as glycolysis while the additional steps are known as fermentation. In yeast, glycolysis and alcohol fermentation can be written as follows: 

Glucose ------- > 2PGAL ---------- > 2Pyruvic acid ---------- > 2Ethanol + 2CO2 (+ 2ATP)

                     GLYCOLYSIS                        FERMENTATION

In muscle cells, glycolysis and lactic acid fermentation can be written as follows:

Glucose ------- > 2PGAL ---------- > 2Pyruvic acid ---------- > 2 lactic acid (+ 2ATP)

                   GLYCOLYSIS                          FERMENTATION

Aerobic Metabolism

In most eukaryotic cells, when oxygen is present, pyruvic acid enters the mitochondrion instead of being fermented in the cytoplasm. There a series of reactions known as the Krebs or citric acid cycle and electron transport occurs. These mitochondrial reactions ultimately require oxygen (hence the aerobic name), and the two pyruvic acid molecules (from the one original glucose molecule) are completely disassembled to CO₂ and H₂O. The Krebs cycle reactions convert the carbons and oxygens in pyruvic acid eventually to CO₂ and the hydrogens to NAD2H or FADH2. The electron transport reactions, which occur on the inner mitochondrial membrane, reoxidize the NAD2H to NAD and then pass the hydrogens along in a type of "bucket brigade" ultimately to oxygen to form H₂O. As a result of this hydrogen (electron) pass-along, many ATP molecules are generated. The glucose molecule releases 686,000 calories of energy if it is completely broken down to CO₂ and H₂O.  36 ATP contain 270,000 calories:  thus about 40% of the energy in the glucose molecule is stored as ATP and the rest escapes as heat.

Overall we can visualize cellular respiration as depicted in the following diagram. Note that the electron transport reactions occur on the inner mitochondrial membrane and are precisely ordered spatially - very much like those electron transport reactions involved in photosynthesis. The importance of this three dimensional relationship within the membrane will be discussed in lecture.

Compare the amount of energy stored in ATP from anaerobic vs. aerobic metabolism.

Record your answer on the answer sheet at the end of the Lab Topic ____________a) 

The overall general equation for aerobic respiration is written above.   

Compare the products of the anaerobic metabolism of glucose to the products of the aerobic metabolism of glucose. ______________________________________  b)

EXERCISE 8.1  Anaerobic versus Aerobic Cellular Respiration