1 GENERAL BIOLOGY LAB 1 (BSC1010L) Lab #6: Cellular Respiration OBJECTIVES: • Understand the major events of glucose catabolism (cellular respiration): glycolysis, the citric acid cycle and oxidative phosphorylation. • Compare and contrast the processes involved in aerobic and anaerobic respiration. • Demonstrate carbon dioxide production during anaerobic respiration. • Determine oxygen consumption during aerobic respiration. • Measure the relative production of carbon dioxide by plants and animals. ______________________________________________________________________________ INTRODUCTION: All living organisms have evolved mechanisms to obtain the energy needed to fuel basic biological functions, including growth, metabolism and maintenance. These mechanisms include a series of biochemical reactions, collectively referred to as cellular respiration. During this process, organic molecules (e.g. glucose) are enzymatically broken down, releasing energy that is stored in the negatively charged bonds of adenosine triphosphate or ATP, which is used by cells to perform essential metabolic functions. Energy flow through biological systems occurs through oxidation-reduction or redox reactions where electrons are transferred from one molecule to another. Recall from the Biologically Important Molecules lab (Lab 4) that reduction is defined as the gain of electrons or hydrogen atoms while oxidation involves the loss of electrons or hydrogen atoms. During cellular respiration, electrons are removed from glucose (i.e., glucose is oxidized) and some of the released energy is stored in the form of ATP. The dozens of redox reactions that take place during this process use electron acceptors for energy transfer. Two of the most important electron acceptors are nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD+), derived from niacin (Vitamin B3) and riboflavin (Vitamin B2), respectively. These molecules are reduced to NADH and FADH2 when they acquire electrons, which they then transfer to other molecules to generate ATP. Depending on which molecule serves as the final electron acceptor, the entire process is considered aerobic or anaerobic. In aerobic respiration, the final electron acceptor is oxygen while in anaerobic respiration the final electron acceptor can be inorganic compounds (other than oxygen) such as nitrates and sulfates or organic molecules such as ethanol or lactate (Fig. 1).2 Figure 1. Comparison of redox reactions in aerobic and anaerobic respiration Cellular respiration (Fig. 2) can be divided into four stages: (1) Glycolysis (2) Pyruvate oxidation (3) Kreb’s cycle (4) Electron Transport Chain and Chemiosmosis Figure 2. An Overview of Aerobic Respiration3 Glucose catabolism begins with glycolysis in the cytoplasm (Fig. 3). Glycolysis includes a series of reactions where each entering glucose (6 carbons) molecule is split into 2 molecules of pyruvate (3 carbons). In total, glycolysis yields 4 ATP molecules, however, 2 ATPs are used for the priming reactions that initiate glycolysis. Thus, a net of 2 ATPs are generated for the entire process. In addition, 2 NADH molecules are reduced from NAD+ during this stage. If oxygen is present, then the processes of aerobic respiration will begin with pyruvate oxidation. During this phase, each pyruvate molecule generated from gylcolysis enters the mitochondria and is converted into carbon dioxide (CO2), which is released as a side-product, and acetyl, a 2 carbon sugar that joins with coenzyme A to form acetyl-CoA. More importantly, this process also reduces NAD+ to NADH, which can be used to generate ATP. During aerobic respiration, acetyl-CoA enters the Krebs cycle (also known as the citric acid cycle or tricarboxilic acid (TCA) cycle). For every turn of the Krebs cycle, one ATP molecule is produced and multiple NAD+ and FAD+ molecules are reduced to NADH and FADH2, respectively. The final products of the Krebs cycle per glucose molecule include: 2 ATP, 2 FADH2, 6 NADH, and 4 CO2. In the final stage of cellular respiration (Fig. 3), the electrons carried by FADH2 and NADH are transferred through a series of transmembrane proteins known as the electron transport chain (ETC), creating a proton gradient that is used to drive ATP synthesis (chemiosmosis). Each molecule of NADH yields 3 ATPs while each FADH2 generates 2 ATPs, resulting in an overall production of 32 ATPs in this stage alone! Figure 3. Overview of ETC and Chemiosmosis Conversely, in the absence of oxygen (anaerobic respiration), the pyruvate molecules produced during glycolysis do not enter the Kreb’s cycle but undergo fermentation instead. Without oxygen, pyruvate cannot enter the remaining steps of aerobic respiration and must be utilized differently. In the process, either ethanol or lactic acid is produced. Along the way, NADH that was created in glucose catabolism is oxidized back to NAD+, pyruvate is reduced and broken down, and a small quantity of ATP is produced. There are two main types of fermentation reactions: (1) ethanol fermentation and (2) lactic acid fermentation. Ethanol Mitochondrial matrixIntermembrane spacePyruvate fromcytoplasmNADHAcetyl-CoAFADH2NADHKrebscycleATP2CO2e–e–1. Electrons are harvestedand carried to the transportsystem.2. Electrons provideenergy to pumpprotons across themembrane.H+H+H+O2O212H2O3. Oxygen joins withprotons to form water.+ 2H+H+ATP324. Protons diffuse backin, driving the synthesisof ATP.ATPsynthaseMitochondrial matrixIntermembrane spacePyruvate fromcytoplasmMitochondrial matrixIntermembrane spacePyruvate fromcytoplasmMitochondrial matrixIntermembrane spacePyruvate fromcytoplasmNADHAcetyl-CoANADHNADHAcetyl-CoAAcetyl-CoAFADH2NADHKrebscycleATP2CO2FADH2FADH2NADHNADHKrebscycleKrebscycleATP2ATPATP2CO2e–e–1. Electrons are harvestedand carried to the transportsystem.e–e–e–e–e–e–1. Electrons are harvestedand carried to the transportsystem.1. Electrons are harvestedand carried to the transportsystem.2. Electrons provideenergy to pumpprotons across themembrane.H+H+H+2. Electrons provideenergy to pumpprotons across themembrane.2. Electrons provideenergy to pumpprotons across themembrane.H+H+H+H+O2O212H2O3. Oxygen joins withprotons to form water.+ 2H+O2O212H2O3. Oxygen joins withprotons to form water.O2O212H2OO2O212H2OO212O212H2O3. Oxygen joins withprotons to form water.3. Oxygen joins withprotons