Download PBIO*3110 – Crop Physiology Lecture #14 Respiration I – Molecular

PBIO*3110 – Crop Physiology Lecture #14 Fall Semester 2008 Lecture Notes for Thursday 23 October Respiration I – Molecular Basis of Respiration How do cells extract biochemical energy from sugars? Learning Objectives 1. Know the functions of glycolysis, the Kreb’s Cycle and mitochondrial electron transport. 2. Understand the parallels between the various phases of aerobic respiration and the analogous phases of oxygenic photosynthesis. Introduction Over the past several lectures, we have been considering the process of photosynthesis at the chloroplast, leaf, and whole­canopy levels. While photosynthesis provides all of the chemical energy and carbon necessary for crop growth, the actual rate of dry matter accumulation is determined by the balance between photosynthesis and respiration. In fact, it has been estimated that typically 50% of all carbon fixed by photosynthesis of field crops is lost to respiratory processes before harvest. Thus, respiration is a very significant portion of the total carbon balance, and altering the efficiency of respiration has the potential to substantially alter the growth and productivity of a crop.
1 In this lecture, we will review the biochemical basis of respiration, and see how chemical energy stored by photosynthesis is eventually made available to cells through the processes of glycolysis, the Kreb’s cycle, and mitochondrial electron transport. Over the next two lectures, we will examine in detail how respiratory processes affect the carbon balance of entire crop canopies. Respiration Defined Aerobic respiration involves the oxidation of complex organic molecules to simpler substances. In the process, energy is released ­ some in the form of heat, but a significant percentage in the form of chemical potential energy which can then be used by the cell in biosynthetic reactions. We will examine the oxidation of glucose in our consideration of plant respiration. The complete oxidation of glucose can be summarized as: 6 C6H12O6 + 6 O2 ! 6 CO2 + 6 H2O + energy Note that this equation could also apply to simple "burning" of glucose, in which case all of the energy would be released as heat (and perhaps light). Respiration in living cells allows for the controlled oxidation of carbohydrates and other substances, so that much of the energy can be retained in a useable form, such as ATP. Glycolysis The first steps of the respiration of glucose occur not in the mitochondrion, but in the cell cytosol. The process of glycolysis (literally, "sugar splitting"), results in the conversion of one molecule of glucose (6C) to two molecules of pyruvate (3C). The main steps of glycolysis are summarized in the following figure. Note that two ATP are used in the early stages of the pathway, but that four ATP and two NADH are made in the latter stages of the pathway; thus, the overall process can be summarized as: GLUCOSE + 2 NAD + 2 ADP + 2 Pi ! 2 PYRUVATE + 2 NADH + 2 ATP + 2 H2O No O2 is consumed and no CO2 is released in glycolysis.
2 The reactions of glycolysis (simplified). All reactions below the dashed line are multiplied by two. You may recognize some of the intermediates of the glycolytic pathway from the Calvin cycle, and from the C4 photosynthetic pathway. It is important to realize that glycolysis occurs in the cytosol, where many other biochemical pathways also exist. Thus, the substrate pools (and enzymes) of glycolysis are shared with other processes, and "carbon­skeletons" may be exported from glycolsis at any point to enter other pathways. The functions of glycolysis are, therefore: 1. partial oxidation of glucose to produce a small amount of NADH 2. production of a small amount of ATP 3. conversion of one molecule of glucose to two molecules of pyruvate, which can be further oxidized in the Kreb’s cycle to produce additional NADH and ATP 4. provision of carbon skeletons that can be used by other biochemical pathways.
3 The Mitochondrion The remaining steps of respiration of glucose occur in the mitochondrion. Before considering these reactions in detail, it is useful to consider the general structure of the mitochondrion itself. Structural Features A typical mitochondrion is a few µm long (similar in size to some bacteria), and most plant cells contain several hundred mitochondria. Mitochondria in many ways resemble chloroplasts, both structurally and functionally. The entire organelle is enveloped in an outer membrane. There is also an inner membrane, which at multiple points is invaginated towards the center of the organelle. Each such invagination is known as a crista (pl. cristae). The area between the two membranes, including the interiors of the cristae, is known as the inter­membrane space. The remainder of the interior of the mitochondrion is called the matrix. Left: Structural features of the mitochondrion. Right: A mitochondrion, as seen under an electron microscope. Parallels between mitochondria and chloroplasts Chloroplasts and mitochondria have many important features in common: Just as the thylakoid membrane of the chloroplast contains the various components of the thylakoid electron transport chain, so too the inner membrane of the mitochondrion houses the various electron carriers of the mitochondrial electron transport chain.
4 The physical separation between the inter­membrane space and the matrix is important for the energetics of ATP production in the mitochondrion; recall that the separation between the lumen and the stroma of the chloroplast has a similar purpose. In a final parallel, the CO2­evolving reactions of the Kreb’s cycle occur in the matrix of the mitochondrion, just as the CO2­fixing reactions of the Calvin cycle occur in the stroma of the chloroplast. The Kreb's Cycle This series of reactions occurs in the matrix of the mitochondrion. Here, pyruvate donates reducing electrons for the production of NADH and FADH, and chemical energy for the production of ATP. In the process, the three carbons of each pyruvate are released as CO2. A summary of the Kreb’s cycle (also called the citric acid cycle, and the tricarboxylic acid (TCA) cycle) is shown below. The Kreb's Cycle (simplified).
5 The overall process may be summarized as: PYRUVATE + 4 NAD + FAD + ADP + Pi + 2 H2O ! 3 CO2 + ATP + 4 NADH + 2 FADH Here, we see the source of CO2 evolved during aerobic respiration; however, still no O2 has been consumed. We will see that the consumption of O2 results from the final stage of respiration, the mitochondrial electron transport chain. The Kreb’s cycle, like glycolysis, shares certain substrates with other important biochemical pathways, including amino acid synthesis. The main functions of the Kreb’s cycle are, therefore: 1. Oxidation of pyruvate to CO2, and reduction of NAD and FAD to NADH and FADH 2. Production of a small amount of ATP 3. Provision of carbon skeletons for other biochemical pathways, including amino acid synthesis. Mitochondrial Electron Transport and Oxidative Phosphorylation The mitochondrial electron transport chain provides a means of using the reducing potential of NADH and FADH to produce ATP. Electrons are donated from NADH at the beginning of the electron transport chain (ETC), and are finally accepted by O2 at the end of the ETC, along with two protons, to produce H2O. Note that this is essentially the opposite process that occurs in the light reactions of photosynthesis, where electrons are abstracted from H2O and used to reduce NADP to NADPH. In photosynthesis, the electrons move from a molecule with very low reducing potential (H2O) to one with high reducing potential (NADPH); this is energetically unfavourable, and thus requires an energy input from light. In the mitochondrial ETC, the opposite is occurring; that is, the electrons are moving down a reducing potential gradient, and no energy input is required. Reducing electrons from succinate (see Kreb’s Cycle) are passed to FAD at a different point in the chain, but ultimately are also used to reduce O2 to H2O. At several points in the ETC, protons are moved across the inner membrane from the matrix into the inter­membrane space. The resulting trans­membrane pH gradient can be used to generate ATP, as the protons return to the matrix via the ATPase. Thus, ATP is phosphorylated in a process that utilizes O2 as the terminal electron acceptor, giving rise to the term "oxidative phosphorylation". This reaction is the reason for consumption of O2 in aerobic respiration.
6 Oxidation of NADH results in more protons being moved across the membrane than does oxidation of succinate; thus NADH oxidation by the electron transport chain produces more ATP than does succinate oxidation via FADH. The mitochondrial electron transport chain. Also, recall that some NADH produced during respiration of glucose ­ that released during glycolysis ­ is produced outside the mitochondrion. This NADH can also be oxidized by the electron transport chain to produce ATP, but this also yields fewer ATP than oxidation of NADH from the Kreb's cycle. This is because there is an energy cost associated with moving NADH from the cytosol into the mitochondrial matrix. Considering this in more detail, it is generally assumed that as a pair of electrons passes through a site of proton translocation (i.e., one of the complexes), it results in the translocation of exactly enough protons to drive the phosphorylation of one ADP molecule. The number of molecules of ADP phosphorylated in the formation of ATP per atom of oxygen reduced in the formation of water is called the P/O ratio. As can be seen from the figure below, the P/O ratio for mitochondrial NADH is 3 if electrons pass through Complexes I, III and IV. The P/O ratio for FADH 2 and cytosolic NADH is 2 because proton translocation does not occur at Complex II and, consequently, electrons pass only through Complexes III and IV. The actual P/O ratios, however, may be lower than these theoretical estimates.
7 The potential energy yield of the complete oxidation of a glucose molecule by the reactions of glycolysis and the TCA cycle, coupled to oxidative phosphorylation, is 36 ATP molecules. This results from:
· 6 ATP molecules in glycolysis: o 2 ATP directly o 4 ATP following NADH oxidation via the ETC, each with a P/O ratio of 2
· 30 ATP molecules in the Krebs cycle: o 2 ATP directly o 24 ATP via mitochondrial NADH oxidation, each with a P/O ratio of 3 o 4 ATP from the 2 FADH 2, each with a P/O ratio of 2 This theoretical yield of respiration may be overestimated – the actual yield is likely closer to 30 ATP per glucose.
8 9