Chapter 9

When an electron is added to a molecule the process is referred to as reduction. A reduced molecule contains more energy than it does in an unreduced state. Usually in biological reactions adding hydrogen has the same effect as adding an electron. So if hydrogen is added then a molecule is being reduced. Oxidation is the reverse of reduction. It involves the removal of an electron (or hydrogen). When a molecule is oxidized it releases energy. (Fig. 9.3)

Usually the processes of oxidation and reduction are coupled in reactions called Oxidation-Reduction (Redox) reactions. A chemical necessary to reduce another substance is called a reducing agent. A reducing agent must have a lesser attraction for electrons than the substance it reduces. A chemical necessary to oxidize another substance is called an oxidizing agent. An oxidizing agent must have a greater attraction for electrons than the substance that it oxidizes. In the following assume e^- represents an electron.

Ae^- + B à A + Be^- + energy

A is a reducing agent and B is an oxidizing agent. Redox reactions that take place spontaneously release energy.

What we are discussing in this chapter is how cells obtain ATP by the catabolic breakdown of organic molecules. ATP is generated by phosphorylating ADP and we recognize two alternative ways this may be done.

A molecule of central importance in this process is Nicotinamide adenine dinucleotide (NAD⁺). (Fig. 9.4)

Substrate level phosphorylation involves a substrate molecule directly passing a phosphate molecule to ADP. (Fig 9.7) Oxidative phosphorylation involves Redox reactions indirectly providing energy that will then be used to drive the endergonic phosphorylation of ADP to ATP.

Aerobic Respiration is the oxidative breakdown of organic molecules to CO₂ molecules using Oxygen in such a way that ATP can be produced. It generally is subdivided into three separate series of reactions: Glycolysis, The Krebs Cycle, and The Electron Transport System.(Fig. 9.6) We will use Glucose as the starting point because, as we will see, much of the breakdown of other organic molecules use biochemical pathways in common the respiratory pathway of glucose. As we study these processes note where they occur in a cell and the inputs and outputs of each stage.

Glycolysis

Glycolysis takes place in the cytosol and does not use oxygen. (Fig. 9.8, 9.9)It begins with a glucose molecule (a 6 carbon molecule) and breaks it down into two simpler three carbon molecules (Glyceraldehyde Phosphates). The first step of this process is to phosphorylate glucose which confers an electrical charge to it and prevents it from leaving the cell across the phospholipid bilayer. ATP is used to drive this phosphorylation. Another ATP is used to add another phosphate. This second phosphate makes the molecule effectively symmetrical and this molecule is then broken down into two three-carbon molecules, each of which becomes Glyceraldehyde Phosphate. Note to this point, we have broken glucose down into two three carbon molecules but have not obtained any ATP and in fact have had to use up two ATP molecules. Every reaction past this point occurs twice because of the two three carbon molecules. To oxidize Glyceraldehyde Phosphate an oxidizing agent is necessary and NAD⁺ is used for this purpose. Following this redox reaction NAD⁺ is reduced to NADH because it pulls away two electrons from Glyceraldehyde phosphate as well as two hydrogen protons (H⁺). One of the protons is released in the cytosol, but the other is bound to the NAD^- forming the NADH. This is a very important step because NAD⁺ is in limited supply in our cells and must somehow be regenerated because NADH is not a good oxidizing agent. If NAD⁺ is unavailable the glucose not only doesn't yield any ATP, but two ATP molecules would be expended to get to this stage. For now we will assume that NAD⁺ is available. The Glyceraldehyde phosphates are dephosphorylated twice each leading to two molecules of pyruvate (3-carbon molecules). The phosphates are past directly to ADP thereby producing 4 ATP molecules via substrate level phosphorylation. The series of reactions describes glycolysis. The input is glucose, the outputs are a net gain of two ATP, Two NADH, and two pyruvates. It is worth noting that NADH is energy rich and if oxidized it can release this energy as well as provide a supply of NAD⁺. We will see that if oxygen is available the oxidation of NADH indirectly can generate some ATP. Without oxygen eukaryotes must still oxidize NADH, but have to do it in ways that do not yield ATP. We will explore these alternatives starting first with what happens when oxygen is available.

When oxygen is available pyruvate is transported in the matrix of the mitochondrion. (Fig 9.10)In the matrix, the carboxyl group of each pyruvate is removed and given off as CO₂. The remaining 2-carbon molecule is oxidized to form acetate (a 2-carbon molecule). Each redox reaction uses NAD⁺ as the oxidizing agent. Each acetate binds with a sulfur-containing compound, coenzyme A, forming acetyl CoA that is the input for the Krebs cycle. Since glucose generates two pyruvates, the generation of 2 acetates produces two NADH and two CO₂ molecules.

The Krebs Cycle

Each acetyl CoA combines with a 4-carbon molecule (Oxaloacetate) and forms Citrate, a 6-carbon atom. This is the beginning of the Krebs cycle. Within the Krebs cycle the Citrate is oxidized back to Oxaloacetate plus two CO₂ molecules. The Oxaloacetate is then available to combine with another acetyl CoA so the cycle can continue. Therefore, for every glucose molecule, two CO₂ are released converting pyruvates to acetates, and four CO₂ are released from the Krebs cycle running twice. These are waste products that we eliminate from our bodies when we exhale during breathing. During each turn of the Krebs cycle,

3 NAD⁺ and an FAD are used as oxidizing agents. The redox reactions therefore produce 3 NADH and 1 FADH₂ each time the Krebs cycle runs (it runs twice for each glucose). In addition, each time the Krebs cycle runs an ATP is generated via substrate level phosphorylation. So for every glucose, in total 6 NADH, 2 FADH₂ and 2 ATP are generated via the Krebs cycle. All of these are energy rich. Remember the Krebs cycle occurs in the Matrix. (Fig. 9.11, 9.12)

The Electron Transport Chain.

Imbedded in the christae of the inner membrane of the mitochondria are a series of carrier molecules. (Fig. 9.13) Along this series of carrier molecules there is a progressive increase in the electronegativity of subsequent carriers. This can be used to oxidize NADH and FADH₂ through a series of redox reactions. The final acceptor of electrons is oxygen gas (O₂). This is why we need oxygen. Energy is released from each redox reaction. The interesting thing about these redox reactions is that some oxidizing agents will only accept electrons with an H⁺ while others will only accept electrons without an H⁺. The carrier molecules are organized such that when electrons must be picked up from the environment for the redox reaction to occur the carriers are near the matrix so remove H⁺ from the matrix, but when H⁺ must be given off those respective carriers are near the intermembrane space. (Fig. 9.14) This process leads to a reduction of H⁺ in the Matrix which makes it negative with respect to the intermembrane space as well as acidic. This creates a free energy gradient across the inner membrane of the mitochondrion. The phospholipid bilayer of the inner membrane blocks the diffusion of H⁺ back into the matrix except through channels lined with ATP synthase. When H⁺ passively diffuse through these channels the ATP synthase causes the phosphorylation of ADP to ATP. This process is called chemiosmosis. The complete oxidation of an NADH pumps enough H⁺ to phosphorylate a little under 3 ATPs and the complete oxidation of FADH₂ pumps enough H⁺ to generate 2 ATPs. All the NADHs produced (i.e., 2 from glycolysis , 2 from the production of acetate, and 6 from the Krebs cycle are oxidized this way) as are the two FADH2 from the Krebs cycle. This yields 34 ATP all of which are said to be produced by oxidative phosphorylation because they are products of these redox reactions. The net gain of 2 ATPs from glycolysis and the other two from the Krebs cycle are produced by substrate level phosphorylation and give a total of 38 ATPs from the aerobic respiration of glucose (Fig. 9.17).

If oxygen is unavailable, then the electron transport system cannot operate (Fig. 9.19). Pyruvate is not transported into the mitochondria. But, NADH must still be oxidized to provide a source for NAD⁺. This is done differently in different groups of organisms. Fermentation is an anaerobic process that includes glycolysis as a component, but continues so that NAD⁺ is regenerated. Different groups use different molecules as oxidizing agents for NADH. In yeast and many bacteria each 3-carbon pyruvate is converted to a 2-carbon acetaldehyde with the remaining carbon given off as CO₂. Each acetaldehyde is then used as the oxidizing agent for NADH and the acetaldehyde is reduced to ethanol (and alcohol). This is the basis for making wine and is called Alcoholic Fermentation. Animals and some other bacteria use pyruvate itself as the oxidizing agent for NADH. The pyruvate is reduced to lactate during the redox reaction. This is called Lactic Acid Fermentation (Fig. 9.18).

We do not simply eat glucose, yet the catabolism of complex carbohydrates, proteins and fats can be understood by the reactions we studied above. Polysaccharides are hydrolyzed to monosaccharides so their breakdown is rather clear. Proteins are broken down first to amino acids that then are deamminated in the liver. The nitrogenous waste in humans is voided from the body as urine. Since there are 20 different amino acids in biological proteins there is no single path that they all take after deammination. But they enter the aerobic pathway as any of the following: pyruvate, acetyl CoA, or Krebs cycle intermediates. Fats are hydrolyzed into glycerol and fatty acids. The glycerol is converted to glyceraldehyde phosphate. The fatty acids are broken down into two-carbon fragments and enter as acetyl CoA. (Fig. 9.20)

The rate at which aerobic respiration takes place is to a large extent dictated by our need for ATP. This is regulated primarily through an enzyme (phosphofructokinase) that operates early in glycolysis. Phosphofructokinase can be inhibited allosterically by ATP as well as citrate; it can be allosterically stimulated by ADP. (Fig. 9.21)