KEY CONCEPT QUESTIONS IN METABOLIC REDOX REACTIONS

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1 Redox Reactions in Metabolism Supplemental Reading Key Concepts - Reduction potentials are a measurement of electron affinity - Coenzymes provide reactive groups that function in enzyme catalysis - The pyruvate dehydrogenase complex is a metabolic machine KEY CONCEPT QUESTIONS IN METABOLIC REDOX REACTIONS: What does the ΔE value of a coupled redox reaction tell you about electron transfer potential? What are coenzymes and how do they function in the pyruvate dehydrogenase reaction? Biochemical Applications of Coenzyme Biochemistry: Thiamin, also called vitamin B 1, is an important enzyme cofactor required for a variety of metabolic reactions. Beriberi is a disease caused by thiamin deficiency resulting in severe weight loss and neurological symptoms. People that eat polished white rice as a sole source of nourishment can develop beriberi because polished rice lacks thiamin. Foods rich in thiamin include watermelon, sunflower seeds, black beans and thiamin enriched grains and breads. Reduction potentials are a measurement of electron affinity Before we begin discussing the Citrate Cycle (lecture 28), we need to describe several biological redox reactions (oxidation-reduction) that represent a form of energy conversion involving the transfer of electron pairs from organic substrates to the carrier molecules NAD + and FAD. The energy available from redox reactions is due to differences in the electron affinity of two compounds and is an inherent property of each molecule based on molecular structure. Since electrons do not exist free in solution, electrons must be passed from one compound to another in a coupled redox reaction. Coupled redox reactions consist of two half reactions, 1) an oxidation reaction (loss of electrons) and 2) a reduction reaction (gain of electrons). Compounds that accept electrons are called oxidants and are reduced in the reaction, whereas compounds that donate electrons are called reductants and are said to be oxidized by loss of electrons. Redox reactions in biochemistry rarely involve molecular oxygen (O 2 ) directly, but rather are characterized by the loss and gain of electrons from carbon. The terminology of biochemical redox reactions is the same as that used in inorganic chemistry. Namely, each half reaction consists of a conjugate redox pair represented by a molecule with and without an electron (e-). For example, Fe 2+ /Fe 3+ is a conjugate redox pair in which the ferrous ion (Fe 2+ ) is the reductant that loses an e- during oxidation to become a ferric ion (Fe 3+ ): Fe 2+ <--> Fe 3+ + e- reductant oxidant Similarly, the reductant cuprous ion (Cu + ) can be oxidized to form the oxidant cupric ion (Cu 2+ ) plus an e- in the reaction: Cu + <--> Cu 2+ + e- reductant oxidant 1 of 12 pages

2 The two conjugate redox pairs in these reactions are Fe 2+ /Fe 3+ and Cu + /Cu 2+. We can now combine these two half reactions into a coupled redox reaction by reversing the direction of the Cu 2+ reduction reaction such that the e- functions as the "common intermediate" shared by the Fe 2+ oxidation and Cu 2+ reduction half-reactions: Fe 2+ <--> Fe 3+ + e- (oxidation of Fe 2+ ) Cu 2+ + e- <--> Cu + (reduction of Cu 2+ ) Fe 2+ + Cu 2+ <--> Fe 3+ + Cu + (coupled redox reaction) The oxidation of Fe 2+ and reduction of Cu 2+ is a coupled redox reaction we will see in lecture 29 when we examine the function of the cytochrome c oxidase complex in the electron transport system. Figure 1. The combination of glycolysis, the citrate cycle and the electron transport system result in the complete oxidation of glucose to form CO 2 and H 2 O by a process called aerobic respiration. The e- donor is glucose which functions as the reductant, and O 2 is the e- acceptor (oxidant) that is reduced in the last step of the electron transport system to form H 2 O. The two conjugate redox pairs NAD + /NADH and FAD/FADH 2 serve as the e- carriers linking glycolysis to the citrate cycle and electron transport chain. It is useful to think of glucose as biochemical "battery" containing stored energy in the form of electrons that can be used to synthesize ATP in the mitochondria as a result of proton motive force and oxidative phosphorylation. Figure 1 illustrates the relationship between methane (CH 4 ), the most highly reduced form of carbon which has 8 e- that can be donated, and carbon dioxide (CO 2 ) in which all of the e- shared by the C and O are tightly associated with the more electronegative O. The more electrons a carbon atom has available to donate in a redox reaction, the more reduced (less oxidized) it is. Hydrogen is less electronegative than carbon, and therefore electrons in C-H bonds are considered "owned" by the carbon. Similarly, since oxygen is more electronegative than carbon, the electrons in C-O and C=O bonds are all "owned" by the oxygen atom. Note that in biological redox reactions, often (but not always), an increase in oxidation state of a carbon is associated with a decrease in the number of hydrogen atoms. Unlike the oxidation of Fe 3+ which simply involves the transfer of one e- to Cu 2+, redox reactions in the citrate cycle (and indeed most all enzyme-catalyzed redox reactions) involve the transfer of electron pairs (2 e-) to the electron carrier molecules NAD + and FAD. The reduction of NAD + to NADH involves the transfer of a hydride ion (:H - ), which contains 2 e- and 1 H +, and the release of a proton (H + ) into solution NAD e- + 2 H + <--> NADH + H + 2 of 12 pages

3 In contrast, FAD is reduced by sequential addition of one hydrogen (1 e- and 1 H + ) at a time to give the fully reduced FADH 2 product FAD + 1 e- + 1 H + <--> FADH + 1 e- + H + <--> FADH 2 Oxidations can also involve a direct combination with oxygen which oxidizes the carbon by pulling e- toward the more electronegative O atom. Enzymes that catalyze biochemical redox reactions are strictly called oxidoreductases, however, since most oxidation reactions involve the loss of one or more hydrogen atoms, they are often called dehydrogenases. We will look at the reduction of the coenzymes NAD + and FAD by dehydrogenases in more detail later. The two primary energy conversion reactions in metabolism are 1) phosphoryl transfers involving ATP, and 2) redox reactions that transfer pairs of electrons between organic compounds and the electron carriers NAD + /NADH and FAD/FADH 2. As we discussed in lecture 24, the change in standard free energy of a reaction under biochemical conditions (ΔGº') is a measure of the spontaneity of the reaction in kj/mol and reflects the tendency of compound A to be converted to compound B (A --> B). A negative ΔGº' (ΔGº' < 0) means the reaction is favored in the direction written from left to right (product B will accumulate), whereas, a positive ΔGº' (ΔGº' > 0) means the reverse reaction is favorable (A will accumulate). Figure 2. In redox reactions, we use the term reduction potential (E), measured in volts (V), to represent the electron affinity of a given conjugate redox pair. Analogous to biochemical standard conditions that define Gibbs Free Energy, Gº', (25ºC, ph 7 and 1 M initial concentration of substrates and products), the term Eº' refers to the biochemical standard reduction potential under the same conditions. Figure 2 illustrates how Eº' values are determined in the laboratory using an apparatus called an electrochemical cell that measures the relative e- affinity of a test redox pair, compared to that of the hydrogen half-reaction (2H + + 2e- <--> H 2 ), which has been chosen as the standard. For these measurements, two half-cells are connected by a type of voltmeter (galvanometer) and platinum electrodes that measure the movement of electrons from one half cell to the other. Depending on the relative electron affinity of the test oxidant compared to H +, the electrons will either move from the hydrogen half-cell toward the test half-cell, or from the test half-cell toward the hydrogen half-cell. The two half-cells are connected by an agar bridge containing potassium chloride that permits counter-ion movement to balance the charge. The Eº' of oxidants with a higher affinity for electrons than H + are recorded as positive Eº' values (Eº' > 0), and oxidants with a lower affinity for electrons than H + are have negative Eº' values (Eº' < 0). In the example shown in figure 2, under standard conditions of 25ºC and 1 atmosphere of pressure, the hydrogen half cell on the right is assigned the arbitrary Eº' value of It can be seen that the voltmeter registers a Eº' value of volts, meaning that the electrons flow from the hydrogen reference cell toward the Fe 3+ /Fe 2+ test cell demonstrating that Fe 3+ has a higher affinity for electrons than H +. 3 of 12 pages

4 Figure 3 lists the Eº' values that have been measured for a number of conjugate redox pairs in biochemical reactions. It can be seen that O 2 is the most potent oxidant in the table with a Eº' value of and will therefore readily accept electrons from all other conjugate redox pairs shown, whereas, α-ketoglutarate is a very weak oxidant (strongest reductant) and will donate electrons to other conjugate redox pairs in the table. By convention, standard reduction potentials are expressed as halfreactions written in the direction of a reduction reaction. Therefore, when a oxidation reaction is written in the reverse direction, the sign of the Eº' value needs to change accordingly (e.g., from + to -). The table highlights some of the redox reactions we will encounter in metabolism. Figure 3. The amount of energy available from a coupled redox reaction is directly related to the difference between two reduction potentials and is defined by the term ΔEº'. By convention, the ΔEº' of a coupled redox reaction is determined by subtracting the Eº' of the oxidant (e- acceptor) from the Eº' of the reductant (e- donor) using the following equation: ΔEº' = (Eº' e- acceptor ) - (Eº' e- donor ) Moreover, the ΔEº' for a coupled redox reaction is proportional to the change in free energy ΔGº' as described by the equation: ΔGº' = -nfδeº' in which n is the number of electrons transferred in the reaction (usually 2 in biochemical redox reactions), and F is the Faraday constant (96.48 kj/v mol). As can be seen by this equation, when the difference in reduction potentials for a coupled redox reaction is positive (ΔEº' > 0) then the reaction is favorable since ΔGº' will be negative. Looking back at the definition of ΔEº', this means that for a coupled redox reaction to be favorable, the reduction potential of the e- acceptor needs to be more positive than that of the e- donor. 4 of 12 pages

5 To see how ΔGº and ΔEº are related, we can use the biochemical standard reduction potentials (Eº') in figure 3 to calculate the change in biochemical standard free energy (ΔGº') for the citrate cycle isocitrate dehydrogenase reaction. Note that in a spontaneous coupled redox reaction the e- flow is from the reductant in the conjugate redox pair with the lower Eº' value (more negative) toward the oxidant in the conjugate redox pair with the higher Eº' value (less negative). The two standard half reactions from figure 3 are written below as reduction reactions. Note that NAD + is the e- acceptor (less negative) and α-ketoglutarate is the e- donor (more negative): α-ketoglutarate + CO e- + 2 H + --> Isocitrate (Eº' = V) NAD+ 2 e- + 2 H + --> NADH + H + (Eº' = V) We can calculate ΔEº' for this coupled redox reaction using the equation below: ΔEº' = (Eº' e- acceptor ) - (Eº' e- donor ) ΔEº' = (Eº' NAD+ ) - (Eº' isocitrate ) ΔEº' = (-0.32 V) - (-0.38 V) = V and then convert this ΔEº' value to ΔGº' using the relationship: ΔGº' = -nfδeº' ΔGº' = -2 (96.48 kj/mol V) V ΔGº' = kj/mol showing that the conversion of isocitrate to α-ketoglutarate is a favorable reaction (ΔGº' < 0) under standard biochemical conditions. Note that since biochemical conditions inside the mitochondrial matrix are not standard, in order to calculate the actual reduction potentials for conjugate redox pairs, we need to take into account the concentration of the oxidant (e- acceptor) and reductant (e- donor) using an equation described by Walther Nernst in 1881: E = Eº' + RT ln [e- acceptor] nf [e- donor] In the Nernst equation, R is the gas constant (8.314 J/Kº mol), T is the absolute temperature in Kelvin (Kº), n is number of electrons transferred and F is the Faraday constant (96.48 kj/v mol). Coenzymes provide reactive groups that function in enzyme catalysis Pyruvate is a three carbon metabolite derived from glucose or amino acids that must be transported from the cytosol into the mitochondrial matrix before it can serve as a source of reducing power for the cell (citrate cycle) or as a precursor for glucose synthesis (gluconeogenesis). Pyruvate that is destined for the citrate cycle (or fatty acid synthesis) is converted to acetyl CoA by the enzyme pyruvate dehydrogenase. As shown in figure 4, acetyl- CoA has only two metabolic fates in the cell, 1) it can be metabolized by the citrate cycle to convert redox energy to ATP by oxidative phosphorylation, or 2) it can be used as a form of stored energy by conversion to fatty acids that are transported to adipocytes (fat cells) as 5 of 12 pages

6 triglycerides. Since the pyruvate dehydrogenase reaction is irreversible (ΔGº = kj/mol), production of acetyl-coa by the pyruvate dehydrogenase reaction is tightly controlled to coordinate energy needs of the cell with the production of acetyl-coa. Figure 4. This is especially important in animals which lack the necessary enzymes to convert fats to carbohydrates, and therefore, cannot reutilized acetyl-coa for glucose production when carbohydrate levels are low. Because of this, the pyruvate dehydrogenase complex is only fully active in animal cells when carbohydrate sources are plentiful. The pyruvate dehydrogenase complex catalyzes the oxidative decarboxylation of pyruvate to form CO 2 and acetyl- CoA using a five step reaction mechanism that requires three distinct enzymes and five different coenzymes. We begin by first looking at the important role of coenzymes in metabolic reactions, specifically NAD +, FAD, CoA, thiamine pyrophosphate (TPP) and lipoic acid which are all utilized in the pyruvate dehydrogenase reaction. Amino acids can only provide a finite number of chemical groups for enzyme reactions, and moreover, structural constraints of the polypeptide backbone restrict the precise positioning (orientation) of amino acid side groups within the active site. However, enzyme complexes that bind reactive biomolecules through covalent or non-covalent interactions provide additional reactive groups for catalytic mechanisms. These biomolecules are called enzyme cofactors or coenzymes and are often complex organic compounds that are obtained as nutrients in the diet. The term vitamin describes organic molecules that are required in the diet, but which do not contribute directly to energy conversion through catabolism, or do not provide a structural role to other biomolecules. Figure 5 lists six coenzymes in metabolism and their role in enzymatic reactions. Figure 5. Coenzyme Vitamin Types of reactions Nutrient source Nicotinamide adenine Niacin (B 3 ) Redox reactions Poultry, fish, dinucleotide (NAD + ) (transfer of hydride ion) vegetables Symptoms of dietary deficiency Causes the disease pellagra Flavin adenine dinucleotide (FAD) Riboflavin (B 2 ) Redox reactions (transfer of electrons) Dairy, almonds, asparagus Causes cheilosis (swelling,cracked lips) Coenzyme A (CoA) Pantothenic acid (B 5 ) Acyl group transfer Chicken, yogurt, avocados Rarely observed Thiamin pyrophosphate (TPP) Thiamin (B 1 ) Aldehyde transfer Lentils, brown rice, fortified cereals Causes beriberi α-lipoic acid (lipoamide) Not a vitamin Acyl group transfer Tomatoes, broccoli, spinach None reported Biocytin (biotin-lysine) Biotin Carboxyl group transfer Breads, cooked eggs, vegetables skin rash, hair loss, 6 of 12 pages

7 Nicotinamide adenine dinucleotide (NAD + ) - is derived from the water-soluble vitamin niacin which is also called vitamin B 3. NAD +, and its phosphorylated form NADP +, are involved in over 200 redox reactions in the cell which are characterized by the transfer of 2 e- in the form of hydride ions (:H - ). Catabolic redox reactions primarily use the conjugate redox pair NAD + /NADH and anabolic reactions use NADP + /NADPH. The structure of the oxidized and reduced forms of NAD(P) + and NAD(P)H, respectively, are shown in figure 6. Note that the "+" charge does not refer to the overall charge of the NAD molecule, but rather only to the charge on the ring N in the oxidized state. Severe niacin deficiency causes the disease Figure 6. pellagra which was first described in Europe in the early 1700s amongst peasants who relied on cultivated corn as their primary source of nutrition. Although it was initially thought that pellagra was caused by an infectious agent in contaminated corn, nutritional studies showed that it was due to insufficient levels of bioavailable niacin in a corn-rich diet. Interestingly, pellagra is rare in Mexico because corn used for tortillas is traditionally soaked in lime solution (calcium oxide) prior to cooking and this releases niacin from its bound form upon heating. Flavin adenine dinucleotide (FAD) - is derived from the Figure 7. water-soluble vitamin riboflavin which is also called vitamin B 2. Riboflavin is the precursor to FAD and to the related molecule flavin mononucleotide (FMN), both of which are often tightly associated with enzymes that catalyze redox reactions. FAD is a coenzyme in the pyruvate dehydrogenase complex and is also covalently bound to a histidine residue in the citrate cycle enzyme succinate dehydrogenase. FAD is reduced to FADH 2 by the transfer of two electrons in the form of hydrogen atoms (figure 7). Unlike NAD, FAD can accept one electron at a time and form a partially reduced intermediate called a semiquinone (FADH ). Foods that have been found to be high in riboflavin include dairy products (milk, cheese, eggs), almonds and asparagus. Riboflavin, like several other vitamins, is destroyed by light which is one reason why milk is no longer stored in clear containers. 7 of 12 pages

8 Figure 8. Bioc Dr. Miesfeld Spring 2008 Coenzyme A (CoA) - is derived from the water-soluble vitamin pantothenic acid which is also called vitamin B 5. CoA is absolutely essential for life as it is required for energy conversion by the citrate cycle, it is also a cofactor in fatty acid, acetylcholine, heme and cholesterol biosynthetic pathways. The primary role of CoA is to function as a carrier molecule for acetate units in the form of acetyl-coa. Figure 8 shows the structure of acetyl-coa which consists of a central pantothenic acid unit that is linked to a functional β-mercaptoethylamine group derived from cysteine, and to adenosine 3,5-diphosphate. The acetate unit is covalently attached to CoA through an activated thioester bond which has a high standard free energy of hydrolysis making it an ideal acyl carrier compound. As such, attachment of acetate units to the reduced form of CoA (CoA-SH) requires reactions with high ΔGº' values, for example, pyruvate dehydrogenase (ΔGº' = kj/mol) and α-ketoglutarate dehydrogenase (ΔGº' = kj/mol). Thiamin pyrophosphate (TPP) - is derived from the water-soluble vitamin thiamin (or thiamine) which is also called vitamin B 1. The structure of TPP is shown in figure 9 where it can be seen that a carbon atom on the thiazole ring is the functional component of the coenzyme involved in aldehyde transfer. Thiamin is absorbed in the gut and transported to tissues where it is phosphorylated by the enzyme thiamin kinase in the presence of ATP to form thiamin pyrophosphate (TPP) and AMP. Figure 9. Thiamin deficiency was first described in Chinese literature over four thousand years ago and is the cause of beriberi, a disease characterized by anorexia, cardiovascular problems and neurological symptoms. Beriberi has been found in populations that rely on white polished rice as a primary source of nutrition (milling rice removes the bran which contains thiamin) and diets rich in foods that contain the enzyme thiaminase which degrades thiamin during digestion. Raw fish contains thiaminase, as does African silkworms a favorite food in some Nigerian cultures. Cooking these foods destroys the thiaminase and alleviates the symptoms of beriberi. α-lipoic acid (lipoamide) - is a coenzyme synthesized in plants and animals as a 6,8- dithiooctanoic acid. The role of α-lipoic acid in metabolic reactions is to provide a reactive disulfide that can participate in redox reactions within the enzyme active site. Lipoamide, the naturally occurring form of α-lipoic acid, is a covalent linkage of α-lipoic acid to a lysine ε-amino group on proteins. The structure of lipoamide is shown in figure 10 where it can be seen that the long hydrocarbon chain bridging -lipoic acid and lysine provides a flexible extension to the reactive thiol group. 8 of 12 pages

9 As illustrated in figure 10, the E2 subunit of the pyruvate dehydrogenase complex contains the lipoamide at the end of a polypeptide tether which functions as a "ball and chain" that moves the lipoamide back and forth across a 50 Å span in the interior of the complex. α- Lipoic acid is not considered a vitamin because it is synthesized at measurable levels in humans, however, because of its potential to function as an antioxidant in the reduced form, α-lipoic acid is promoted as a nutritional supplement. High levels of α-lipoic acid are in broccoli, liver, spinach and tomato. Figure 10. The pyruvate dehydrogenase complex is a metabolic machine The conversion of pyruvate to acetyl-coa by the pyruvate dehydrogenase complex is an oxidative decarboxylation reaction that represents another amazing example of protein structure and function. The eukaryotic pyruvate dehydrogenase complex contains multiple subunits of three different catalytic enzymes that work together as a metabolic machine to carry out the following net reaction: Pyruvate + CoA + NAD+ ---> acetyl-coa + CO2 + NADH ΔGº = kj/mol Coenzymes perform a critical role in the pyruvate dehydrogenase complex by providing a chemical platform for the Figure 11. catalytic reactions. Three of the coenzymes are covalently linked to enzyme subunits, with TPP attached to the E1 pyruvate dehydrogenase subunit, lipoamide is the functional component of the E2 dihydrolipoyl transacetylase subunit, and FAD is covalently bound to the E3 dihydrolipoyl dehydrogenase subunit. The two 9 of 12 pages

10 Bioc Dr. Miesfeld Spring 2008 other coenzymes, CoA and NAD+, are transiently associated with the E2 and E3 complexes, respectively. The pyruvate dehydrogenase reaction can be broken down into five distinct catalytic steps in which steps 1, 2 and 3 lead to the formation of acetyl-coa, with steps 4 and 5 serving to regenerate the oxidized from of lipoamide and in the process, transfer 2 e- to NAD+ (figure 11). Step 1 - The E1 subunit (pyruvate dehydrogenase) binds pyruvate and catalyzes a decarboxylation reaction resulting in the formation of hydroxyethyl-tpp and the subsequent release of CO2. Step 2 - The hydroxyethyl-tpp of E1 reacts with the disulfide of the lipoamide group on the Nterminal domain of the E2 subunit (dihydrolipoyl transacetylase) to generate acetyldihydrolipoamide through a thioester bond. Step 3 - The E2 lipoamide domain carries the acetyl group from the E1 catalytic site across a ~50 Å gap in the complex to the E2 catalytic site where it reacts with CoA-SH to yield acetyl-coa and fully reduced dihydrolipoamide. Step 4 - The lipoamide domain then swings over to the E3 subunit (dihydrolipoyl dehydrogenase) where it is reoxidized to the disulfide by a transfer of 2 e- and 2 H+ to a disulfide contained on the E3 subunit; the dithiol is reoxidized by transferring 2 e- and 2 H+ to the E3-linked FAD moiety to transiently form E3-FADH2. Figure 12. Step 5 - The E3-FADH2 coenzyme intermediate is reoxidized in a coupled redox reaction that transfers the 2 e- to NAD+ as a hydride ion (:H-) leading to the formation of NADH + H+. As shown in figure 12, the E1, E2 and E3 subunits of the mammalian pyruvate dehydrogenase are packed together in a huge ~400 Å diameter sphere with a combined molecular weight of ~7800 kda. The colored E1 (yellow), Figure 13. E2 (green) and E3 (red) pyruvate dehydrogenase complex subunits are labeled, and the linker region connecting E2 to E1 is shaded gray. The stoichiometry of the E1:E2:3 subunits (22:60:6) is consistent with there being 60 active sites in the pyruvate dehydrogenase complex. Figure 13 shows that the lipoamide moiety of the E2 subunit is attached near the end of a ~200 amino acid long segment of the protein that functions as both a structural linker connecting the E2 and E1 subunits (gray region in figure 16), and as a type of lipoamide "ball and chain." An important component of the linker region is the E1-binding domain that serves as a "pivot" for the ball and chain. The hydrocarbon extension on the lipoamide 10 of 12 pages

11 moiety itself is sometimes referred to as a "swinging arm." Because of the relative positioning of E2 and E1 subunits with the pyruvate dehydrogenase complex, a single E2 subunit can "harvest" hydroxyethyl groups from multiple E1 catalytic sites and deliver them to the E2 subunit. The lipoamide group is the workhorse in this catalytic machine, and without a fully functional pyruvate dehydrogenase complex, the link between glycolysis and the citrate cycle would be broken. A naturally occurring inhibitor of lipoamide coenzyme function is the element arsenic (As) which in the form of arsenite (AsO 3 3- ) creates bidentate adducts on dihydrolipoamide as shown in figure 14. Inadvertent ingestion arsenite can lead to an untimely death by irreversibly blocking the catalytic activity of lipoamide-containing enzymes such as the pyruvate dehydrogenase and α- ketoglutarate dehydrogenase complexes. Figure 14. Figure 15. Chronic arsenic poisoning can come from environmental sources such as arsenic-contaminated drinking water or household paints and results in the appearance of ulcerous skin lesions and an increased risk of a variety of cancers. While stories abound that arsenic poisoning was used routinely to kill off kings and queens in the Middle Ages, and may have even been involved in the death of the exiled French general Napoleon Bonaparte, a more tragic example is that of accidental arsenic poisoning of thousands of people in Bangladesh, India, which has occurred over the last 20 years. Since the 1990s it has been documented that millions of people in India have been chronically exposed to toxic levels of arsenic in contaminated drinking water obtained from shallow hand-pumped wells (figure 15). During the 1970s and 1980s, UNICEF and other relief organizations helped drill thousands of wells in small Indian villages as an humanitarian effort to circumvent public water supplies that had become biologically contaminated. About ten years later when large numbers of villagers in the Ganges delta region developed skin lesions and cancers, it was realized that these wells contained water with toxic levels of arsenic. Massive efforts were undertaken to close down contaminated wells and to develop purification systems to reduce the arsenic to safe levels in other water supplies. Arsenic-contaminated drinking water has also been found in Southeast Asia and South America, usually near areas that had been extensively mined. 11 of 12 pages

12 ANSWERS TO KEY CONCEPT QUESTIONS IN THE CITRATE CYCLE: The difference in reduction potential (ΔE) for a coupled redox reaction represents the tendency of the reductant in one conjugate redox pair to donate electrons to the oxidant of the other conjugate redox pair. Since the ΔE value of a redox reaction is proportional to the change in free energy (ΔG) as described by the equation ΔG = -nfδe, redox reactions with positive ΔE values (ΔG<0) are more favorable than reactions with negative ΔE values (ΔG>0). The reduction potential for a given conjugate redox pair is determined experimentally using an apparatus that measures electron flow from one half-cell to another. Electrons move through platinum wires from the half cell containing a reductant with low electron affinity toward the half-cell containing an oxidant with high electron affinity. In many biochemical redox reactions, electrons are transferred in pairs (2 e-) from one molecule to another, sometimes as hydride ions (:H-), as is the case with the coenzyme NAD + (NAD + + H e- <--> NADH). Coenzymes are biomolecules that provide additional functional groups to enzyme active sites and participate in catalytic mechanisms. The pyruvate dehydrogenase complex consists of multiple copies of three protein subunits (E1, E2, E3) and requires five coenzymes (thiamin pyrophosphate; TPP, lipoamide, coenzyme A; CoA, flavin adenine dinucleotide; FAD, and nicotinamide adenine dinucleotide; NAD + ) to mediate a five-step reaction mechanism. Most coenzymes are classified as vitamins and nutritional deficiencies in some of these important biomolecules can lead to human disease such as beriberi due to thiamin deficiency, and pellagra, a disease caused by niacin deficiency which leads to decreased levels of NAD + /NADH. 12 of 12 pages