In the Classroom
Assigning and Using Oxidation Numbers in Biochemistry Lecture Courses Christopher J. Halkides Department of Chemistry, University of North Carolina at Wilmington, Wilmington, NC 28403-3297;
[email protected] The purpose of this article is to illustrate the uses of assigning oxidation numbers to metabolic intermediates and end products in biochemical equations. Oxidation–reduction reactions, reactions in which electrons are transferred, comprise a major class of biochemical processes. For example, why anaerobic metabolism produces some products but cannot produce others is easily understood by the need to conserve the number of electrons. Assigning oxidation numbers is a useful bookkeeping device to help one account for all electrons, notwithstanding some ambiguities (1, 2). It is especially useful to have a set of rules to deal with large biomolecules. Redox reactions can be identified when one compares oxidation numbers between the products and reactants. Therefore, if a biochemical reaction involves an increase in the oxidation numbers between substrate and product, one can infer that the oxidized form of a redox cosubstrate or coenzyme1 must be reduced concomitantly. Oxidation numbers are used in many chemistry courses, and adopting them in biochemistry courses would serve to reinforce the concept and to integrate biochemistry more tightly into the chemistry curriculum. Achieving the latter goal is especially timely, now that the American Chemical Society has decided to require biochemistry as part of the undergraduate curriculum. Yet few biochemistry textbooks present oxidation numbers in a formal way (3). In my experience, moreover, even the best students struggle to apply their knowledge about oxidation and reduction to biochemistry, if they are not provided guidance. When the instructor repeatedly assigns oxidation numbers to the carbon atoms of metabolites over the whole course, it generates simple and explicit definitions of the concepts of oxidation and reduction. It also impels the student to focus on only the reactive portions of large, complicated metabolites, cosubstrates, and coenzymes. When students identify a redox reaction by assigning and comparing oxidation numbers, they can make two inferences about the reaction: that the redox cosubstrate or coenzyme is probably either NAD(P)+ or FAD on one side of the balanced equation, and that the enzyme will probably be named as a dehydrogenase or a reductase. These inferences may help the student learn the reactions and names of enzymes in pathways such as glycolysis and the TCA cycle. Assigning Oxidation Numbers Oxidation of carbon can be defined as the making of bonds to more electronegative atoms (principally O and N) or the breaking of bonds to more electropositive atoms (usually H). Reduction can be defined as the making of bonds to more electropositive atoms or the breaking of bonds to more electronegative ones. We know that electrons are shared unequally in most chemical bonds owing to electronegativity differences between the two atoms in the bond. Therefore, oxidation has the effect of making carbon more positively 1428
O C
H3C Number of Bonds to O 0 2 − Number of Bonds to H 3 1 Oxidation # of Carbon = −3 +1
O H
C H3C 0 3 3 0 −3 +3
O
CH3 1 3 −2
Figure 1. Calculation of the oxidation numbers of the carbon atoms in acetaldehyde and ethyl acetate. For each carbon the number of bonds to hydrogen is subtracted from the number of bonds to oxygen to obtain the oxidation number of that carbon atom. Note that two methyl carbons in ethyl acetate do not have the same oxidation number.
charged (decreasing its electron density), and reduction has the effect of making carbon more negatively charged (increasing its electron density). Oxidation numbers are assigned as if the more electronegative atom in a bond owned the electrons completely. We can make a simple set of rules to write oxidation numbers for carbon atoms, slightly modified from those presented in general and organic chemistry textbooks (4–6 ). 1. To obtain the oxidation number of a carbon atom, subtract the number of bonds to hydrogen from the number of bonds to oxygen. This means for example that for a carbonyl carbon, which has a C=O double bond, you must count both bonds. So for acetaldehyde, the carbonyl carbon atom has an oxidation number of +1, and the methyl carbon has an oxidation number of ᎑3 (Fig. 1). Note that two methyl carbons in ethyl acetate do not have the same oxidation number (Fig. 1). 2. Bonds between carbon and nitrogen are treated exactly like bonds between carbon and oxygen because nitrogen and oxygen are both more electronegative than carbon. In a bond between two atoms of equal electronegativity such as two carbon atoms, the electrons are treated as being shared equally. 3. The oxidation number of a free element (O2 or H2, for example) is zero. 4. The oxidation number of oxygen in compounds with other elements is ᎑2 except in peroxides, where it is ᎑1, and in compounds with fluorine. 5. The oxidation number of hydrogen is +1 in C–H bonds, O–H bonds, or N–H bonds. 6. The algebraic sum of all of the oxidation numbers in an ion or molecule is equal to its charge. 7. In a balanced chemical equation, the total oxidation number is conserved (is equal between reactants and products). If one atom decreases in oxidation number as it reacts, another atom must increase in oxidation number. The reason for this is that electrons are not created or destroyed in a chemical reaction. How can we apply oxidation numbers to biochemical equations? Although rules 6 and 7 apply to the sum of oxida-
Journal of Chemical Education • Vol. 77 No. 11 November 2000 • JChemEd.chem.wisc.edu
In the Classroom
tion numbers of all atoms, it is usually faster to sum up the oxidation numbers of the carbon atoms only. By assuming that oxygen, nitrogen, and hydrogen have the same oxidation number in reactants as they do in the products, we greatly simplify the calculation and, in effect, give students far fewer rules to learn. This assumption is often true; however, some exceptions are given below. Using Oxidation Numbers
numbers of 0, and carbon 6 has an oxidation number of ᎑1, giving glucose a net oxidation number of 0. The methyl carbon atom of ethanol has an oxidation number of ᎑3 and the hydroxymethyl carbon has an oxidation number of ᎑1, and we need to take into account that there are two moles of ethanol per mole of glucose. The four carbon atoms of the two molecules of ethanol have a total oxidation number of ᎑8. 2 × ᎑1 = ᎑ 2 2 × ᎑3 = ᎑ 6 — ᎑8
Let us first examine the oxidation of succinate to fumarate On the other hand, glucose has an oxidation number of 0. (Fig. 2). The oxidation number of each of the two methylene Clearly, we have not yet balanced either carbon atoms or carbons of succinate is ᎑2 and the oxidation number of each oxidation numbers. There are two carbon atoms still to be of the carboxylate carbon atoms is +3; therefore, the net accounted for and they must each have an oxidation number oxidation number of the four carbon atoms is +3 + (᎑2) + of +4, to sum to +8 and balance the charge and the number (᎑2) +3 = +2. The sum of the oxidation numbers for the four of atoms. Therefore, we know that two molecules of CO2, carbon atoms of fumarate is +3 + (᎑1) + (᎑1) +3 = +4. According each of which has an oxidation number of +4, must also be to rule 7, the sum of the oxidation numbers must be equal produced. between products and reactants for any chemical reaction. Three final points about anaerobic metabolism should be Therefore, a second molecule, a cosubstrate or coenzyme, stated. must oxidize succinate by two electrons producing fumarate First, oxidation numbers can be used to decide whether and reducing the coenzyme by two electrons. In this case the putative products of a fermentation process are possible or coenzyme FAD is reduced to FADH2 (see Fig. 5b for structures). impossible from the standpoint of leading or not leading to Let us look at the hydration of fumarate to create malate a balanced equation. Yet oxidation numbers cannot be used as a second example from the TCA cycle (Fig. 2). The net to discriminate between several alternative products if all the oxidation number of the carbon atoms of fumarate is +4 (see above) and the net oxidation number of the carbon atoms of malate is +3 + 0 + (᎑2) +3 = +4. Since one carbon is oxidized and one is reduced in the hydration, no net oxidation or reO O− O O− O O− duction has taken place (each of the hydrogen atoms of water C C C +3 +3 +3 has an oxidation number of +1, and the oxygen atom has a H 2O FAD FADH CH CH HC OH 2 −1 0 −2 2 value of ᎑2, the same values as they have in the product). − 1 HC −2 CH2 CH2 −2 The reaction catalyzed by glutamate synthase is a nice illustration of a reaction that may not appear to be a redox +3 +3 +3 C C C process at first glance (Fig. 3). Suppose a student knew that O O− O O− O O− glutamine plus α -ketoglutarate yields two molecules of +4 +4 +2 glutamate but forgot that the reaction also uses NADPH, a Fumarate Malate Succinate 2-electron reducing agent. Could the student determine that Figure 2. Conversion of succinate to fumarate and then to malate. NADPH is necessary just by knowing the structures of The oxidation number of each carbon atom is given to the left of it, glutamine, glutamate, and α-ketoglutarate? Yes. In α-ketoand the sum of the oxidation numbers of each compound is given glutarate the keto carbon (*) has an oxidation number of +2, above its name. Assigning oxidation numbers to the carbon atoms but the corresponding α-carbon atom in the glutamate moldemonstrates that the first reaction requires the 2-electron reduction of ecule on the right (*) has an oxidation number of 0. This a cofactor and that the second reaction does not. The cofactor is carbon has been reduced by two electrons. Since all of other FAD, which is reduced to FADH2; however, assigning oxidation carbon atoms retain the same oxidation numbers from reacnumbers cannot establish the identity of the cofactor. tion to product, there must be a 2-electron reducing agent in the balanced equation, − − − − which is NADPH in this case. O O O O O O O O Oxidation numbers are quite useful in C C C C +3 +3 +3 +3 understanding microbial fermentation, a HC NH3+ HC NH3+ C O HC NH3+ +2 0 0 0 process in which an organic molecule is deCH2 CH2 CH2 CH2 −2 −2 −2 −2 graded to drive ATP synthesis without net CH2 CH2 CH2 −2 −2 −2 −2 CH2 oxidation or reduction (7 ). It is often diffiNADPH NADP + cult to decide whether the products of fer+3 +3 +3 +3 C C C C H − − − mentation are in redox balance with the reO O O O O O O H2N actant by inspection because the molecules are complex. Consider the fermentation of Glutamine α-Ketoglutarate Glutamate Glutamate glucose to two molecules of ethanol Figure 3. Reaction catalyzed by glutamate synthase. Glutamine donates its amide (C6H12O6 → 2CH3–CH2OH + ?), shown nitrogen to α-ketoglutarate making 2 molecules of glutamate. The α-keto carbon (*) is in Figure 4a. Carbon 1 has an oxidation reduced by 2 electrons in this process. This reduction is balanced by the oxidation number of +1, Carbons 2–5 have oxidation of NADPH to NADP+. +
+
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alternatives lead to balanced equations. Which products are actually formed depends of course on the enzymes present within a particular organism. For one example, the fermentation of glucose can lead either to ethanol and carbon dioxide or to lactate (Fig. 4). Homofermenting lactic acid bacteria produce lactate almost exclusively, whereas heterofermenting lactic acid bacteria produce an equimolar mixture of lactate, ethanol, and carbon dioxide. For another example, oxidation numbers cannot be used to show why some heterofermenting lactic acid bacteria can produce mannitol from fructose but not glucose (Problem 5), since both glucose and fructose have a net oxidation number of 0. For a final example, three moles of pyruvate can be fermented to one mole of propionate, and to two moles each of acetate and carbon dioxide, or to three moles each of succinate and acetate (7 ). Second, some bacteria produce H2 in their fermentations, or even use a small amount of oxygen (7). Although it is relatively straightforward to assign oxidation numbers to hydrogen and oxygen to balance the equations for these processes, a formal treatment is outside the scope of this paper. Third, the conversion of glucose to lactate by heavily exercising muscle in the Cori cycle can be seen as being in redox balance using oxidation numbers (Figure 4b).
(a)
−1 CH2OH 0
H
O H OH
0
OH
+4 C
−3 −1
+1
H
2 H3C CH2 OH + O
O
H
OH 0
0 H
OH
(b) CH2OH O
H H OH
OH
H
H
0
+3
2 H3 C
H C
C
OH
H
OH
+3
O O-
OH
Figure 4. (a) Fermentation of glucose to two molecules of ethanol and carbon dioxide. The oxidation number is given next to each carbon. (b) The conversion of glucose to lactate. This process is seen both in bacteria and in heavily exercising muscle.
(a) NADPH + H+
NADP+
Extensions to Atoms Other Than Carbon E•FAD
With respect to oxidation–reduction processes of atoms other than carbon, those of sulfur and oxygen are among the most important. These processes can be analyzed quantitatively by assigning redox numbers to these heteroatoms using the rules given above. The reduction of a disulfide bond to two thiol groups is a 2-electron process. An example of this is the reduction of glutathione disulfide (GCH2SSCH2G) to two molecules of glutathione (GCH2SH), where G stands for the cysteine-containing tripeptide glutathione (Fig. 5a). Since carbon and sulfur have about the same values for electronegativity, each sulfur atom is assigned an oxidation number of 0 in the reactant and ᎑1 in the product.2 The reducing agent is NADPH, and the enzyme glutathione reductase transiently reduces the tightly bound FAD cofactor to FADH2 (Fig. 5b), which then reduces glutathione disulfide. Reduced glutathione (GCH2SH) can in turn reduce protein disulfide bonds to protein thiol groups, in addition to its other roles (8, 9). The reduction of O2 to 2 molecules of H2O is a 4-electron process. It takes only 2 electrons to reduce O2 to hydrogen peroxide (H2O2), in which each oxygen atom has an oxidation number of 0 in the reactant and ᎑1 in the product. It also takes two electrons to reduce hydrogen peroxide to water. The latter process is catalyzed by glutathione peroxidase and the reducing equivalents are supplied by GCH2SH. Using Oxidation Numbers in the Classroom The instructor should make the students aware that comparing oxidation numbers between reactants and products will determine whether a given reaction is a redox reaction and will specify the number of electrons involved. Yet assigning and comparing oxidation numbers cannot determine which redox molecule is the cosubstrate or coenzyme, nor does it guarantee that a particular organism possesses the enzyme(s) necessary to effect a given transformation (see above and 1430
2 GCH2S-H −1
E•FADH2
GCH2S-SCH2G 0
(b) R N
R N
O
N
H N
NH N
O NH
N H O
FAD
O FADH2
Figure 5. (a) Reduction of glutathione disulfide to glutathione. The FAD-containing enzyme glutathione reductase uses NADPH as the reducing agent. The sulfur atom of glutathione is reduced. (b) Structures of FAD and FADH2.
problem 5). The instructor is more likely to teach students to think in terms of oxidation numbers by returning to this subject repeatedly over the course of the semester, rather than simply discussing it once at the beginning of the semester and hoping that students apply this analysis themselves. As elementary as it may seem to the instructor, he or she should point out that an enzyme catalyzing a redox process is probably named as a reductase or a dehydrogenase. For example, consider the oxidation of carbon atom #1 in ethanol (oxidation number ᎑1) to acetaldehyde (oxidation number +1) with the concomitant reduction of NAD+ to NADH. The enzyme is alcohol dehydrogenase, but the instructor might ask the students to propose a name for this enzyme as part of a lecture. Oxidation numbers can be brought into discussions of glycolysis (lactate dehydrogenase), the TCA cycle (to define the term oxidative decarboxylation), fatty acid degradation (which
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In the Classroom
strongly resembles the sequence of reactions involving succinate shown in Fig. 2) and amino acid metabolism (for example, the conversion of aspartate to homoserine). Oxidation numbers can also be brought into discussions of the electron transport chain (by assigning the oxidation numbers of quinones), electron shuttle systems between the mitochondria and cytoplasm (for example, dihydroxacetone phosphate and glycerol-3phosphate) or shuttle systems between cells (as in the C-4 pathway) (8, 9). Questions for Students 1. What are the maximum and minimum oxidation numbers that carbon can attain? Name two molecules for which carbon has these oxidation numbers. Answer: +4 in CO2 or bicarbonate ion, and ᎑4 in CH4. 2. Can a mole of glucose be fermented to 2 molecules of lactic acid (CH3–CHOH–COOH)? Answer: Yes, because the sum of the oxidation numbers of lactic acid is 0, the same as glucose (Fig. 4b). Homofermenting lactic acid bacteria produce lactic acid by this route; heterofermenting lactic acid bacteria produce one mole each of lactic acid, ethanol, and carbon dioxide (7). 3. Can a mole of glucose be fermented to two moles of pyruvate and no other compound? Answer: No, the process is in carbon balance but not redox balance. Another way to think about this question is that NADH, which is also produced in the conversion of glucose to pyruvate, must be reoxidized to NAD+ for anaerobic metabolism to operate. Pyruvate, which is the product of glycolysis, can be reduced by NADH to lactate or can be decarboxylated to ethanal and CO2 (the ethanal is then reduced to ethanol, Fig. 4), or can suffer other fates that depend on the organism (7). 4. Can three moles of glucose be fermented to 2 moles of butanediol, two moles of glycerol, and four moles of CO2? Answer: Yes, the products are in redox balance and carbon balance, and this fermentation is seen in the Bacillus genus (7 ). Although an undergraduate biochemistry student would not be expected to be aware of this particular fermentation, he or she should be able to determine its possibility. 5. Is the following fermentation possible? 3 fructose → lactate + acetate + CO2 + 2 mannitol Answer: Yes, the sum of the oxidation numbers of the carbons of fructose, lactate, and acetate are all 0. The sum of the oxidation numbers of mannitol, CH2OH–(CHOH)4–CH2OH, is ᎑2; when this is multiplied by its stoichiometric coefficient of 2 it exactly cancels the +4 oxidation number of carbon dioxide. Some heterofermenting lactic acid bacteria can use this fermentation when fructose, but not glucose, is the carbon source because they possess a mannitol dehydrogenase that accepts fructose as a substrate (7 ). Again, an undergraduate biochemistry student would not be expected to be aware of the existence of this enzyme; that is why the question is framed as a hypothetical. 6. Malolactic acid fermentation reduces the acidity of wine made from grapes, especially those grown in cool weather, giving the wine a smoother taste (7, 10). This process (Fig. 6) converts one molecule of malate to one molecule of lactate and one other carbon-containing compound. What is the other compound, and why? Answer: The other compound
O−
O +3
C C
0 H −2
OH
C O
C
0 H
CH2
+3
O−
O +3
−3
C
OH
+
?
CH3
O−
+4
0
Lactate
Malate
Figure 6. Malolactic acid fermentation in winemaking. This process gives wine a smoother taste. Malate is converted into lactate and one other carbon-containing compound (see problem 6). The oxidation number of each carbon atom is given to the left of it, and the sum of the oxidation numbers of each compound is indicated.
H2N
N
H N N 5-Methytetrahydrofolate
N N
H2N
OH
CH3 NH R *
N
H N N 5,N10-Methenyltetrahydrofolate
N N OH
HC *
N R
H2N
N
H N N 5,N10-Methylenetetrahydrofolate
N N OH
H2C *
N R
Figure 7. Three derivatives of tetrahydrofolate at three different oxidation states of carbon (see problem 7).
is CO2, having an oxidation number of +4, which brings both carbon atoms and redox numbers into balance. NOTE: It could be argued that a more complete answer would include a water molecule on the reactant side and would have either CO2 and OH᎑, or (equivalently) HCO3᎑ as the other product. This balances all atoms. 7. Assign oxidation numbers to the carbon with the asterisk for the three derivatives of tetrahydrofolate shown in Figure 7. Which is most oxidized? Which is most reduced? Using oxidation numbers that you assigned, write a balanced equation for the conversion of N 5,N 10-methylenetetrahydrofolate (bottom structure) to N 5-methyltetrahydrofolate (top structure), assuming that NAD+ is the cofactor. Give a plausible name for the enzyme that carries out this process. Answer: N 5-methyltetrahydrofolate (᎑2) is most reduced, N 5,N 10-methylenetetrahydrofolate (0) is intermediate, and N 5,N 10-methenyltetrahydrofolate (+2) is most oxidized. The balanced equation is N 5,N 10-methylenetetrahydrofolate + NADH → N 5-methyltetrahydrofolate + NAD+. The enzyme is named N 5,N 10-methylenetetrahydrofolate reductase, although it could also be plausibly named N 5-methyltetrahydrofolate dehydrogenase.
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Acknowledgments
Literature Cited
I would like to thank my students and colleagues at the University of North Carolina at Wilmington for many helpful suggestions.
1. Woolf, A. A. J. Chem. Educ. 1988, 65, 45–46. 2. Calzaferri, G. J. Chem. Educ. 1999, 76, 362–363. 3. Abeles, R. H.; Frey, P. A.; Jencks, W. P. Biochemistry; Jones and Bartlett: Boston, 1992. 4. Brown, T. L.; LeMay, H. E. Jr.; Bursten, B. E. Chemistry the Central Science; Prentice Hall: Upper Saddle River, 1997. 5. Dickerson, R. E.; Gray, H. B.; Haight, G. P. Jr. Chemical Principles; Benjamin/Cummings: Menlo Park, CA, 1979. 6. McMurry, J. Organic Chemistry; 4th ed.; Brooks/Cole: Pacific Grove, CA, 1996. 7. Stanier, R.; Ingraham, J. L.; Wheelis, M. L.; Painter, P. R. The Microbial World; 5th ed.; Prentice-Hall: Englewood Cliffs, NJ, 1986. 8. Zubay, G. Biochemistry; 4th ed.; Wm. C. Brown: Dubuque, IA, 1998. 9. Voet, D. G.; Voet, J. G. Biochemistry; 2nd ed.; Wiley: New York, 1995. 10. Fox, M. A.; Whitesell, J. K. Organic Chemistry; Jones and Bartlett: Sudbury, UK, 1997.
Notes 1. A cosubstrate is an organic molecule that is bound and released during the catalytic cycle of an enzyme. A coenzyme is an organic molecule that remains tightly bound to an enzyme over many catalytic cycles. Thus NAD+ and NADH are cosubstrates, and FAD and FADH2 are coenzymes. 2. It would be convenient to treat sulfur as if it were more electronegative than carbon to stress its similarity to oxygen (esters versus thioesters, for example). This would lead us to assign oxidation numbers to sulfur of ᎑1 and ᎑2 in the reactant and product of this reaction, respectively. Yet the advantage of simply using the electronegativities as given in standard textbooks is, arguably, the overriding principle.
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