Dr Bengt Ingvarsson Department of Math and Science Kristianstad University Sweden

Teaching cell respiration as a redox reaction

Key words: Cell metabolism, conceptualisation, Lewis electron structure formulas, meaningful knowledge, oxidation numbers, redox reaction, student understanding, teaching strategy.

Background
I have been working as a teacher in biology and chemistry at the upper secondary school and at the university for almost 20 years. One area that has fascinated me in my teaching is cellular metabolism. I can still remember when I once learned this area mostly by rote. I could write all the reactions in the glycolysis, citric acid cycle and respiration chain, but I had very little understanding of what actually happened in these complicated reactions and why they occur! I believe that most of the students leave this specific area in a similar way as I did. This is of course not satisfactory because cell metabolism is a fundamental and important part of the syllabus in biochemistry and cell biology in the upper secondary school and at the university. I think, looking back on my own studies and on my years as a teacher, that the students need a lot of support in order to understand this complicated process. The choice of a teaching strategy and the way the subject matter is presented in the textbooks are in my opinion of greatest importance in order to optimise the learning process. During the years of teaching I have gradually developed my own understanding of cellular metabolism. One major step in my conceptualisation occurred when I understood that the cell respiration process is a redox reaction, and can be treated in the same way as redox reactions are treated in inorganic chemistry. This paper presents a teaching strategy for the cell respiration process based on the redox model. The strategy treats, in my opinion, the complicated cell respiration process in a comprehensible and concrete way. Neither the textbooks nor my teacher colleagues taught, as far as I know, cell respiration in this way. This is not surprising because oxidation numbers are normally not used in organic chemistry or in biochemistry. The redox model can also be used in teaching other parts of the cell metabolism such as the beta-oxidation of fatty acids and photosynthesis. I will in this paper refer to the following three textbooks: Alberts et. al.: Molecular Biology of the cell (1994), Campell et. al.: Biology (1999) and Mauseth: Botany. An introduction to plant biology (1998). These textbooks are at the moment used in cell biology courses at the University of Kristianstad, where I am working.

The teaching sequence of the cell respiration process as a redox reaction

1. Lewis electron structure formulas and oxidation numbers. Lewis electron structure formulas and oxidation numbers are discussed for the carbon dioxide -, oxygen - and water molecules, as well as for the functional groups in the different organic compounds that are involved in the redox process. See figure 1. The chemical concepts used in this discussion (electronegativity, Lewis formula, oxidation number, oxidation, reduction) have already been introduced in earlier studies. It is important that the students understand how to calculate the oxidation numbers within a given Lewis formula.

2. The redox model applied to the overall reaction. The transfer of 24 electrons for each glucose molecule oxidised. Cell respiration is the process where carbon atoms in carbohydrate molecules are oxidised to carbon atoms in carbon dioxide molecules, and oxygen atoms in oxygen molecules are reduced to oxygen atoms in water molecules. The process is very complex, and occurs in many small steps. The formula (with oxidation numbers for the atoms involved in the redox process) can be written: 0 C6 H12 O6 0 +IV * 6 O2 + 6 H2 O + 36 ADP + 36 P 6 CO2 -II 12 H2 O* + 36 ATP

This is, in my opinion, the best way to write the overall formula, as all the twelve oxygen atoms in the six oxygen molecules are involved in the redox process. These twelve oxygen atoms are finally found in the twelve water molecules produced in the process (*)! It is easy to find out from this formula and from the oxidation numbers for the atoms that are involved in the redox process, that the six carbon atoms in one glucose molecule are oxidised in 24 steps (6 x 4). Furthermore it can be seen that the 12 oxygen atoms in the six oxygen molecules are reduced 24 in steps (12 x 2). Thus, 24 electrons are in the redox model transferred from one glucose molecule to six molecules of oxygen to give twelve molecules of water.

3. The 24 electrons are transferred in pairs in NADH or FADH2 . The identification of two more electrons in the Lewis electron structure formulas of NADH and FADH2 compared to NAD+ and FAD. The 24 electrons involved in the oxidation of one glucose molecule are in the cell respiration process treated in pairs. The electrons are bound and transferred within complicated organic molecules such as the coenzymes nikotinamid-adenin-dinukleotid (NAD+) and flavin-adenindinukleotid (FAD) according to the following formulas:

NAD+ + 2 e- + H+ FAD + 2 e- + 2 H+

NADH FADH2

It is easy to calculate from the Lewis electron structure formulas that two more electrons are bound in NADH and FADH2 compared to NAD+ and FAD (figure 2). 4. The 24 electrons makes 10 NADH and 2 FADH2 . The identification of 10 NADH and 2 FADH2 for each glucose molecule completely oxidised. The oxidation of one glucose molecule to carbon dioxide and water yields 24 electrons. This means that you will look for 12 reduced coenzymes, since each NAD+ or FAD binds 2 electrons. Actually, 10 NADH and 2 FADH2 are found in the process, according to the following schedule:

Glucose (C 6 ) ____________________________ Glyceraldehyde 3-P (C 3 ) Glyceraldehyde 3-P (C 3 ) NAD+ NADH Pyruvate (C 3 ) NAD+ NADH CO2 Acetyl CoA (C 2 ) citrate cycle 3 NAD+ 3 NADH FAD FADH2 2 CO2 NAD+ NADH Pyruvate (C 3 ) NAD+ NADH CO2 Acetyl CoA (C 2 ) citrate cycle 3 NAD+ 3 NADH FAD FADH2 2 CO2

5. The production of each NADH or FADH2 in the glycolysis and in the citrate cycle is coupled to a two step oxidation of carbon atoms. The production of each NADH or FADH2 molecule in the glycolysis and in the citrate cycle is coupled to a two step oxidation of carbon atoms. This can easily be shown with the use of oxidation numbers, as can be seen in the different reactions below.

6. The reactions in the glycolysis in a redox perspective. The glucose molecule is in the first part of the glycolysis rearranged and split into two molecules with three carbon atoms each. One of them, glyceraldehyde 3-P, is then oxidised in the second part of the glycolysis to the corresponding carboxylic acid with the formation of NADH. The reaction in the glycolysis where NADH is produced is normally written: CHO (+I) CHOH CH2 OP COOP (+III) CHOH CH2OP

+ Pi + NAD +

+ NADH + H+

Glyceraldehyde 3-phosphate

1,3-diphosphoglycerate

Oxidation numbers are written in brackets for the carbon atoms that are involved in the redox process. The production of the NADH molecule is in this reaction coupled to a two steps oxidation of the aldehyde carbon atom in glyceraldehyde 3-phosphate to the carboxylic acid carbon atom in 1,3-diphosphoglycerate.

7. The reaction that converts pyruvate to acetyl-CoA. The next time NADH is produced in the cell respiration process is in the reaction where pyruvate is transformed into Acetyl- CoA. This reaction can be written: COO - (+III) C = O (+II) CH3 Pyruvate S-CoA + HS-CoA C = O (+III) CH3 Acetyl-CoA

+ NAD +

+ CO2 (+IV) + NADH

The formation of NADH is in this reaction coupled to an oxidation of two carbon atoms that are oxidised one step each. The carbon atom in the carboxylic acid group is oxidised one step to the carbon atom in carbon dioxide, whereas the carbon atom in the carbonyl group is oxidised one step to the carbon atom in the carbonyl group which bind to the sulphur atom.

8. The reaction that converts pyruvate to lactate. Muscle cells produce lactate in the absence of oxygen in order to regenerate NAD+. The reaction can be written:

COO C = O (+II) CH3 Pyruvate

NADH

+ H+

COO CHOH (0) CH3 Lactate

+ NAD +

This reaction is in the redox perspective the opposite of the other reactions discussed in this paper, because NADH is consumed and not produced. The oxidation of NADH to NAD+ is in the reaction coupled to a two step reduction of the carbon atom in the carbonyl group.

9. The Citrate cycle in an overall perspective. We can identify the production of 3 NADH and 1 FADH2 in the citrate cycle. This is equivalent to 8 electrons and a total increase in oxidation numbers of 8 steps. This can be seen in an overall perspective in the following way: S-CoA C = O (+III) + 3 NAD + + FAD 2 CO2 (+IV) + HSCoA + 3 NADH +3 H+ + FADH2 CH3 (-III) Acetyl-CoA The two carbon atoms in acetyl- CoA are oxidised to two carbon dioxide molecules in the citrate cycle. The carbon atom in the methyl group is oxidised seven steps, and the carbon atom in the carbonyl group is oxidised one step.

10. The specific reactions in the citrate cycle. The production of each NADH or FADH2 in the citrate cycle is coupled to a two step increase in oxidation numbers of carbon atoms. This can be seen in the following four reactions: COO H-C-OH (0) H-C-COO - (-I ; +III) H-C-H COO Isocitrate COO C = O (+II) CH2 (-II) + CH2 COO -ketoglutarate

+ NAD +

CO2 (+IV) + NADH + H+

COO - (+III) C=O (+II) CH2 + NAD + + HS-CoA CH2 COO -ketoglutarate

S-CoA C=O (+III) CH2 + CO 2 (+IV) + NADH CH2 COO succinyl-CoA

COO CH2 (-II) CH2 (-II) COO Succinate

+ FAD

COO HC (-I) CH (-I) COO Fumarate

+ FADH2

COO CHOH (0) + CH2 COO Malate

NAD +

COO C=O (+II) CH2 COO Oxaloacetate

+ NADH + H+

11. The electron transport chain and the final placement of the transported electrons in the water molecules. The 24 electrons obtained from the oxidation of one glucose molecule are transferred to the electron transport chain within 10 NADH and 2 FADH2 . These electrons are in the electron transport chain transported between different redox pairs. It is possible, with the use of Lewis formulas, to identify two more electrons in the reduced form of each redox pair, for example FMNH2 and QH2 , compared to the corresponding oxidised form. The 24 electrons are finally transferred into six molecules of oxygen with the following formation of 12 water molecules according to the following formula: 24 e- + 6 O2 + 24 H+ 12 H2 O

The six oxygen molecules have 72 (6 x 12) valence electrons (se figure 1) and the twelve water molecules have 96 (12 x 8) valence electrons (se figure 1). This makes the difference of the 24 electrons.

Discussion
Cell metabolism is a difficult domain to teach, partly because the process is very complicated and complex by itself, and partly because it is difficult to connect the process to everyday experiences. The teaching strategy presented in this paper is in my opinion useful in teaching cell respiration of the following three reasons: 1. The teaching strategy treats the complicated process in a comprehensible and concrete way and can serve as a conceptual framework for the students. 2. The teaching strategy helps the students to find a proper progression in their learning process, with a start in the overall perspective. 3. The teaching strategy includes chemical concepts that already have been introduced in earlier studies, such as electronegativity, oxidation numbers, redox reaction and Lewis electron structure formulas. Furthermore, the teaching strategy treats the cell respiration process in the same way as a redox reaction is treated in inorganic chemistry according to the oxidation number method. Research in science education has shown that it is important to start the education sequence in the students world. Ausubel states that the most important single factor that influences the learning process is what the learner already knows. Try to ascertain this he says, and teach him accordingly (4). The only everyday experiences that most students might have concerning cell metabolism is that food is necessary to get energy, and that muscle cells can produce lactic acid when they are working hard. It is therefore particularly important to work with concepts that are familiar to the students. Modern textbooks in Chemistry and Biology present in my opinion the cell respiration process in a similar way, and little is normally written concerning the redox perspective of the process. I have not found any textbook that discuss the cell respiration process in terms of oxidation numbers and Lewis electron structure formulas as is done in the teaching strategy described in this paper. Alberts et. al. (1994) discuss in the section Cells obtain energy by the oxidation of biological molecules the successive oxidation of organic compounds to carbon dioxide in a similar way as is shown in figure 1. The great difference is that Alberts et. al. do not use oxidation numbers in their teaching model. Campell et. al. (1999) compare in the two sections Redox reactions release energy when electrons move closer to electronegative atoms and Electrons fall from organic molecules to oxygen during cellular respiration the cell respiration process with redox processes from inorganic chemistry. The cell respiration process is in the redox perspective a similar reaction to the reaction between Sodium and Chlorine. They also discuss the movement of the covalently shared electrons that occur in the redox process, but do not use oxidation numbers. Campell et.al. (1999) write the overall formula for the cell respiration process with the net production of six water molecules. This is not surprising, because the only reason to write the overall formula with another 6 water molecules as reactants and products is if you will treat the reaction as a redox reaction. All the twelve oxygen atoms in the six oxygen molecules that react with the glucose molecule are in the process reduced to oxygen atoms in water molecules, which means that 12 water molecules are produced.

Mauseth (1998) is discussing the complicated photosynthesis process. He uses in the section Energy and reducing power the oxidation numbers for carbon atoms in carbon dioxide and in carbohydrate in order to find out that four electrons must be added to each carbon atom in carbon dioxide to lower the oxidation state from +4 to zero. This is in fact the only textbook I have seen that uses oxidation numbers for different carbon atoms in the discussion of the cell respiration- or the photosynthesis process. The teaching strategy presented in this paper will be tested in further research in science education settings both at the upper secondary level and at the university level. The usefulness of the redox model will be evaluated by using questionnaires and interviews with students and teachers. In order to make such an investigation successful it is necessary to know more about the students thinking about the process. Finally, it is important to state that this teaching strategy only treats the cell respiration from the redox perspective, and that many other perspectives must be taken into consideration in order to achieve a meaningful learning in this specific domain.

Acknowledgements
The author would like to thank Dr Gustav Helldn, Dr Gte Johansson and Dr Hans-Erik kerlund for their helpful comments.

References
1. Alberts B. Bray D. Lewis J. Raff M. Roberts K. and Watson J. (1994) Molecular biology of the cell, 3 ed (ISBN 0-8153-1620-8). 2. Campell N. Reece J. and Mitchell L. (1998) Biology, 5 ed (ISBN 0-8053-3044-5). 3. Mauseth J.D. (1998) Botany. An introduction to plant biology, 2 ed (ISBN 0-7637-07465). 4. Ausubel D. Novak J. and Hanesian H. (1978) Educational phychology: A cognitive view, 2 ed. New York: Holt, Rinehart and Winston.

Figure 1: Oxidation numbers for atoms that are involved in the redox process of the cell respiration. The organic substances are ordered in terms of increasing oxidation numbers, with the oxidation number for the carbon atom in the functional group given in brackets. +IV (carbon atom) O C O 0 O 0 O H Carbon dioxide molecule Oxygen molecule -II (oxygen atom) O H

Water molecule

H H C C C H O C C C H O C C H

Fat (-II)

Carbohydrate (0)

Aldehyd (+I)

O C C C

O C C O H

Keton (+II)

Carboxylic acid (+III)

10

Figure 2: The Lewis electron structure formulas for NADH compared to NAD+ and FADH2 compared to FAD. H H O H C NH2 H H

O C NH2

N R

N R

NAD+ H N CH3 CH3 N H R N O N H O

NADH

FAD

H CH3 CH3

H N

O N

H O

N H R

N H

FADH2

11