Supplemental Lecture (97/10/07 update) by Stephen T. Abedon (abedon.1@osu.edu)

  1. Chapter title: Cellular Respiration
    1. A list of vocabulary words is found toward the end of this document
    2. Cellular respiration describes various biochemical pathways that convert all available (reduced) chemical bonds of an energy-containing substrate into energy stored as ATP. For glucose (a very common energy containing substrate) and in eucaryotes, the overall process of glycolysis followed by aerobic respiration looks like this: C6H12O6 + 6O2 + 36ADP + 36Pi à 6CO2 + 6H2O + 36ATP (or 38 ATP, for example, in certain procaryotic systems).
    3. Note that all of the C-H bonds and C-C bonds have been lost. Only bonds having minimal energy have been spared or created (C-O or H-O). Note also how many more ATP are produced from a single glucose via aerobic respiration than via fermentation (i.e, 36 or 38 versus 2).
    4. This generation of bonds with oxygen is specifically called oxidation and chemically it is a process in which the electrons found in the C-H and C-C bonds are donated to oxygen atoms during bond breaking and formation. The general process of electron donation to an electron acceptor is also referred to as oxidation even though the electron acceptor may not be oxygen. Particularly, the process of cellular respiration consists of a series of oxidation and reduction reactions occurring within a complex electron transport system.
    5. In this lecture we will discuss of the oxidation of carbon-containing substrates via such processes as the Kreb's citric acid cycle, electron transport, and, finally, the formation of ATP via chemiosmosis.
  2. Lecture review

Overview of Cellular Respiration
(with per Glucose ATP Accounting)

# ATP

step

-2

  • priming glycolysis

+4

  • substrate level phosphorylation (glycolysis)

+6

  • 2 NADH produced (glycolysis)

-2

  • transportation of two NADH into mitochondria

+6

  • 2 NADH produced in conversion of pyruvate to acetyl-CoA

+18

+4

+2

+36

  • total ATPs produced from one glucose from aerobic respiration in eucaryotes. Compare with total from glycolysis alone (i.e., 2 ATP).

 

  1. Cellular respiration
    1. Cellular respiration is a complex mechanism whereby large numbers of ATPs are produced via the utilization of an inorganic final electron acceptor and an electron transport chain.
    2. Aerobic and anaerobic:
      1. Cellular respiration comes in two general varieties distinguished by the nature of their inorganic final electron acceptor: oxygen versus everything else.
      2. These two forms of cellular respiration are dubbed aerobic and anaerobic respiration (or cellular respiration), respectively.
  2. Aerobic respiration
    1. Cellular respiration in which oxygen serves as the final electron acceptor.
    2. Aerobic respiration is by far and away the more common form of cellular respiration.
  3. Anaerobic respiration
    1. No oxygen, yes electron transport chain:
      1. Cellular respiration in which something other than oxygen serves as the final electron acceptor.
      2. Examples of non-oxygen final electron acceptors include:
      3. nitrate, NO3-, which is converted to nitrite, nitrous oxide, or nitrogen [NO21-, N2O, and N2, respectively] in the process
      4. sulfate, (SO42-, which is converted to hydrogen sulfide, H2S
      5. carbonate, CO32-, which is converted to methane, CH4
    2. Anaerobic respiration doesn't result in as much ATP production as when oxygen is the final electron receptor.
    3. Nevertheless, organisms (i.e., bacteria) that use anaerobic respiration occupy important niches and, indeed, exploit niches that organisms which require aerobic respiration cannot exploit and therefore cannot compete for.
  4. Products of glycolysis
    1. Glycolysis is cytoplasmic:
      1. Glycolysis occurs in the cytoplasm of eucaryotic cells.
      2. Glycolysis also occurs in the cytoplasm of bacteria.
    2. NADH and pyruvate:
      1. Besides ATP, the two important products of glycolysis are NADH and pyruvate.
      2. When oxygen is lacking, both products are sacrificed to the process of fermentation in order to regenerate NAD+.
      3. In organisms capable of undergoing cellular respiration, however, both NADH and pyruvate may be further oxidized to generate additional ATP.
      4. These further steps are performed within mitochondria in eucaryotes.
    3. Bacteria do it all cytoplasmically:
      1. Within bacteria possessing electron transport systems, both glycolysis and cellular respiration take place in their cytoplasms.
      2. This differs from the doings of eucaryotes because eucaryotes have literally co-opted bacteria--a.k.a. mitochondria--to perform their cellular respiration).
  5. NADH into mitochondria
    1. Active NADH transport into mitochondria:
      1. The NADH product of glycolysis may be utilized in cellular respiration given transport of the NADH into mitochondria (procaryotes, of course, don't have this problem).
    2. This transfer costs eucaryotic cells one ATP.
  6. Acetyl-CoA [coenzyme A]
    1. Central metabolic intermediate:
      1. A central molecule in cellular respiration, one to which all proteins, lipids, and carbohydrates must be converted prior to participation in cellular respiration.
      2. The fate of acetyl-CoA is dependent upon ATP needs. When ATP is prevalent, acetyl-CoA serves as the basis for fatty acid synthesis, which forms the basis of your body's long-term energy storage: triglycerides (i.e., fat).
      3. Alternatively, acetyl-CoA may enter the Kreb's citric acid cycle.
    2. Structure of acetyl-CoA:
      1. Structurally, acetyl-CoA consists of a two carbon group attached to a coenzyme (coenzyme A). That is, an acetyl group has the following structure:
    |  
    H3C-C=O
  7. Note the unpaired electron on top of the first carbon (i.e., that attached to the double-bonded oxygen) which is bound to coenzyme A in acetyl-CoA. Coenzyme A is far more complex and based upon an ADP core. See figure 1100.1.
  8. Conversion of pyruvate to acetyl-CoA
    1. Pyruvate is transported into mitochondria at no apparent energy cost.
    2. There it is converted into acetyl-CoA with the following stoichiometry: pyruvate + NAD+ + CoA à acetyl-CoA + NADH + CO2
  9. Kreb's (citric acid, tricarboxylic acid) cycle
    1. A multi-step reaction during which an acetyl group is completely oxidized to CO2 . . .
    2. . . . and reducing hydrogens.
    3. This oxidation is analogous, and in some ways mechanistically similar to the oxidation of glucose in glycolysis through pyruvate and acetyl-CoA formation. That is:
      1. reducing hydrogens are accumulated in NADH
      2. some ATP is generated
      3. there is a reduction in carbon number, though here this is effected through liberation of carbon dioxide
    4. In the Kreb's cycle there is an electron acceptor in addition to NAD+ called FAD (flavin adenine dinucleotide; which is converted to FADH2 in the course of reduction).
    5. Overall, the Kreb's cycle has the following stoichiometry:
    
    oxaloacetate + acetyl-CoA + 2H2O + ADP + Pi + 3NAD+ + FAD à oxaloacetate + 2CO2 + CoA + ATP + 3NADH + 3H+ + 2"FADH2

  10. Biochemistry of Kreb's cycle (simplified outline)
    1. Below is a greatly simplified outline of the Kreb's cycle to give you a very basic idea of the reactions involved0:
      1. C2-CoA + C4 + H2O à C6 + CoA
      2. C6 + NAD+ à C5 + NADH + H+ + CO2
      3. C5 + 2NAD+ + FAD + ADP + Pi + H2O à C4 + 2NADH + 2H+ + 2"FADH2 + ATP + CO2
      4. C2-CoA + C4 + H2O à C6 + CoA
      5. etc. . . .
    2. Note that:
      1. C4 in its final form (as shown above) is oxaloacetate (recall that the above is a gross over-simplification and thus C4 compounds in the Kreb's cycle also include, in order, succinyl-CoA, succinic acid, fumaric acid, and malic acid)
      2. C6 is citric acid (as well as a number of other compounds as it is successively converted to including cis-aconitic acid, isocitric acid, and oxalosuccinic acid)
      3. C2-CoA is, of course, acetyl-CoA.
      4. C5, on the other hand, represents just a-ketoglutaric acid.
  11. Structures of Kreb's cycle intermediates
    1. C6: citric acid (note: H2O is gained, acetyl-CoA is added to oxaloacetate, and coenzyme-A is lost in the making of this intermediate):
    2.      H   
           |   
      HOOC-C-H 
           |   
      HOOC-C-OH
           |   
      HOOC-C-H 
           |   
           H   
      
    3. C6: cis-aconitic acid (cis-acontate) (note: H2O is lost to make this intermediate):
    4.      H  
           |  
      HOOC-C  
           || 
      HOOC-C  
           |  
      HOOC-C-H
           |  
           H  
      
    5. C6: isocitric acid (isocitrate) (note: H2O is gained to make this intermediate):
    6.      H   
           |   
      HOOC-C-OH
           |   
      HOOC-C-H 
           |   
      HOOC-C-H 
           |   
           H   
      
    7. C6: oxalosuccinic acid (oxalosuccinate) (note: NAD+ is reduced in the making of this intermediate):
    8.      H  
           |  
      HOOC-C=O
           |  
      HOOC-C-H
           |  
      HOOC-C-H
           |  
           H  
      
    9. C5: a-Ketoglutaric acid (alpha-ketoglutarate) (note: CO2 is lost in the making of this intermediate):
    10. HOOC-C=O
           |  
         H-C-H
           |  
      HOOC-C-H
           |  
           H  
      
    11. C4: succinyl-CoA (note: CO2 is lost, NAD+ reduced, and coenzyme A added in the making of this intermediate--this step should remind you of the conversion of pyruvate to acetyl-CoA):
    12.      coenzyme-A
           |         
           C=O       
           |         
         H-C-H       
           |         
      HOOC-C-H       
           |         
           H         
      
    13. C4: succinic acid (succinate) (note: in the making of this intermediate sufficient energy is liberated to phosphorylate ADP thus producing one ATP):
    14.      H  
           |  
      HOOC-C-H
           |  
      HOOC-C-H
           |  
           H  
      
    15. C4: fumaric acid (fumarate) (note: FAD is reduced in the making of this intermediate):
    16.      H 
           | 
      HOOC-C 
           ||
      HOOC-C 
           | 
           H 
      
    17. C4: malic acid (malate) (note: H2O is gained to make this intermediate):
    18.      H   
           |   
      HOOC-C-H 
           |   
      HOOC-C-OH
           |   
           H   
      
    19. C4: oxaloacetic acid (oxaloacetate) (note: NAD+ is reduced in the making of this intermediate):
         H  
         |  
    HOOC-C-H
         |  
    HOOC-C=O
         |  
         H  
    
  12. Regeneration of NAD+ and FAD
    1. As with glycolysis, continued cycling of the Kreb's cycle depends on the regeneration of NAD+ and FAD.
    2. Unlike fermentation, cellular respiration has a means of extracting the energy in the reduced molecules (NADH and 2"FADH2).
    3. In fact, this energy extraction procedure employs an electron transport system, has a molecular oxygen terminal electron acceptor, and is employed to generate ATP via a process called chemiosmosis.
  13. Electron transport system [chain]
    1. A series of oxidizing and reducing molecules (proteins and other) found in the inner membrane of mitochondria as well as all bacteria capable of undergoing cellular respiration.
    2. Electron transport chains convert the energy associated with reduced electrons, liberated in the conversion of NADH to NAD+ (and 2"FADH2 to FAD), to protons (H+) pumped from the mitochondria matrix (for example) to the mitochondria outer compartment.
    3. high [H+] out, low in (matrix)
    4. As electrons are transported along this chain of electron donors and acceptors they incrementally lose energy (which, as noted, is harnessed in the pumping of protons out of the mitochondria matrix).
    5. The final acceptor of the electrons, in aerobic respiration, is molecular oxygen, a matrix reaction which serves to increase the proton concentration gradient across the mitochondria inner membrane: 4H+ + 4e- + O2 à 2H2O
    6. Absolute dependence on final electron acceptor:
      1. Note that in the absence of a final electron acceptor, cellular respiration stops.
      2. Note also that for humans and other aerobic respirators, this means that cellular respiration cannot occur in the absence of oxygen.
  14. Chemiosmosis
    1. The pumping of protons from the mitochondria matrix into the mitochondria outer compartment establishes a concentration gradient.
    2. Influx coupled to ATP synthesis:
      1. This concentration gradient may be harnessed though coupling with the facilitated diffusion of these protons back into the matrix.
      2. In fact, this facilitated diffusion is harnessed (via a proton-pump running in reverse; for an idea of how a proton-pump might work see how a sodium-potassium pump pumps sodium ions) to generate ATP in a process known as chemiosmosis.
    3. The stoichiometry of chemiosmosis and electron transport chain (ETS) pumping is:

    
    9H+ (matrix) + NADH
    
    --(ETS)-- 
    
    NAD+ + 9H+ (outer compartment)
    
    6H+ (matrix) + 2"FADH2
    
    --(ETS)-- 
    
    FAD + 9H+ (outer compartment)
    
       3H+ (outer compartment) + ADP + Pi   
    
    --(with concentration gradient)--
    
    3H+ (matrix) + ATP

  15. Vocabulary
    1. Acetyl-CoA
    2. Active transport of NADH into mitochondria
    3. Aerobic respiration
    4. Anaerobic respiration
    5. Biochemistry of Kreb's cycle
    6. Cellular respiration
    7. Chemiosmosis
    8. Citric acid
    9. Conversion of pyruvate into acetyl-CoA
    10. Electron transport chain
    11. FAD
    12. 2"FADH2
    13. Kreb's cycle
    14. Kreb's citric acid cycle
    15. Oxaloacetate
    16. Regeneration of FAD
    17. Regeneration of NAD+
    18. Tricarboxylic acid cycle
  16. Practice questions
    1. Which is the least reduced (circle correct answer): [PEEK]
    (i)               (ii)             (iii)  
                                           
                       H               H   
                       |               |   
HOOC-C=O          HOOC-C-OH       HOOC-C-H 
     |                 |               |   
HOOC-C-H    or    HOOC-C-H   or   HOOC-C-OH
     |                 |               |   
HOOC-C-H          HOOC-C-H        HOOC-C-H 
     |                 |               |   
     H                 H               H   
                                           
                                           
              (iv) no difference
            
    1. What's the next reaction (i.e., involving C4)? (circle correct answer) [PEEK]
    2. 	(1) C2-CoA + C4 + H2O à C6 + CoA
      
      	(2) C6 + NAD+ à C5 + NADH + H+ + CO2
      
      	(3) C5 + 2NAD+ + FAD + ADP + Pi + H2O à C4 + 2NADH + 2H+ + FADH2 + ATP + CO2
      
      	(4) ???
      
    3. What two products of glycolysis are transported into the mitochondria thus allowing eucaryotes to generate more than two ATP per glucose? [PEEK]
    4. What happens to acetyl-CoA if a cell already has sufficient quantities of ATP? [PEEK]
    5. Fill in the missing numbers to produce a stoichiometrically correct overall Kreb's cycle: [PEEK]
    6. 	oxaloacetate + acetyl-CoA + H2O + 
      
      		___ ADP + ___ Pi + ___ NAD+ + ___ FAD à
       
      
      		oxaloacetate + ___ CO2 + CoA + ___
      
      		ATP + ___ NADH + ___ H+ + ___ FADH2
    7. The following molecule is? (circle correct answer) [PEEK]
    8.   OH    
        |     
        C=O   
        |     
      H-C-H   
        |     
        C=O   
        |     
        C=O   
        |     
        O- Na+
      
      1. Oxaloacetate
      2. NADH
      3. glucose 6-phosphate
      4. citric acid
      5. FADH2
      6. pyruvate
      7. all of the above
      8. none of the above
    9. Draw citric acid. [PEEK]
    10. In eucaryotes, how many gross (i.e., ignore priming and transport costs) additional ATPs are produced per glucose via chemiosmosis? (circle correct answer) [PEEK]
      1. 1
      2. 11
      3. 22
      4. 34
      5. 36
      6. 38
      7. 40
      8. 42
    11. Based on your limited knowledge of the mitochondrial electron transport chain, and considering only those electrons which are routinely lost and gained, which of the following compounds would you say is the most reduced? (circle correct answer) [PEEK]
      1. NADH
      2. NAD+
      3. FADH2
      4. FAD
      5. all are equivalent
    12. What is the following: [PEEK]
     H   
     |   
HOOC-C-H 
     |   
HOOC-C-OH
     |   
HOOC-C-H 
     |   
     H   
      1. pyruvic acid
      2. ascorbic acid
      3. citric acid
      4. benzoic acid
      5. oxaloacetic acid
      6. none of the above
    1. I mentioned in class that certain Pseudomonas species are responsible for the conversion of fixed nitrogen to molecular nitrogen in anaerobic soils, particularly those soaked with water. They do this by employing nitrate (NO3-) as a final electron acceptor. What is a general name for this process whereby an inorganic substance is employed as a final electron acceptor in an anaerobic environment? [PEEK]
    2. Name two differences between respiration and fermentation.[PEEK]
    3. In aerobic respiration, what is the final electron acceptor[PEEK]
    4. Which two of the following are composed of, in part or in whole, of a three carbon core such as that found in glycerol (shown below)? [PEEK]
    5.     OH  OH  OH   
          |   |   |    
      H - C - C - C - H
          |   |   |    
          H   H   H    

      (circle both correct answers below)

      1. Glycolysis
      2. Kreb's cycle
      3. phospholipids
      4. tricarboxylic acid
      5. inorganic phosphate
      6. sterols
    6. Start with one Glucose. At the end of glycolysis but prior to acetyl CoA production (and prior to the Kreb's cycle, and prior to electron transfer, and prior to chemiosmosis, i.e., in solving this problem ignore all other aspects of cellular respiration other than the transport of glycolytic products into the mitochondria), if all of the glycolytic products which normally find their way into the mitochondria are now there, what is the up to this moment net yield of ATP? (circle one correct answer) [PEEK]
      1. 0.
      2. 1.
      3. 2.
      4. 3.
      5. 4.
      6. more than four.
    7. How many carbon dioxides are produced per individual turn of the Kreb's citric acid cycle (i.e., starting with one acetyl-CoA and one oxaloacetate and ending with one oxaloacetate)? (circle one correct answer) [PEEK]
      1. 0.
      2. 1.
      3. 2.
      4. 3.
      5. 4.
      6. 5.
      7. 6.
      8. more than 6.
    8. Just considering numbers of carbon atoms (and ignoring ADPs, ATPs, NAD+, NADH, FAD, FADH2, various enzymes, and other structural components of the cell), glycolysis and then the Kreb's cycle progress via (non-carbon dioxide) carbon containing compound intermediates having __________ carbon atoms each (note: ignore isomerizations as well as other changes in compounds which do no result in changes in carbon number). (circle one correct answer) [PEEK]
      1. 6, 5, 4, 3, 2, and then 1.
      2. 6, 4, 3, 6, 4, and then 2.
      3. 6, 7, 6, 4, 3, 6, 4, and then 2.
      4. 6 and then 2.
      5. 6, 3, 2, 6, 5, and then 4.
      6. 6, 4, 2, 6, and then 4.
      7. 6, 2, 4, and then 6.
    9. The total ATP produced (i.e., gross--ignore priming costs and transport costs), as a consequence of both substrate level phosphrolylation and chemiosmosis, as a consequence of (i) glycolysis, (ii) acetyl-CoA production, (iii) Kreb's cycle, (iv) substrate-level phosphorylation-only, and (v) chemiosmosis-only, all per one starting glucose, is __________ ATPs, repectively. (circle one correct answer) [PEEK]
      1. 4, 12, 18, 3, and 24.
      2. 8, 4, 22, 5, and 30.
      3. 6, 2, 20, 4, and 28.
      4. 10, 6, 24, 6, and 34.
      5. 2, 8, 16, 2, and 20.
      6. 0, 6, 14, 1, and 16.
    10. Name two ways fermentation differs from anaerobic respiration. [PEEK]
    11. Name three possible byproducts of ATP generation. [PEEK]
    12. "__________," is one similarity between fermentation and anaerobic respiration. [PEEK]
      1. both occur purely in the cytoplasm.
      2. both are a feature of obligate aerobes.
      3. both occur only in eucaryotes.
      4. both regenerate NAD+.
      5. both require oxygen.
      6. humans are capable of doing both.
  1. Practice question answers
    1. i is the answer (structure on left). These are all Kreb's cycle intermediates. The one in the middle is isocitric acid while the one on the left is oxalosuccinic acid. Citric acid is on the far right. Note that, in the course of the Kreb's cycle, citric acid is converted to isocitric acid which is then converted to oxalosuccinic acid, the latter reaction results in NAD+ being reduced to NADH + H+. Thus, the most oxidized (i.e., least reduced) is the compound on the left, oxalosuccinic acid. Regardless, the intermediates to oxalosuccinic acids (middle and far right) both have more hydrogens thus suggesting a greater level of reduction.
    2. Same as first reaction (1), C2-CoA + C4 + H2O à C6 + CoA
    3. pyruvate and NADH
    4. storage fats are made
    5. all are ones except in front of CO2 which is a two and in front of NAD+, NADH, and H+ which are all threes.
    6. i, oxaloacetate
    7. tricarboxylic acid:
     H   
     |   
HOOC-C-H 
     |   
HOOC-C-OH
     |   
HOOC-C-H 
     |   
     H   
    1. iv, per pyruvate there is 1 NADH + H+ from glycolysis + 1 NADH + H+ from acetyl-CoA formation + 3 NADH + H+ from the Kreb's citric acid cycle + 1 FADH2 from the Kreb's citric acid cycle. Given 3 ATP per NADH and 2 ATP through FADH2 following electron transport and chemiosmosis, that's (5 * 3) + 2 ATP = 17 ATP per pyruvate or a 34 ATP per glucose gross yield from chemiosmosis. Of course we lose 2 ATP for the transport of 2 NADHs into the mitochondria, so the net gain is 32 ATP from chemiosmosis. Note that two additional ATPs come from glycolysis and substrate level phosphorylation during the Kreb's cycle bring the total net ATP yield to 36 per glucose in eucoryotes.
    2. i, NADH. The easy way to figure this out is to note how many ATPs each species is worth upon access to an electron transport chain in a mitochondria: 3, 0, 2, and 0 for NADH, NAD+, FADH2, and FAD.
    3. iii, citric acid
    4. anaerobic respiration
    5. Respiration makes lots of ATP, uses inorganic final electron acceptors (such as molecular oxygen), and uses an electron transport chain. Fermentation makes only a little ATP, does not use an electron transport chain, and uses organic final electron acceptors. Note that in either case "waste" molecules are given off into the environment (CO2 is the waste molecule in the case of aerobic respiration).
    6. Oxygen
    7. i and iii, glycolysis (a number of intermediates) and phospholipids (a glycerol derivative forms the central core)
    8. i, 0; recall that only two net ATP are produced by glycolysis, but that these are both employed in the transport of NADH into the mitochondria.
    9. iii, 2; remember, each turn of the Kreb's cycle converts a six carbon molecule to a four carbon molecule with each carbon lost as a CO(2).
    10. v, 6, 3, 2, 6, 5, and then 4.
    11. iv, 10, 6, 24, 6, and 34.
    12. organic final electron acceptor rather than inorganic, less ATP versus more, production of organic waste versus lack of production of organic waste, lack of use of electron transport system versus use of, ATPs generated by substrate level phosphorylation versus ATPs mostly generated by chemiosmosis, lack of chemiosmosis versus chemiosmosis, etc.
    13. H2O, various non-oxygen inorganic electron acceptors including nitrogen gas, NADH, fermentation products including: lactic acid, ethanol, CO2, acetone, formic acid, H+.
    14. iv, both regenerate NAD+.
  1. References
    1. Raven, P.H., Johnson, G.B. (1995). Biology (updated version). Third Edition. Wm. C. Brown publishers, Dubuque, Iowa. pp. 153-178.