Biochemistry: The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

How electrons encounter oxygen, how ATP is generated in the process, and related phenomena.
Oxygenases and hydroxylases
Desaturation of fatty acids

Enzymatic activity associated with the Endoplasmic reticulum and requiring O₂ has been detected, which converts saturated Fatty acids into cis-monounsaturated acids (e.g., stearoyl-CoA or oleoyl-CoA; Table 2-7) [160]. Hydroxylation is likely the initial step in these reactions. Cytochrome b₅ appears to act as a specific electron carrier in this system, although the exact Nature of the hydroxylase itself remains unknown.

Questions and Problems

1. You are provided with 50 mg samples of two pure iron-sulfur Proteins: rubredoxin and ferredoxin. Describe a simple chemical test to distinguish between these two substances.

2. What will be the difference in the net charge at pH 7 between clostridial apoferredoxin (i.e., the iron-free protein) and reconstituted ferredoxin prepared by adding 4 ferrous ions, 4 ferric ions, and 8 molecules of H₂S to the apoprotein? Hint: The pK_a of the cysteinyl SH group is ~9.

3. Consider two reactions of The Tricarboxylic Acid Cycle:

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a. How many ATP molecules can be produced in The Cell as a result of each of these oxidative reactions?

b. How many ATP molecules can be generated via the cytoplasmic oxidation of glyceraldehyde-3-phosphate by NAD⁺?

In answering question (b), consider various shuttle mechanisms that transport reducing equivalents into the Mitochondria.

4. a) Calculate ΔG' (pH 7) for the Oxidation of Pyruvate to CO₂ and Water by NAD⁺. b) Compare this value with ΔG' for oxidation by O₂. c) How large will the difference be if the oxygen pressure is only 0.01 atm? d) Would such a low intracellular O₂ pressure lead to a significant slowdown of pyruvate oxidation in the mitochondria?

5. Assume that a membrane-bound Hydrogenase has equilibrated H₂ with H⁺ and Other components of the Organism's Respiratory Chain. a) At pH 7 and an H₂ pressure of 0.1 atm, what will be The values of the following ratios: [NADH]/[NAD⁺]; [F_d (reduced)]/[F_d (oxidized)] (for clostridial ferredoxin); [Cytochrome c (reduced)]/[Cytochrome c (oxidized)]? b) Will these ratios change if electron flow through respiratory chain site 1 is tightly coupled to the synthesis of one ATP molecule? If so, how large will this change be at a phosphorylation potential R_p = 10^-5 M^-1?

6. The electrode potential of the NAD⁺/NADH pair in intact mitochondria was determined by equilibrating the mitochondria with the acetoacetate–β-hydroxybutyrate pair, which has a standard electrode potential E⁰' of –0.266 V. When the [NAD⁺]/[NADH] ratio, determined spectrophotometrically, was equal to 1, the [acetoacetate]/[β-hydroxybutyrate] ratio was found to be 0.16 [Erecinska et al., ABB, 160, 412 (1974)].

a. What is the E⁰' of the NAD⁺/NADH pair under these experimental conditions?

b. The E⁰' value for the free NAD⁺/NADH pair is –0.32 V. Assuming that the discrepancy between this figure and the result obtained in (a) is due to the binding of NAD⁺ and NADH to a mitochondrial protein P with dissociation constants K₁ and K₂, and assuming that the spectrophotometric method does not distinguish between free and bound forms, show that the E⁰' determined in (a) exceeds the free-state E⁰' by

Which of the constants, K₁ or K₂, is larger according to this interpretation?

7. Consider the reaction for methane production from H₂ and CO₂ [Equation (10-42)]. Outline all the steps of this process in detail. Can you suggest how this reaction might be coupled to ATP generation? Are there any steps in this process that require ATP?

8. a) Estimate the total amount in grams of nicotinamide, pantothenic acid, and thiamine in the body of a 70-kg human. b) Calculate The amount of ATP (in millimoles and grams) required for such a person to climb from the street to the fourth floor of a building (a height of 10 m), assuming an ATP utilization efficiency of 50% and that the phosphorylation potential R_p remains constant at 10³ M^-1. c) What amount of ATP can be provided by The transfer of a phosphate group from creatine phosphate stored in Muscles? Assume that muscles constitute 40% of the total body mass. d) Calculate the average turnover number for each molecule of nicotinamide—i.e., how many times it is reduced and reoxidized—during the climb to the fourth floor mentioned in (b). Perform the same calculation for pantothenic acid (transition between acyl-CoA and free CoA and back) and for thiamine.

9. Dehydrated firefly tails are now supplied by at least one biochemical reagent company. What are they used for in biochemical assays?

10. Assume that the creatine phosphate content in Muscle is 20 mM and that it can drop to 10 mM As a result of muscle activity. What will be the resulting phosphorylation potential R_p if the creatine kinase reaction reaches equilibrium?