Biochemistry Chapter 9-10

Outline of Aerobic Metabolism

  • Citric Acid Cycle:

    • Conversion of pyruvate to Acetyl-CoA

    • Reactions of Citric Acid Cycle

    • The amphibolic Citric Acid Cycle

    • The Glyoxylate Cycle

    • Regulation of Citric Acid Cycle

  • Electron Transport Chain:

    • Electron transport and its components

    • Electron transport inhibitors

  • Oxidative Phosphorylation:

    • The chemiosmotic theory

    • ATP synthase

    • The complete oxidation of fatty acids

Introduction to Aerobic Metabolism

  • Aerobic metabolism involves organisms that can utilize oxygen to generate energy. The key biochemical processes involved include:

    1. The citric acid cycle

    2. The electron transport pathway

    3. Oxidative phosphorylation

  • In eukaryotes, these processes occur within the mitochondria.

Citric Acid Cycle

  • The citric acid cycle is a series of biochemical reactions that aerobic microorganisms utilize to release energy stored in Acetyl-CoA, produced from the catabolism of carbohydrates, lipids, and amino acids.

  • High energy electrons are removed from intermediates of the citric acid cycle and transferred to NAD and FAD, forming reduced coenzymes NADH and FADH2.

Reactions in the Citric Acid Cycle

  1. Condensation of Acetyl-CoA and Oxaloacetate:

    • The two-carbon Acetyl-CoA reacts with the four-carbon oxaloacetate to form the six-carbon citrate.

    • The enzyme responsible for this reaction is Citrate synthase.

  2. Isomerization of Citrate:

    • Citrate is reversibly converted to isocitrate through the action of Aconitase. This is an isomerization reaction.

  3. Oxidative Decarboxylation of Isocitrate:

    • Isocitrate is converted to alpha-ketoglutarate by Isocitrate dehydrogenase, generating NADH. The rapid conversion of isocitrate promotes the formation of isocitrate in the previous reaction.

  4. Conversion of alpha-Ketoglutarate to Succinyl-CoA:

    • Alpha-ketoglutarate dehydrogenase complex catalyzes this reaction, requiring cofactors like CoASH and NAD. This enzyme's activity can be inhibited by succinyl-CoA, NADH, ATP, and GTP.

  5. Cleavage of Succinyl-CoA:

    • The thioester bond of Succinyl-CoA is cleaved to form succinate, catalyzed by Succinate thiokinase. This reaction is coupled with the formation of GTP (or ATP in some microorganisms).

  6. Oxidation of Succinate:

    • Succinate dehydrogenase catalyzes the oxidation of succinate to fumarate, tightly associated with the mitochondrial inner membrane. It is activated by high concentrations of succinate, inorganic phosphate (Pi), and ADP, but inhibited by oxaloacetate.

  7. Hydration of Fumarate to Malate:

    • This reversible reaction is catalyzed by Fumarase or Fumarase hydratase.

  8. Oxidation of Malate to Oxaloacetate:

    • Malate dehydrogenase catalyzes this last step, also producing NADH. This reaction is endergonic and is driven forward by the subsequent conversion of oxaloacetate to citrate.

  • During the cycle, three molecules of NADH and one molecule of FADH2 are generated, which enter the electron transport chain to generate ATP.

The Amphibolic Citric Acid Cycle

  • The citric acid cycle functions both as a catabolic and anabolic pathway.

    • Catabolic aspect: Acetyl-CoA is oxidized to CO2, storing energy in NADH and FADH2.

    • Anabolic aspect: Many intermediates serve as precursors for biosynthesis.

    • Example: Oxaloacetate is used in gluconeogenesis for glucose synthesis and amino acid synthesis; alpha-ketoglutarate serves as a precursor for amino acid synthesis.

  • Acetyl-CoA is a precursor for lipid biosynthesis but cannot enter the inner mitochondrial membrane directly. It is converted to citrate, which can then be transported to the cytoplasm, where it is converted back to Acetyl-CoA and oxaloacetate by Citrate lyase.

  • The biosynthesis of porphyrins requires succinyl-CoA. Thus, citric acid cycle intermediates are essential for many biosynthetic pathways.

Glyoxylate Cycle

  • Some organisms, including plants, certain fungi, algae, protozoans, and bacteria, metabolize two-carbon compounds such as ethanol, acetate, and Acetyl-CoA through the Glyoxylate cycle, a modified version of the citric acid cycle.

  • In plants, the cycle occurs in glyoxysomes, while in other eukaryotes and bacteria it takes place in the cytoplasm.

  • The glyoxylate cycle consists of five reactions:

    • The first two reactions are similar to those in the citric acid cycle.

    • Isocitrate is split into succinate and glyoxylate via Isocitrate lyase.

    • Succinate is subsequently converted to malate using mitochondrial enzymes.

    • Glyoxylate reacts with a second Acetyl-CoA molecule, forming malate through a reaction catalyzed by Malate synthase.

    • The cycle concludes with the conversion of malate to oxaloacetate by Malate dehydrogenase.

  • Ultimately, two molecules of Acetyl-CoA produce one molecule each of oxaloacetate and succinate.

Regulation of Citric Acid Cycle

  • Regulation ensures that the citric acid cycle meets the cellular demand for energy and biosynthesis. Key enzymes are modulated based on substrate availability and energy status:

    • Citrate synthase:

    • Stimulated by Acetyl-CoA and oxaloacetate.

    • Inhibited allosterically by high concentrations of citrate and succinyl-CoA, as well as NADH and ATP, which indicate high energy status.

    • Isocitrate dehydrogenase:

    • Stimulated by high concentrations of ADP and NAD.

    • Inhibited by ATP and NADH, driving the conversion of isocitrate to alpha-ketoglutarate forward.

    • Alpha-ketoglutarate dehydrogenase:

    • Strictly regulated since it plays a role in multiple pathways.

    • Stimulated by low concentrations of NADH.

    • Inhibited by high concentrations of NADH.

Electron Transport Chain (ETC)

  • The mitochondrial electron transport chain (also known as the electron transport system) allows the transfer of electrons derived from reduced coenzymes to oxygen, with oxygen serving as the final electron acceptor. This process is termed aerobic respiration.

  • During electron transfer, energy released is coupled with ATP synthesis. The principal sources of electrons are reduced coenzymes produced during glycolysis, the citric acid cycle, and fatty acid oxidation.

Components of the Electron Transport Chain

  • Located in the mitochondrial inner membrane (in eukaryotes) or the inner membrane of bacteria.

  • Organized into four complexes consisting of multiple proteins and prosthetic groups.

  • Other components include:

    • Coenzyme Q (Ubiquinone, UQ)

    • Cytochrome C (cyt c)

Complexes of the ETC

  1. Complex I: NADH dehydrogenase complex

    • Transfers electrons from NADH to UQ.

    • Composed of over two dozen polypeptides, one FMN, and seven iron-sulfur centers.

    • Electrons are first transferred to FMN, forming FMNH2.

    • Electrons are then transferred to iron-sulfur centers and ultimately to UQ.

  2. Complex II: Succinate dehydrogenase complex

    • Acts as a citric acid cycle enzyme, transferring electrons from succinate to UQ.

    • Contains two iron-sulfur proteins and a covalently bound FAD.

  3. Complex III: Cytochrome bc1 complex

    • Transfers electrons from UQH2 to cytochrome C.

    • Composed of two b-type cytochromes, one cytochrome C1, and one iron-sulfur center.

  4. Complex IV: Cytochrome oxidase

    • Catalyzes the four-electron reduction of oxygen to form H2O.

    • Membrane-spanning complex may contain between six and thirteen subunits.

    • Contains copper atoms that alternate between +1 and +2 oxidation states, facilitating electron transfer.

Inhibitors of the ETC

  • Several molecules inhibit the electron transport chain at different stages:

    • Antimycin A: Inhibits Cyt b (Complex III)

    • Rotenone and Amytal: Inhibit NADH dehydrogenase (Complex I)

    • Carbon monoxide (CO), Azide (N3-), cyanide (CN-): Inhibit cytochrome oxidase

Oxidative Phosphorylation

  • The process of oxidative phosphorylation involves the phosphorylation of ADP to ATP, driven by the energy generated during electron transport.

  • Chemiosmotic coupling theory (proposed by Peter Mitchell in 1961):

    1. As electrons pass through

ATP Synthase - ATP synthase is a crucial enzyme that catalyzes the formation of ATP from ADP and inorganic phosphate (Pi) during oxidative phosphorylation. - The enzyme harnesses the proton gradient generated by electron transport to drive the phosphorylation reaction. - It consists of two major components: the F0 (membrane-embedded part) and F1 (the catalytic portion). - Proton flow through F0 causes a conformational change that enables ADP and Pi to combine and form ATP in the F1 region.
Complete Oxidation of Fatty Acids

  • The complete oxidation of fatty acids occurs through a process called beta-oxidation, which takes place in the mitochondria.

  • During beta-oxidation, fatty acids are broken down into acetyl-CoA units, which then enter the citric acid cycle.

  • Each cycle of beta-oxidation generates one molecule of FADH2 and one molecule of NADH, which contribute to the electron transport chain for ATP production.

  • The overall process efficiently converts the energy stored in fatty acids into usable ATP, making fats a significant energy source for aerobic metabolism.