Chapter 7

Key Concepts

• Overview of Cellular Respiration

  • Process by which living cells obtain energy from organic molecules, typically glucose, to synthesize ATP.

  • Primarily uses glucose, but can also utilize other organic molecules.

  • The primary goal is to generate ATP, NADH, and FADH2.

  • Distinction between Aerobic respiration (uses oxygen and is highly efficient) and Anaerobic respiration (no oxygen, less efficient but crucial in oxygen-limited environments).

• Major Stages of Cellular Respiration:

  1. Glycolysis

  2. Breakdown of Pyruvate

  3. Citric Acid Cycle (Krebs Cycle)

  4. Oxidative Phosphorylation

• Additional Topics:

  • A closer look at ATP Synthase

  • Metabolic connections among carbohydrates, proteins, and fats

  • Anaerobic Respiration and Fermentation

Overview of Cellular Respiration

• Living cells require energy to perform various functions.

• Cellular respiration is a multi-step process:

  • Aerobic respiration:

    • Oxygen is consumed and carbon dioxide (CO2) is released.

  • Anaerobic respiration:

    • Does not involve oxygen.

Metabolic Pathways in Aerobic Respiration

• There are four major stages in aerobic respiration:

  1. Glycolysis

  2. Breakdown of pyruvate

  3. Citric Acid Cycle

  4. Oxidative Phosphorylation

Glycolysis

• Glycolysis represents the first stage of cellular respiration.

• Can occur with or without oxygen.

• Takes place in the cytoplasm outside of mitochondria for both prokaryotic and eukaryotic cells.

• Comprises 10 steps organized into three phases:

  1. Energy investment phase

  2. Cleavage phase

  3. Energy liberation phase

Phases of Glycolysis

• Phase 1: Energy Investment

  • Involves the hydrolysis of 2 ATP molecules to produce fructose-1,6-bisphosphate, priming the molecule for cleavage by phosphorylating glucose and fructose-6-phosphate.

• Phase 2: Cleavage

  • The 6-carbon molecule, fructose-1,6-bisphosphate, is split into two 3-carbon glyceraldehyde-3-phosphate (G3P) molecules. Initially, one G3P and one dihydroxyacetone phosphate (DHAP) are formed, and DHAP is then isomerized to G3P.

• Phase 3: Energy Liberation

  • Each G3P molecule transforms into pyruvate through a series of redox reactions and substrate-level phosphorylation.

  • This phase generates 4 ATP (gross yield) via substrate-level phosphorylation and 2 NADH from the reduction of NAD+.

  • The net yield from glycolysis is 2 ATP per glucose (4 produced minus 2 consumed in Phase 1).

Detailed Steps of Glycolysis

1. Energy Investment (Steps 1-3)

   • Step 1: A phosphate group from ATP is transferred to glucose to form glucose-6-phosphate, aided by an enzyme, Hexokinase. This phosphorylation traps glucose in the cell.

   • Step 2: Glucose-6-phosphate is converted to fructose-6-phosphate by Phosphoglucoisomerase (an isomerization reaction).

   • Step 3: Another ATP donates a phosphate to convert fructose-6-phosphate to fructose-1,6-bisphosphate, facilitated by Phosphofructokinase (PFK). PFK is a key regulatory enzyme of glycolysis.

2. Cleavage (Steps 4-5)

   • Step 4: Aldolase splits fructose-1,6-bisphosphate into dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P).

   • Step 5: Triose Phosphate Isomerase rapidly converts DHAP into another G3P, ensuring both 3-carbon molecules proceed through the energy liberation phase.

3. Energy Liberation (Steps 6-10)

   • Step 6: Each G3P is oxidized, reducing NAD+ to NADH, and a phosphate group is added to form 1,3-bisphosphoglycerate, generating a high-energy phosphate bond.

   • Step 7: A high-energy phosphate is transferred from 1,3-bisphosphoglycerate to ADP, forming ATP, catalyzed by Phosphoglycerate Kinase (the first substrate-level phosphorylation).

   • Following steps involve rearrangements of the molecule, eventually leading to the formation of phosphoenolpyruvate (PEP).

   • Step 10: PEP donates its phosphate to ADP, forming ATP and pyruvate, catalyzed by Pyruvate Kinase (the second substrate-level phosphorylation).

Breakdown of Pyruvate

• Representing Stage 2 of cellular respiration, pyruvate molecules (3 carbons each) are converted into acetyl CoA (2 carbons each). This crucial link reaction connects glycolysis to the citric acid cycle.

• Involves the removal of CO2 and reduction of NAD+ to NADH, catalyzed by the pyruvate dehydrogenase complex.

• This conversion occurs in the mitochondrial matrix.

Process of Pyruvate Breakdown

1. Transport into Mitochondria:

   • Pyruvate enters the mitochondrial matrix from the cytoplasm via a specific pyruvate translocase protein in the inner mitochondrial membrane through facilitated transport and symport mechanisms.

2. Conversion:

   • Each 3-carbon pyruvate molecule undergoes oxidative decarboxylation:

     • A carboxyl group is removed as CO2.

     • The remaining two carbons are oxidized, and NAD+ is reduced to NADH.

     • The resulting 2-carbon acetyl group attaches to Coenzyme A, forming acetyl CoA.

   • Yields: 1 NADH for every pyruvate, totaling 2 NADH per glucose.

Citric Acid Cycle (Krebs Cycle)

• Represents Stage 3 of cellular respiration.

• A cyclical metabolic pathway that combines acetyl CoA (2C) with oxaloacetate (4C) to create citrate (6C).

• The cycle operates through a series of redox reactions generating per turn (per pyruvate):

  • Two carbons are progressively oxidized and released as 2 CO2.

  • A single ATP molecule is generated per turn (via a GTP intermediate) through substrate-level phosphorylation.

  • Three NAD+ molecules are reduced to 3 NADH.

  • One FAD molecule is reduced to 1 FADH2.

  • Oxaloacetate is regenerated to continue the cycle.

Key Features of the Citric Acid Cycle

• The products for one glucose (as it produces two acetyl CoA) become 4 CO2, 2 ATP (or GTP), 6 NADH, and 2 FADH2.

• Each of these reduced coenzymes (NADH and FADH2) will be utilized in the final oxidative phosphorylation phase for high ATP yield.

• The cycle is highly regulated, primarily at the steps catalyzed by citrate synthase, isocitrate dehydrogenase, and $ext{alpha}$-ketoglutarate dehydrogenase.

Oxidative Phosphorylation

• Stage 4 of cellular respiration focusing on high ATP yield.

• Involves two components: the electron transport chain (ETC) and ATP synthase, both primarily embedded in the inner mitochondrial membrane.

• Key is the use of NADH and FADH2, which are oxidized in the ETC, leading to the phosphorylation of ADP to produce a large amount of ATP.

The Role of the Electron Transport Chain

1. NADH is oxidized to NAD+, which donates high-energy electrons to Complex I of the electron transport chain.

2. FADH2 donates electrons at a lower energy level to Complex II of the chain.

3. As electrons move sequentially through protein complexes of the ETC (Complexes I, III, and IV pump protons), protons (H+) are pumped from the mitochondrial matrix into the intermembrane space, generating an electrochemical gradient (also known as the proton-motive force).

4. The electrons are ultimately transferred to oxygen, the final electron acceptor, forming water. This step is crucial for maintaining electron flow through the ETC.
   $2H^+ + 2e^- + rac{1}{2}O<em>2 ightarrow H</em>2O$

ATP Synthesis via ATP Synthase

• The flow of H+ ions down their concentration gradient, from the intermembrane space back into the mitochondrial matrix through ATP synthase, is the energy source for ATP synthesis, termed chemiosmosis. This proton-motive force represents stored potential energy.

• ATP Synthase is a multi-subunit protein complex (consisting of F0 and F1 components) that acts as a molecular motor. Its subunits undergo conformational changes during synthesis.

• The high H+ concentration in the intermembrane space drives H+ to flow into the mitochondrial matrix through the F0 component of ATP synthase, which synthesizes ATP from ADP and Pi through rotary catalysis in the F1 component.

Connections Among Metabolism

• Carbohydrates, proteins, and fats can all feed into the glycolysis or citric acid cycle at distinct points, demonstrating metabolic flexibility and interconversion.

• Carbohydrates: Polysaccharides are hydrolyzed to glucose, which directly enters glycolysis.

• Fats: Triglycerides are broken down into glycerol (which can be converted to glyceraldehyde-3-phosphate and enter glycolysis) and fatty acids. Fatty acids undergo $\beta$-oxidation, a process that breaks them down into two-carbon units of acetyl CoA, which then enters the citric acid cycle.

• Proteins: Proteins are hydrolyzed into amino acids. After deamination (removal of the amino group), the carbon skeletons of amino acids can enter cellular respiration at various points – as pyruvate (entering pyruvate breakdown), as acetyl CoA, or directly as intermediates of the citric acid cycle (e.g., $ext{alpha}$-ketoglutarate, succinyl CoA, fumarate, oxaloacetate).

• This convergence of metabolic pathways enhances efficiency, enabling the cell to adapt fuel usage based on availability and energy demands.

Anaerobic Respiration & Fermentation

• In environments devoid of oxygen, organisms can still generate ATP through anaerobic methods. These processes are distinct from aerobic respiration primarily in their final electron acceptor.

1. Anaerobic respiration occurs when organisms use alternatives to oxygen as the final electron acceptor in an electron transport chain. Examples include nitrate ($NO<em>3^-$), sulfate ($SO</em>4^{2-}$), or ferric iron ($Fe^{3+}$) used by some prokaryotes. This still uses an ETC but is generally less efficient than aerobic respiration.

2. Fermentation leads to ATP production solely via substrate-level phosphorylation from glycolysis. The primary purpose of fermentation pathways is to regenerate NAD+ for glycolysis to continue, as glycolysis is the sole ATP-producing pathway in these conditions.

Fermentation Processes

• Both types of fermentation start with glycolysis, producing 2 ATP (net) and 2 NADH. The subsequent steps regenerate NAD+ from NADH.

1. Lactic Acid Fermentation:

   • Glycolysis converts glucose to pyruvate. Pyruvate is then directly reduced to lactate by lactate dehydrogenase, simultaneously oxidizing NADH back to NAD+. This regeneration of NAD+ allows glycolysis to continue.

   • The overall net process from glucose is:
     $ext{Glucose} <br>ightarrow 2 ext{Pyruvate} <br>ightarrow 2 ext{Lactate} + 2 ext{ATP}$ (net ATP from glycolysis)

   • This pathway occurs in animal muscle cells during strenuous exercise when oxygen supply is limited, and in some bacteria (e.g., in yogurt production).

2. Ethanol Fermentation:

   • In a two-step process, pyruvate is converted to acetaldehyde and then to ethanol.

     • First, pyruvate is decarboxylated by pyruvate decarboxylase to produce acetaldehyde and carbon dioxide (CO2).

     • Second, acetaldehyde is reduced to ethanol by alcohol dehydrogenase, re-oxidizing NADH to NAD+ to ensure glycolysis can continue.

   • The overall net process from glucose is:
     $ext{Glucose} <br>ightarrow 2 ext{Pyruvate} <br>ightarrow 2 ext{Acetaldehyde} + 2 ext{CO}_2 <br>ightarrow 2 ext{Ethanol} + 2 ext{ATP}$ (net ATP from glycolysis)

   • This pathway is common in yeast and some bacteria, utilized in the brewing of alcoholic beverages and in baking.

Experimental Confirmation of ATP Synthase Function

• ATP synthase captures free energy by allowing H+ ions to flow through and harnessing the proton motive force to generate ATP. Subsequent experiments have definitively confirmed its rotary nature.

• Experiments, pioneered by Nobel laureates Paul Boyer and John E. Walker, revealed that the F1 component of ATP synthase has a rotating central stalk within a stationary headpiece.

• These experiments established the connection between the flow of protons, the rotation of the central stalk, and the conformational changes in the F1 catalytic sites, indicating its pivotal role in ATP synthesis through a process known as the binding change mechanism. This mechanism states that the binding of substrates (ADP and Pi), ATP synthesis, and ATP release occur in different conformations induced by rotation.