Biochemistry: TCA Cycle and Oxidative Phosphorylation

Aerobic cells extract energy from nutrients by coupling oxidation reactions to ATP production. Two linked systems sit at the center of this process: the tricarboxylic acid (TCA) cycle, which harvests high-energy electrons from acetyl-CoA, and oxidative phosphorylation, which converts those electrons into ATP through the electron transport chain and chemiosmosis. Together they explain how carbohydrates, fats, and many amino acids converge on a common energy pathway and how oxygen ultimately enables efficient ATP synthesis.

Where the TCA Cycle Fits in Aerobic Metabolism

The TCA cycle (also called the citric acid cycle or Krebs cycle) is not primarily an ATP-producing pathway. Its central role is to oxidize a two-carbon acetyl group to carbon dioxide while capturing energy in reduced electron carriers, mainly NADH and FADH₂. Those carriers then feed oxidative phosphorylation, where most ATP is generated.

Metabolism funnels diverse fuels into acetyl-CoA:

• Glucose is converted to pyruvate via glycolysis; pyruvate is then oxidized to acetyl-CoA by the pyruvate dehydrogenase complex.
• Fatty acids are broken down by beta-oxidation into acetyl-CoA units.
• Several amino acids enter as acetyl-CoA or as TCA intermediates after deamination and carbon skeleton rearrangement.

Because of this convergence, the TCA cycle sits at a junction between catabolism (energy extraction) and anabolism (biosynthesis), which is why it is often described as an amphibolic pathway.

The Citric Acid Cycle: Purpose, Location, and Key Outputs

In eukaryotes, the TCA cycle runs in the mitochondrial matrix; in most bacteria, it occurs in the cytosol, with the electron transport chain located on the plasma membrane.

The Core Logic of the Cycle

The cycle begins when acetyl-CoA (2 carbons) condenses with oxaloacetate (4 carbons) to form citrate (6 carbons). Through a series of reactions, citrate is rearranged and oxidized, releasing two molecules of CO₂ and regenerating oxaloacetate to accept another acetyl group.

Per acetyl-CoA oxidized, the cycle produces:

• 3 NADH
• 1 FADH₂
• 1 GTP (or ATP, depending on organism/tissue)
• 2 CO₂

These products explain the cycle’s energy strategy: a modest amount of substrate-level phosphorylation (GTP) and a large yield of reducing equivalents (NADH, FADH₂) destined for the electron transport chain.

Notable Steps and Why They Matter

Several reactions carry particular biochemical weight:

• Citrate synthase commits acetyl-CoA to the cycle; it is commonly regulated by substrate availability and energy status.
• Isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase are major oxidative steps producing NADH and releasing CO₂. They are also frequent control points because they respond to cellular indicators such as ATP, NADH, and ADP.
• Succinyl-CoA synthetase generates GTP (substrate-level phosphorylation), highlighting that ATP can be formed directly in the cycle without the electron transport chain.
• Succinate dehydrogenase produces FADH₂ and is unique because it is embedded in the inner mitochondrial membrane as part of Complex II of the electron transport chain, physically linking the TCA cycle to oxidative phosphorylation.

Integration With Other Metabolic Pathways

The TCA cycle is continuously replenished and tapped because its intermediates serve as building blocks:

• Citrate can exit mitochondria and support fatty acid and cholesterol synthesis.
• Alpha-ketoglutarate and oxaloacetate are precursors for amino acid synthesis (glutamate and aspartate families, respectively).
• Succinyl-CoA contributes to heme synthesis.

When intermediates are drawn off for biosynthesis, anaplerotic reactions refill the cycle. A classic example is pyruvate carboxylation to oxaloacetate, a reaction that helps maintain TCA flux when acetyl-CoA supply is high or intermediates are depleted.

Oxidative Phosphorylation: From Electrons to ATP

Oxidative phosphorylation occurs across the inner mitochondrial membrane (or bacterial plasma membrane) and couples electron transfer to ATP synthesis. It has two tightly linked components:

1. Electron transport chain (ETC): transfers electrons from NADH and FADH₂ to oxygen through a series of complexes.
2. Chemiosmosis: uses the resulting proton gradient to drive ATP formation by ATP synthase.

Electron Transport Chain: Complexes and Electron Flow

Electrons enter the ETC mainly by two routes:

• Complex I (NADH dehydrogenase) accepts electrons from NADH and transfers them to ubiquinone (coenzyme Q). This process is coupled to proton pumping from the matrix to the intermembrane space.
• Complex II (succinate dehydrogenase) accepts electrons from FADH₂ generated during succinate oxidation. It passes electrons to ubiquinone but does not pump protons, which is why FADH₂ yields less ATP than NADH.

Ubiquinone delivers electrons to Complex III, which passes them to cytochrome c, a mobile carrier on the outer surface of the inner membrane. Cytochrome c then transfers electrons to Complex IV (cytochrome c oxidase), where oxygen serves as the terminal electron acceptor and is reduced to water. Complexes III and IV also contribute to proton pumping.

Oxygen’s role is decisive: by accepting electrons at the end of the chain, it keeps electron flow moving. Without oxygen, the chain backs up, NADH accumulates, and pathways that depend on NAD⁺ regeneration (including pyruvate oxidation and the TCA cycle) slow dramatically.

The Proton Motive Force and Chemiosmosis

As electrons move through the ETC, protons are pumped across the inner mitochondrial membrane, creating an electrochemical gradient called the proton motive force. This gradient has two components:

• A membrane potential (charge separation)
• A pH gradient (difference in proton concentration)

A useful way to summarize this stored energy is:

$$\Delta p=\Delta \psi -\frac{2.303RT}{F}\Delta pH$$

where $\Delta p$ is the proton motive force, $\Delta \psi$ is membrane potential, and the second term reflects the pH contribution.

The inner mitochondrial membrane is normally highly impermeable to ions. That tight barrier is essential because it prevents the proton gradient from dissipating before it can be used.

ATP Synthase: Turning a Gradient Into Chemical Energy

ATP synthase (Complex V) allows protons to flow back into the matrix, and the energy released drives ATP formation from ADP and inorganic phosphate. This is the core idea of chemiosmosis: energy stored as a gradient is converted into a covalent bond in ATP.

The mechanism is often explained as rotational catalysis: proton flow causes parts of the enzyme to rotate, triggering conformational changes that bind ADP and phosphate, synthesize ATP, and release it. While the exact proton-to-ATP ratio depends on the organism and specific mitochondrial architecture, the major concept remains that the gradient, not a soluble high-energy intermediate, is the immediate driver of ATP synthesis.

Efficiency, Control, and Practical Implications

The TCA cycle and oxidative phosphorylation are regulated by energy demand. When ATP is plentiful and NADH is high, key dehydrogenases slow, decreasing electron delivery to the ETC. When ADP rises, oxidative phosphorylation accelerates because ATP synthase activity increases proton flow back into the matrix, which in turn supports continued electron transport and proton pumping.

This coupling explains several real biological outcomes:

• High aerobic capacity tissues (heart, oxidative skeletal muscle) contain dense mitochondria and rely heavily on fatty acid oxidation feeding acetyl-CoA into the TCA cycle.
• Hypoxia or ischemia disrupts oxygen availability, limiting Complex IV activity, reducing ATP generation, and forcing cells toward less efficient anaerobic ATP production.
• Uncoupling (whether physiologic, as in thermogenesis, or pathologic) dissipates the proton gradient as heat, increasing oxygen consumption without proportional ATP synthesis.

The Big Picture

The TCA cycle extracts electrons from acetyl-CoA and packages them into NADH and FADH₂. The electron transport chain uses those electrons to pump protons and build a proton motive force. ATP synthase then converts that gradient into ATP via chemiosmosis. Understanding this flow, carbon to electrons to gradient to ATP, clarifies why aerobic respiration is so efficient and why mitochondria are central to both energy production and metabolic integration.