9 - Fuel Oxidation and the Generation of ATP

Why are electrons from carbon-hydrogen (C–H) bonds mostly used to make ATP, and what happens to these electrons?

C–H bonds have high-energy electrons because carbon and hydrogen share electrons equally. These electrons are easy to remove and transfer to carriers like NAD⁺ and FAD, which help make ATP.

What is the general direction of electron transfer during fuel oxidation?

Electrons move from glucose, fatty acids, and amino acids to acetyl-CoA, then to the TCA cycle(e- stripped), and finally to the electron transport chain (ETC), where they help make ATP.

What does the TCA cycle complete, and how?

It completes the aerobic oxidation of acetyl-CoA by removing carbons as CO₂ and transferring its electrons to NAD⁺ and FAD, which become NADH and FADH₂ for use in the ETC.

What happens in the first step of the TCA cycle?

Acetyl-CoA (2C) combines with oxaloacetate (4C) to form citrate (6C), using the enzyme citrate synthase.

What happens in the two decarboxylation steps of the TCA cycle? (And what is crucial about carbon after these steps?)

• Isocitrate dehydrogenase removes 1 CO₂ from isocitrate → α-ketoglutarate (makes 1 NADH).

• α-Ketoglutarate dehydrogenase removes 1 CO₂ from α-ketoglutarate → succinyl-CoA (makes 1 NADH).

• No more carbon is lost after succinyl-CoA formation

What happens in the GTP-forming step of the TCA cycle?

Succinyl-CoA becomes succinate, releasing energy that attaches a phosphate to GDP, forming 1 GTP.

What happens in the FADH₂-forming step of the TCA cycle?

Succinate becomes fumarate. This releases enough energy to reduce FAD to FADH₂ (but not enough for NADH).

What happens in the final step of the TCA cycle?

Malate is converted back to oxaloacetate, producing 1 NADH and completing the cycle.

True or false: An acetyl CoA added to the citric acid cycle requires one round to be used up

False; 2

What is the total product yield of one TCA cycle turn (from 1 acetyl-CoA)?

3 NADH, 1 FADH₂, 1 GTP, and 2 CO₂.

How is the TCA cycle regulated by substrate availability, and how is the PDC involved here?

More acetyl-CoA and oxaloacetate (OAA) leads to more TCA activity. Acetyl-CoA is made from pyruvate by the pyruvate dehydrogenase complex (PDC), which is inhibited by NADH, ATP, and acetyl-CoA.

How does NADH vs NAD⁺ affect TCA cycle direction?

• High NADH: reaction shifts backward, OAA decreases, and TCA slows (resting state).

• High NAD⁺: reaction shifts forward, OAA increases, and TCA speeds up (active state)

(Since malate + NAD+ = OAA + NADH)

What are the main feedback inhibition points of the TCA cycle?

NADH inhibits key enzymes:

1. Citrate synthase

2. Isocitrate dehydrogenase

3. α-KG dehydrogenase

4. Pyruvate dehydrogenase complex (not in the cycle but controls acetyl-CoA entry)

What does it mean that the TCA cycle is amphibolic? (Use Citrate and alpha KG as examples)

It means the cycle is both catabolic (breaks down fuels) and anabolic (provides building blocks).

• Citrate can leave to make fatty acids.

• α-KG can leave to make amino acids.

What is reduction potential (E°), and how does it guide electron movement?

Reduction potential shows how badly a molecule wants to gain electrons. Electrons move from molecules with low E° to high E°, measured in volts.

How is reduction potential measured(What is the reference for RP level), and what does ΔE tell us?

• Measured using a voltaic cell with 2 substances with their own E.

• Reference: (0V for H+ + e- → 1/2H2)

• ΔE = E°(acceptor) − E°(donor).

• A positive ΔE means the reaction is spontaneous.

How is reduction potential (E°′) linked to free energy (ΔG)?

• ΔG = −nFΔE

• Positive ΔE → negative ΔG → spontaneous reaction.

What are the three types of electron transfer in cells and give examples?

• Direct (e.g., Fe²⁺ → Fe³⁺)

• Hydrogen atom transfer (1 e⁻, 1 H⁺, FAD gains 2 hydrogen atoms to become FADH2)

• Hydride ion transfer (2 e⁻, 1 H⁺, e.g., NAD⁺ → NADH)

Where does the ETC happen, and how is it linked to the TCA cycle? (Why does this not happen in RBCs)

ETC occurs in the inner mitochondrial membrane. H⁺ is pumped into the intermembrane space. The TCA cycle (in the matrix) makes NADH/FADH₂, which donate electrons to the ETC.

• RBCs want to conserve oxygen

What happens in the ETC and what is is the main purpose?

To transfer electrons through complexes I–IV, pumping H⁺ into the intermembrane space. This creates a gradient used by ATP synthase to make ATP.

What happens in Complex I of the ETC?

NADH is oxidized, donating 2 e⁻ to Complex I. It pumps H⁺ and passes electrons to ubiquinone (Q), forming QH₂ (ubiquinol).

What is the role of Complex II in the ETC?

Oxidizes FADH₂ (from the TCA cycle) and transfers electrons to Q, forming QH₂. It does not pump protons.

What is the role of Coenzyme Q(Aka ubiquinone)?

A mobile carrier that transfers electrons from Complexes I/II to III in the form of QH₂. Also gets electrons from sources like β-oxidation and glycerol-3-phosphate.

What does Complex III do in the ETC?

Accepts electrons from QH₂ and transfers them to cytochrome c. It pumps H⁺ and requires 2 cycles to pass both electrons.

What happens in Complex IV of the ETC, and what makes it different from the previous complexes?

Accepts electrons from cytochrome c and gives them to O₂, forming H₂O. It pumps H⁺ and contains Cu instead of Fe-S.

How does ATP synthase work, and what are its structural components?

ATP synthase has two main parts:

• F₀ unit (in the inner membrane): forms a proton channel. H⁺ flow causes rotation of the c-ring and γ (gamma) shaft.

• F₁ unit (in the matrix): made of a hexamer of 3 α and 3 β subunits. The rotating γ shaft changes the shape of the β subunits, driving ATP synthesis.

• Each 360° rotation of the γ shaft = 3 ATP (one per β subunit).

Why does 1 NADH yield ~2.5 ATP and 1 FADH₂ yield ~1.5 ATP?

Because NADH and FADH₂ donate electrons to different parts of the ETC, resulting in different amounts of H⁺ pumping:

• NADH enters at Complex I → pumps a total of 10 H⁺

• FADH₂ enters at Complex II (which doesn't pump) → pumps 6 H⁺
  It takes 4 H⁺ to make 1 ATP, so:

• NADH: 10 ÷ 4 = ~2.5 ATP

• FADH₂: 6 ÷ 4 = ~1.5 ATP

Why does it take about 4 H⁺ to make 1 ATP in the mitochondria?

• 3 H⁺ are used by ATP synthase to rotate and make 1 ATP.

• 1 extra H⁺ is needed to transport inorganic phosphate (Pᵢ) and ATP across the inner mitochondrial membrane.
  So in total, ~4 H⁺ are needed per ATP made.

What are the two main NADH transport shuttles into the mitochondria?

• Malate-Aspartate Shuttle – Most efficient; turns cytoplasmic NADH into mitochondrial NADH.

• Glycerol 3-Phosphate Shuttle – Less efficient; turns NADH into FADH₂ (Complex I is skipped).

What are three ways the ETC or ATP synthesis can be inhibited?

• ETC inhibitors stop H⁺ pumping → no gradient → no ATP.

• ATP synthase inhibitors block H⁺ flow, gradient doesn’t change so ETC slows since it doesn’t want to worsen the gradient.

• Uncoupling lets H⁺ leak, making heat instead of ATP.

What are the two main uncouplers of oxidative phosphorylation?

• Chemical uncoupler (e.g., 2,4-dinitrophenol): allows H⁺ to leak across the membrane → heat is made instead of ATP.

• UCP1 (Uncoupling Protein 1): found in brown adipose tissue, naturally leaks H⁺ to generate heat for thermoregulation.

What are reactive oxygen species (ROS), and how are they formed?

ROS form when O₂ gains electrons prematurely in the ETC. Examples include:

• Superoxide (O₂•⁻)

• Hydrogen peroxide (H₂O₂)

• Hydroxyl radical (OH•)

How can ROS be helpful or harmful?

• Helpful: signaling, immune defense, growth.

• Harmful: damage lipids (e.g., LDLs), proteins (wrong bonds), and DNA (mutations).

How does the body defend against ROS?

• Using glutathione, regenerated through this reaction, because GSH + ROS = GSSG + H2O

• GSSG + NADPH → GSH + NADP⁺

How can we get OXPHOS defects, what happens in them, and how can they be treated?

• Can be inherited (mtDNA mutation) or acquired.

• Affects high-energy organs (brain, heart).

• Treated with antioxidants or mitochondrial gene therapy (from healthy donors).

What is Leber’s Hereditary Optic Neuropathy (LHON)?

• Cause

• Mechanism

• Result

• Treatment

LHON is a mitochondrial disorder caused by a mtDNA mutation that affects complex I of the ETC. It leads to optic nerve (which requires a lot of ATP) degradation, causing central vision loss, especially in young males.

• It's treated with gene therapy or antioxidants.