01 / 22

Citric Acid Cycle &
Oxidative Phosphorylation

Master the energy-harvesting core of aerobic respiration: from acetyl-CoA entry into the TCA cycle to the electron transport chain and ATP synthesis.

MCAT Biochemistry

The Big Picture of Aerobic Respiration

Aerobic respiration converts glucose into ATP through three major stages. The citric acid cycle and oxidative phosphorylation are stages 2 and 3, accounting for the vast majority of ATP produced per glucose molecule.

Stage 1
Glycolysis: glucose to pyruvate in the cytoplasm
Stage 2
TCA Cycle: acetyl-CoA oxidized in the mitochondrial matrix
Stage 3
Oxidative phosphorylation: ETC + chemiosmosis at the inner membrane

Pyruvate Oxidation: The Gateway Step

Before entering the TCA cycle, pyruvate must be converted to acetyl-CoA in the mitochondrial matrix by the pyruvate dehydrogenase complex (PDC).

Pyruvate (3C) PDC
\( \text{CO}_2 \) released
\( \text{NAD}^+ \rightarrow \text{NADH} \)
 Acetyl-CoA (2C) TCA Cycle

The Citric Acid Cycle (TCA/Krebs)

An 8-step cyclic pathway in the mitochondrial matrix that fully oxidizes the 2-carbon acetyl group, generating electron carriers and one high-energy phosphate per turn.

Citrate (6C) Isocitrate (6C)
\( \alpha \)-Ketoglutarate (5C)
 Succinyl-CoA (4C) Succinate (4C) Fumarate (4C) Malate (4C) Oxaloacetate (4C) Acetyl-CoA (2C) NADH NADH GTP
\( \text{FADH}_2 \)
 NADH
\( \text{CO}_2 \)
\( \text{CO}_2 \)

TCA Cycle: Products Per Turn

Each acetyl-CoA that enters produces the following. Since one glucose yields 2 acetyl-CoA, multiply by 2 for per-glucose totals.

\( 3 \text{ NADH} \)
At isocitrate dehydrogenase, \( \alpha \)-ketoglutarate dehydrogenase, and malate dehydrogenase
\( 1 \text{ FADH}_2 \)
At succinate dehydrogenase (Complex II of ETC)
\( 1 \text{ GTP} \)
Substrate-level phosphorylation at succinyl-CoA synthetase (equivalent to 1 ATP)

Regulation of the TCA Cycle

Three irreversible steps are rate-limiting and tightly regulated by energy charge and substrate availability.

1
Citrate synthase: inhibited by ATP, NADH, and citrate; activated by substrate availability (OAA + acetyl-CoA).
2
Isocitrate dehydrogenase: inhibited by ATP, NADH; activated by ADP and \( \text{NAD}^+ \).
3
\( \alpha \)-Ketoglutarate dehydrogenase: inhibited by succinyl-CoA, NADH; activated by \( \text{Ca}^{2+} \).

The Electron Transport Chain

Located on the inner mitochondrial membrane, the ETC consists of four protein complexes plus mobile carriers that transfer electrons from NADH and \( \text{FADH}_2 \) to \( \text{O}_2 \).

Inner Mitochondrial Membrane Complex I NADH DH
\( \text{NADH} \rightarrow \)
 Complex II SDH
\( \text{FADH}_2 \rightarrow \)
 CoQ Complex III Cyt bc1 Cyt c Complex IV Cyt c Ox
\( \rightarrow \text{H}_2\text{O} \)
 H+ H+ H+ Intermembrane Space Mitochondrial Matrix ATP Synthase (Complex V) H+ back
\( \text{O}_2 \text{ is final } e^- \text{ acceptor} \)

Chemiosmosis and ATP Synthase

As electrons pass through Complexes I, III, and IV, protons (H+) are pumped from the matrix into the intermembrane space, creating an electrochemical gradient (proton-motive force).

1
Proton gradient forms: H+ concentration becomes much higher in the intermembrane space than in the matrix.
2
H+ flows back through ATP synthase (Complex V), a molecular turbine embedded in the inner membrane.
3
Rotational catalysis: the flow of H+ drives conformational changes that catalyze \( \text{ADP} + \text{P}_i \rightarrow \text{ATP} \).

Substrate-Level vs Oxidative Phosphorylation

Substrate-Level Oxidative
Mechanism Direct transfer of phosphate from substrate to ADP Proton gradient drives ATP synthase
Location Cytoplasm (glycolysis) and matrix (TCA) Inner mitochondrial membrane
\( \text{O}_2 \) Required? No Yes (final electron acceptor)
ATP Yield per Glucose 4 ATP (net 2 from glycolysis + 2 GTP from TCA) ~26-28 ATP
Enzymes Kinases (e.g., pyruvate kinase) ETC Complexes I-IV + ATP Synthase

Total ATP Yield Per Glucose

Stage NADH \( \text{FADH}_2 \) ATP/GTP
Glycolysis 2 0 2 (net)
Pyruvate Oxidation 2 0 0
TCA Cycle (x2) 6 2 2 GTP
Total Carriers 10 2 4

Why Does NADH Yield More Than \( \text{FADH}_2 \)?

The difference comes down to where electrons enter the electron transport chain and how many protons are pumped.

NADH
Donates electrons at Complex I. Protons pumped at Complexes I, III, and IV: ~10 H+ total. Yields ~2.5 ATP.
\( \text{FADH}_2 \)
Donates electrons at Complex II. Protons pumped at Complexes III and IV only: ~6 H+ total. Yields ~1.5 ATP.

Inhibitors and Uncouplers

Understanding how poisons disrupt oxidative phosphorylation is a high-yield MCAT topic.

ETC Inhibitors
Block electron flow at a specific complex. Examples: rotenone (I), antimycin A (III), cyanide/CO (IV). Both ETC and ATP synthesis stop.
Uncouplers
Dissipate the proton gradient by carrying H+ across the membrane. ETC runs fast, but ATP synthase makes no ATP. Energy released as heat. Example: 2,4-DNP, thermogenin (UCP1).
ATP Synthase Inhibitors
Block ATP synthase directly. Example: oligomycin. The proton gradient builds up, and ETC slows to a halt (no place for H+ to go).

Worked Example: Carrier Counting

Problem: How many NADH molecules are produced from one glucose molecule through glycolysis, pyruvate oxidation, and the TCA cycle?

1
Glycolysis: 2 NADH (at glyceraldehyde-3-phosphate dehydrogenase)
2
Pyruvate oxidation: 2 pyruvates each produce 1 NADH = 2 NADH
3
TCA cycle: 3 NADH per turn x 2 turns = 6 NADH
4
Total: \( 2 + 2 + 6 = \) 10 NADH per glucose

Worked Example: ATP Calculation

Problem: If a cell oxidizes 3 molecules of glucose completely, approximately how many ATP are produced?

1
Per glucose: \( 10 \text{ NADH} \times 2.5 = 25 \text{ ATP} \)
2
\( 2 \text{ FADH}_2 \times 1.5 = 3 \text{ ATP} \)
3
\( 4 \text{ ATP/GTP from substrate-level phosphorylation} \)
4
Per glucose total: \( 25 + 3 + 4 = 32 \text{ ATP} \)
5
For 3 glucose: \( 32 \times 3 = \) 96 ATP (theoretical max)

Worked Example: Cyanide Poisoning

Problem: A patient is exposed to cyanide. What happens to NADH levels, \( \text{O}_2 \) consumption, and ATP production?

1
Cyanide inhibits Complex IV, blocking the transfer of electrons to \( \text{O}_2 \).
2
Electrons cannot flow through the chain, so Complexes I-III become fully reduced (backed up).
3
NADH accumulates (cannot donate electrons), \( \text{NAD}^+ \) drops.
4
Proton pumping stops, gradient dissipates. ATP synthase stops: ATP production collapses.
5
TCA cycle slows (needs \( \text{NAD}^+ \) and FAD). \( \text{O}_2 \) consumption drops to zero.

Common Mistakes

✕ Confusing GTP and ATP
The TCA cycle produces GTP (not ATP) at succinyl-CoA synthetase. GTP is energetically equivalent to ATP but they are different molecules.
✕ Wrong \( \text{FADH}_2 \) count
Only 1 \( \text{FADH}_2 \) per turn of the TCA cycle (at succinate dehydrogenase), not 2. Per glucose: 2 \( \text{FADH}_2 \) total.
✕ Uncoupler vs inhibitor
Uncouplers increase ETC activity and \( \text{O}_2 \) consumption (electron flow speeds up). Inhibitors stop both. Students often reverse these effects.

Step-by-Step Review Checklist

1
\( \text{Pyruvate} \rightarrow \text{Acetyl-CoA} \): PDC in matrix. Produces \( \text{CO}_2 \) + NADH. Irreversible.
2
TCA Cycle: 8 steps. Per turn: 3 NADH, 1 \( \text{FADH}_2 \), 1 GTP, 2 \( \text{CO}_2 \). Regulated by energy charge.
3
ETC: Complexes I-IV pass electrons from NADH/\( \text{FADH}_2 \) to \( \text{O}_2 \). Protons pumped at I, III, IV.
4
Chemiosmosis: H+ flows back through ATP synthase (Complex V), driving ATP production.
5
ATP yield: ~30-32 per glucose. NADH = ~2.5 ATP, \( \text{FADH}_2 \) = ~1.5 ATP.

Question 1 of 5

How many molecules of NADH are produced per single turn of the citric acid cycle?

Question 2 of 5

Which of the following is the final electron acceptor in the electron transport chain?

Question 3 of 5

A researcher adds 2,4-dinitrophenol (DNP), an uncoupler, to isolated mitochondria. What is the expected result?

Question 4 of 5

\( \text{FADH}_2 \) yields fewer ATP than NADH because \( \text{FADH}_2 \):

Question 5 of 5

Which enzyme is allosterically activated by ADP and serves as a key regulatory step of the citric acid cycle?