How Do Mitochondria Generate ATP Through Oxidative Phosphorylation?

Inside nearly every one of your cells, billions of tiny molecular “turbines” spin every second, minting the energy currency that keeps you alive. The factory that powers those turbines is oxidative phosphorylation—a beautifully orchestrated relay that turns food into a proton-powered flow, and that flow into ATP.

Key ideas Electron Transport Chain Proton Motive Force ATP Synthase Chemiosmosis

What oxidative phosphorylation actually is

Oxidative phosphorylation (often shortened to OxPhos) is the final stage of cellular respiration. It couples the oxidation of high-energy electron carriers—NADH and FADH₂—to the phosphorylation of ADP → ATP. The process unfolds across the inner mitochondrial membrane, using an electron transport chain (ETC) to pump protons and build a proton motive force (PMF). The enzyme ATP synthase then taps that PMF to synthesize ATP.

The cast: complexes and carriers

Main protein complexes

• Complex I (NADH:ubiquinone oxidoreductase): accepts electrons from NADH, pumps H⁺.
• Complex II (succinate dehydrogenase): accepts electrons from FADH₂ (via succinate), does not pump H⁺.
• Complex III (cytochrome bc₁): Q-cycle; pumps H⁺.
• Complex IV (cytochrome c oxidase): reduces O₂ to H₂O; pumps H⁺.
• Complex V (ATP synthase): uses H⁺ flow to make ATP.

Mobile electron carriers

• Ubiquinone (CoQ): lipid-soluble shuttle within the inner membrane.
• Cytochrome c: small protein carrying electrons between Complex III and IV.
• NADH / FADH₂: high-energy carriers produced by glycolysis, pyruvate oxidation, and the TCA cycle.

Step-by-step: from electrons to ATP

Step 1: Electrons enter the chain

NADH donates two electrons to Complex I. As these electrons travel through redox centers inside the complex, energy released is used to pump protons from the matrix to the intermembrane space. FADH₂ enters via Complex II, which passes electrons to CoQ but does not pump protons. Because of this, NADH yields more ATP than FADH₂ on average.

Step 2: The Q-pool and the Q-cycle

Reduced CoQ (ubiquinol) ferries electrons to Complex III. Here, the Q-cycle splits and recombines electrons in a way that both advances them to cytochrome c and pumps additional protons, amplifying the gradient.

Step 3: Oxygen—the final electron acceptor

Complex IV collects electrons from cytochrome c and uses them to reduce molecular oxygen to water. This step is crucial: by providing a “deep well” for electrons to fall into, O₂ drives the entire chain forward. Complex IV also pumps protons, further strengthening the PMF.

Step 4: Building the proton motive force

As Complexes I, III, and IV pump protons, the intermembrane space becomes more acidic and positively charged relative to the matrix. The resulting electrochemical gradient has two components: a pH gradient (ΔpH) and a membrane potential (ΔΨ). Together, they form the proton motive force, a store of potential energy across the inner membrane.

Step 5: ATP synthase: a rotary nano-motor

ATP synthase (Complex V) spans the inner membrane. Protons flow down their gradient through its Fo channel, causing a ring of c-subunits to rotate. This rotation twists the central stalk (γ-subunit) inside the F₁ catalytic head, driving conformational changes that bind ADP and inorganic phosphate (Pi), form ATP, and release it—mechanochemistry at work.

Rule of thumb: about 3–4 protons flowing through ATP synthase are required per ATP produced (exact values vary by organism and coupling).

Where the electrons come from

• Glycolysis (cytosol) → NADH, pyruvate.
• Pyruvate oxidation (mitochondrial matrix) → NADH, acetyl-CoA.
• TCA cycle (matrix) → NADH, FADH₂, and GTP/ATP.
• Fatty acid β-oxidation → NADH and FADH₂ directly in the matrix.

Note: Cytosolic NADH from glycolysis must be “shuttled” into the mitochondrion (e.g., malate–aspartate or glycerol-3-phosphate shuttles), which can slightly affect ATP yield.

ATP yield: why numbers differ

You’ll often see ranges for ATP yield per glucose. That’s because yield depends on shuttle systems, proton leak, and tissue-specific coupling efficiency. A commonly taught contemporary estimate:

Carrier	Entry point	Relative H+ pumping	Approx. ATP
NADH	Complex I	High (I, III, IV)	~2.5 ATP / NADH
FADH2	Complex II	Moderate (III, IV)	~1.5 ATP / FADH2

For one glucose oxidized completely, a typical total is roughly ~30–32 ATP, recognizing biological variability.

Why membranes matter

The inner mitochondrial membrane is densely packed with proteins and is impermeable to most ions, which prevents protons from leaking back to the matrix except through ATP synthase or specific channels. Its unique lipid, cardiolipin, stabilizes complexes and supercomplex assemblies, supporting efficient electron flow and reducing short circuits.

Coupling and uncoupling

Oxidative phosphorylation is “coupled” when electron transport and ATP synthesis are tied together by the proton gradient. If protons return to the matrix without passing through ATP synthase, the process is “uncoupled.”

• Physiological uncoupling: Brown adipose tissue expresses UCP1, a proton channel that dissipates the gradient as heat—vital for thermogenesis.
• Pathological or chemical uncoupling: Compounds like DNP shuttle protons across the membrane, ramping up oxygen consumption but collapsing ATP output.

Regulation: matching supply to demand

The system self-regulates: when ADP levels rise (during exercise, for example), ATP synthase speeds up, protons flow, the gradient drops slightly, and the ETC accelerates to rebuild it—boosting oxygen consumption. When ATP is plentiful and ADP is low, the gradient accumulates and the ETC slows.

• Substrate availability: NADH/FADH₂, O₂, ADP, and Pi set the pace.
• Allosteric control: TCA cycle enzymes and substrate shuttles tune upstream supply.
• Mitochondrial dynamics: fission and fusion help maintain function and distribute mitochondrial DNA and proteins.

Reactive oxygen species (ROS): a necessary hazard

A small fraction of electrons “leak” from the ETC (notably Complex I and III) and reduce O₂ prematurely, forming superoxide. Cells counter with antioxidants (SOD, catalase, glutathione systems). Mild ROS can signal adaptations (like mitochondrial biogenesis), but chronic excess damages lipids, proteins, and DNA.

Classic inhibitors and what they teach us

Target	Inhibitor (example)	Effect
Complex I	Rotenone	Blocks NADH entry; decreases proton pumping and ATP yield.
Complex III	Antimycin A	Blocks electron flow to cytochrome c; collapses chain downstream.
Complex IV	Cyanide, CO	Prevents O2 reduction; halts ETC entirely.
ATP synthase	Oligomycin	Stops H+ flow through Fo; electron transport slows as gradient spikes.
Uncouplers	DNP, FCCP	Dissipate gradient; ETC races, ATP synthesis drops, heat rises.

Tissue differences and adaptation

Not all mitochondria are alike. Heart and slow-twitch muscle pack membranes with ETC complexes for endurance. Brown fat specializes in heat production via UCP1. Training and cold exposure can increase mitochondrial content and tweak coupling, shifting how calories become ATP or heat.

From molecules to medicine

• Mitochondrial disorders: Mutations in mtDNA or nuclear genes that encode ETC components can reduce ATP output, especially harming high-demand tissues (brain, heart, muscle).
• Aging and metabolic disease: Cumulative mitochondrial dysfunction, altered coupling, and ROS handling are linked to insulin resistance, neurodegeneration, and sarcopenia.
• Cancer metabolism: Many tumors rewire fuel use. Even when glycolysis is elevated, mitochondrial OxPhos often remains active and targetable.
• Drugs and toxins: Some pharmaceuticals inadvertently inhibit ETC components or increase proton leak, leading to fatigue or organ-specific side effects.

Common misconceptions—quick fixes

• “Oxygen makes ATP.” Oxygen is the final electron acceptor; ATP is made by ATP synthase using the proton gradient.
• “FADH₂ is equal to NADH.” FADH₂ bypasses Complex I and generally yields less ATP.
• “All cells have the same yield.” Yield varies with tissue, shuttles, and coupling efficiency.

Putting it all together (the one-sentence summary)

Oxidative phosphorylation uses electrons from NADH/FADH₂ to power proton pumps (Complex I, III, IV), building a proton motive force across the inner mitochondrial membrane that drives ATP synthase to convert ADP and Pi into ATP.

FAQ

Is oxygen always required? For the ETC, yes—oxygen (or a similar terminal acceptor in some microbes) pulls electrons through the chain. Without it, the chain stalls and cells rely more on anaerobic pathways like fermentation.

Why does exercise increase ATP production? Muscle contraction raises ADP and Pi, accelerating ATP synthase and ETC activity; training also increases mitochondrial number and capacity.

Can the gradient be “too strong”? If ATP use is low, the gradient climbs, making further pumping energetically costly and slowing the ETC until demand returns.

Cheat sheet: Electrons flow NADH → I → CoQ → III → cyt c → IV → O₂ (and FADH₂ → II → CoQ). Protons pumped at I, III, IV. Protons return via ATP synthase to make ATP.