ATP synthase as a molecular mill
The cell does not have an ATP charging socket, so the energy for its synthesis is extracted using a special molecular mechanism located in the inner mitochondrial membrane. This mechanism is the enzyme ATP synthase, which works on the windmill principle.
ATP synthase consists of two parts: the membrane-bound F0 (the rotor through which protons pass) and the catalytic F1 (where inorganic phosphate is attached to ADP). The flow of protons through F0 rotates the rotor: every three protons turn it by 120°, and with each such turn, one ATP molecule is synthesized on one of the subunits. A full rotation yields approximately three ATP molecules.
Proton gradient and intermembrane space
For ATP synthase to rotate, a high concentration of protons H+ must be maintained in the intermembrane space of the mitochondria, and a low concentration in the matrix. This difference creates an electrochemical gradient, denoted as ΔμH+, and consists of two components: the chemical potential (concentration difference) and the electrical potential (charge difference across the membrane sides).
The sides of the inner membrane are referred to as:
- P-side (positive) — faces the intermembrane space, positively charged due to an excess of protons;
- N-side (negative) — faces the matrix, negatively charged.
Protons tend to return from the P-side to the N-side, but the membrane is impermeable, and the only path is through the ATP synthase rotor.
Electron sources: NADH and FADH2
Protons are pumped into the intermembrane space using the energy released during electron transfer. The sources of electrons are reduced coenzymes generated in the Krebs cycle and other reactions:
- NADH + H+ — effectively carries a hydride ion (H−) with two electrons;
- FADH2 — also serves as a donor of two electrons and two protons.
Electrons are transferred sequentially through protein complexes of the inner membrane, losing energy that is used to pump protons.
Complexes I and II of the respiratory chain
Complex I — NADH-ubiquinone oxidoreductase (NADH dehydrogenase). It accepts two electrons from NADH via flavin mononucleotide (FMN) and transfers them to ubiquinone (Q). Upon receiving two electrons and two protons from the matrix, ubiquinone is converted into ubiquinol (QH2). Simultaneously, 4 protons are pumped from the matrix into the intermembrane space.
Complex II — succinate dehydrogenase. Does not pump protons. Its task is to maintain the ubiquinol pool by oxidizing succinate to fumarate (a Krebs cycle reaction) with the formation of FADH2 and subsequent electron transfer to ubiquinone. Heme B within the complex prevents electron leakage to oxygen, which prevents the formation of hydrogen peroxide and reactive oxygen species.
Complex III and the Q-cycle
Complex III — ubiquinol-cytochrome-c oxidoreductase. It utilizes the energy from the re-oxidation of ubiquinol. It has two active sites: one on the P-side and the other on the N-side. Electrons from QH2 are split: one goes to cytochrome c via the Rieske iron-sulfur protein, and the second goes to heme B and then back to ubiquinone, reducing it on the N-side (Q-cycle).
The cleavage of covalent bonds during electron redistribution releases energy, enabling the complex to pump 4 protons into the intermembrane space. Cytochrome c is a mobile carrier that delivers electrons to the next complex.
Complex IV and water formation
Complex IV — cytochrome c oxidase. Accepts one electron at a time from four molecules of cytochrome c. Contains a bimetallic cluster with iron and copper atoms in its active center. Electrons are sequentially transferred to molecular oxygen O2.
Controlled reduction of oxygen prevents peroxide formation: protons from the matrix are gradually added to it, resulting in the formation of two H2O molecules. The energy of the process is used to pump 2 protons into the intermembrane space. Thus, the respiratory chain safely "dumps" electrons onto the final acceptor — oxygen.
Energy Balance
Total number of pumped protons:
- Oxidation of one NADH molecule: 4 (Complex I) + 4 (Complex III) + 2 (Complex IV) = 10 protons;
- Oxidation of one FADH2 molecule: 4 (Complex III) + 2 (Complex IV) = 6 protons.
Since about 3–4 protons are required for the synthesis of one ATP, the yield is approximately 2.5 ATP per NADH and 1.5 ATP per FADH2 (rounded to 3 and 2, respectively).
Key points
- ATP synthase is an F0F1-enzyme that synthesizes ATP using the energy of the proton gradient.
- The electrochemical H+ gradient is generated by complexes I, III, and IV of the respiratory chain.
- NADH donates electrons to Complex I, and FADH2 to Complex II via succinate dehydrogenase.
- Ubiquinone (Q) and cytochrome c are mobile electron carriers.
- Complex II does not pump protons; it merely maintains the ubiquinol pool.
- Complex IV reduces O2 to H2O, preventing peroxide formation.
- NADH oxidation yields ~2.5 ATP, while FADH2 oxidation yields ~1.5 ATP.
- Electron leakage to oxygen leads to the formation of reactive oxygen species and cell damage.