Oxidative Phosphorylation

How is a proton gradient used to provide the energy for ATP synthesis?

• The chemiosmotic theory refers to the conversion of energy from redox reactions or light into potential energy in the form of an electrochemical proton gradient across a proton-impermeable organelle membrane. Proteins in the inner mitochondrial membrane allow the passage of protons across this membrane.

• The proton circuit consists of both a chemical gradient (ΔpH) and a membrane potential (Δψ), which together constitute proton-motive force and provide the necessary energy for ATP synthesis.

• The electron transport system consists of four protein complexes (complexes I–IV) embedded in the inner mitochondrial membrane and two mobile electron carriers (coenzyme Q and cytochrome c).

• Peter Mitchell correctly predicted that chemiosmosis is a process that couples redox and light energy to ATP synthesis without the need for a high-energy phosphate intermediate (A~X).

• Proof for the chemiosmotic theory came from experiments with a reconstituted artificial membrane system, showing that the protein bacteriorhodopsin is able to capture light energy and convert it into a proton gradient, which generates ATP by use of a membrane-embedded ATP synthase complex from bovine heart mitochondria.

• Flow of electrons through the electron transport system is facilitated by the sequential arrangement of electron carriers, primarily proteins with Fe-containing prosthetic groups that are arranged in order of increasing reduction potentials (E°′); that is, in order of more positive values.

How does the electron transport system translocate H⁺ and reduce oxygen?

• The donation of 2 e⁻ from NADH to the electron acceptor in complex I initiates a series of redox reactions through the rest of the electron transport system, resulting in the reduction of molecular oxygen to form water and translocation of 10 H⁺ across the inner mitochondrial membrane.

• Proton translocation across the inner mitochondrial membrane occurs by two mechanisms: (1) a redox loop in which a separation of the H⁺ and e⁻ occurs on opposite sides of the membrane; and (2) redox-driven conformational changes, which alter pK_a values of functional groups located on the inner and outer faces of the membrane.

• Complex I (NADH–ubiquinone oxidoreductase) accepts two electrons from NADH in the form of a hydride ion (:H⁻) and, through a series of redox reactions involving iron–sulfur (Fe–S) centers, reduces Q to form QH₂. In the process, 4 H⁺ are translocated from the matrix side of the membrane (N; negative side) to the intermembrane space (P; positive side).

• Complex II (succinate dehydrogenase) is a citrate cycle enzyme that also functions as a redox enzyme in the electron transport system. The FADH₂ moiety in complex II donates 2 e⁻ to Q to form QH₂ without translocating any protons across the inner mitochondrial membrane.

• Glycerol-3-phosphate dehydrogenase and ETF-Q oxidoreductase also donate 2 e⁻ from FADH₂ to Q and function as components of other energy-converting pathways.

• Complex III (ubiquinone–cytochrome c oxidoreductase) is the docking site for QH₂ and transfers electrons one at a time to the heme center of the mobile electron carrier protein cytochrome c. In the process, it translocates 4 H⁺ across the inner mitochondrial membrane.

• The conversion of a two-electron carrier (QH₂) to a one-electron carrier (cytochrome c) within complex III uses a four-step reaction called the Q cycle, which uses two unique binding sites, Q_P and Q_N, to oxidize one net QH₂ and transfer 2 e⁻ to two cytochrome c carriers.

• Two models have been proposed to explain the mechanism by which an oxidized Q molecule enters the Q_N site: (1) the Q channel model proposes a hydrophobic channel within the complex III protein that allows the oxidized Q molecule to diffuse from the Q_P site to the Q_N site, whereas (2) the exit/entry model proposes that the oxidized Q molecule in the Q_P site diffuses back to the membrane and a different Q molecule enters the Q_N site. These two models may reflect evolutionary differences between complex III structures in bacteria and mitochondria.

• Cytochrome c is localized to the intermembrane space and is responsible for transporting 1 e⁻ from complex III to complex IV by using an iron-containing heme prosthetic group. Cytochrome c is a highly conserved protein in nature and has a critical role in both the electron transport system and the intrinsic apoptotic pathway.

• Complex IV (cytochrome c oxidase) accepts electrons one at a time from cytochrome c and donates them to oxygen to form water. In the process, it translocates 2 H⁺ across the inner mitochondrial membrane.

• In total, when starting with 2 e⁻ from NADH, 10 H⁺ are translocated from the matrix to the intermembrane space by the electron transport system: 4 H⁺ in complex I, 4 H⁺ in complex III, and 2 H⁺ in complex IV (complex II does not translocate any protons).

How does H⁺ flow through the ATP synthase complex drive ATP synthesis?

• The mitochondrial ATP synthase complex consists of numerous protein subunits distributed among two large structural components, called F₁ and F_o. F₁ contains the catalytic activity, whereas F_o functions as the proton channel crossing the inner mitochondrial membrane.

• The ATP synthase complex contains three functional units: (1) the rotor rotates as protons enter and exit the c ring, (2) the catalytic headpiece is responsible for ATP synthesis in the intact complex, and (3) the stator contains two half-channels for protons to enter and exit the c ring.

• The catalytic headpiece consists of an α₃β₃ hexamer, with each β subunit containing a catalytic site for ATP synthesis. The catalytic sites have three occupancy states: T, tight with ATP bound; L, loose with ADP + P_i; and O, open with no reactants or products bound. Each state depends on the β-subunit conformation.

• The binding change mechanism model explains how conformational changes in β subunits control ATP production. The rate-limiting step is release of the newly formed ATP when the β-subunit conformation goes from T → O as a function of γ-subunit rotation in the rotor.

• Proton flow through F_o rotates the γ subunit such that with each ~120° rotation, the β subunits sequentially undergo a conformational change from L → T → O → L. A 360° rotation of the γ subunit produces ~3 ATP as a result of ~9 H⁺ binding to the c ring and crossing the inner mitochondrial membrane back into the matrix.

• Structural flexibility in the c-subunit ring and the γ-subunit rotor likely accounts for the noninteger number of protons that was experimentally measured for ATP synthesis (~3.3 H⁺ per ATP).

• Protons cross the membrane by entering and exiting through half-channels in the a subunit, where they come in contact with negatively charged Asp59 residues in the c subunit. Because of polarity differences between protonated and unprotonated Asp59 residues with respect to the hydrophobic membrane, the binding of 1 H⁺ to Asp59 in one c subunit results in a ~36° rotation of the c-ring.

How do mitochondrial transport systems determine ATP yields from oxidation?

• A key element of the chemiosmotic theory is that the membrane must be impermeable to the free diffusion of ions to establish the proton gradient. Therefore, biomolecules must be shuttled in both directions across the mitochondrial and chloroplast membranes by transporter proteins.

• The ADP/ATP carrier protein is an antiporter that exports 1 ATP for every 1 ADP that is imported. Similarly, phosphate translocase translocates 1 P_i and 1 H⁺ into the matrix by an electrically neutral import mechanism when functioning as a symporter or exchanges 1 P_i for 1 OH⁻ when it functions as an antiporter.

• NADH cannot cross the inner mitochondrial membrane. Instead, it must be oxidized in the cytosol to donate electrons via shuttle systems to NAD⁺ inside the mitochondrial matrix. One shuttle is the reversible malate–aspartate shuttle, which functions in liver cells; the other is the irreversible glycerol-3-phosphate shuttle, which functions in muscle cells.

• The malate–aspartate shuttle reduces oxaloacetate in the cytosol with NADH to generate NAD⁺ and malate, which is shuttled into the matrix and oxidized to produce NADH and oxaloacetate. In contrast, the glycerol-3-phosphate shuttle oxidizes cytosolic NADH to yield FADH₂, which donates the electron pair directly to Q.

• The ATP currency exchange ratio is based on (1) the number of H⁺ translocated across the inner mitochondrial membrane after NADH (10 H⁺) or FADH₂ (6 H⁺) oxidation by the electron transport system, and (2) the number of H⁺ that must flow through the ATP synthase complex to generate each ATP (~3 H⁺ for each 120° rotation + 1 H⁺ for the P_i translocase = 4 H⁺ per ATP). Therefore, ~2.5 ATP per NADH are generated (10 H⁺/4 H⁺ = 2.5), and ~1.5 ATP per FADH₂ are generated (6 H⁺/4 H⁺ = 1.5).

• The net ATP yield from oxidizing 1 mole of glucose in liver cells is 32 moles of ATP, considering the ATP exchange ratio of ~2.5 ATP per NADH and ~1.5 ATP per FADH₂ and use of the malate–aspartate shuttle. Oxidizing 1 mole of glucose in muscle cells yields 30 moles of ATP, because the 2 NADH generated by the glycolytic pathway are used to produce 2 FADH₂ by the glycerol-3-phosphate shuttle.

How is oxidative phosphorylation regulated to meet the energy needs of the cell?

• High flux through catabolic pathways leads to increased levels of ATP and NADH. In contrast, when flux through anabolic pathways is high, ATP is consumed, and levels of ADP, AMP, and P_i increase. ATP and NADH are allosteric inhibitors of enzymes in energy-converting pathways, whereas ADP, AMP, and P_i are allosteric activators.

• Three classes of inhibitors decrease rates of mitochondrial ATP synthesis: (1) inhibitors of the electron transport system, (2) uncouplers that allow protons to cross the mitochondrial membrane without going through the ATP synthase complex, and (3) inhibitors of the ATP synthase complex and ADP/ATP carrier protein.

• The chemical uncouplers FCCP and DNP dissipate the proton gradient by diffusing across the inner mitochondrial membrane as protonated species; in addition, DNP binds to the uncoupler protein UCP1 and stimulates proton translocation through the channel.

• Mitochondrial diseases originate in neuronal cells and skeletal muscle, which contain large numbers of mitochondria and rely on aerobic metabolism for ATP synthesis. Many of these diseases are due to mutations in mitochondrial genes, which are inherited maternally because mitochondria are derived from egg cells.

Everyday example: CO-14: Contaminated Tempeh Cakes