Oxidative Phosphorylation

What is the Chemiosmotic Theory?

● 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 (delta pH) and a membrane potential (delta ψ), 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).

● 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 using a membrane-embedded ATP synthase complex from bovine heart mitochondria.

How does the electron transport system convert redox energy into proton motive force?

● 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.

● 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 (Eº’); that is, in order of more positive values.

● Proton translocation across the inner mitochondrial membrane occurs by two mechanisms: (1) a redox loop in which a separation of the H1 and e2 occurs on opposite sides of the membrane; and (2) redox-driven conformational changes, which alter pKa values of functional groups located on the inner and outer faces of the 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 2 cytochrome c carriers.

● 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 the mitochondrial ATP synthase complex synthesize ATP in response to a proton gradient?

● 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.

● 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 c-ring rotation of ~36Å.

How do mitochondrial transport systems contribute to the ATP currency exchange ratio?

● The ATP/ADP translocase 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 malate–aspartate shuttle, which functions in liver cells; the other is the glycerol-3-phosphate shuttle, which functions in muscle cells.

● 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 120Å 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 ATP), and ~1.5 ATP per FADH₂ are generated (6 H⁺/4 H⁺ = 1.5 ATP).

● The net ATP yield from oxidizing one molecule of glucose in liver cells is 32 molecules 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 one molecule of glucose in muscle cells yields 30 molecules 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 in response to metabolic flux?

● When flux through catabolic pathways is high, it 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 Pi increase. ATP and NADH are allosteric inhibitors of enzymes in energy-converting pathways, whereas ADP, AMP, and Pi 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 ATP/ADP translocase.

● 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.