Mitochondria respiration – State 3/4 and Uncoupling



Douglas C. Wallace

The mitochondria generate energy by oxidizing hydrogen derived from our dietary carbohydrates (TCA cycle) and fats (β-oxidation) with oxygen to generate heat and ATP (Figure 2). Two electrons donated from NADH + H+ to complex I (NADH dehydrogenase) or from succinate to complex II (succinate dehydrogenase, SDH) are passed sequentially to ubiquinone (coenzyme Q or CoQ) to give ubisemiquinone (CoQH) and then ubiquinol (CoQH2). Ubiquinol transfers its electrons to complex III (ubiquinol:cytochrome c oxidoreductase), which transfers them to cytochrome c. From cytochrome c, the electrons flow to complex IV (cytochrome c oxidase, COX) and finally to 1/2 O2 to give H2O. Each of these electron transport chain (ETC) complexes incorporates multiple electron carriers. Complexes I, II, and III encompass several iron-sulfur (Fe-S) centers, whereas complexes III and IV encompass the b + c1 and a + a3 cytochromes, respectively. The mitochondrial TCA cycle enzyme aconitase is also an iron-sulfur center protein (234, 235, 237).

The energy released by the flow of electrons through the ETC is used to pump protons out of the mitochondrial inner membrane through complexes I, III, and IV. This creates a capacitance across the mitochondrial inner membrane, the electrochemical gradient (ΔP = ΔΨ + ΔpH). The potential energy stored in ΔP is coupled to ATP synthesis by complex V (ATP synthase). As protons flow back into the matrix through a proton channel in complex V, ADP and Pi are bound, condensed, and released as ATP. Matrix ATP is then exchanged for cytosolic ADP by the adenine nucleotide translocator (ANT) (Figure 2).

Because the ETC is coupled to ATP synthesis through ΔP, mitochondrial oxygen consumption rate is regulated by the matrix concentration of ADP. In the absence of ADP, the consumption of oxygen is regulated by proton leakage across the inner membrane and thus is slow (state IV respiration). However, when ADP is added, it binds to the ATP synthase and is rapidly converted into ATP at the expense of ΔP. As protons flow through the ATP synthase proton channel, the proton gradient is depolarized. Stored fats and carbohydrates are then mobilized to provide electrons to the ETC, which reduce oxygen to water and pump the protons back out of the mitochondrial matrix. The resulting increased oxygen consumption on addition of ADP is known as state III respiration. ΔP is also used for the import of cytosolic proteins, substrates, and Ca2+ into the mitochondrion. Drugs such as 2,4-dinitrophenol (DNP) and nDNA-encoded uncoupler proteins 1, 2, and 3 (Unc1, 2, and 3) render the mitochondrial inner membrane leaky for protons. This short-circuits the capacitor and “uncouples” electron transport from ATP synthesis. This causes the ETC to run at its maximum rate, causing maximum oxygen consumption and heat production, but diminished ATP generation (Figure 2).

The efficiency by which dietary calories are converted to ATP is determined by the coupling efficiency of OXPHOS. If the ETC is highly efficient at pumping protons out of the mitochondrial inner membrane and the ATP synthesis is highly efficient at converting the proton flow through its proton channel into ATP, then the mitochondria will generate the maximum ATP and the minimum heat per calorie consumed. These mitochondria are said to be tightly coupled. By contrast, if the efficiency of proton pumping is reduced and/or more protons are required to make each ATP by the ATP synthase, then each calorie burned will yield less ATP but more heat. Such mitochondria are said to be loosely coupled. Therefore, in an endothermic animal, the coupling efficiency determines the proportion of calories utilized by the mitochondrion to perform work versus those to maintain body temperature.

As a toxic by-product of OXPHOS, the mitochondria generate most of the endogenous ROS. ROS production is increased when the electron carriers in the initial steps of the ETC harbor excess electrons, i.e., remain reduced, which can result from either inhibition of OXPHOS or from excessive calorie consumption. Electrons residing in the electron carriers; for example, the unpaired electron of ubisemiquinone bound to the CoQ binding sites of complexes I, II, and III; can be donated directly to O2 to generate superoxide anion (O2). Superoxide O2 is converted to H2O2 by mitochondrial matrix enzyme Mn superoxide dismutase (MnSOD, Sod2) or by the Cu/ZnSOD (Sod1), which is located in both the mitochondrial intermembrane space and the cytosol. Import of Cu/ZnSOD into the mitochondrial intermembrane space occurs via the apoprotein, which is metallated upon entrance into the intermembrane space by the CCS metallochaperone (166, 207). H2O2 is more stable than O2 and can diffuse out of the mitochondrion and into the cytosol and the nucleus. H2O2 can be converted to water by mitochondrial and cytosolic glutathione peroxidase (GPx1) or by peroxisomal catalase. However, H2O2, in the presence of reduced transition metals, can be converted to the highly reactive hydroxyl radical (OH) (Figure 2). Iron-sulfur centers in mitochondrial enzymes are particularly sensitive to ROS inactivation. Hence, the mitochondria are the prime target for cellular oxidative damage (241, 242).

For tightly coupled mitochondria, in the presence of excess calories and in the absence of exercise, the electrochemical gradient (ΔP) becomes hyperpolarization and the ETC chain stalls. This is because without ADP the ATP synthase stops turning over, thus blocking the flow of protons back across the mitochondrial inner membrane through the proton channel of the ATP synthase. However, the ETC will continue to draw on the excess calories for electrons to pump protons out of the mitochondrial inner membrane until the electrostatic potential of ΔP inhibits further proton pumping. At this point, the ETC stalls and the electron carriers become maximally occupied with electrons (maximally reduced). These electrons (reducing equivalents) can then be transferred to O2 to generate O2 and thus increased ROS and oxidative stress.

By contrast, in individuals who actively exercise and generate ADP, the ATP synthase keeps ΔP hypopolarized and electrons continue to flow through the ETC to sustain ΔP. Consequently, the electron carriers retain few electrons (remain oxidized). The low electron density of the ETC electron carriers limits ROS production and reduces oxidative stress.

This same effect can be achieved if the mitochondria become partially uncoupled, either by decreasing the number of protons pumped per electron pair oxidized or by permitting protons to flow back through the inner membrane without making ATP. Uncoupling can be achieved by disconnecting electron transport from proton pumping by alterations of complexes I, III, or IV; by increasing the number of protons required by the ATP synthase to make an ATP; or by expression of an alternative proton channel such as an uncoupling protein (UCP).

mitochondrial permeablility transition pore

The mitochondria are also the major regulators of apoptosis, accomplished via the mitochondrial permeability transition pore (mtPTP). The mtPTP is thought to be composed of the inner membrane ANT, the outer membrane voltage-dependent anion channel (VDAC) or porin, Bax, Bcl2, and cyclophilin D. The outer membrane channel is thought to be VDAC, but the identity of the inner membrane channel is unclear since elimination of the ANTs does not block the channel (98). The ANT performs a key regulatory role for the mtPTP (98). When the mtPTP opens, ΔP collapses and ions equilibrate between the matrix and cytosol, causing the mitochondria to swell. Ultimately, this results in the release of the contents of the mitochondrial intermembrane space into the cytosol. The released proteins include a number of cell death-promoting factors including cytochrome c, AIF, latent forms of caspases (possibly procaspases-2, 3, and 9), SMAD/Diablo, endonuclease G, and the Omi/HtrA2 serine protease 24. On release, cytochrome c activates the cytosolic Apaf-1, which activates the procaspase-9. Caspase 9 then initiates a proteolytic cascade that destroys the proteins of the cytoplasm. Endonuclease G and AIF are transported to the nucleus, where they degrade the chromatin. The mtPTP can be stimulated to open by the mitochondrial uptake of excessive Ca2+, by increased oxidative stress, or by deceased mitochondrial ΔP, ADP, and ATP. Thus, disease states that inhibit OXPHOS and increase ROS production increase the propensity for mtPTP activation and cell death by apoptosis (Figure 2) (241, 242).


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