The actual synthesis of ATP resulting from energy transfer of the ETS is catalyzed by proton translocating ATP synthase (Complex V). One clue to the mechanism of the synthesis is that Complex V is physically separate from the ETS. Remember, however, that the ETS and oxidation phosphorylation are tightly coupled. The ETS must build a potential energy that Complex V harvests to make ATP. The exact mechanism by which this is accomplished has been the subject of extensive research.
Definition: The P/O ratio is the number of ATPs produced per pair of electrons traveling through the electron transport system (ETS). Depending upon the point at which electrons enter the chain, the P/O ration will change (See Figure 15.12). As discussed earlier, electrons entering from NADH produce 3ATPs per pair of electrons, from FADH2 produce 2 ATPs per pair of electrons, and from ascorbate produce 1 ATP per pair of electrons. Since the production of ATP is not directly linked to each step in the transport of electrons, but rather by the gradient arising from pumping of protons during the electron transport, it is not necessary that an exact integral number of ATPs be produced per entry point. In fact, it appears that it is not exactly 3, 2, or 1 ATP produced per pair of electrons, but it is useful conceptually to think in this way.
Let us consider three mechanisms of oxidative phosphorylation that have been considered over the years.
This is the oldest (and simplest) of three models. It simply states that the ETS produces reactive intermediates that can directly phosphorylate ADP + Pi to ATP in a mechanism like substrate level phosphorylation in glycolysis. No suitable reactive intermediates as hypothesized have ever been found.
This hypothesis states that electron transport generates energized proteins that, when interacting with ATP synthase, release their energy, driving ATP synthesis. Again, there is little evidence for this model.
Peter Mitchell proposed that the ETS generates potential energy by pumping protons out of the mitochondrial matrix into the intermembrane space. The energy of the proton gradient is used to synthesize ATP. This method is believed to be correct for several reasons.
It is interesting to note a couple of ionophores which affect mitochondria. Valinomycin, as noted previously, can transport K+ ions across a mitochondrial membrane. By doing so, it can balance the charge difference across the membrane without affecting the H+ difference. It has little effect alone on oxidative phosphorylation. Similarly, nigericin acts as an H+/K+ antiport (See Antiport). It destroys the H+ gradient without affecting the charge gradient. It too does not affect oxidative phosphorylation significantly. When the two compounds are used together, however, oxidative phosphorylation is uncoupled from ETS.
The antibiotic Oligomycin disrupts phosphorylation by binding to a specific protein in Complex V
The ETS pumps protons across the inner membrane as part of its operation. This action serves to store energy from the ETS process as potential energy in the form of a proton gradient (high H+ in the intermembrane space, low H+ inside the mitochondria). As noted, pumping protons from a region of low concentration to a region of high concentration requires energy, and this energy is supplied by the ETS. Remember that protons are charged, so the action of pumping positive charges outside of the mitochondria causes the inner membrane's inner surface to become negatively charged relative to its outer surface. The energy required to pump out protons is 21.5 kJ/mole. The energy required to synthesize ATP from ADP + Pi is 40-50 kJ/mole. ATP is synthesized as protons "fall" back into the mitochondria, reaping the energy "saved" by pumping them out. Though in theory, fall of two protons back into the mitochondria could synthesize an ATP, it probably takes 3 protons to accomplish this (remember that energy transfer processes are not 100% efficient).
The enzyme that uses the energy of the proton gradient to synthesize ATP from ADP + Pi is called proton-translocating ATP synthase (PTAS). This complex system is composed of two major components and numerous other subunits. Close examination of the mitochondrial cristae reveals a knobby set of structures (See Figure 15.13a) sitting on top of what appear to be stalks. The knobs are known as F1 spheres. The stalk and its base are referred to as F0 (See Figure 15.14). F0 itself is composed of many subunit proteins. It is transmembranous (insoluble in water) and creates a channel for protein translocation.
F1 is water soluble and a peripheral membrane protein. It has several subunits and interacts with F0 weakly. Solubilized F1 can hydrolyze ATP, but cannot synthesize it. SP from which F1 has been removed no longer exist as knobby "lollipops" and further cannot synthesize ATP. Addition back of F1 to SP containing F0 restores both the lollipop appearance and the ability to synthesize ATP. The antibiotic oligomycin inhibits ATP synthase by binding one of F0's subunits and preventing H+ transport through F0. Dicyclohexylcarbodiimide (DCCD), a lipid soluble compound also inhibits H+ transport through F0.
The mechanism of ATP synthesis consists of three phases; 1) movement of the protons by F0; 2) Catalysis of formation of ATP by F1; 3) Coupling of the proton gradient dissipation energy with ATP synthesis - occurs through interaction between F1 and F0.
The observed mechanism supports a model in which ATP synthesis occurs by a model depicted in Figure 15.19 (A better figure from the second edition is available here). Basically, subunit F1 has three adjustable binding sites for ATP, called L, T, and O, which bind respectively ATP loosely, tightly, or not at all. The configuration of each of the binding sites can be switched to the other with concomitant conversion of the remaining sites to alternate conformations. One site can be T, one O, and one L at any given time. Binding of ADP + Pi occurs at a site in the L conformation. The T site is always bound with ATP, and the O site is not bound with ATP or ADP at all. Energy passed because of H+ transport causes the three binding sites in the F1 subunit to flip conformations. The T site is converted to an O site (causing release of ATP bound there), the L site is converted to a T site (catalyzing conversion of ADP + Pi to ATP), and the O site is converted to an L site (where the next ADP + Pi will bind).
ATP is a limiting nutrient. The total needs of the body for ATP in a day are over 2000 times the amount present at any given time. Moreover, the needs for ATP over a 24 hour period in the body vary up to 100 fold between sleep and strenuous exercise. Clearly, control of synthesis of ATP in the body is extremely important.
Remember that most metabolic reactions occur under conditions where the reactions are freely reversible. Steps, such as the phosphofructokinase (PFK) reaction of glycolysis, that are essentially irreversible under cellular conditions, are tightly controlled (usually allosterically) and actually serve as control points for entire pathways. One essentially reversible step of oxidative phosphorylation/electron transport occurs in the transfer of electrons from NADH to cytochrome c, with coincident conversion of ADP + Pi to ATP. Addition of product (ATP) favors reversal of the reaction (formation of NADH). Addition of NADH favors the forward reaction (production of ATP). The last step of electron transport (cytochrome oxidase), however, is essentially irreversible and can act as an excellent control point for electron transport. Cyt c oxidase is exclusively controlled by the availability of its substrate, reduced cyt c. Remember that reduced cyt c is produced as a result of the forward reaction. Thus, high NADH/low ATP concentrations favors formation of reduced cyt c, and the cyt oxidase reaction (increased electron transport). Resting individuals have high ATP concentrations, thus low reduced cyt c concentrations, and little electron transport. This control mechanism is called Acceptor Control, meaning that ultimately, one of the controls is ADP, the phosphoryl group acceptor. One complicating factor is that the critical ADP/ATP ratio is inside the mitochondrial matrix, whereas most ATP is used in the cytoplasm, and the inner mitochondrial membrane is impermeable to adenine nucleotides and Pi. Shuttle mechanisms must operate in order to move ADP and Pi inside the mitochondrion. It may be that transport of ADP and Pi across the mitochondrial membrane is the ultimate rate limiting step of oxidative phosphorylation.
Control points of glycolysis (PFK), TCA cycle (pyruvate DH, citrate synthase, isocitrate DN, and alpha keto glutarate DH) are responsive to adenine nucleotides (ADP, ATP), NADH, or both as well as other metabolites. All the major paths for production of ATP (glycolysis, TCA cycle, oxidative phosphorylation) must be coordinated for production of ATP as needed. Oxidative phosphorylation is dependent on the ETS, which is dependent on electrons carried by NADH/FADH2. Glycolysis and the TCA cycle are readily producers of NADH and FADH2. It is easy to see how what I call feed forward activation of ATP synthesis (glycolysis turns on TCA, and both turn on oxidative phosphorylation by turning on the electron transport system) is coordinated. Feed backwards deactivation of ATP synthesis is also important, because cells need to turn off ATP synthesis, as well as turn it on. One important metabolite that helps to turn off the coordinated feed forward system is citrate, which acts allosterically to deactivate PFK, the major control point of glycolysis. When the body moves from exercising to rest, the concentration of ATP increases (because the ATP being produced rapidly by oxidative phosphorylation is not being burned to ADP + Pi). Increasing ATP inhibits alpha keto glutarate DH and slows isocitrate DH (activated by ADP). The combined effects of the inactivation or slowing of these enzymes slows the TCA cycle, and increases citrate concentration. Increasing citrate in the inner mitochondrion is transported to the cytosol where it interacts with PFK, shutting down carbohydrate breakdown (glycolysis).
Remember that under anaerobic conditions the ETS is stopped (no oxygen). The end products of glycolysis then do not enter the TCA cycle. In mammals, muscle cells often encounter anaerobic conditions because oxygen from the blood supply cannot keep up with the metabolic rate of the cell. When this happens, pyruvate from glycolysis is converted to lactate (instead of Acetyl-CoA), using the two NADH generated during glycolysis. TCA is shut down, since there is not a ready source of AcCoA. The net products of anaerobic glycolysis then are 2 lactates, two protons, two H2Os, and 2 ATPs. Under aerobic conditions, pyruvate is converted to AcCoA, which enters the TCA cycle, and the energy from all the NADHs and FADH2s of glycolysis and the TCA cycle are reaped by ETS/oxidative phosphorylation. The net products of metabolism under aerobic conditions are 6 CO2s, 44 H2Os, and 38 ATPs. Thus, aerobic metabolism is 19 times more efficient that anaerobic metabolism at producing ATP. Remember above how ATP production from ETS can shut down the TCA cycle, which yields excess citrate, which turns off PFK. Not surprisingly then, shifting cells from anaerobic conditions to aerobic conditions results in lowering of PFK activity. Thus, under aerobic conditions, glycolysis is slowed, which makes sense, considering the increased efficiency of metabolism under aerobic conditions. One limit to anaerobic glycolysis, though, is the inability of PFK to function much below pH 7. Remember that lactate production generates protons, reducing the pH. Another important point to remember is that the enzymes of glycolysis are present in a great enough concentration that, when they are turned on, they can produce ATP much more rapidly than oxidative phosphorylation.
Remember that oxidative phosphorylation is tightly coupled to electron transport. This means that blocking of the ETS system will prevent oxidative phosphorylation and, importantly, that the reverse is true also - blocking oxidative phosphorylation stops electron transport (See also Figure 15.23). Thus, at a resting state, oxidative phosphorylation is slowed considerably, and the ETS system too stops - because the ever increasing gradient of H+ becomes too difficult to pump more H+ against. It is possible to "uncouple" these two processes as noted above, using agents that permeabilize the inner mitochondrial membrane to protons. Two such agents are 2,4 dinitrophenol (DNP) and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Remember that permeabilizing the inner mitochondrial membrane to proton dissipates the electrochemical gradient in which energy from electron transport is stored. Uncoupling the ETS system from oxidative phosphorylation is potentially lethal. In an uncoupled system oxidative phosphorylation does not occur. In addition, the cell's energy needs turn on the electron transport system even further in an attempt to generate ATP. Electron transport occurs furiously, but no ATP is generated. (Remember also that normally oxidative phosphorylation serves to modulate the ETS - resting states turn off the ETS/active states turn it on).
Uncoupling the ETS from oxidative phosphorylation speeds metabolism, and generates heat. Some mammals lacking fur use this function in brown adipose tissue as a way of generating heat. One such process is called nonshivering thermogenesis that occurs in cells in the neck and upper back. The mitochondria of brown adipose tissue cells contain a protein called thermogenin (or uncoupling protein - UCP). Thermogenin acts as a channel to permeabilize these cells' inner mitochondrial membrane to protons. Normally, ADP, ATP, GDP, and GTP are present in high enough concentrations to block the flow of protons through it. However, thermogenin in the mitochondria of these cells is activated to uncoupling by the presence of free fatty acids. Free fatty acids can be generated in these cells by the hormone norepinephrine, which through second messengers (including cAMP) activates hormone-sensitive triacylglycerol lipase to cleave fats to release fatty acids. Thus, brown adipose tissue cells respond to norepinephrine by uncoupling the ETS from oxidative phosphorylation, speeding metabolism and generating heat, at the expense of metabolic energy.
Figure 15.24 depicts a number of the transport systems specific for molecules that must cross the inner mitochondrial membrane.
Approximately 90% of the oxygen in cells is used for oxidative phosphorylation. Some of the other reactions of oxygen are listed below.
Oxidases and Oxygenases: Oxidases catalyze oxidation of substrate without using molecular oxygen. One reaction shown at the bottom of page 551 of the book involves replacement of an amino nitrogen on a D amino acid by a D-amino acid oxidase. The oxygen comes from water, and FAD is a cofactor. By contrast, oxygenases incorporate oxygen directly from O2. One example is tryptophan 2,3 dioxygenase, which catalyzes a reaction in tryptophan catabolism. Dioxygenases, of course, utilize both atoms of O2 in a reaction. Monooxygenases, on the other hand, use only one atom of O2 in a reaction. The product of this type of a reaction often ends up hydroxylated. A family of heme containing proteins perform numerous hydroxylation reactions. This class of proteins is referred to collectively as the Cytochrome P-450 enzymes. Reactions they catalyze involve steroid hormones, fatty acids, and fatty acid epoxides. Numerous foreign substances (called xenobiotics) including drugs and carcinogens are also hydroxylated by the Cytochrome P450 systems. Cytochrome P450 works by splitting O2, resulting in one oxygen atom binding to the iron atom of the heme group. This intermediate, called a perferryl ion, is extraordinarily reactive. Hydroxylation via this intermediate results in transfer of reducing equivalents being transferred from NADPH, as shown in Figure 15.25.
Reduction of O2 to water takes place sequentially as additions of four electrons to O2, one at a time. Each of the intermediates of the reduction, superoxide (O2-), hydrogen peroxide (H2O2), and OH radical/hydroxyl are very reactive substances. Hydroxyl radicals, for example, can cause oxidative damage to DNA, resulting in strand breakage. Cellular protection against oxidative damage include biological antioxidants, such as Vitamins C & E, glutathione, and uric acid. Enzymes, such as superoxide dismutase and peroxidases protect respectively against superoxide radicals and hydrogen peroxide.
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Inhibitors and Artificial Electron Acceptors