The real key to understanding oxidative phosphorylation is understanding the energy in electrons and how this is expressed in terms of oxidation and reduction. Oxidation is the process of giving up an electron. Gaining an electron makes an oxidized molecule reduced. Focus on gaining expertise with the terms oxidized and reduced. Don't attempt to master the terms oxidant and reductant or oxidizing agent and reducing agent; these seem to cause more confusion than help.
It may be useful to think of oxidation and reduction as a concept similar to deprotonation. For a given weak acid, one can write the equation for protonation and define the pKa.
HA A- + H+
The pKa defines the strength of the acid and whether it is likely to donate or accept protons at any given solution pH. We can similarly write equations for oxidation and reduction reactions. The following is the equation for the reduction of ferric iron to ferrous iron.
Fe3+ + e- –> Fe2+
Just as most protonation/deprotonation equations are written as deprotonations, most oxidation and reduction reactions are written left to right as the reduction. Fe3+ is the oxidized species and Fe2+ is the reduced species. The strength of iron's ability to give off or accept an electron is expressed as the standard redox potential (Eo'). This is the solution potential at which iron will be half Fe3+ and half Fe2+. The lower (more negative) the Eo' of a molecule, the more energy there is in its electrons and the more tendency for it to donate them to other molecules. The higher (more positive) the Eo' of a molecule, the more tendency for it to accept electrons. If you mix two molecules of different Eo', electrons will tend to flow from the one with the lower Eo' to the one with the higher Eo'. Stated differently, molecules with lower Eo' values tend to become oxidized, whereas those with higher Eo' values tend to become reduced. Let's examine two elements--iron and oxygen, the components of rust.
The oxidized form of each is written on the left and the reduced form is written on the right. Reduction occurs right to left. Notice that the Eo' for the reduction of O2 to water is greater than the Eo' for the reduction of Fe3+ to Fe2+.
The electrons will tend to flow from the molecule with the lower Eo' (+0.771) to the molecule with the higher Eo' (+0.816). Thus we can predict that the iron will tend to accumulate as Fe3+ (rust) and the oxygen will accept these electrons and be reduced to H2O. The extent to which electrons will flow from one molecule to another is dependent on the difference in Eo', which is related to Go'.
The greater the difference in the Eo' of two molecules, the greater the amount of free energy that is released in the electron transfer and the greater the extent to which one molecule will be come oxidized and the other reduced. Why don't electrons normally flow from water to Fe3+? Because moving to molecules with lower Eo' values represents a positive change in free energy.
When mapping electron transfer components, we normally draw the Eo' scale with (-) on the top and (+) on the bottom. As electrons 'flow downhill' the free energy is released and it is possible to capture some of it for biochemical work.
Before the evolution of oxidative phosphorylation, life went on at a slower pace for millions of years. Life was based on anaerobic mechanisms and substrate-level phosphorylation to produce ATP. Reduced organic compounds were converted to slightly more oxidized molecules and the free energy released could be used to stay alive. If you struck a match on this world, it would not burn.
Then some organisms developed photosynthesis and finally were able to extract energy from sunlight to excite electrons and tear the electrons from molecules. The revolution had started once they evolved a mechanism to tear the electrons from water. A byproduct of this process was O2. As atmospheric O2 concentrations increased, it created an opportunity for nonphotosynthetic organisms. The standard reduction potential for O2 is a very large number (~+0.8 V). If O2 could be used as an electron acceptor for partially oxidized organic molecules, they could be completely oxidized and a large amount of free energy would be available to cells.
One problem with using O2 to oxidize organic molecules is that the amount of free energy released is so large, that it is dangerous. It's like having a nuclear power plant in your kitchen. Much as humans decided to centralize power production and distribute it by transduction to electrical currents, cells centralized the production of free energy from oxidation and distributed it via transduction to ATP. The actual transduction machinery is locked away in a membrane for safety.
Prologue: Much of biochemical energy for microbes and animals comes in the form of organic molecules in the environment (proteins, amino acids, lipids, polysaccharides, sugars). For the present, consider reduced carbon in the form of D-glucose. A process called glycolysis converts glucose to pyruvate. Pyruvate is then oxidized to CO2 and most of the electrons are transferred to NAD+ and stored as molecules of NADH [local]. This latter process involves the TCA Cycle and one of its intermediate metabolites is succinate, which is oxidized by the enzyme succinate dehydrogenase. Oxidative phosphorylation is the process cells use to extract the stored free energy in NADH and succinate.
The components [local] of oxidative phosphorylation must be viewed in three separate ways: First as a linear assembly [local] of electron transfer components [local]; Second as a series of components ordered according to their standard reduction potentials; and third in terms of their location in the membrane [local].
The linear assembly:
NADH –>FMN –>FeS –>Q –>[FeS-Cyt b] –>Cyt c1 –>Cyt c –> Cyt a –>Cyt a3 –>O2 Succinate –>FAD –>FeS^
The sequence ordered in terms of standard redox potential
Complex I transfers 2e- from NADH to Q, making QH2; some protons are pumped to the outside
Complex II transfers 2e- from succinate to Q, making QH2
QH2 functions as a hydrophobic mobile electron and proton carrier
Complex III transfers e- from QH2 to Cytochrome c; this is the site of the Q cycle
Cytochrome c functions as a soluble mobile electron carrier
Complex IV transfers e- from Cytochrome c to O2
The secret of life: The transfer of electrons from NADH and succinate to oxygen releases massive amounts of free energy. Because of the supramolecular arrangement of the machinery in the membrane, the free energy is transduced to a proton gradient. The proton gradient [local] produced across the membrane is released and the free energy produced is used to reverse an ATPase activity (ATP + H2O -–> ADP + Pi).
Thus energy goes through several different manifestations before it is in a useful form.
The supramolecular architecture of oxidative phosphorylation
ATP synthase (Complex V) is composed of two parts, the F0, which is an integral membrane complex which functions as a proton pore, and F1 which can catalyze ATP hydrolysis. When the relative proton concentration on the outside of the membrane in increased by electron transfer reactions (proton pumping by complexes I, III, and IV, Q, proton annihilation by complex IV, etc.) the F0 complex is a proton channel and can let the protons run down the concentration gradient, releasing free energy. The close association of F1 with F0 causes the released free energy to be used to reverse the hydrolysis of ATP, with the net synthesis of ATP from ADP and Pi.
The F1 complex has a threefold axis of symmetry and three binding sites for ATP or ADP. As the protons flow back into the inner compartment, the free energy released is used to rotate the protein complex. As the complex rotates, its conformation undergoes successive changes. These facilitate the conversion of ADP and Pi to ATP + H2O. This part does not seem to require much energy. The energy from the proton gradient seems to be most required for forcing the F1 to dissociate the ATP and free the active site so that it can bind another ADP and Pi.
This linking of ATP production to the oxidation of organic molecules via a proton gradient is called chemiosmotic coupling. ATP can be synthesized without electron transport. Artificial generation of a trans membrane difference in pH is sufficient to produce ATP
Inhibitors of Oxidative Phosphorylation [local]
Inhibitors of oxidative phosphorylation have been useful tools in understanding its mechanism of action. For obvious reasons, these inhibitors are also highly toxic poisons. These have one of two general mechanisms. Some, like rotenone bind to components in the middle of the electron transfer chain and halt transfer. Upstream components become more reduced, whereas downstream components of the chain become more oxidized, helping to identify their position in the overall chain. Rotenone has been used to paralyze fish, causing them to rise to the surface and be easily caught. Rotenone blocks electron flow from NADH to coenzyme Q.
Other inhibitors like cyanide, azide and carbon monoxide bind to hemes, like those in Complex IV. These block the terminal steps in electron transfer, causing most of the electron transfer chain to become more reduced. Carbon monoxide also binds to the heme in hemoglobin and can be lethal in causing suffocation. Interestingly, H2S is more toxic than cyanide, but we generally don't worry about being poisoned by this gas because we can detect its stench-odor at even lower concentrations than cause toxicity.
Other inhibitors are classified as uncouplers. These are generally slightly soluble molecules that are able to move protons across the membrane and release them on the other side. Addition of these molecules to an ATP-generating system causes a collapse of the trans-membrane proton gradient. With the uncoupler present, ATP synthesis is halted, but the rate of electron transfer may actually be increased (respiration continues). A classic uncoupler is 2,4-dinitrophenol. At one point in the 1930s it was thought that molecules such as 2,4-DNP might be useful for weight loss, because they would lower the efficiency of energy capture from respiration. While this is true in theory, they are too dangerous to be practical. What would you do if someone accidentally ingested 2,4-DNP? Their body temperature would increase, because energy not coupled to ATP production would be lost as heat. You could try putting them into a cold bath and hope for the best.
Animal mitochondria can contain an uncoupling protein called thermogenin that can make metabolism less efficient. Plants also contain a similar uncoupler protein, but it is not used in the skunk cabbage. Plant mitochondria also contain an alternate oxidase system that transfers electrons from NADH to O2, but does not pump protons or contribute to ATP production. This system is used in skunk cabbage and vodoo lillies to volatilize awful-smelling amines that attract flies for pollination. If you travel in the mountains in early spring, you will note many wildflowers that seem to 'burn' their way through the snowbanks to reach the open air. These also use the alternate oxidase pathway to heat their shoots and melt the snow as they grow.
The ability to bury electrons in O2 means that aerobic organisms have a big advantage in biochemical efficiency. Bacteria forced to grow without oxygen get a meager 2 ATP per glucose and have a large amount of toxic waste that must be disposed. An identical organism in an aerobic environment can get 30 or more ATP per glucose and the toxic waste (CO2) is a gas and takes care of itself.
