The coupling of metabolic and light energy to the performance of transmembrane work through the intermediary of electroosmotic gradients. Processes include synthesis of adenosine triphosphate (ATP) by oxidative phosphorylation or by photosynthesis, production of heat, accumulation of small molecules by active transport, movement of bacterial flagella, uptake of deoxyribonucleic acid (DNA) during bacterial conjugation, genetic transformation and bacteriophage infection, and insertion or secretion of proteins into or through membranes.
During the 1940s and 1950s, mitochondria were shown to be the powerhouses of the eukaryotic cell and the site of synthesis of ATP by oxidative phosphorylation. In the oxidation portion of ATP synthesis, reductants, such as reduced nicotinamide adenine dinucleotide (NADH) and succinate, are generated during metabolism of carbohydrates, lipids, and protein. These compounds are oxidized through the series of redox reactions performed by membrane-bound complexes, called electron transport or respiratory chains. The respiratory chains have common features. All are found associated with the inner mitochondrial membrane, and each chain pumps protons from inside the mitochondrion to the cytosol. Each begins with a membrane-bound dehydrogenase enzyme which oxidizes the soluble reductant. The dehydrogenase complex transfers electrons to a lipid-soluble quinone coenzyme, such as ubiquinone, which in turn passes electrons to a series of cytochromes. Cytochromes are proteins that have a heme moiety. Unlike the oxygen-carrier protein hemoglobin, which maintains its heme iron in the reduced state, the irons of cytochromes undergo a cycle of oxidation-reduction, alternating between the ferrous and ferric states. During reduction, a cytochrome accepts an electron from the reduced side of the chain and passes it to the cytochrome on the oxidized side of the chain. The terminal cytochrome interacts with molecular oxygen, forming water. The overall reaction for the mitochondrial NADH oxidase is expressed in reaction (1):
The phosphorylation half of oxidative phosphorylation is catalyzed by a specific H+-translocating adenosine triphosphatase (ATPase) enzyme. The ATPase is also a membrane-bound enzyme located in the inner mitochrondrial membrane, catalyzing reaction (2),
where ADP is adenosine diphosphate and Pi is inorganic phosphate. The reaction occurs with a standard free-energy change, ΔG°′, of +7.3 kcal/mol. The two processes, oxidation and phosphorylation, are metabolically coupled, with mitochondria continually synthesizing ATP by using energy derived from the respiratory chain. In the case of NADH oxidase, the net reaction is shown as reaction (3):
In the overall biological reaction, three ATPs are synthesized for each NADH reduced, even though enough energy was available for six or seven ATPs. This assumes that the actual free-energy change, ΔG, is approximately the same as the standard free-energy change at pH 7, ΔG°′. Calculations suggest that the free energy of formation of ATP is probably greater than −7.3 kcal/mol, perhaps as much as −10 to −15 kcal/mol, but it is difficult to determine this parameter in a living system. The stoichiometry is a function of the molecular mechanism of coupling.
Most early postulates about the mechanism by which the redox energy of NADH is converted into the energy-rich phosphoric anhydride bond of ATP involved the formation of a high-energy chemical intermediate during the flow of electrons down the respiratory chain. This chemical intermediate could then interact with ADP, forming a high-energy nucleotide which could finally interact with Pi, producing ATP. For 2 decades, this chemical intermediate proved elusive. The search culminated in the formulation of the chemiosmotic hypothesis by Peter Mitchell in 1961. For this contribution, Mitchell was awarded the Nobel Prize in medicine in 1978.
Mitchell postulated that chemical energy, that is, the energy of a chemical bond, can be reversibly converted into electroosmotic energy. The reactions are catalyzed by enzyme complexes which function as primary ion pumps. From the work of Mitchell and of others, the respiratory chain and the ATPase have both been shown to be proton pumps. The respiratory chain transports protons out of the mitochondrion into the cell cytosol, creating an electrochemical gradient of protons. When the ATPase hydrolyzes ATP, it also pumps protons out of the mitochondrion, hence the name H+-translocating ATPase. Since the free energy produced by respiration is so much greater than that derived from ATP hydrolysis, proton translocation by the respiratory chain creates a force which drives the uptake of protons by the ATPase and consequently the synthesis of ATP. A diagram of the coupling of oxidation and phosphorylation is shown in the illustration.
Mitchell termed the force derived from proton translocation the proton motive force (pmf), by analogy with the electromotive force (emf) of electrochemical cells. Because the pmf is produced by pumping of H+ ions across the mitochondrial membrane, it has both electrical and chemical components. The electrical or membrane potential (Δψ) is due to the separation of positive and negative charges across the mitochondrial membrane. The chemical component results from the separation of H+ and OH−. Since pH = −log [H+], [H+] being the concentration of H+ ions, the chemical proton gradient is measured as a pH difference, ΔpH. Thus, pmf = Δψ − (2.3RT/F) · ΔpH and pmf = ΔG/F, where R is the gas constant, T is the temperature, and F is the Faraday constant.
The concept of chemiosmosis is now well accepted, although some still question whether the pmf is the primary source of energy for phosphorylation. Most effort is concentrated on the molecular mechanisms of chemiosmotic coupling. It is clear that respiratory chains pump out protons, but questions such as which components directly transport protons, how many protons are translocated per pair of electrons, and what is the catalytic mechanism of proton translocation are still not adequately answered. It is clear that the ATPase transports protons outward during ATP hydrolysis and inward during synthesis, but the number of protons per ATP is controversial. The protons may directly interact with substrate within an active site, participating directly in catalysis. Conversely, binding of protons may change the shape or conformation of the ATPase enzyme in such a way as to alter its affinity for ATP. These are not trivial distinctions. How these macromolecular complexes work, how chemical energy is transformed into electrochemical and back into chemical energy, cannot be understood without the answers to the more basic questions. See also: Adenosine triphosphate (ATP); Biological oxidation
Bacterial oxidative phosphorylation
Bacteria do not contain mitochondria, but many of the functions of the mitochondrial membrane are carried out by the bacterial cytoplasmic membrane. All bacteria contain a H+-translocating ATPase in their cytoplasmic membrane. This enzyme is nearly identical to that of mitochondria. In fact, the genes for the bacterial and mitochondrial versions are clearly derived from a single evolutionary precursor. Many bacteria also use respiratory chains. Oxygen is not always the terminal electron acceptor of those chains. In some, nitrate or other oxidants can serve this role. But all of the chains are proton pumps. Thus, oxidative phosphorylation in bacteria is also chemiosmotic in nature. This resemblance to mitochondria is more than chance. The evidence, although mostly circumstantial, suggests that mitochondria, chloroplasts, and perhaps other eukaryotic organelles were originally free-living bacteria. These bacteria and larger proto-eukaryotic cells became mutually symbiotic, so that neither was complete or viable without the other. The animal and plant kingdoms arose from these endosymbiotic events. See also: Bacterial physiology and metabolism
Photosynthesis is the conversion of light energy into chemical energy. Overall photosynthetic bacteria and the chloroplasts of eukaryotic plants capture sunlight or other light and use that energy to generate both ATP and a reductant for use in biosynthesis. The mechanism of photophosphorylation, that is, the use of light energy to drive the phosphorylation of ADP to ATP, resembles that of oxidative phosphorylation. Chloroplasts use a H+-translocating ATPase for phosphorylation. Again, the genes for the chloroplast ATPase are derived from the same evolutionary precursor as the mitochondrial and bacterial genes. Electron transport chains are also components of membranes found within chloroplasts. These chains have components similar to those of mitochondria, but the direction of electron flow is reversed. Water is oxidized to produce oxygen. Nicotinamide adenine dinucleotide phosphate (NADP+) is reduced to NADPH, which is used for CO2 fixation and the synthesis of carbohydrates. The energy to drive these endergonic reactions is derived from light through the absorption of photons by chlorophyll-containing photosystems. The chloroplast electron transport chain comprises a proton pump, generating a pmf that drives the phosphorylation of ADP to ATP by the ATPase. See also: Photosynthesis
Not all of the energy of the proton motive force is coupled productively. It was noted above that enough chemiosmotic energy is generated by respiration to allow for phosphorylation of six or seven ATP. Yet, at best, only three are produced. If not coupled to the performance of work, the excess energy may be lost as heat. In some cases, heat production or thermogenesis through the dissipation of the pmf is regulated. Some mammals, for example, cold-adapted animals, hibernating animals, and infants of hairless species (including humans), have a specialized form of adipose tissue called brown fat. The inner membrane of brown-fat mitochondria has unique proton channels which open or close depending on the need for heat. When open, the channels allow for the return of protons extruded by respiration. This cycling of protons uncouples the pmf from ATP synthesis. The energy from proton cycling becomes heat energy. By controlling the rate of the cycle, animals can regulate body temperature. See also: Adipose tissue; Thermoregulation
Other chemiosmotic systems
Oxidative phosphorylation and photophosphorylation are but specialized examples of chemiosmotic energy coupling. In the broader sense, the pmf is an ion current analogous to an electric current. Electric generators are machines that convert stored energy into a current of electrons. Among the forms of useful energy are chemical energy, such as that derived from fossil fuels, and light energy in the case of solar cells. Electricity is transmitted to motors, which couple electrical energy to the performance of work. Bacterial cells, mitochondria, and chloroplasts have protonic generators and protonic motors. Respiratory and photosynthetic electron transport chains are generators of proton currents pmf's, which then drive the various motors of the cell or organelle. When the H+-translocating ATPase is “plugged in,” the proton current drives phosphorylation. There are other motors present in the cell. Most membranes contain specific transport systems for small molecules, such as ions, sugars, and amino acids. Many of these transport systems are protonic; that is, they use the energy of the pmf to drive the accumulation or extrusion of their substrate. To give one example, in many bacteria the transport system for the sugar lactose consists of a single membrane protein which catalyzes the simultaneous co-transport of both lactose and a proton. Entry of protons through this carrier protein is driven by the pmf. Since a molecule of lactose enters the cell with each proton, entry and accumulation of lactose is thus coupled to the pmf. See also: Biopotentials and ionic currents
Other metabolic events have been found to use the pmf. Flagellar rotation in bacteria is coupled to the pmf. The flagellum is a motor which uses a flow of protons to do mechanical work, just as a paddle wheel uses a flow of water to turn it. Another example of a membrane event which uses the pmf is the uptake of DNA by bacterial cells. Foreign DNA can enter cells by sexual conjugation, by direct uptake (transformation), or by injection of viral DNA by bacteriophage. Similarly, some proteins destined to be secreted by cells require a pmf to be inserted into, or translocated through, the cytoplasmic membrane. Exactly how the pmf functions in the transport of DNA or protein is not clear. In conclusion, chemiosmosis is a universal phenomenon involved in many membrane and transmembrane events. See also: Bacterial genetics; Bacteriophage; Cell membrane