The Inner Mitochondrial Membrane of the microbial eukaryote and the cytoplasmic membrane of the prokaryote are the key sites where energy available from processes such as the oxidation of nutrients or from light is converted into other forms that the cell needs. Most prominent among these other forms is ATP and thus these types of membrane are concerned with oxidative phosphorylation (or photophosphorylation).
I. INTRODUCTION
Energy-transducing membranes share many common components, but most importantly they operate according to the same fundamental chemiosmotic principle. This states that energetically downhill reactions that are catalyzed by the components of these membranes are coupled to the translocation of protons (or more rarely sodium ions) across the membranes. The direction of movement is outward from the matrix of the mitochondria or the cytoplasm of bacteria. The consequence of this translocation is the establishment of a proton electrochemical gradient. This means that the matrix of the mitochondria or cytoplasm of the bacteria tends to become both relatively negatively charged (thus, called the N side) and alkaline relative to the other side of the membranes, the intermembrane space in the mitochondria and the periplasm in gramnegative bacteria (and equivalent zone in gram-positive bacteria and archae), which is thus called the P side (Fig. 33.1). This electrochemical gradient is in most circumstances dominated by the charge term, which means that there is often a substantial membrane potential across the membranes, frequently estimated to be on the order of 150–200mV. In most circumstances, the pH gradient generated by the proton translocation is small, 0.5 unit would be an approximate average value. The membrane potential is added to the pH gradient to give the total gradient, which is usually called the proton- motive force if it is given in millivolts. The conversion factor is such that 0.5 pH unit is approximately equivalent to 30mV. Strictly speaking, the expression of the gradient as an electrochemical potential requires that units of kJ/mol be used; in practice this is rarely done, which sometimes causes confusion. I use the term proton-motive force in this article.
II. MITOCHONDRIAL ENERGETICS
The best-known machinery for generating the protonmotive force is the mitochondrial respiratory chain. The standard mitochondrial respiratory chain is found, at least under some growth conditions, in eukarytoic microbes. The key point is that as a pair of electrons traverses the chain from NADH to oxygen there are three segments (formerly called sites, but this term is inappropriate because it implies equivalence and relates to a very old idea that ATP is made at three sites within the electron-transport chain) where protons can be translocated across the membrane. The first and last of these segments move four protons per two electrons, while the middle segment moves only two (Fig. 33.2) (consideration of the mechanisms of these proton translocations is beyond the scope of this article). Thus, 10 protons are moved per two electrons moving along the chain from NADH to O2. Electrons may enter the chain such that they miss the first proton-translocating segment of the chain; succinate and the intermediates generated during fatty-acid oxidation are the most prominent examples of electron sources for this. In these cases, six protons are translocated per two electrons.
The entry of electrons at the third segment would obviously give a translocation stoichiometry of four. The proton-motive force generated can then be used to drive various uphill reactions. Most prominent is ATP synthesis. This is achieved by protons flowing back across the membranes and through the ATP synthase enzyme, often called FoF1ATP synthase. There is increasing insight into the mechanism of this enzyme; it appears to function akin to a rotary motor in which the flow of protons through the Fo is coupled to rotation and structural changes in the F1 part of the molecule, events that are somehow linked to ATP synthesis. It is not settled how many protons must pass through the ATP synthase to make one ATP molecule; a consensus value adopted here, even though it is not fully confirmed, is three. On the basis of “what goes one way across the membrane must come back the other,” it might therefore be thought that the stoichiometry of ATP production per pair of electrons (called the P/O or P/2e ratio) flowing from NADH to oxygen would be 10/3 (i.e. 3.3) for NADH and 6/3 (i.e. 2) for succinate. However, matters are a little more complicated. The combined process of entry of ADP and Pi (phosphate) into mitochondria and the export of ATP to the cytoplasm involves the movement of one proton into the matrix (Fig. 33.3). Thus for each ATP made, the expected stoichiometry is 10/(3_1)_2.5 for NADH and 6/(3_1)_1.5 for succinate. These values differ from the classic textbook values of 3 and 2, respectively, but they are rapidly becoming accepted. It was generally thought that eukaryotes were only capable of aerobic respiration. However, there is now evidence for a form of mitochondrial anaerobic respiration in which nitrate is reduced to nitrous oxide (more typically a prokaryotic characteristic, see the following) and for a novel type of mitochondrion from the ciliate protist Nyctotherus ovalis that reduces protons to hydrogen. In both these examples, electrons are derived from NADH.
III. BACTERIAL ENERGETICS
Many species of bacteria employ a respiratory chain similar to that found in mitochondria in order to generate a proton-motive force. However, there are many more types of electron donor and acceptor species that can be used by bacteria (eukaryotes are restricted to the oxidative breakdown of reduced carbon compounds), and various forms of anaerobic respiration are widespread. A further general difference between bacteria and microbial eukaryotes is that in the former the protonmotive force can drive a wider range of functions and be generated in more diverse ways than in the latter. Thus, functions alongside ATP synthesis (for which the enzyme is very similar to that found in mitochondria), such as driving of many active transport processes and the motion of the flagella, are important processes that depend on the protonmotive force in many organisms (Fig. 33.4). A common mode of active transport is known as the symport; the classic example of this is the lactoseproton symporter coded for by the lacY gene of the lac operon of E. coli. In this case, a transmembrane protein translocates together a proton down its electrochemical gradient and a lactose molecule up its concentration gradient (Fig. 33.5); the exact mechanism is presently unknown. There are cases known where Na is the translocated ion. A second type of transport system is the antiport (Fig. 33.5). Here the movement via a protein of the proton down its electrochemical gradient is obligatorily linked to the movement of another species, typically an ion, in the opposite direction and up its electrochemical gradient. The third type, uniport, is the case where an ion moves in direct response to the membrane potential and is probably rarer than the other two examples in the prokaryotic world. It is important to appreciate that not all transport processes across the bacterial cytoplasmic membrane are directly driven by the proton-motive force. Some transport reactions are driven directly by ATP. Notable among such systems are ABC (ATP binding cassette) transporters. A more subtle aspect of prokaryotic energetics is that in some species of bacteria the proton-motive force must drive reversed electron transport under some circumstances (see later). A common misconception when multiple functions of the proton-motive force are discussed is that this force can be divided (e.g. 100 mV for ATP synthesis and 50 mV for flagella motion). This notion is incorrect because the proton-motive force across a membrane has a single value at any one time and it is the magnitude of this force that is acting simultaneously on all energy-transducing units, be they ATP synthases, active transporters, or flagella.
IV. PRINCIPLES OF RESPIRATORY ELECTRONTRANSPORT LINKED ATP SYNTHESIS IN BACTERIA
In principle, energy transduction on the cytoplasmic membrane is possible if any downhill reaction is coupled to proton translocation. The most familiar examples are probably those that also occur in mitochondria, for example, electron transfer from NADH to oxygen or from succinate to oxygen. In these cases, the electrons pass over a sizeable redox drop (Table 33.1). In contrast to mitochondria, various species of bacteria can use a wide variety of electron donors and acceptors. The fundamental principle is that the redox drop should be sufficient for the electron transfer to be coupled to the translocation of protons across the cytoplasmic membrane. Table 33.1 shows that such sizeable drops are associated with the aerobic oxidation of hydrogen, sulfide, carbon monoxide, and methanol, to cite just a few electron donors. Anaerobic respiration is also common with many suitable pairings of reductants and oxidants (e.g. Table 33.1). Thus NADH can be oxidized by nitrate, nitrite, nitric oxide, or nitrous oxide. The flow of electrons to these acceptors, each of which (other than nitrate) is generated by the reduction of the preceding ion or molecule, is the process known as denitrification. In E. coli under anaerobic conditions, formate is frequently an electron donor, and nitrate and nitrite are the acceptors, with the latter being reduced to ammonia (Table 33.1) rather than to nitric oxide, as occurs in denitrifying bacteria. A wide variety of electron-transport components, including many different types of cytochrome are involved in catalyzing these reactions. The mechanisms whereby electron transport is linked to the generation of the proton-motive force are frequently complex. However, nitrate respiration (Fig. 33.6) provides an example of one of the simplest mechanisms that corresponds to Mitchell’s original redox loop mechanism. An important point is that the consideration of the energy drop between the donor and acceptor (Table 33.1) is only a guide as to whether proton translocation, and thus ATP synthesis, can occur and, if so, with what stoichiometry. Thus while many bacterial species can form a respiratory chain with considerable similarity to that found in mitochondria, others vary from this pattern. Notable here is Escherichia coli, which always lacks the cytochrome bc1 complex, and which, following some growth conditions, has cytochrome bo as the terminal oxidase, but which under others has cytochrome bd. The consequence is that when the former proton-pumping oxidase is operating only eight protons are translocated per pair of electrons flowing from NADH to oxygen, while with the latter oxidase the stoichiometry would be six. The corresponding stoichiometry for mitochondria is ten. This example illustrates the important point that it is not just the energy drop between a donor and an acceptor that is important, but also the details of the components (or molecular machinery) in between. Another example is methanol to oxygen. Periplasmic oxidation of methanol feeds electrons into the electron-transport chain close to the terminal oxidase, yet energetic considerations alone would indicate that electrons could span more protontranslocating sites, just as they do when succinate is the electron donor (compare the redox potentials for fumarate–succinate and methanol–formaldehyde; Table 33.1). A final example to consider is the case in which both the electron donor and acceptor are in the periplasm and they are connected purely by periplasmic components. In such a case, which applies to methanol (as donor) and nitrous oxide (N2O as acceptor), the electrons do not pass through any protontranslocating complex. Thus, no proton translocation would occur no matter what the redox drop between the two components. It is not necessary for electrons to flow over such a large energy drop as they do when they pass from NADH to oxygen (Table 33.1) in order to generate a proton-motive force. Thus, if the driving force associated with a reaction was very small, it might still be energetically be possible for the passage of two electrons from a donor to an acceptor to cause the translocation of just one proton. If three protons are required for the synthesis of ATP, then the ATP yield stoichiometry would be 0.166 per electron flowing from electron donor to acceptor.
This seemingly bizarre stoichiometry is not only energetically possible but also mechanistically possible because the chemiosmotic principle involves the delocalized proton-motive force that is generated by all the enzymes of the membrane and also consumed by them all. There is no case known that matches this extreme; nevertheless, there may well be organisms yet to be discovered that have such low stoichiometries of ATP synthesis. One example of the lowest known stoichiometries of ATP synthesis per pair of electrons reaching the terminal electron acceptor (oxygen) occurs in Nitrobacter. Table 33.1 shows that the redox drop is small between nitrite and oxygen. This organism also illustrates the versatility and subtlety of the chemiosmotic mode of energy transduction. Nitrobacter species oxidize nitrite to nitrate at the expense of the reduction of oxygen to water in order to sustain growth. The energy available as a pair of electrons flows from nitrite to oxygen is sufficient to translocate two protons (a more detailed consideration of how this is done is outside the scope of this chapter). This means, recalling the current consensus that three protons are needed for the synthesis of one ATP molecule, that the ATP yield stoichiometry would be 0.66/2e. Nitrobacter also illustrates another important facet of energy transduction in the bacterial world. The organism is chemolithotrophic, which means that it grows on nitrite as the source not only of ATP but also of reductant (NADPH), which is required for reducing CO2 into cellular material. Energetic considerations immediately show that nitrite cannot reduce NADP directly. What happens in the cell is that a minority of the electrons originating from nitrite are driven backward up the electrontransfer system to reduce NAD(P) to NAD(P)H. This is achieved by the inward movement of protons reversing the usual direction of electron movement (Fig. 33.7). This reversed electrontransport process is an important phenomenon in a variety of bacteria, especially those growing in the chemolithotrophic mode. Most studies of electron transport-linked ion translocation have been done with species of eubacteria. However, the same fundamental process also occurs in archaebacteria, although with some novel features that reflect some of the extreme growth modes tolerated by these organisms. For example, a key step in methane formation by methanogenic bacteria is electron transfer from hydrogen or other reductant to a small molecule contains a disulfide bond. The latter is reduced to two sulfides and the overall process is coupled to the translocation of protons across the cytoplasmic membrane. The proton-motive force thus set up can be used to drive ATP synthesis. Interestingly the ATP synthase in archaebacteria shows significant molecular differences from its counterpart in eubacteria and mitochondria, but is believed to function according to the same principle.
V. GENERATION OF THE ION ELECTROCHEMICAL GRADIENT OTHER THAN BY ELECTRON TRANSPORT
A. ATP hydrolysis Organisms that are incapable of any form of respiration still require an ion electrochemical gradient across the cytoplasmic membrane for purposes such as nutrient uptake. One way in which this requirement can be met is for some of the ATP synthesized by fermentation to be used for ATP hydrolysis by the FoF1 ATPase. This means that this enzyme works in the reverse of its usual direction and pumps protons out of the cell. Thus there are many organisms that can prosper in the absence of any electron-transport process, either as an option or as an obligatory aspect of their growth physiology. B. Bacteriorhodopsin A specialized form of light-driven generation of proton-motive force, and hence of ATP, occurs in halobacteria; these organisms are archaebacteria. The key protein is bacteriorhodopsin, which is a transmembrane protein with seven _-helices that has a covalently bound retinal. The absorption of light by this pigment initiates a complex photocycle that is linked to the translocation of one proton across the cytoplasmic membrane for each quantum absorbed. Bacteriorhodopsin is one of a family of related molecules. Another, halorhodospin, is structurally very similar and yet catalyzes the inward movement of chloride ions driven by light. C. Methyl transferase One step of energy transduction in methanogenic bacteria involves an electron-transfer process (see earlier). Another important process in methanogens is the transfer of a methyl group from a pterin to a thiol compound. This exergonic (energetically downhill) reaction is coupled to ion, in this case sodium, translocation across the cytoplasmic membrane. D. Decarboxylation linked to ion translocation In the bacterial world, the electrochemical gradients can be generated by diverse processes other than electron transport or ATP hydrolysis. For example, Propionegenium modestum grows on the basis of catalyzing the conversion of succinate to propionate and carbon dioxide. One of the steps in this conversion is decarboxylation of methyl-malonyl coenzyme A (CoA) to propionyl CoA. This reaction is catalyzed by a membrane-bound enzyme that pumps sodium out of the cells, thus setting up a sodium electrochemical gradient (or sodium-motive force). This gradient in turn drives the synthesis of ATP as a consequence of sodium ions reentering the cells through a sodiumtranslocating ATP synthase enzyme. Apart from illustrating that sodium, instead of proton circuits, can be used for energy transduction in association with the bacterial cytoplasmic membrane, this organism also illustrates that the stoichiometry of ATP synthesis can be less than one per CO2 formed. It is believed that each decarboxylation event is associated with the translocation of two sodium ions and the synthesis of ATP with three. Thus non-integral stoichiometry is consistent with the energetics of decarboxylation and ATP synthesis. This is an important paradigm to appreciate; the underpinning growth reaction for an organism does not have to be capable of supporting the synthesis of one or more integral numbers of ATP molecules. E. Metabolite ion-exchange mechanisms Another example of the generation of a proton-motive force is ion exchange across the membrane. For example, in fermenting bacteria there is evidence that under some conditions an end-product of metabolism, lactic acid, leaves the cell together (i.e. in symport) with than one proton; this results in the generation of a proton-motive force (Fig. 33.8). A second example is provided by Oxalobacter formigenes, in which the entry of the bivalent anion oxalate is in exchange for the exit of the monovalent formate ion generated by decarboxylation of the oxalate, leading to the net generation of membrane potential (Fig. 33.8). This seems to be the principal mode of generating membrane potential in this organism.
VI. PHOTOSYNTHETIC ELECTRON TRANSPORT
Prokaryotic photosynthesis involves a cyclic electrontransport process in which a single photosystem captures light energy and uses it to drive electrons around the cycle (Fig. 33.9). The consequence of this cyclic electron flow is the generation of the proton-motive force. There are two types of photosystem found in prokaryotes. One is related to the water-splitting photosystem that is found is plants; typically this bacterial photosystem is found in organisms such as Rhodobacter sphaeroides. The second type of photosystem is closely related to the second photosystem of plants, the one that is concerned with the generation of NADPH. Heliobacter is an example of an organism carrying this type of center. Some microorganisms have both of these photosystems, arranged to operate in series as in plants. In this group are the prokaryotic blue-green algae and the eukaryotic algae. VII. ALKALIPHILES An interesting unresolved problem relates to energy transduction in the alkaliphilic bacteria. The problem is straightforward. These organisms can grow in an environment with a pH as high as 11 or 12. A cytoplasmic pH even as high as 9 means that the pH gradient could be as much as 3 units (equivalent to 180 mV) the wrong way around in the context of the chemiosmotic mechanism. The membrane potential always seems to be larger than 180mV, but the total proton-motive force can be very low (e.g. around 50 mV). For some organisms that use a conventional proton-translocating respiratory chain and ATP synthase, it is not understood how they survive energetically. In other organisms, there is evidence for the role of a sodiummotive force. This would sidestep the problem of the adverse proton concentration gradient.
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