Proton circuit

The mechanism of ATP synthesis discussed here assumes that protons extruded during electron transport are in the bulk phase surrounding the inner mitochondrial membrane (intermembrane and extramitochondrial spaces). An alternative view is that there are local proton circuits within or close to the respiratory chain and complex V, and that these protons may not be in free equilibrium with the bulk phase (Williams, 1978), although this has not been supported experimentally (for references see Nicholls and Ferguson, 1992). The chemiosmotic mechanism is both elegant and simple and explains all the known facts about ATP synthesis and its dependence on the structural integrity of the mitochondria, although the details may appear complex. This mechanism will now be discussed in more detail. 

Figure 12-8. Principles of the chemiosmotic theory of oxidative phosphorylation. The main proton circuit is created by the coupling of oxidation in the respiratory chain to proton translocation from the inside to the outside of the membrane, driven by the respiratory chain complexes I, III, and IV, each of which acts as a protonpump. Q, ubiquinone C, cytochrome c F Fq, protein subunits which utilize energy from the proton gradient to promote phosphorylation. Uncoupling agents such as dinitrophenol allow leakage of H" across the membrane, thus collapsing the electrochemical proton gradient. Oligomycin specifically blocks conduction of H" through Fq.

The uptake of potassium by microorganisms has been well studied. In the case of E. coli, kinetic investigations on different strains have demonstrated the presence of three or four transport systems. The presence of inducible pathways with widely different Km values for binding K+ allows the cell to accumulate K+ to a constant level under different environments. These transport pathways include those linked to the proton circuit and an example linked to a pump and ATP hydrolysis. Thus the Kdp system is a high affinity pathway with Km = 1 mol dm-3, and involves three proteins in the inner membrane of E. coli, including a K+-stimulated membrane ATPase. The KHA (i.e. K+-H+ antiport) path is driven by proton motive force, while the low affinity system TrKA depends on both ATP and the proton motive force.80,81,82 S. cerevisiae accumulates K+ by K+/H+ exchange.83 Potassium transport may thus be used to control intracellular pH. 

This chapter is concerned with the proton circuit which in the chemiosmotic scheme links the generators and utilizers of proton electrochemical potential (A/if ) [1,2]. Since the topic has been covered extensively in a recent monograph [3], this chapter will attempt to avoid repetition by concentrating on the ionic circuitry found in association with energy conserving organelles, and no attempt will be made to discuss the structures of the black boxes of the membrane. 

The uncouplers which abolish the coupling of respiratory rate to ATP synthesis act as proton translocators, inducing net proton translocation across the membranes. In this way the proton circuit can be short-circuited , allowing the protons translocated by the generator of to cross back across the membrane without passing through the ATP synthase and producing ATP. The majority of the uncouplers are protonatable, lipophilic compounds with an extensive pi-orbital system which allows the electron of the anionic, de-protonated form to be delocalized [11]. This enhances the permeability of the anionic form in the hydrophobic membrane, and allows the proton translocators to permeate in both their neutral (protonated) and anionic (deprotonated) forms. In this way they can catalyze the net transport of protons... 

Fig. 2.2. The chemiosmotic proton circuit, a, during the synthesis of matrix ATP (State 3) b, during the synthesis of extra-mitochondrial ATP (State 3) c, during State 4 respiration d, in the presence of proton translocator. R, respiratory chain A, ATP synthase P, phosphate carrier U, uncoupler. The respiratory chain is simplified to a single proton pump.

As discussed in Chapter 1, an accurate treatment of energy flows in a chemiosmotic system demands the use of non-equilibrium thermodynamics to relate flux to net driving force. However, since the same laws control the flow of energy in complex systems such as mitochondria and simple systems such as electrical or hydraulic circuits, the use of the latter as analogues for the proton circuit has considerable attractions to the non-mathematical bioenergeticist. 

The mitochondrial respiratory chain consists of three proton pumps which act in series with respect to the electron flow and in parallel with respect to the proton circuit (Fig. 2.2a). Two limiting states are frequently referred to for isolated mitochondria - State 4 in which the proton current is limited by the inhibition of proton re-entry through the ATP synthase (due to either actual inhibition of the synthase or to the attainment of equilibrium), and State 3 in which there is ready proton re-entry into the matrix and hence brisk respiration. The State 3 condition can be due to an induced proton leak in the membrane or to the maintenance of AG tp below that required to equilibrate with AfiH+ (by either removing ATP, or following the addition of ADP. 

As with any other energy flow circuit, the proton circuit has inter-related potential, flow and conductance terms. These will now be considered in turn. 

After discussing the generation and quantitation of the potential term of the proton circuit, we shall now turn to the proton current, and examine the factors which control the flux of protons around the circuit. Although it is not possible to determine the proton current under steady-state conditions directly, the parameter may be calculated indirectly from the respiratory rate and the stoicheiometry of proton extrusion by the respiratory chain. It is outside the scope of this chapter to discuss the contentious issue of the proton stoicheiometries of the complexes, but the important feature is that, unless the complexities of variable stoicheiometry are invoked, respiration and proton current vary in parallel. 

When the mitochondria are allowed to produce ATP, for example by adding ADP and Pj to an incubation, the potential and current parameters of the proton circuit both change in the direction which indicates an increase in the proton conductance of the membrane due to proton re-entry through the ATP synthase. Thus, falls and this lowers the thermodynamic back pressure upon the respiratory chain, which therefore respires more rapidly. 

Fig. 2.3 also shows that the transfer of energy from the respiratory chain to the proton circuit can be extremely efficient, in that a slight thermodynamic disequilibrium results in a considerable energy flux. The actual disequilibrium between the respiratory chain and the proton electrochemical potential is even less than appears from the drop in the latter, since the redox span across the respiratory chain proton pumps also contracts [24],... 

Reversed electron transfer and the proton circuit driven by A TP hydrolysis... 

The three proton pumps of the mitochondrial respiratory chain normally function in parallel with respect to the proton circuit. It is however possible to manipulate the conditions such that the proton electrochemical potential generated by two of the pumps can be used to reverse the third proton pump (but not cytochrome oxidase). 

Fig. 2.4. Further variants of the proton circuit, a, reversed electron transfer in Complex I driven by Aft from succinate oxidation, b, reversed electron transfer in Complex I driven by Afin from ATP hydrolysis.

Since the ATP synthase is reversible, it is also possible in the absence of respiration to drive a proton circuit by ATP hydrolysis (Fig. 2.4). The + achievable by this means is identical to that supported by respiration [39] and can be utilized to drive ion transport, as well as to reverse electron flow through Complex I (Fig. 2.4). 

While an ATP-driven proton circuit is an experimental device with mitochondria, anaerobic mitochondria such as Strep, faecalis use the hydrolytic mode of the ATP synthase to maintain a Aju,H+ across their membrane for metabolite transport, the ATP being supplied by anaerobic glycolysis. 

Coupling of the proton circuit to the transport of divalent cations... 

Since mitochondrial Ca transport is discussed in depth in Chapter 9, this section will be restrict to a brief summary of the way in which the proton circuit can be diverted into accumulating and regulating the transport of the cation, and how the permeation of weak acids is linked indirectly to net Ca transport. 

This net proton extrusion results in a net acidification of the external medium and an alkalinization of the matrix (Fig. 2.5), and should be distinguished from the steady-state cycling which occurs during the operation of the proton circuit for ATP synthesis. Clear, alkalinization of the matrix cannot continue indefinitely, and the limitation which is set is essentially thermodynamic - the respiratory chain is incapable of maintaining a proton electrochemical potential in excess of 200-230... 

Fig. 2.5. Interaction of Ca transport with the proton circuit, a, Ca uptake alone discharges the membrane potential b, Ca uptake in exchange for protons extruded by the respiratory chain generates a pH gradient c, Ca uptake together with a weak acid such as acetate does not build up a pH gradient d, Ca cycling in heart mitochondria driven by the proton circuit. R, respiratory chain C, calcium uniport NH, sodium/proton antiport NC, sodium/calcium antiport.

Is the proton circuit in equilibrium with the bulk aqueous phases on either side of the membrane ... 

In the proton circuit discussed so far in this chapter, it has been assumed that the protons, after being translocated across the membrane by the respiratory chain or... 

Fig. 2.6. Hypothetical localized variants on the proton circuit, a, fully delocalized circuit b, proton current flows along surface of membranes (note that circuit is still in equilibrium with the bulk phases) c, one leg of the proton circuit is conducted through a lateral channel insulated from the bulk phase d, both legs of the proton circuit are conducted through lateral channel insulated from the bulk phases R, respiratory chain A, ATP synthase. Note the necessity for both outward and return legs in all models.

Before considering this information in detail, it is worthwhile to summarize briefly the implications of a localized proton circuit. One possibility is that the major part of the proton current flows not through the bulk aqueous phase (Fig. 2.6a) but along the two surfaces of the membrane (Fig. 2.6b). Note that in this model there is no insulating barrier between the surfaces of the membrane and the bulk phases. Therefore, under steady-state conditions, the electrochemical potential of the protons on the surfaces of the membrane must be the same as in the bulk phases, since otherwise there would be a net flow of protons down the supposed gradient from surface to bulk. This model does not therefore represent true localized chemiosmosis, since the bulk-phase potential measured experimentally will accurately reflect the true potential driving ATP synthesis. 

The next stage in developing a model of localized chemiosmosis is to assume that there is a substantial resistance between the local circuit and the bulk phase [43,44]. One could devise hypothetical models in which one (Fig. 2.6c) or both (Fig. 2.6d) of the portions of the proton circuit parallel to the membrane were insulated from the bulk phases. It is important, however, to appreciate that such models still require the presence of a highly insulating phase separating the outward and return limbs of the proton circuit, with the addition of one or two further substantial resistances to protons. 

With these observations in mind we shall now consider the nature of the thermodynamic and kinetic discrepancies which have led a number of groups to consider the possibility of localized proton circuits. 

These discrepancies could be due to experimental problems in the measurement of the potentials, or to the existence of micro-circuits out of equilibrium with the bulk phases. These would possess a significant resistance between the localized proton circuit and the bulk phase. The respiratory chain would see the localized... 

The next few years will decide whether the present anomalies in the quantitation of the proton circuit are merely due to experimental problems in the precise measurement of the parameters, or whether they will demand a wholesale revision in our understanding of the proton electrochemical coupling of respiration to ATP synthesis. In the author s opinion the evidence is not yet adequate for the second alternative, but there is a need for proponents of delocalized chemiosmosis to propose straightforward and unambiguous explanations for these phenomena. 

The above estimates of the minimum pmf in State 4 are important also insofar as they are independent of the present uncertainty of whether protonic coupling is localised [15,19-22,35-38] with direct protonic circuits between respiratory complexes and ATP-synthase, or entirely delocalised between the bulk aqueous compartments on each side of the membrane. Clearly, any reUable measurement of a bulk phase pmf in State 4 that yields values lower than 200 mV is indicative of localised coupling (as may be the case above). 

At present much evidence is quoted in favour of the idea [19-22,25,26,38] that the proton circuitry between respiration and ATP synthesis is confined to the membrane proper or its interphases with the aqueous media transversal localisation). Special high conductance pathways of the protons have been postulated along the membrane, with resistive and capacitative barriers against delocalisation of translocated protons into the bulk media. Typical of this idea is the prediction that the functionally relevant pmf (across a restricted membrane domain) is higher than that between the bulk aqueous phases (cf., above). However, other sets of experiments based on inhibitor titrations [35-37,89,90] suggest lateral localisation of protonic circuits. This implies that a particular respiratory chain complex would be able to drive ATP synthesis only in a limited membrane domain containing one or very few ATP synthase complexes. These two modes of localisation are, of course, not mutually exclusive. Transversal localisation does not necessarily require lateral localisation, but the latter is difficult to envisage unless the former is also true. 

Slater, E.C. (1981) In Chemiosmotic Proton Circuits in Biological Membranes (Skulachev, V.P. and Hinkle, P.C., eds.) pp. 69-104, Addison-Wesley, London and Massachusetts. 

Ernster. L. and Schatz, G. (1981) Mitochondria A historical review. J. Cell Biol. 91, 227s-255s. Skulachev, V.P. (1981) The proton cycle History and problems of the membrane-linked energy transduction, transmission, and buffering. In Chemiosmotic Proton Circuits in Biological Membranes. (Skulachev, V.P. and Hinkle, P.C., eds.) pp. 3-46. Addison-Wesley, Reading. MA. 

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