Proton free energy differences in membranes drive ATP formation

TIBS - October 1976

N 222

IXSCUSSION FORM The mechanism of oxidative phosphorylation Harold Morowitz - stored energy lies in the electrochemical potential of HS

R. J. P. Williams - proton free energy differences in membranes drive ATP formation

Harold J. Morowitz is Professor of Molecular Biophysics and Biochembtry at Yale University New Haven, Connecticut, U.S.A. He is the author of Energy Flow in Biology and Entropy for Biologists. His researches have been in the fields of thermodynamic foundations of biology, mycoplasma as minimal cells, and membrane structure. He is currently working in problems of energy transduction in membrane systems.

A dialog had best commence by trying to set forth areas of general accord before proceeding to those items of a debatable nature. In the case of oxidative phosphorylation by mitochondria it is usually agreed that the process may be divided into three stages: (a) the energy-yielding oxidation of NADH or other reduced metabolites at the inner mitochondrial membrane, (b) the transfer and storage of this energy in some potential form in which it must be kept from being degraded to thermal energy, (c) the use of this intermediate energy to drive the endergonic synthesis of adenosine triphosphate from its direct precursors. Most workers also accept the idea that the reactions are reversible and therefore near-to-equilibrium. While a number of theories have been put forward to account for the preceding generalizations, they appear to fall into two broad groups which may be roughly classified as chemical and thermodynamic. In the chemical theories the stored energy is localized in specific molecules while in the thermodynamic theories it is associated with the entire system in a form that may be described by thermodynamic variables. Among the most widely discussed chemical theories are the high energy intermediate which associates energy with specific covalent bonds and the conformational intermediate which distributes the energy over the degrees of freedom of a macro-molecule. A crucial question behind this debate hinges on which major class of theories best accounts for the experimental results. The thermodynamically oriented electrochemical coupling hypothesis set forth by Peter Mitchell [I] postulates that the intermediate storage form occurs as a transmembrane separation of H+ and anions. The chemical potential thus has an enthalpic contribution due to charge separation and an entropic contribution due to separation of chemical entities. An important feature of this mode of storage is that it is an overall property of the mitochondrial membrane system as distinguished from local storage sites near both the oxidation-reduction and the phosphorylation centers. As a consequence the system is subject to analysis by macroscopic thermodynamics in spite of the small size domain of the organelle. Since both Peter Mitchell and R. J. P. Williams agree that there is an asymmetric generation of protons [2], the area of conflict centers on the

R. J.P. Williams is Napier Research Professor of the Royal Society at The Inorganic Chemistry Laboratory at Oxford, U.K. His research interests are in the role of metal ions in biological systems (he is sometimes known as the grandfather of bio-inorganic chemtitry), protein structure in solution and bio-energetics. With C. S. G. Phillips, Williams is the author of Inorganic

Chemistry.

I consider the following points to be outside the divergence of opinion between my ideas and those of Morowitz (Chemiosmosis?). None of these points belong originally to chemiosmosis, but all are incorporated into it, and I believe them to be essentially correct. (1) The initial act of energy reactions, light or oxidation, is the production of a charge gradient. (2) The charge gradient is converted to protons separated from electrons (and OH -?) and it is restriction on their diffusion which keeps them apart. (3) These reaction sites are in the membrane and are the initial energy which can be used for ATP, transport etc. (4) There is a final step in which the ATP is produced through proton flow also in the membrane and the ATPase carries out the chemistry of the reaction. (5) There is no osmotic component in any of these steps. (6) Uncouplers are reagents which equilibrate H+ in the membrane (Mitchell, who adds that they are H + transports). (7) No chemical intermediates are known (or necessary?) Divergence of opinion begins and ends with the discussion of what happens between Step 3 and Step 4. Chemiosmosis . Inserts one extra step: (8) A release of protons and hydroxide ions to different aqueous phases to generate an osmotic pH plus a ‘potential’ gradient across the membrane. The osmotic pH and potential components must then be found (by measurement) and shown to be kinetically required for ATP synthesis. The following experiments show that in fact no or a very small pH gradient is required to be present in many of these systems (I take the systems described by Morowitz in turn). (a) At environmental pH zz8 ATP synthesis occurs in the purple membrane of halo-bacteria with no or a reversed pH gradient

PI. (b) The chlorophyll membrane requires no pH gradient to make ATP [2]. (c) pH Gradients associated with mitochondria are energetically small (all workers). (d) pH Gradients are not generally required across bacterial membranes to link ATP to other energies or energies to trans-

N 223

YBS - October 1976 Harold Morowitz storage mode and the related formation of ATP which Klingenberg calls ‘a major problem hindering elucidation of the mechanism of oxidative phosphorylation’ [3]. The chief methodological difficulty in resolving varying views on oxidative phosphorylation stems from the fact that problems of heterogeneous thermodynamics are less well understood than the normal homogeneous cases. The experimental problems in dealing with amphipathic materials have proven to be quite severe. Finally, it must be realized that essentially cooperative properties of a structure cannot be exhibited by homogeneousphase constituents in isolation. Thus, the need for intact structures for phosphorylation argues for the Mitchell model with a thermodynamic requirement for the separation of phases. A large corpus of experimental material now exists associating ATP synthesis with transmembrane potentials in a variety of biological systems. The last few years have witnessed a growing realization that the energy transduction mechanisms at the prokaryotic cell plasma membrane have great similarity to those at the inner organelle membrane of eukaryotes. As a consequence, the studies on the bacterium Halobacterium halobium by W. Stoeckenius and his co-workers have a strong bearing on our discussion. Under anaerobic culture conditions these cells synthesize purple patches of bacteriorhodopsin-containing membrane which, when radiated by a flux of visible light, generate a transmembrane electrochemical potential by the transport of H +. This stored energy is then used by the cell for the production of ATP at sites on the membrane outside of the photosensitive patches. The fact that the two energy conversion processes (electromagnetic to intermediate stored form and intermediate stored form to ATP) are on the same membrane but spatially separated argues for a system property which in this case can be little other than the electrochemical potential of H + maintained by the low ionic conductivity of the membrane. In a related experiment Racker and Stoeckenius [4] have incorporated the ATPase-containing hydrophobic protein of beef heart mitochondria into vesicles with Halobacter membrane, and have demonstrated a light-driven ATP synthesis. Other bacterial experiments indicate that the same energy intermediate that is used in ATP synthesis is used in transport and in the rotation of flagella. Again, there is a requirement for spatial delocalization of the stored energy. Additional support for the electrochemical coupling hypothesis has come from studies on chloroplasts. The application, in the dark, of a pH gradient across phosphorylating membranes leads to a synthesis of ATP related to the pH change. In this case, studied by Jagendorf and Uribe [5], the thermodynamic nature of the proton-motive force is apparent. The similarity of transduction mechanisms in mitochondria, chloroplasts and bacteria is a unifying feature of contemporary bio-energetics. There appears to be a consensus that ATP synthesis in mitochondria is localized on ATP synthase complexes which are observed in the electron microscope as spherical particles on the interior face of the inner mitochondrial membrane. A major difficulty that biochemists have had with the Mitchell viewpoint is being able to visualize conceptually the molecular hardware used in the energy transduction. At this point we can suggest such a visualization and at the same time try to rationalize Williams’ view of a proton concentration in a localized space with Mitchell’s view of a transmembrane difference of electrochemical potential [6]. For if we assume that the part of the ATP synthase complex that is embedded in the membrane acts as a proton conductor in much the same way that a metallic wire acts as an electron conductor, then the surface of the proteins facing into the innermost mitochondrial space will be a site where the m.acroscopic electrochemical potential postulated by

R. J. P. Williams port [3-51. Some bacteria make ATP at pH above 11.O! (e) pH Buffers do not have their expected effects generally. I take it that the pH osmotic component is not required as a mechanistic intermediate and at best chemiosmosis is a misnomer. I accept that in models man-made pH gradients can make ATP (see Racker [6]) but this has always been my position and will not distinguish the two cases of interest (letters to Mitchell early 1961). With Avron and Witt~(refs 7 and 2) I believe that pH gradients seen in chloroplasts are energy stores not essentially at equilibrium with ATP synthesis, that is they are not kinetically required. Now I turn to the idea that the ‘potential’ of the chemiosmotic proton-motive force drives ATP formation. Firstly it must be made clear what the chemiosmotic potential is for it is used uncritically as a ‘grab-all’ by some (not Mitchell but here I am not sure where Morowitz stands). The potential in chemiosmosis is given by the charge difference, q, between two aqueous phases across the membrane (i.e. the charge is in the aqueous media of a vesicle or in the aqueous solution outside it.) The classical equation is potential, (v) =q/C

where C is the membrane capacitance and the membrane is nothing but an insulating layer of given thickness. I reject any inclusion of membrane-bound charges as specifically outside this chemiosmotic potential for otherwise my ideas can be incorporated into chemiosmosis (e.g. as in micro-chemiosmosis). Charges in membranes can not contribute to any osmotic component. Continuing with chemiosmosis its proton motive force is P= V+6pH but as 6pH is not required (see above) we ask is this Vessential? Now firstly there is great difficulty in measuring V(Pressman [8], Tedeschi [9]) and keeping it separate from the overall potential which includes in-membrane charge assymetry. Note however that if V can only be built via 6pH (chemiosmosis), the scheme of kinetic order must be energy and then 6pH before V could make ATP. This is kinetically testable (e.g. by Witt) and there is no 6pH before charge separation (not V) makes ATP. Again as we do not need any 6pH to drive ATP formation we clearly need not form osmotic V when we can get ATP. (In Morowitz language I consider that there is an over-voltage at his platinum electrode and unlike his electron the H + and e circuits are both in the electrode (membrane)). In which other ways could ATP be formed from Step 3? Chemiosmosis gives P= V+GpH but this can not be the total ‘proton-motive force’ at all. In fact P should be just a way of stating the difference in free energy (in volts) of the H + between two parts of space divided by a line across the ATPase-active site. Now the assumed parallel is with the equation of theoretical diffusion (junction) potentials, as seen for ions in nerve and muscle, which have no membrane but only a notional dividing line, AG z=

AE=f$ln[ion]+Y

However, this parallel does not hold as it is only true for an inert (non-existent) membrane. But the biological membrane is not inert and is known to be asymetric and energised asymetrically. Thus, the true dG (I prefer to use free energies not volt equivalents as I believe the latter unit misleads biochemists to think about electrical energies which are. only part of dG in volts) for protons across the membrane ATPase is AG

6AGo

z=nF+V+GpH

where GAGO/nFis the energy required to transfer a proton from its retained site in the membrane across the ATP forming site

N 224

TIBS - October 1976

Harold Morowitz Mitchell can be converted into the local proton concentration .equired by Williams. The situation is analogous to the surface of a platinum black :lectrode connected to the anode of an external battery being i site of locally high electron activity. If surface enzymes use :his proton activity to protonate ADP and phosphate then the ;ynthase catalyzes the synthesis of H,ATP2- which will subsequently undergo two acid-base dissociations to ATP4-. The process can proceed near-to-equilibrium as the positive free mergy of synthesis is balanced by the negative free energy of Icid-base dissociation. The equilibrium feature means that the mergy of oxidation is not dissipated but is effectively conserved :ven though the storage mode involves bulk phases on both sides of the membrane. Solid state proton conductors are known in non-biological systems. Among the more actively studied cases are normal ice and crystalline monobasic ammonium phosphate. The existence of such structures suggests the possibility of driving coupled acid-base reactions with proton potential in much the same fashion that oxidation-reduction reactions are driven with electron potential. Biological systems appear to have discovered this principle a long time ago. The detailed mechanism of ATP synthesis is certainly not known at this time, but the newly obtained results on bacterial systems as well as the elaboration of very sound thermodynamic foundations make it difficult to avoid the conclusion that intermediate stored energy is indeed in the electrochemical potential ofH+. References 1 Mitchell, P. (1966) Biol. Rev. 41, 445 2 Energy Transport and Energy Conservation (1970) Adriatica Editrice, Bari 3 4 5 6

Ciba Foundation Symposium (1975) Vol. 31, Elsevier, Amsterdam Racker, E. and Stoeckenius, W. (1974) J. Biol. Chem. 249, 662 Jagendorf, A.T. and Urihe, E. (1966) Proc. Nat. Acad. Sci. U.S.A. 55, 170 Morowitz, H. J. (1976) in preparation

R. J. P.

Williams

References 1 Bogomolni, R.A., Baker, R.A., Lazier, R.H. and Stoeckenius, W. (1976) Biochim. Biophys. Actu 440,68-88 (see Fig. 12) 2 Witt, H. (1977) 10th IUB International Congress on Biochemistry, July, 1976, in press, Springer-Verlag, Heidelberg 3 Luria, S.E. (197S)Sci. Am. 233,30-37 4 Brewer, G.J. (1976) Biochemistry 15,1387-1395 5 Ramos, S., Schuldinger, S. and Kaback, H.R. (1976) Proc. N&l. Acad. Sci. USA. 73,1892-1896 6 Racker, E. (1975) Trans. Biochem. Sot. 3,27-45 7 Avron, M. (1975) in Molecular Aspects of Membrane Phenomena (Kaback, H.R; et al., eds.), pp. 297-305, Springer-Verlag, Heidelberg 8 Pressman, B.C. and Field, J. (1976) Biophys. J. (Abstr.) 16,19a 9 Tedeschi, H. (1976) Biophys. J. (Abstr.) 16,18a-19a 10 Junee. W. and Aushinder. W. (1975) in Electron Transfer Chains and Oxi&ive Phosphorylation (Qua&uidllo, E., et al., eds.): pp. 243-250, North-Holland, Amsterdam 11 Lehninger, A.L., Brand, M.D. and Reynafarje, B. in Electron Transfer Chains und Oxidutive Phosphotyhztion (Quagliariello, E., et al., eds.). nn. 329-334, North-Holland, Amsterdam - -- _12 van Dam. K. (1975) in Electron Transfer Chains and Oxidative Phosphorylation (Quagliariello, E., et al., eds:), pp. 335-342, North-Holland, Amsterdam 13 Yaguzhinsky, L.S., Boguskavsky, L.I., Volkov, A.G. and Rakhmaninova, A.B. (1976) Nature 259,494-495 14 Ferguson. S. (1976) to be published

Williams in the absence of osmotic V. V is not due to protons alone and any field across the membrane will still act on the bound protons or in chemiosmosis (but 6pH may or may not contribute). If there is diffusion restriction between the aqueous and membrane phases 6pH is not effective. (Junge [lo] has shown there is a diffusion restriction in chloroplasts.) Obviously in principle every possibility exists between Mitchells’s position (V+ SpH drives ATP formation, V formed from 6pH) and mine (GdGO/nF( + v) drives ATP formation) I consider however that experimental evidence is now against any osmotic H + component as part of the required path of ATP formation. (I agree with Morowitz that even if there are circumstances where osmotic terms are involved in the sequence of reactions the general mechanism will still be much as I have stated it. Note I require intact two phase structures (see Morowitz) but chemiosmosis absolutely demands vesicles.) In this article I have avoided mention of such matters as loops and stoichiometry for here the evidence at present is against any simple view (Lehninger [ 1l] and Van Dam [ 12]), but I must stress that even thermodynamic competence of chemiosmosis has not been proven (Nicholls, Rottenberg) unless the number of protons used is ;3 proving that a considerable local membrane store of protons is required. I have also avoided discussion of ATP synthesis by enzymic mechanisms (pathways) but here I must add that I believe that the conformation mechanisms described by Slater and Boyer are possible (gratuitously I put my own mechanism in that category too but I am inclined to put that of Mitchell as impossible or extremely improbable). Morowitz and I agree fully about proton transport in ice-like channels (proton circuits), see my abstract at the last meeting at Bari, but note that such diffusion is faster than through water itself and could easily short circuit any aqueous phase diffusion. Sometimes it is said to me that really Mitchell (Morowitz) says the same things as I do. This is true as far as all the points in the introduction are concerned but it has nothing to do with chemiosmosis. My view is that the mechanism of ATP formation has no connection with osmosb. Two phases suffice and the third phase is not a requirement, though it could be connected as a store and this has never been in doubt. Model experiments have proved that ATP can be formed without the third phase (refs 6, 13, [Ca-ATPase]). Again my’proposals stress kinetic barriers in membranes which in the absence of lateral diffusion of components would make it a localised model in the strictest sense. Evidence that localised regions of the membrane and not general energisation is all that is required has been often mentioned in the literature but most experimentalists do not try to test for this. When I proposed this model lateral diffusion was not recognised by me but it is unfortunately (for the examination of ideas) the case that rapid lateral diffusion can limit the ability to distinguish delocalised from local membrane reactions but neither of these should be confused with chemiosmosis. I conclude that proton-free energy differences in membranes drive ATP formation but osmotic components are not kinetically required and therefore even where they exist they are outside the main path of synthesis. (My points of difference with Morowitz are seen to be small but my disagreement with the part called chemiosmosis (Mitchell) is fundamental and testable as I have indicated. I do not agree with Morowitz (a) that the systems are necessarily near equilibrium or reversible in general [ 141or (b) that conformational intermediates can be distinguished from charged membrane intermediates in a general way but they are separable from chemiosmosis). (See opposite for references.)

R. J. P.