Biochimica et Biophysica Acta, 505 (1978) 1 - 4 4 @Elsevier/North-Holland Biomedical Press

BBA 86046 THE MULTIFARIOUS COUPLINGS OF ENERGY TRANSDUCTION

R.J.P. WILLIAMS

Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR {U.K.) (Received July 28th, 1977)

Contents I. II.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical bond coupling and ATP formation . . . . . . . . . . . . . . . . . . . . . . . . . . A. Glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The possibility of chemical intermediates in oxidative phosphorylations . . . . . . . . . III. The charged species of coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The energy to be coupled . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Stoicheiometry and energy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Efficiency and stoicheiometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Summary of coupling energy of protons . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Couplings using charges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Electron/electron coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Proton/proton coupling steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Pathways for protons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Electron/proton coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Multiple couplings of charges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Structural features of the coupling devices . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Proton/ATP coupling devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Direct proton coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Indirect proton coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Gradients of cations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XII. Ion stoicheiometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Addendum (July 1978) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 2 4 8 13 15

17 18 18 19 21 21 22 24 25 29 30 34 35 39 40 40 40 41 44 44

I. Introduction This article describes o n e a p p r o a c h t o t h e discussion o f t h e c o u p l i n g o f oxidative a n d p h o t o n energies t o t h e f o r m a t i o n o f c h e m i c a l e n e r g y , e.g. ATP, in b i o l o g y . In o r d e r t o see the p r o b l e m s I have s t a t e d in the first s e c t i o n m y w a y o f l o o k i n g at c h e m i c a l c o u p l i n g in the g l y c o l y t i c p a t h w a y and w h y this t y p e o f coupling, c h e m i c a l b o n d coupling, is so

unlikely to be the path in the Krebs cycle oxidations. The alternative and almost accepted explanation of the coupling is that the coupling is not due to such chemical intermediates. There is general concurrence that in place of an orthodox chemical couple which drives ATP formation there is the movement of an energised proton through sonde energy drop. The evidence for this intermediate form of energy at some step in the coupling is very strong and need not be repeated here. Two difficulties remain: (a) to show that this intermediate is energetically competent, which means that its energy must be carefully analysed; (b) to show that there is a kinetic path which converts the energised proton to a deenergised proton plus ATP (or some other form of chemical energy). Under (a) a proper inspection of energy in the systems being analysed will require much more experimental work. Under (b) there have been a number of ideas as to pathway but none have been proved. We fall short of an understanding of coupling by some long way whilst (a) and (b) remain in so confusing a state. At the end of the article I have given seven survey references which when read together illustrate the degree of accord and discord between experts. There is little need to stress our lack of understanding of coupling. II. Chemical bond coupling and ATP formation

IIA. Glycolysis In order to appreciate the merits of chemical-bond coupling (which still may be found to be a part of oxidative phosphorylation) we can look first at substrate level phosphorylation. The overall energy for the production of ATP from sugars is apparently not oxidation for the reaction C6H1206 -+ 3 C2H402

does not involve a general oxidation or reduction of carbon. However it is an internal oxidation/reduction as is to be seen in the following change. -HC--CH-

I I OH OH

~ H3C-CO2H

Here every second carbon is oxidised and the first carbon is reduced. If we take the oxidation states of all carbons in glucose to be zero, the two carbons of acetic acid are in oxidation states - 3 and +3 respectively. This is then an internal disproportionation, oxidationreduction, reaction. It has a favourable standard state free energy change of say - x kJ. As the convertible form of energy in biological systems is the pyrophosphate link it is necessary to couple this internal redox reaction to the reaction P - O H + H O - P ~ P - O - P + H20 (ADP + Pi ~ ATP + H20) This reaction has an unfavourable standard state free energy change of +y kJ. We need to show how the reactions are coupled. The following scheme of chemical coupling (not quite glycolysis, see Fig. 1) does this. The first reaction is phosphorylation of the sugar -CH--CH-

I OH

I OH

+ Pi - C H - - C H -

r OP

r OH

+ H20

Glucose

(Glucosyl)n

, Glucose-l-P ~ Glucose-6-P Fructose-6-P

(Glucosyl)n_ 1

Fructose-1,6-P 2 Dihydroxyacetone-P ~ Glyceraldeh;de-3-P

//

NADH

1,3-diphosphoglycerate

3-phosphoglycerate Lactate*--' 2-phogphoglycerate

2-enolpyruvate

•

pyruvate

Fig. 1. The glycolytic series of reactions.

This is a phosphorylation o f an alcohol hydroxyl o f a sugar and for this reaction AG is small and we shall put AG as effectively zero. The enzymes used are phosphorylases. The second step is -CH--CH-

J OH

I OH

+ H20 -~- H C H - C H - O H

f I OP OH

This is the splitting o f a sugar to a smaller molecule (by aldolases) but involves the first assymetric addition o f oxygen (OH) and hydrogen (H) to two linked carbon atoms and is therefore an inte~al oxidation-reduction. The third step is the isomerisation of the compound on the right hand side to HOCH-CH2 .

I

I

OP OH The rearrangement requires an immerase. Both the second and third steps have a small AG for their overall reactions. Again we put this at zero. The next reaction is dehydration HOCH-CH~ -~ H O - C = C H 2 + H20

I OH I OP

t~P

which is followed by the rearrangement HO-C=CH2 -~ O=C-CHa OP

OP

Note that an internal redox reaction occurs again in this step. The reactions of enolases are of this kind and again go with a small AG. Now during these reactions, which as written have avoided oxidation and then reduction by NAD ÷ and NADH (which is actually used in glycolysis), two major changes have occurred. Firstly the C/H/O distribution has been changed, although the stoicheiometry is always C2HzO2P, from - C H - C H - to O=C-CH3

I

I

OP OH

I

OP

This reaction has a favourable free energy change for carbon compounds as the disproportionation of a sugar C2H402 to acetic acid CH3CO2H has a favourable standard state change, AG = - x kJ. At the same time the phosphate has been transferred from an alcohol oxygen to an acid anhydride oxygen - C H • OP-* - C O • OP and the P - O bond has become weaker costing roughly +x kJ. Therefore the overall reaction so far has roughly zero free energy change but the phosphate binding has been made weaker, i.e. energised. The reaction O=C-CH3 + ADP ~ O=C-CH3 + ATP

I

OP

I

OH

has a standard state transfer energy of ( - x + y ) kJ where x and y are approximately equal and so it is possible to transfer Pi to ADP making ATP by chemical coupling of redox disproportionation with intermediate P i - O - C bond energisation. The reaction is highly efficient as the chemical pathway gives a single final step which matches ATP formation in free energy. Analysis of this scheme shows that it could be truly reversibly coupled, that it has a fixed stoicheiometry, and that it is highly energy efficient. The explanations are entirely satisfactory. It was then tempting to carry over such a chemical scheme to oxidative and photo phosphorylation but on closer inspection it is not possible to see how this could have been done even in principle without some serious modification and additional coupling.

IIB. The possibility of chemical intermediates in oxidative phosphorylations We must write the two reactions to be coupled (acetic acid as starting material) CH3CO~H + 2 O2 ~ 2 CO2 + 2 H20 ( - z kJ) which has a favourable free energy of - z kJ and ADP + Pi -+ ATP + H~O (+y kJ) Now here z is approXimately twelve times y so that if we are to get efficient use of energy we must make some twelve ATP for each acetic acid, i.e. 3 per 2 electrons or 3 per oxygen atom. Again if we are to use direct chemical coupling as in the glycolysis we must bind Pi to the acetic acid as it goes through oxidation and we must energise successively twelve O-Pi bonds. This is clearly a chemical impossibility for not only can we not make

CH3COCOOH

H++DPNH~

COCOOH [ ,.,~ CH2COOH

+ A malic DPN ~ ' l dehydrogenase HOCHCOOH

H20--~ ~"~ CO2+ 9H+ + 2e [CL CH~COOH [ ~ HOCCOOH [ CH2COOH aconitase

[ CH2COOH__ H 2° " ~

H20

CHCOOH [} CCOOH

fumarase

I

CHCOOH

CH2COOH

CHCOOH 2H++ 2 e ~

HOCHCOOH [ CHCOOH

succlnic dehydrogenase

CH2COOH

I

I isocitric dehydrogenaaeL

CO2+ 2H++ 2e

Fig. 2. The

COCOOH [ CH~ H20 [ CH2COOH

~

oxal. . . . . inic decarboxylase ¢ •

CO~

COCOOH j [ CHCOOH [ CH2COOH

f ~ TPNH + H+

Krebs citric acid cycle of reactions.

the first C - O - P i bond without using an energy equal to that of ATP (the compound must be CHaCO'OPi) but we have to find twelve intermediate states of oxidation between CH3COOH and CO2 + H20. A very simple partial solution has been found to this problem by the evolution of the Krebs cycle which is an oxidative cycle designed to supply a whole series of intermediate, equal changes of oxidation states Fig. 2. The average oxidation states are: oxaloacetic + acetic acid = citric acid or isocitric acid, +1.0, oxalo-succinic acid, +1.33; succinic acid +2 CO2, +1.67; fumaric acid, +2 CO2 = malic acid + 2 CO2, +2.0; oxaloacetic acid + 2 CO2, +2.33. This means that there has been a total change of oxidation state for the six carbons of 6 × 1.33 = 8, i.e. 2 02 are required, which of course is the reaction CH3CO2H + 2 02 ~ 2 CO2 + 2 H20. The complication of fractional oxidation states arose as a six-carbon fragment was utilised to break-down the oxidation of acetic acid into four equal steps each involving one oxygen atom or H2 (2e). Thus the Krebs cycle produces an oxidation route giving four equal steps. Quick chemical inspection shows that of all the four possible intermediates only one or two could be phosphorylated at all easily and therefore only a very few direct phosphate transfer steps could be achieved using this path. Thus the process would be inefficient. We need then a different group as a phosphate carrier. Let us write the group as X, now not a

part of the substrate X + Pi ~ XP

XP -~ X'P + 2e(oxidation) ADP + X'P ~ X' + ATP X' + 2 e ~ X The cycle has to be coupled twelve times per oxidation of acetic acid as only in this way will it generate sufficient free energy for 12 ATP and we must couple X to X' to oxidation of Krebs cycle intermediates by oxygen. We then write (O) + malate ~ oxaloacetate + H20, for example, in the following way: X + Pi -~ XP

(0) + XP ~ X'P ADP + X'P ~ X' + ATP malate + X' -+ X + oxaloacetate Sum (O) + ADP + malate ~ ATP + oxaloacetate + H20 and we see that the chemical part of this scheme is totally satisfactory and we need only uncover suitable groups X to carry out the coupling. However this scheme fails to give the correct energy stoicheiometry of 3 ATP for each oxygen atom used. Thus we have only reduced the problem of getting 12 ATP from acetate to one of getting 3 ATP from each of four steps without finding any chemically plausible transfer steps. Now in principle the path from malate to oxaloacetate could be made fantastically complicated in an organic chemistry sense so as to give three sub-steps for each of the above major steps but an alternative solution has been found. For each of the four Krebs cycle oxidations a new coupling of three (approximately) turnovers of groups X has been achieved within a separate (cytochrome) chain. That is the reactions XP -~ X'P, X'P ~ ATP + X' and X' ~ X occur three times using apparently three different reaction centres for X and maybe in fact three different X groups. Thus the coupling is

-U (ox)X'"r"

"D" X r e d ~

~X'

(ox) ~'"

substrate -~ (I)

(2)

(3)

Much of the complexity of our problems can be recognised now for it is necessary to couple these redox sub-steps in a pattern so that they follow one another and each one is coupled to give ATP, three in all. Coupling is not just making ATP from chemicals it is the coupling of several ATPforming steps and coupling the passage of reducing equivalents to oxygen in an appropriate series. Rather obviously each group X if it exists as a chemical coupler must be a part of an enzyme or a co-enzyme for it carries out a catalysed transfer. The search for such groups X within the cytochrome chain which could covalently bind phosphate and transfer it, has not met with success but this should not blind us to the way in which such groups could be used. Given the nature of the reactions it should be a compound which undergoes 2e-reactions, that can bind Pi and therefore can transfer it. The redox potential of the reaction should be close to that of the coupling oxidations. It could be that in very

primitive systems this was the manner of ATP formation. Possible chemical groups X are hydroquinones and disulphide bridges but at other potentials NAD ÷ and ravin might be used and at yet other potentials histidine (very unlikely in my opinion) could be used. It is extremely unlikely that any common amino-acid side chain of a protein could be used except the thiol and imidazole groups. On the whole amino-acids are of very little use in redox reactions and that is one reason that most redox reactions in biology require cofactors and coenzymes, usually metal ions. Let us suppose that such a set of compounds, X, is found [1]. In principle we now have an energy-efficient, stoicheiometric scheme of considerable chemical complexity. Proof rests in establishing that the pieces exist just as it did for glycolysis and the Krebs cycle. In order to see the chemical coupling of this scheme in more detail we note next the exact way in which the Krebs cycle intermediates have been oxidised. The immediately and startlingly significant feature of the cycle is that acetic acid is made into citric acid before oxidation is started, i.e. we start by building bigger molecules having just broken six carbon sugar to two carbon acetate in glycolysis.

~°2H C - - CH2CO2H O//

~H2CO2H +

CH3COOH~

C(OH)CO2H

L

CH2CO2H

Thus a larger molecule is made and the reason for this, apart from the potential for coupling discussed above, becomes apparent in the next two steps to isocitric acid and then to oxalo-succinic acid. The first step, a rearrangement, makes it possible to abstract H2 in the second step. The subsequent shuffling of C/H/O groups allows this to happen four times in the cycle. It is then never the case that the oxygen molecule acts on any organic molecule directly in the Krebs cycle. The reaction 2 02 + CH3CO2H -~ 2 CO2 + 2 H:O is then done as four hydrogen abstraction steps 4 X H2 = 2 × 02. From here onwards we have only to consider that coupling is linked to H2/O2 reactions, with the H2 at a series of different potentials. This analysis gives a complete rationalisation of the compounds which appear in the Krebs cycle, which now becomes the way to drop acetate through 4 ×H2 dehydrogenation steps. The steps are required to get to 12 ATP. Thus we do not need to follow the fate of substrates in an effort to understand coupling but we must follow the fate of H~. We also know, even for chemical bond coupling, that the H2 is transferred as 2H ÷ + 2e down the cytochrome chain for the electrons are 'seen' passing along the cytochrome chain. While great effort has been expended in following the fate of electrons in the chain of components of mitochondria very little knowledge has been obtained as to the whereabouts of the associated protons. We know that the following protonated materials are in the membrane at various stages but we have no clear knowledge of the path of transfer between the sites, NADH, FlavinH, QH2, (QH'), H20. This is a gap in knowledge which will require great skill to fill. Proof of a chemical coupling scheme then requires three agents X (H2) to couple to ATP formation in the chain. As we are putting the case for chemical coupling we have to maintain that the protons are passed strictly to centre after centre within the chain giving stoichiometry to any coupling reactions of necessity. I take

it as obvious that photophosphorylation could be written in the same way. Initially the light would give bound H2 and from then on the reactions would follow those in oxidative phosphorylation. There is no direct evidence against these schemes but the evidence for them is also absent. The fact that energised H ÷ is a required intermediate is not contrary to the scheme. The views of Griffith [ 1] are not necessarily wrong and stand or fall, like all other approaches, on evidence, i.e. no theoretical judgement can be expressed about them. III. The charged species of coupling I wish now to approach coupling in quite a different way, i.e. to search for charged intermediates or if they are illusive at least to search for their properties. The first change in this stage of the discussion is that we reject all substrates from the analysis, see above, and the species which interest us are positive and negative centers derived by photoionisation or by the reaction H2 ~ 2 H ' + 2e. Thus we can ignore the physical-chemical origin of the charges and concentrate upon their properties, their energy and location in space. Amongst those who accept this approach much ground now appears to be common in the discussion of energy conservation and therefore I shall assume in this article that some stages in the production of energy of mitochondria and chloroplasts are supported by considerable evidence [2-4]. (1) There is then initial production of an electronic charge separation where the charges are not far apart, say 5 - 1 0 A. The energy source could be light or the reaction of hydrogen. The charges are initially associated with metal complexes. (2) There is also diffusion (down-hill energetically) of the charges of opposite sign away from one another in space due to the movement of electrons toward (the movement of positive charge) and away from (movement of negative charge) the initial charge centres via electron-transport centres of different redox potential. We shall analyse this process here as it has a large number of metal centres, giving the system some considerable charge storage, which must be coupled into multi-electron steps in some of the redox reactions, and which must be a controlled path so as to capture energy efficiently. (3) At some point in the membrane the positive charge becomes a proton rather than residing on a metal-ion centre and further movement of this charge, the proton, is through hydrogen-bond networks in the membrane. (I here could include diffusion through the bulk aqueous phases, an additional postulate by Mitchell [3] (see also ref. 5)

membrane

I I

Q

I

f I I

H+

\ ×x

%.,f I

aqueous

H

÷

H30 - P - - O - - P

P-OH ÷ P-OH

Fig. 3. The 'Proton-in-the-membrane' scheme for energy transduction. It is the unit XX with which this article is mainly concerned. The membrane phase is defined in the text.

aqueous I

rnembfane

aqueous 11

2® H

H30 +

I

+ HaO + P - - O - - P

H30+ XX

P-OH + P-OH

Fig. 4. The chemiosmotic scheme for energy transduction which differs from Fig. 3 in the pathway of the proton but faces the same problems of proton coupling to ATP formation at XX. as a possibility though I do not consider it of great significance.) The simplest proton diffusion is associated with groups such as-OH, and - N H within water and acids and bases of the membrane. Proton/proton movement is required to be coupled before ATP formation for more than one proton is required in ATP formation, see Figs. 3 and 4. (4) At some point in space the negative charge may become the hydroxyl ion and it can now diffuse in an opposite sense to the proton by proton transfer in hydrogen bond networks. (Again an aqueous phase could be included when the diffusion path must not mix with the aqueous phases of (3) until work is done). (5) Interaction (binding) of the charge-separated pair, or several pairs, with the ATPcoupling factor. This step needs very careful analysis, see (3). (6) Production of an energised state of some other molecular units than H÷ and OH-. For example a group A could be bound or released at a membrane site, the binding or release requiring a change of the interaction of the coupling device with A on interaction of the same device with H ÷ or OH-. An alternative would be a change in the energy of two or more components in the coupling device, e.g. A + B-* 'AB', on binding H ÷ or OH- to the coupling device. This step is usually drawn (OH-)

H+ high e n e r g y ~

A

(OH-) H + low energy ~ "

x--p. NA

We shall identify A -~ ~ A with any process which raises the energy of a carrier system or chemically transforms a compound which requires energy to form it e.g. ADP + Pi-* ATP + H20, and this does not as yet exclude any high energy chemical or physical 'intermediate'. The interesting feature of biological coupling is not just that it makes ATP, for ATP is merely a biologically interesting energy form, but that it transfers energy from separated charges in membranes to other chemical or physical forms. Other possibilities than ATP using phosphate would be acyl phosphates and pyrophosphate and others not using phosphate would be almost any acid anhydride, or any physically strained system, or any establishment of a new concentration gradient, e.g. of a cation, within the membrane. (In some cases this would be a type of chemical intermediate). (7) Release of ~ A with the formation of say free ATP. (8) There is general agreement that some but not all of these steps can go backwards.

10 Excess ATP can generate reducing equivalents in mitochondria or chloroplasts. However ATP cannot be used to produce oxygen in mitochondria and in general it cannot produce light from chloroplasts. Thus the system is not fully capable of going backwards. (In the above I have deliberately avoided the use of the words 'reversible' and 'irreversible' because these words have often been used in a very confusing manner in the discussion of energy capture. In a thermodynamic sense, and since none of the procesges proceed at equilibrium, all the processes are irreversible. However in the sense that particles H ÷, e, ATP, etc. flows through a cycle of reactions, that flow can be 'reversed' in the schemes of Mitchell (chemiosmosis) or my own, Figs. 3 or 4. The two uses of irreversible, i.e. thermodynamic and 'capable of going backwards' are so confused in ref. 3 as to make a discussion of some points in that paper impossible without clarification). Let us now suppose that the above account describes the basis scheme of coupling and that chemical intermediates if there are any are included by the reference to " A in paragraphs 6 and 7 above. There would appear to be general acceptance that this is in fact a correct picture. We can then turn to the problem of coupling knowing that there is an area of unresolved difficulty: whether or not the energised protons enter into general equilibria with aqueous phases as in chemiosmosis Fig. 3 or whether they are involved in local membrane schemes as in Fig. 4. The difficulty will only be mentioned from time to time in this article (see especially sections VIIIC and IX) since this is not the main purpose of the presentation. The major purpose of this article is therefore to direct attention away from the exchange of views between myself and Mitchell, about whether the protons or hydroxyl ions are in aqueous spaces or not, to the question as to how membrane charges can bring about coupling. We need a mechanism to stand beside that of substrate level coupling. We choose to describe the ATP formation mechanisms in terms of protons only. There are limitations of four kinds to take into account. (a) The energy of the high energy protons, i.e. the energy competence of protons. (b) The several multi-electron steps of reduction e.g. 02 ~ H20, i.e. electron coupling. (c) The mechanisms which can be driven by a Lewis acid such as a proton. (d) The control of protons in space i.e. control of diffusion [5], so that they are capable of multi-proton coupling. The chemistry here is much like that of an electric circuit in which the diffusion and storage of charge is controlled, see Fig. 5, but the circuits are partly electronic and partly electrolytic (ion migration) in biology, Fig. 6. While referring to the proton it must be clear that the hydroxide ion could be of equal importance. Again while making comparison with electronic coupling devices we must note that electrons cannot be used directly to drive biological condensation reactions such as ADP + Pi --> ATP + H20, [5] and that they will be coupled only in redox reactions. The reason is obvious enough in that the redox potentials of the components of this condensation reaction, ADP, P, and ATP, are not accessible so that these chemicals cannot pick up electrons readily. It is then necessary to switch the redox energy to a coupling element which can drive condensations of such reactants. The most obvious reaction switch is to redox reactions of organic molecules rather than redox reactions of metal ions (the normal form of electron energy in chemistry). Organic redox reactions cause gross changes in acid/base dissociation constants of the organic moieties. For example consider hydroquinones OH2 ~ Q + 2 H + + 2 e

(1)

where QH2 is a weak acid but Q has a low proton affinity. Oxidation gives a corre-

11 E~

\ ATP ~

ATP

T [TT

REDox META'S PROTO"A . D AND .J." ON-REDOX ' I GRADIENTS E2

MEMBRANE

I

AQUEOUS

Fig. 5. An elementary electrical (electronic) circuit. Note the features of controlled diffusion (along wires), intermediate storage of separated charge (condenser), source of energy (chemical cell El)trap of transduced energy (chemical cell E~) and control devices (switches). Feed-back loops could be constructed to the controls. All the features are present in the coupling in biological membranes, where the circuits are electronic and electrolytic, as shown on the right (see text). Fig. 6. The parallel circuit diagram to Fig. 5 for coupling in biological membranes. sponding liberation o f nH ÷. The reaction is in marked contrast with a metal ion reaction 2 Fe 2 ÷ g 2 Fe3÷+ 2e

(2)

where there need be no corresponding concentration change in any other components than metal ions. Putting together redox couples o f type 2 leads to electron transport and charge separation but putting 2 and 1 together leads additionally to the production of a general purpose acid, H ÷ and/or a base, OH-. Thus this is one essential coupling feature: coupling o f redox to acid/base changes. The proton can be used in a great many reactions in which the electron cannot take part, for example, it can bind directly to all the components in the equation ADP + Pi ~ ATP + H~O. Thus we take it that the fundamental coupling scheme is: Energy (hu, O2/H 2, etc.)

i Electron charge ) separation at metal centres

ii Proton/hydroxide ) acid/base separation

iii Chemically ) useful energy forms (ATP)

As ii is generally thought to be understood and accepted [ 2 - 4 ] , i and iii require greatest discussion. I stress four points again so that confusion can be avoided in the subsequent analysis: (1) Many intermediates can arise at each state o f i or iii and the first chemistry ofiii for example need n o t be that o f ADP + Pi- We shall examine such possibilities. (2) We do not need to consider any o f the (chemiosmotic) aqueous phases for at best

12 they could provide communication or storage forms and at worst they are not involved. Many other communication paths and minor storage systems are available. We can treat the later stages of the coupling problem as a one-phase problem if we wish, i.e. we need not consider any other components than those in the membrane since that is where coupling occurs. (3) As is is a further requirement of biology that it needs to maintain aqueous concentration gradients, and that these could be coupled, it is an obvious point that the assymetry of separation at any point along the chain, charge-separation ~ acid/base separation ~ chemical separation, within the membrane phase must be linked at some stage to chemical separation across a membrane but this is at a different stage than the high energy intermediate of oxidative phosphorylation. We shall avoid discussion of such coupling steps until the end of the article. (4) A final point is that it is necessary to have a closed system of reaction of all intermediate forms of energy including charge separation and proton/hydroxide separations if possible. If this cannot be achieved then efficiency is lost. Given these points of clarification we can turn to how coupling using simple electron and proton charges only can occur within membrane phases where the fact that it is a membrane we are considering is totally incidental. The membrane is now only an organised local region of space in which diffusion paths of chemicals are restricted. In the past I have usually drawn a diagram to include one aqueous region, see Fig. 3, since the initial chemical source of hydrogen in biology is from aqueous-phase small substrates, but this aqueous phase is not important for the aqueous phase is only required to get reactants and products in and out of the system, not for coupling. Fig. 4 gives the chemiosmotic scheme too for the sake of completeness. (If it should be asked, 'How do we know which phase a proton is in?' then the only answer I know is by combination of measurements of proton concentration with kinetic measurement of proton movements in phases I, II and III. We cannot suppose that phase boundaries in biological systems are seeable structures or can be represented by lines (discontinuities) drawn in space (said to represent membranes) for there is no such clear discontinuity of the macroscopic kind such as we use between ice and water. There will be however an energy barrier to free diffusion between membrane and solution phases and this permits kinetics to distinguish the 'phases'. We draw membrane lines in diagrams such as Figs. 3 and 4 to indicate these kinetic discontinuities but they can hardly represent space discontinuities in a real sense. Such is the nature of a biological membrane. A more thorough analysis of the membranes in question is given in Section IX). We see that the exact coupling step is not described by this analysis and that it could be (a) chemical but the chemistry would be acid/base reactions in or close to the ATPforming site (b) conformational in which the proton did not reach the site of ATP at all (c) physical in that through the operation of 'fields' chemical reactions might be driven. I shall not discuss (c) although it is often written in chemi-osmosis mechanisms since these mechanisms have no membrane bound protons. I do not understand these proposed field-driven devices and I know of no chemically comparable systems. A field can bring about chemistry only if there is binding. I believe (a) or (b) or some combination of them to be plausible and of great interest. We must try to follow the protons in the membrane for I believe they are chemically attached there in some way.

13 j energy source ~

charge separation tl

- ~ conforrnational changes ~

chemical gradients (1)

~"~"~'~ ~ chemical gradients(2) x~ ch¢rnical synthzsis

Fig. 7. The connection of energy to gradients within and outside the membrane. The proton and electron gradients are indicated under charge separation. ATP and other ion gradients are shown separately. For ions such as calcium and magnesium the binding to the membrane will be strong and to

some extent they should be included with the proton and the electron.

The final coupling model must include all the transients and their energies but today we cannot hope to give such a complete picture. Even so the devices in the chloroplast and mitochondrial membranes are so complex that we must consider at least coupling under the headings electron/electron, proton/proton, electron/proton, chemical bond, proton]ATP, proton/ion gradients, ATP]ion gradients to obtain even a first approximation to a true picture, Fig. 7. It is almost certain that any reduction in the number of coupled events beneath these headings will omit some process vital to the overall conservation. In order to approach such a task simplification is inevitable and I shall adopt the approach of describing features of the overall events separately despite the fact that it is extremely likely that all the parts are cooperatively involved in the whole. IV. The energy to be coupled The total possible energy released into the system by oxidation or light has to be in some general form such as AG = A(AG °) + if' + Z A p H

(3)

where we define all energies before coupling to ATP etc. in terms of proton energies and we do not assume equilibration between all phases of Fig. 3 or 4. Thus, AG is the total energy per proton which can be released after energisation and during the relaxation of the system back to the ground state; ZApH represents the part of this energy stored in proton pH gradients across the membrane as recorded by pH electrodes inserted into phases I and II of Fig. 4 (and which arose from energisation); t~' is the sum of all potentials which arise from lack of charge balance across (~k) and internal to the organic phase and which arose from energisation. (This is not the potential of chemiosmosis (if)). A(AG °) is then the sum of all additional excess chemical potential energies in the membrane, per proton, and will include all conformational and chemical potential changes other than those in if' and ZApH. Coupling cannot go through any reaction which does not go through t~' or A(AG °) for that would involve -action at a distance. Thus ZApH, which is often trivially small, is also inappropriate and we shall ignore it in the later discussion of coupling. It could become a storage device and it could work as a connecting wire between energy generation and energy coupling (see above) but it is not important in energy coupling, which must be due to protons in the membrane. Now let us consider if, that part of if' which is the electrical potential from phase I to phase II ignoring bound charges. This potential is very difficult to isolate and even though direct measurement with electrodes suggest that it is small, [22-24] we note that the additional contribution from a Gouy-Chapman double layer potential will always act on

14 charges moving transversely in a membrane. However the charges of the double layer or the charges in phases I and II which contribute to ff can not constitute a coupling device. In order to couple we must have bound species in the membrane. The energy drop of these species, if charged, through the coupling device can depend on if, where such a potential exists, but clearly we can look at the mechanics not the energetics of the coupling mechanism without examining this energy. If necessary we can then add field energies from ff to help to drive the coupling. As pointed out by many authors, the clear and fundamental weakness of chemiosmosis as a hypothesis was, and is, that it stated energy equivalences between different energy modes but provided no connection between the modes except through osmotically developed fields [4]. This statement is made without prejudice as to the presence or absence of the osmotic energy terms, ZApH or if, themselves. This brings us to the obvious position that if the coupling is through protons they are in close contact with the coupling device and they are the protons associated with A(AG °) and i f ' - ft. As all energies are being stated in terms of relaxation of the energisation of protons within the membrane we have included of necessity the conformational changes in the membrane on energisation in A(AG °) + (if' - i f ) . We also necessarily include the energy of any potentials arising from ions other than protons but derived from proton gradients. We must note explicitly that while the energy of A(AG)+~'+ZApH must be competent in coupling, each of four terms A(AG), ZApH, if, or ¢' - if, could contain sufficient energy to drive coupling. This is competence in the sense of 'having quantitatively a sufficient energy' to drive say ATP formation. This energy is a large number ~40 kJ in the steady state and smaller energies are inadequate. The situation is quite different from 'having sufficient energy to produce a cation gradient' for such a gradient can be of any energy magnitude (/XG) only greater than zero, see Section XI. I wish to suppose at this stage of the article that the form of the energised state has been correctly described and that the events which have not been described in coupling are in the membrane itself. At the end of this section those who have read the experiments of Jagendorf, Racker, Skulachev and others on the production of ATP by manmade pH gradients may feel that this work proves the contrary to my statements. It does not. It is obvious in those experiments that: (1) the energy is supplied artificially by manmade gradients of large capacity, (2) the protons must be taken up into the coupling device, (3) that while the arrangement of Fig. 4 would inevitably be consistent with those experiments it is obvious that the scheme of Fig. 3 would also be consistent if there is any connection from aqueous phases to the membrane. Model experiments have shown that a two phase system can be used to drive ATP formation. Experiments by Witt [23] show conclusively that the process of ATP formation does not need the aqueous phase protons shown in Fig. 4. I quote: T h e experiments show that an artificial electric potential difference alone is sufficient to generate phosphorylation. The field is based on polarisation and is n o t accompanied by a pH gradient. The p r o t o n s m a y be taken n o t from the inner a q u e o u s phase b u t from the m e m b r a n e near the inner surface

[23], see Fig. 3. Finally it has always been the author's opinion that chemiosmotic model systems would generate ATP as was stated clearly in 1961 (personal communications between Mitchell, P. and Williams, R.J.P.; see also refs. 5 and 6) but I do not believe that these models represent the way in which membranes work in intact biological systems.

15 There are conditions in which the three energised phases of Fig. 4 could be in an 'equilibrium' steady state. This would mean that the bound charges of the membrane must equilibrate with free charges in the aqueous phases. It is this condition which is assured by the methods of energisation used by Racker, Jagendorf and Skulachev, but these are not the methods of biology, I would maintain. Under these artificial conditions transfer of charge from phase ! to the membrane at side I would result in no free energy change and we need not distinguish ~k + ZApH from ~b' + ZApH. There should be however a term added to both which is represented by A(AG °) to include all other energies which contribute to the total energisation. Chemiosmosis stands or falls on (i) equilibration of the phases and (ii) no such A(AG °) terms. It could be re-written to take into account the extra A(/XG°) energies, but not local effects. Micro-chemiosmosis has no sense. In the localised proton theories there is no equilibrium between three phases and no osmotic terminology can be satisfactory. Since 1970 there has been great volume of evidence to show that there is in fact a heavy local binding of protons to membranes, which is inconsistent with the osmotic view. (See particularly the change in emphasis in the writings of Rumberg, ref. 22.) I have been helped in writing the above section by exchanges of letters and articles with Professors E.C. Slater and H.J. Morowitz. V. Stoicheiometry and energy Given that proton gradients drive ATP formation we might think that we had to write an equation defining the stoicheiometry of nH ÷ required to make one ATP n H ÷ + Pi + ATP +OH~ ATP + H20

(4)

where (OH-) is added as a possible neutralising anion. We do this because we are familiar with coupled chemical changes in test tubes e.g. the following reaction has a stoicheiometry of unity. HC1 + NaOH -~ H~O + NaC1 (or H ÷ + OH- ~ H20) Every simple covalent chemical compound has a stoicheiometry and most biochemists are educated to look for whole number correspondences in compounds AnBm where n and m are small whole numbers, e.g. NH3, H20, CHaCOOH etc. Thus in substrate level phosphorylation the reaction are stoicheiometry, and to make this doubly Clear I refer again to the examination of the reactions in Section II. For instance: CH3CO • OP + ADP ~ ATP + CHaCOOH In such reactions we do not need to look closely at the energetics in order to appreciate the stoicheiometry. Even when we do examine the energetics simple analysis of concentration terms and equilibrium constants allow us to understand the balance position of the reactions

K =K x = ([ATP] / [ADP] [PiI)/([CH3COOP] / [Pi] [CH3COOH]) Ky Now if we convert chemical energy into mechanical or electrical work then these energies are not related to the numbers of atoms in given chemical combination. The

16 disposition in space of the atoms in force fields becomes important, not just the numbers of moles of each chemical. Energy is then a function of position in space and we write equations such as Potential energy of charges - - - ~ ele: (electrical)

P

Potential energy of masses

=~ mxm2 (gravitational) P

Energy changes continuously with position here as there are forces acting on the particles. We must now write our coupling problem in terms o f a machine which is nearer to our problems of coupling proton energies to ATP energy. Chemical change I -~ Electrical + mechanical energy ~ Chemical change II e.g. AG(H2 + ½02 -+ H20) ~ (membrane) ~ AG(ADP + Pi ~ ATP) It is clear that if the membrane is only a transmitter of energy (100%) then we can miss it out and write:

~ ( H 2 + 102) + ~ (ADP + Pi) -"~~ A T P + ~ H 2 0 1 2 2 1 There is however no direct coupling of chemical reactants in this equation and only energy is exchanged. There is then no whole number stoicheiometry in the above sense for all the thermodynamic activities are concentration and some are field dependent. The exact amount of ATP which can be produced per O2 depends on the pressures or activities of all the reactants. A further way in which to see this is to write an electro-chemical cell and to convert chemical energy to electrical energy and back again. The 'stoicheiometry' depends on pressures and concentrations of reactants in the cell compartments ATP-.,~,-P[

+ ADP ,.L

-T

_L. H2 +

-I-

}O2._P..H2 0

There is now no whole number rule as in compounds i.e. as in chemical coupling. Before proceeding we may wish to notice that any real flow of electricity in the system dissipates energy as heat just as any real flow of current in a membrane dissipates heat so that there is a lower conversion (efficiency) than the simple subtraction of AG values suggest. True equilibrium cannot be established in a flow system. This makes the measured stoicheiometry a variable depending upon efficiency. This is not true for strict chemical coupling no matter how energy-efficient it is. This is a major distinction between the theories [5]. Now you may say that these notional schemes are not yet appropriate coupling devices, and this is true, and we must write an equation which shows how the ATP coupling is achieved. However I hope to have shown already for example that the general use of a known phosphate potential to calculate back to a number of protons required in coupling can be quite incorrect. To drive this point home let us take the scheme of Eqn. 5 in the form H ÷ + NADH + Q ~ QH: + NAD ÷ QH~Q+2H

÷

2 H ÷ + ADP + Pi ~ ATP + H~O

17 or generally H~oun0 (NADH) --> NAD + + ADP + Pi ~ ATP If ~ is an absolutely defined set of chemical steps for handling H ÷ then stoicheiometry can arise. If however, H ÷ on being generated produces an energy A(AG °) + ~' across the ATPase site then for the reaction ADP + Pi ~ ATP there is no requirement for a stoicheiometry as the number of protons, n, giving the required energy for ATP synthesis is related to the magnitude of A(AG °) + ~b', which can be varied independently from the H ÷ production step. These points apply to the localised or chemismotic protons. It was concepttLal mistake in chemiosmosis to insist upon schemes of given stoicheiometry. In fact this is just a residue from thinking based upon chemical intermediates. Thus when my views on how ATP is formed are challenged in that they lack stoicheiometry and are therefore less explicit than those of chemiosmosis [3] this challenge is bogus. It is easy to insert any stoichiometry in either scheme but such stoichiometry is just an additional postulate. Moreover any postulate is only likely to hold good under particular conditions. In my writings I have been careful to challenge any written stoicheiometries involving the proton. I shall show that this does not conflict with stocheiometry at a given site for electrons i.e. the 2e/ATP/site. This paragraph must not be taken to say that 2H+/ 2e/ATP/site is right or wrong but it is not an essential feature of my views or of a proper presentation of chemiosmosis. (Note that in addition an initial rate (e.g. a pulse experiment) may not given the same optimum 'stoicheiometry' as a steady state experiment since the pulse must first produce an adequate energy configuration.) There is another large difference between substrate level coupling, where the kinetics are controlled by chemical bonds and enzyme sites, and oxidative or photo-phosphorylation, where it. is impossible to keep the chemical kinetic control of all steps; the system of necessity is one of physical kinetic (diffusion) control. Diffusion control is the great advantage which accrues to systems which have phase boundaries, i.e. interfaces or membranes. Diffusion control needs much investigation.

VI. Efficiency and stoicheiometry A further problem in the discussion of energy conversion is that all the energy from a gradient cannot be used for generating ATP. The following additional energies are required: (i) energy for adjusting the membrane components in cycles (ii) energy for control Off) energy for the export of ATP (mitochondria) (iv) energy for the establishment of other gradients than protons e.g. calcium, magnesium, potassium in mitochondria and chloroplasts. These gradients and that of (iii) are generated so as to establish a link between energisation and metabolism, and they can and do fluctuate with ATP levels. They will always leak to some degree (v) establishment of gradients of other chemicals e.g. sugars, amino-acids, etc., across bacterial membranes, which also leak.

18 VII. Summary of coupling energy of protons It is hoped at this stage that the problem of the energetics of devices which convert proton energy has been directed away from the n to m ratios of chemicai bond exchange and that I have shown that to expect stoicheiometry is wrong but to find it would mean that we must devise strict chemical coupling steps. This is of course part of some schemes of chemiosmosis but it is not a requirement even from them. The only requirement which is actually known (apparently) is for 2 redox equivalents to pass an ATP-forming site for each molecule of ATP formed. Therefore although the overal reaction is:

1

3 Pi + 3 ADP + ~O2 + H2 -+ H20 + 3 ATP we must be very careful not to say that each step must have the stoicheiometry H2X + Pi + ADP -+ ATP + X + 2 H ÷ + 2e The only evidence we have is for the compulsory passage of the two electrons at each of three sites to make ATP as found by examining the partial reactions. Thus each one of the intermediate reactions must be written: HnX + Pi + ATP -~ ATP + X + n H ÷ + 2 e n could be 1, 2, 3 etc. but X -+ HnX cycles via two electrons. Photophosphorylation after the change of photon energy to chemical, bound H, energy has to be distinguished from oxidative phosphorylation in that we have no reason to assume that the energy (hv) can be transferred with high efficiency to ATP formation. Let us suppose the ineffeciciency lies in the initial production of HnX. It follows from the above discussion that in the coupling of HnX to ATP formation the stoicheiometry will probably be with the number of electrons flowing between sites and not at all necessarily with the number of protons n which can now be different from that found in oxidative phosphorylation. Should it prove to be the case, and this seems very probable, that two or more than two protons are involved in ATP formation, then it is inevitable that proton/proton coupling must occur. There is a parallel between coupling within 4 H ÷ + 4 e + O2 -~2 H20 and n H÷(state I) + Pi + ADP ~ ATP + n W(state II) Since the protons act in the membrane to make ATP they must be accumulated in packets of n and this implies coupling of proton movement. Such protons must be 'bound' in the membrane. This concludes the section on energy and we must now go to mechanism, knowing that the energetics have been diffusely described but maybe this is an essential feature of the scheme.

VIII. Couplings using charges The consideration of couplings will cover: electron/electron coupling proton/proton coupling steps pathways for protons

19 electron/proton coupling structural features of the total coupling device proton/ATP coupling My experimental approach to these problems is to examine with as much care as possible the nature of separate proteins involved in coupling. When we understand the properties of proteins we shall have a better chance of understanding coupling of the action of electrons/protons/ions and other chemicals. I stress that an understanding of proteins is far from complete. A summary of the knowledge we have gleaned from NMR studies in recent years is given in reference [7].

VillA. Electron/electron coupling As coupling is now seen to be a very complicated topic it is possibly best to tackle the very simplest type of coupling first. We turn therefore to coupled electron flow which is truly a diffusion restricted flow through known centres e.g. cytochromes. The simplest parallel non-biological device is a chemical cell plus a condenser plus a second chemical cell. The wiring is the diffusion path, very strictly controlled, and the condenser is the coupler as it restricts charge flow until a certain charge is built up when it discharges cooperatively, Fig. 5. The two cells are the source and sink for energy. We now look at such a flow in the electron circuits of mitochondria and chloroplasts to see if the equivalent parts of the circuit can be found. Note that the purpose of the condenser is to build up a sufficient accumulation of electrons to drive the reaction in the sink. The cells of Fig. 5 can be fuel cells [5] and the reactions can be of any kind provided they can be connected to electrons. It is the conductivity and storage of electrons only with which we are concerned, and not the nature of the cells. As we have pointed out above the electron reactions of oxidative phosphorylation and of photophosphorylation can be divided into two groups. There are the one-electron transfer steps of the individual sites of the electron transfer chain: M~ + M2 -~ M1 + M~

(6)

These units constitute :he wiring of the circuit but this is not their only function, see below. There are also the two or more electron steps of the non-metal components of the chain: NADH ~ NAD ÷ + H ÷ + 2e

(7)

4 H ÷ + 4 e + O~ -+ 2 H20 In order to avoid release of organic radicals such as OH" and H" and moieties such as H202 the single electron transfers of step 6 must be made cooperative (coupled) to a high degree in reactions 7. (Note that the biological wiring can act as a charge storage itself, a condenser, in the membrane which is the case for all such hop conductors). This means that there has to be a storage of charge at several single electron centres, cytochromes and ferredoxins. The electrons must now be released cooperatively, that is close to simultaneously, so that a set of single electron transfer centres is not just a requirement of oxidative and photophosphorylation but is common to many complex redox reactions in multi-enzyme systems, prominent amongst which are nitrogen-fixation, the sulphate reactions of cytochrome c3 and the use of oxygen in the P-450 hydroxylation reaction. In these systems reactions, such as N2 -~ NHa and 02 ~ H~O, occur through a store of electrons in ancillary reaction centres connected to the main chemical-reaction site, Table I.

20 TABLE I ELECTRON STORAGE (CONDENSER) PROTEINS Protein

Storage units

Reaction system

Cytochrome oxidase Nitrogenase Cytochromes ca

2Cu, 2Fe (haem) approx. 24Fe (Fe/S) 4Fe (haem)

02 + 2H2 ~ 2H20 N2 + 3H2 ~ 2NH3 Sulphur metabolism

They often involve separate protein subunits in one enzyme. We must ask about the possible mode of electron coupling of these centres for these modes are likely to be very similar to those of the cytochrome chain. It is now necessary to look carefully at the nature of electron transfer proteins as revealed by recent NMR studies [7]. In the cases of the binding of electrons to such proteins as cytochrome c, unit change of charge at the binding site, with the accompanying change of charge of the protein, is relayed to large regions of the protein. The change of redox state of cytochrome c, Fe ~÷ ~ Fe a÷, causes little conformational change in fact in the interior of the protein but the surface properties are changed. The protein has external surface charges, largely of lysines, and no doubt their average extension is changed by the central Fe 2÷ ~ Fe a* switch. The surface change is seen too in the binding of anions to the proteins and in their physical properties such as thermal stability, pH stability, solubility etc. The 'conformational change' is largely an electrostatic, throughspace, effect of charge at the centre of a molecule on the external exposed charges at the periphery. We have also observed the effect of change of charge directly in protein/protein interaction. It is possible to study separately and in a pair, the NMR spectra of cytochrome c and of a ferredoxin. The study of the binding of the two proteins shows that it is dependent upon their oxidation states. Thus reduced cytochrome c binds much less readily to reduced ferredoxin than does oxidised cytochrome c. This is the first element in cooperativity but we must now show that this binding can couple electron-transfer rates. The binding of the two proteins as ferredoxin (reduced)/cytochrome c (oxidised) but not as ferredoxin (reduced)/cytochrome c (reduced) means of course that binding alters the normal redox potential of the cytochrome c. Now consider two redox couples of redox potential E1 (ferredoxin) and E2 (cytochrome c). The rate of electron transfer between them is dependent upon the difference between E1 and E2 especially so if the mechanism of electron transfer is by tunnelling, where for effective tunnelling E1 must equal E2. Suppose that the redox proteins can either be bound together in space or can be held further apart. When they are together the redox potential of couple E1 is dependent upon the redox state of couple E2. Thus we can have the situation that when one of the pair, cytochrome c, discharges an electron, the second immediately binds and discharges its electron and is then released. El~,E2 -~ e sink . Whenever E2 loses an electron to the sink, El immediately transfers an electron to E2 and then to the sink. However if El and E2 are moved a small way apart then when E2 gives an electron to the sink the electron transfer El ~ E2 will not occur. The electron transfer in this chain is compulsory coupled to a state in which El is bound to E2. (This device is of

21 course a conventional switch in electrical circuits). Note that the charging of cytochrome c allows binding and alteration of its potential, and therefore gives discharge. We see that wires and condensors are not so readily disinguishable in hop-type conduction as in metal circuits. In the cytochrome chain, in N2 fixation, in P-450 oxidation, this compulsory coupling of electron transfer to the state of metal atoms further along the chain is also coupled to other events. The regulation can be a requirement for an additional conformational change of the proteins E~ and/or E2 by the binding of additional molecules. These can be substrates (P-450 oxidation), ATP (N2-fixation requires ATP to activate electron flow), or ADP + Pi (ATP formation in the cytochrome chain). In each case it is easily seen that binding which adjusts the E~ or E2 potentials or which prevents the electron transfer proteins from approaching one another will stop reaction. Thus coupling is controllable. Note that here the membrane not the aqueous phases contains all the coupling devices. In N2 fixation only particles are essential.

VIIIB. Proton/proton coupling steps Now coupling has been illustrated for the electron-transfer processes we need to turn to see if proton-transfer steps can be similarly coupled. Here we use the NMR studies of another protein: lysozyme [7]. In lysozyme protons can be bound at sites such as Glu-35, in the active site region or at His-15 on the surface. The histidine is known to be very mobile rather like the lysines of cytochrome c. We find little or no linked protein conformation change associated with protonation of this histidine. When Glu-35 is ionised however there is by contrast a conformational change extending at least over a considerable region of the substrate binding pocket. Other proton binding sites of the protein could therefore be affected to some degree. The parallel with electron/electron coupling is obvious. However if we are to make a parallel with the circuit diagram of Fig. 5, we must also understand the ways in which protons move within a matrix for they can not hop (tunnel) over 10-20 A. Thus proton coupling will demand that proton pathways of a special type are devised, which match the tunnelling devices for electrons.

VIIIC. Pathways for protons I am frequently asked 'What are the pathways which protons follow if they do not move through bulk aqueous phases?' There are so many possibilities here that only new experimental evidence can describe particular motions. Let us divide discussion into a consideration of pure lipid and pure protein systems. One pathway of migration of protons in pure lipid bilayers will be along the surface groups such as -OH, -NH~, (RO)2P(T2, -C(Y2 etc. Such a pathway can be complemented by migration through water closely associated with the bilayers, without any gross leakage of protons to the bulk medium. Experimental observations by Y. Lange, E.K. Ralph and A.G. Redfield [8] are relevant here. They show that in phosphatidylethanolamine vesicles there is fast proton migration over the surface of the vesicle. The interaction of proton charges with vesicle layers requires a complicated analysis and cations such as calcium and magnesium can displace protons from the bilayers while anions such as tetraphenylboride which are strongly bound to bilayers assist cation binding generally [10], see also Fig. 15 ref. 22. The pathway along a surface does not contribute to transverse motion across a lipid membrane but short-circuits any requirement for (bulk) aqueous phases. Lipids may not be able to supply transverse pathways across membranes but the insertion of special channel-forming groups will do this. For example all proteins which cross a bilayer will

22 provide some such path. Proteins show only relatively small regions which are impermeable to protons on a time scale greater than minutes to hours. The qualitative proof of this statement lies in deuterium hydrogen (2H/H) and tritium/hydrogen exchange studies. Precise details of the accessible and inaccessible regions of proteins to protons are becoming more available through NMR studies of 2H/H exchange. It is clear that the centre of many proteins e.g. parvalbumin and cytochrome c, can be virtually impermeable to protons: no H/2H exchange. However this is the case for only a very small number of peptide protons: say less than ten. The vary majority of peptide and other protons exchange relatively quickly and the protons on the very outside of all proteins exchange extremely quickly. Thus a pathway for proton movement is provided by the outside of all proteins. However in the absence of surface water the coherence of this pathway could be lost so that unrestricted diffusion via the surfaces of proteins which cross membranes is limited. The very fact that diffusion is restricted in this way permits defined proton channels to be used. These proton channels can show proton/proton cooperative events (proton/proton coupling) since the conformation of the lining of a channel is dependent upon the number of protons bound in it. The parallel between the effect of electronic and proton charges is complete. (In a somewhat different context this was recognised at about the same time by Mitchell [3] in his use of protomotive force (compare EMF) expressions but he limited analysis to aqueous phases.) Just as electron tunnelling can have an activation. The thermal noise is provided by the rotational/vibrational modes of the carriers e.g. protein, as seen in NMR studies, and can be effective in assisting electron and proton movement. The system for coupling nH ÷ must not allow the entry of Na ÷, K ÷, Mg2+ or Ca 2+. Thus it is a chemically specific space and the most probable device is a region of immobilised water such that only H÷ can travel through it. This local hydration path is different from the channels for Na +, K +, Mg2+ and Ca 2+ which undoubtedly exist as anion based passages associated with some general water movement. In these channels cation competition is established even between H +, Ca 2+ and Na +. Oxidative and photophosphorylation are not inhibited by Na + or other cations. Thus the proton channel can not be just a large well or pore and the simplest device is effectively a solid state system for proton migration in an ice-like matrix [9]. When we come to describe the coupling of this flow of the protons to ATP formation we still have to connect the proton flow to the coupling device. The proton channel will then require some modification. The ideas of proton channels and proton circuits of the kind I have envisaged is not new. In a revealing article Morowitz [4] has shown that many of the great names in electrochemistry have thought about the problem and even foresaw the possible application to biology. For a r~sum~ of these ideas I refer the reader to Morovitz's very interesting article. I have drawn directly on the work of such authors as Bjerrum [9] but was unaware of the contributions of several of the other authors. A known proton path on the surface of an enzyme (i.e. a particle) is shown by the work of Campbell et al. [ 10] on the catalysis of reactions of COz by carbonic anhydrase C.

Villi). Electron/proton coupling This step is well-recognised as mentioned above for it occurs in the chemically coupled reaction of a hydrogen-carrying redox centre such as a quinone: QH2 ~ Q + 2 H ÷ + 2e

23

Q will not pick up electrons without protons and Q can not bind protons without electrons. Readers are reminded of the considerations of stoicheiometry in such a reaction, Section V. Note that there is no required proton/electron coupling in the electron transfer reactions of metal ions, see later, but that using the ideas described for electron/electron and proton/proton coupling it is possible to couple electron and proton movements within proteins. They are coupled in a further way of course as they are both charged. This through-space coupling is discussed by Kemeny [ 111. Now we know that electron transfer proteins are locked in the membrane, we know that the membrane becomes ‘energised’ by initial reaction with light or oxygen and we have postulated consistently that this energisation releases protons in the membrane. Therefore these reactions must change the charge on the electron carriers. It would be sufficient for cooperative coupled events of electrons and protons if the protons in the membrane adjust the association of the electron carriers Er and E2, see above. Thus the proton in the membrane can modulate redox potentials in the energised state (as observed) and can thence control electron transfer rates. This modulation also makes many single electron transfer steps into multi electron-transfer processes. Thus coupling provides a switch and a condenser discharge phenomena, Fig. 5, of both protons and electrons. The reverse coupling is also possible. Again we note that conformational changes of the proteins are involved but they may be very small and largely confined to surfaces. Such changes are very difficult to follow by X-ray methods and would not normally have been classified as conformational changes. Fig. 8 presents an earlier summary of the energisation steps. We see that there are two processes: (1) conformation change of the membrane or protein conglomerate i.e. a change of protein/protein interaction as a whole, Fig. 8(b), see also Fig. 16.

(a)

(b) Fig. 8. The two types of conformational change which act within energy-transducing membranes (a). The gross reorganisation of the membrane on energisation (b). The flux of conformational changes as electrons, protons and ADP + Pi go through reaction steps in the energised state. The change makes coupling possible.

24

(2) fluctuations within individual proteins and for redox steps these may be confined to surfaces of the proteins, Fig. 8(a). In recent publications, e.g. ref. 22, the binding of protons to fixed, charges is represented as the main source of energised protons and this will obviously cause a phase change as in Fig. 8(b). As the fixed charges are local and on the membrane this is a distinctly different effect from chemiosmosis.

VIIIE. Multiple couplings of charges We return first to the discussion of the effects of absorption of oxygen by haem proteins. As we pointed out [12] such absorption causes a change in spin-state of the iron and thence a change in conformation of the iron-haem-protein unit, Fig. 9. Through the work of Hoard [13] and Perutz [14], and their coworkers knowledge of the exact geometry of those changes is available and the initial idea is now proven. It is the spinstate switch which connects the allosteric trigger between O2-binding, diphosphoglycerate binding and proton binding. Putting together the trigger of spin-state change and oxidation-state change there can be cooperativity between all the reactions:

\

protons

l

\

reacton

ts

(ATP)

Moreover we can regulate 02 uptake by having a requirement for uptake such as the prior binding of a substrate which also causes conformation changes in the protein e.g. in P-

N(S)

/- / Fe

(a) Low-spin

Fe(ID

(b) Low-spin

Fe([l])

(c)High-spin

Feal)

N

O \\

.-"

(d) High-spin Fe(W)

N

Fig. 9. One of the local c o n f o r m a t i o n changes in a protein which are now k n o w n to have far-reaching effects. The changes are c o m m o n to haemoglobin and c y t o c h r o m e oxidase.

25 450 cytochromes where 02 is not bound until after substrate is bound. This is conventional compulsory order kinetics. Likewise there can be control of oxygen reduction by electrons in the electron transport chain by substrates (for energy) such as ATP, ADP and Pi which adjust the geometry at the haem iron much as other or even the same phosphates adjust the conformation of haemoglobin (diphosphoglycerate site). We see that coupling of electron and binding reactions can be made to be controlled and cooperative, see also Section VillA. If we may generalise from the above we can link electron/electron, electron/proton and proton/proton coupling to any other event. The link is made above through protein/ protein changes but elsewhere we have shown [15] that this is but a microscopic reflection of a more general macroscopic effect - a phase change. It is then permissible to extend the concept of coupling to the membrane which contains the materials of oxidative phosphorylation and here the lipids are involved as much as the proteins. Moreover, as is obvious enough, the factors bringing about conformation changes are not just electrons and protons but must include all groups such as metal ions and small diffusable organic molecules. Although the exact details of the conformation changes need never be known, we still do not understand them in hemoglobin or lysozyme, the general ideas are clear. There is an inevitable connection between coupling and conformational changes but we have shown more specifically that there is not a required stoicheiometric relationship, Section V. A further feature of biological energy-transducing membranes is the high concentration of quinones which Klingenberg has referred to as a buffering pool of redox equivalents. The quinones have long aliphatic hydrocarbon chains attached to them presumably to prevent them from acting as general H-transport systems which would break down diffusion control. This pool has three oxidation states, Q, QH" and QH2. While QH2 and Q are neutral QH" can form the cation QH2+ when it can act as a link between charge and redox state in the membrane. I have been assisted in work related to this section by the writings of Professor Chance and Dr. DeVault with whom I have exchanged views. IX. Structural features of the coupling devices If the above description of the coupling device is correct then certain structural features are required to control diffusion of ions and protons. It then pays us to examine mitochondria and chloroplast membranes in order to see what structural restrictions are present remembering that only the interior of biological lipid films and proteins prevent proton diffusion. The inner mitochondrial membrane has a roughly fixed lipid : protein mass ratio of 1 : 4. This is probably a fair guide to the volume ratios of the components too. The figure is largely independent of the source of the mitochondria. The membrane can be in no way like the conventional Davson-Danielli model. It is then the proteins which form the membrane in large part. Given that they are frequently large proteins it follows that it is quite inevitable that many of them, especially those of high molecular weight, should span the membrane. After all a lipid membrane is known to be only 50 A across and all proteins of molecular weight approx. 50 000 must have at least one dimension close to this width. As such proteins cannot help but span the membrane (which they form!) we must not jump to conclusions about the functional significance of this spanning. Some preferred orientation of the protein has to be proved and this orientation must be present

26 in the energised state if it is to be useful in energy coupling. As many proteins are much larger than 5 0 0 0 0 daltons some parts o f the membrane will be approx. 200 A across. (This number is derived from approx. 100 A for the ATPase + 50 A for the membrane + 50 A for proteins on the opposite side o f the membrane). Taking it that four/fifths o f the volume o f the membrane is due to protein these proteins must control the ion diffusion in which we are interested although no doubt they are assisted b y the hydrophobic lipids. We have little indication that proteins can rotate in membranes but lateral movements are well known. The next dimension of interest is the overall length of the mitochondria. Curiously size seems to be rather unimportant. There are mitochondria which are very small but there are also giant mitochondria, Recent work summarised by Garland and his coworkers [ 16] indicates that in some cells there may be but one mitochondrion per cell which forms a huge weaving reticulum. There can be no possibility in such systems o f rapid ion equilibration along the whole length of the mitochondrial membrane. Thus while we cannot prove

\

! I

I I

Cd) I

(f)

~)

Fig. 10. Illustrative packing in a two dimensional grid of units where the filled circles are in a minority and the open circles (corners of s q u a r e s ) a r e in a majority. The lattice composition is A B n . (a) n = 4 other n = 2. The different packings can have packing errors (e), (f), (g). In a biological membrane the dark circles could be ATPase and the open circles catalytic units.

27 that there is not equilibrium throughout the entire content of the smallest mitochondria (chemiosmotic theory) we can state with great confidence that this is very unlikely as an essential general feature of the design of mitochondria. Chemiosmotic systems cannot be general. In the giant mitochondfia there have to be localised circuits of energisation and energy conservation. It is then only the precise meaning oflocalisation which is in question. The next structural features can be deduced from the analytical composition of the proteins of the membrane. Roughly 10% of the protein of the ox heart inner mitochondfial membrane is ATPase. The distribution would appear to be uniform over the whole membrane surface as seen by the ATPase knobs in electron microscopy. Again although there are mutants in which the system is deficient in one or several components of the ATPase, and the four oxidation-enzyme containing particles, I, II, III, IV, if one examines normal mitochondria some of these particles are present in close to stoichiometric amounts with the ATPase. There is every reason to suppose that all the 5 units are distributed evenly over the whole surface of the membrane. Local circuits between ATPase and oxido-reductive particles are then exceedingly probable and almost certain in large mitochondria since some 50% of the protein which is 80% of the membrane is in these units. On the basis of this evidence I am tempted to look at possible packing modes of units ABn where A is the ATPase and B the particles I, II, III and IV, Fig. 10. I shall choose below to consider the packing of AB3 and AB2 units and I shall use inorganic chemical systems for this purpose. The analogy between inorganic and biological phases has been described in detail in a previous review [15]. (Reference to such inorganic systems may appear to be curious to some biochemists but I know of no other way in which to approach the packing problems posed by the mitochondrial and other membranes.) We also note that the ATPase is situated on one face of the membrane as it must be if ATP is to be produced inside the mitochondria. The disposition of all other proteins would seem to be uncertain to me. The thylakoid presents a very similar problem. The agreed analytical data indicate a high protein content of the membrane (>50%). Some of the proteins are very large and must have a thickness approaching 100 A (see above). The thylakoid membranes are stacked and the distance between the centres of the membranes is about 200,8, (in vivo, not in the swollen isolated state). There is then a question as to the nature of any internal 'aqueous' space. Does it exist in any real sense or is there just an internally hydrated membrane-protein surface and no aqueous phase? Putting this question aside, since I can find no experimental proof one way or the other, we can continue to look at the thylakoid in vivo. As with giant mitochondria the weight of the evidence suggests that the separated thylakoids found in vitro which are approximately lO 000 A long do not exist in vivo, but that the thylakoid membranes form a very long weaving reticulum. Perhaps there is only one 'thylakoid' in a chloroplast. Once again it is impossible for an osmotic equilibrium to be set up rapidly in such a space even given that there is a free aqueous space in which to set it up, see above. Buffering capacity must be enormous. We turn next to the composition of the thylakoid membrane. Again we find that the ATPase proteins are fairly evently distributed but they are now separated by a larger distance due to the large size of the photon capture units. The membrane is tightly packed with these pigment-carrying molecules in proteins and each set of about 500 to I000 chlorophyll molecules is associated with the electron transfer chain leading to the reduction system and oxygen production. A striking piece of information is given by Olasko and Moudrianakis [17] and Berzborn (privately communicated); there is one

28

(i)

(ii)

(

(iv)

(iU1

(v) Fig. 11. The packing of AB2 and AB3 units in layer inorganic compounds. Does one of these resemble the mitochondrial membrane?

coupling ATPase unit (as measured by the analytical amount ofCF~)per 500-1000 mol of chlorophyll. The extremely high concentration of the ATPase, approximately one per active centre, and the extreme length and convolution of the thylakoid membrane is consistent with a localised proton circuit only, making the thylakoid resemble the large mitochondria. The precise sidedness of the membrane is not known but the ATPase knobs are now on the outer surface and ATP is produced outside the particle. Now we can attempt a packing model for this system as for the mitochondria. There are many lattices of the type ABs amongst inorganic compounds, Fig. I 1, from those of closely associated molecular units (e.g. BFs) to those of continuous lattices of the type A1Fs. Each F(B) in the first case has one special B(A) while in the second case each F(B) sees equally six AI(A). Little change is made to this picture if B can exchange site rapidly. We place an ATPase on each A site and a photo-capture or oxidative chain on each B site. A localised model then says that protons from B go to the nearest A or if there are several, one of the nearest A groups. This lattice model appears eminently reasonable for mitochondria and thylakoids though in the latter there would be a mosaic

29 structure of the three components, active centre, electron transport chain, ATPase. Of course it is foolish to over-stress the regularity of packing as in these models, see Fig. 10, but they serve to show the extreme likelihood that local diffusion paths will overwhelm general chemiosmotic phenomena in a system where the convoluted form of both the mitochondrial inner membrane (note its tightly formed invaginations) and the very close stacking of the thylakoid membranes (perhaps no internal aqueous phase and little free diffusion) force any proton produced by oxidation to be in the immediate very close proximity of an ATPase. In bacterial membranes we shall suppose that the ATPase and the oxidative or photo-producing proton units are in close association in patches. This is not essential but it will prevent losses, see Addendum. The surfaces of membranes are highly likely to be charged. We are particularly interested in the changes of positive charge representing the bound protons and metal ions. These charges will be held in large part by the so-called fixed charges, negative, of the membrane proteins and lipids. The negative charges are not randomly distributed and we can not apply a simple Gouy-Chapman theoretical approach. Thus we know that cytochrome c provides a small positive patch (one per ATPase) bound to cytochrome oxidase and we know that some negatively charged phospholipids are associated with the membrane and surely they will occur in hydrophobic patches. The ferredoxins are usually negatively charged proteins in aqueous solution. These local charge densities build up local fields and they act as local traps for the energised ions e.g. the protons in the membrane. At present we can not describe the synthesised mosaics of charge in real membranes but estimates of the bound local against the free (aqueous) membrane potential, if, have been made [22] and the bound charges dominate by at least 10 to 1. In the halobacteria the membranes are very strongly negative due to the presence of a special phospholipid of high pK a, phosphatidyl glycerophosphate. These bacterial normally live in high salt which provides cations e.g. Na ÷ which may bind these anions at these salt concentrations. The largely different affinity of protons. ~>107, for such phosphate groups will ensure that any protons generated by the energised membrane will be trapped initially in the membrane in local patches of this lipid. Not unexpectedly halo bacteria can make ATP using protons without a change in ApH. I consider that virtually no change in osmotic ff will be found when it is measured properly and assuming that the above account of proton/Na + exchange is correct. Model experiments based on chemiosmosis will obviously work here but obscure the basic in vivo mechanism. I wish to thank Professor Garland (Dundee), Dr. R.J. Berzborn (Bochum)and Professor R. Whatley (Oxford) for help with this section. X.

Proton/ATPcouplingdevices

At this stage let us assume that we know the modes of input of H ÷ and its energy and the output of ATP and its energy. We require a direct or indirect connection between the part of the machine which binds protons and the part that binds ATP. Therefore we need to look at what we know about proton binding and what we know about the binding of the four units involved in the reaction: ADP + Pi ~ ATP + H20

(8)

We shall assume that there are two extreme possible forms of coupling. In the first the proton binding sites and the sites of binding of the four components of reaction 8 are immediately adjacent. The second possibility is that the two re~ions of space are remote

30 and that there must be a connecting bridge between them. There will be possibilities involving all sorts of complex connecting links but analysis of the two cases, adjacent coupling and remote coupling, will reveal all the essential features of coupling. Mechanical analogies might be helpful. If I wish to lift a weight I can supply the potential energy directly by lifting the weight in my hands or I can use a see-saw lever in which the energy is applied at a distance but there is a connecting lever..This distinguishes adjacent action from remote action. A more complex situation is described by the consideration of the movements of the hands of a clock. I can turn the clock hands directly by appling a torque to the hands themselves or I can apply remote power by storing energy first in the winding of the spring of the clock. In this example the energy drive is restricted to one direction by a ratchet-device. It is not certain whether the energy driven reactions of biochemistry are controlled by ratchets or not. While it appears that mitochondria have a reversible relationship between electron transfer and ATP, the reactions cannot be driven to release oxygen. In the bacterial membrane reversibility is in greater doubt, and in the chloroplast membrane there is little evidence that ATP can be used to generate light except under artificial circumstances. All forms of energy can be supplied directly from a source to a sink or through very complicated coupling circuit~ such as those in a mechanical clock but the direct coupling allows less opportunity for smooth continuous operation (there is no intermediate buffering storage) and provides fewer modes of control, see below.

XA. Direct proton coupling An inorganic chemist can illustrate direct coupling very easly. Consider a redox reaction which produces protons in a small volume. The hydrogen ion concentration rises. The reaction could be: 02 + S + H20 -> H2S04 (i.e. 2 H ÷ + SOl-) Let us put into the water a small amount of NaaPO4, If the hydrogen ion concentration rises enough its reactions are as follows, as oxygen is used to oxidise sulphur: PO4a- ~ HPO]- -+ H2POg ~ H 3 P 0 4 and then since the system looses water to form H2S04 the following reaction is driven HaPO4 + H3PO4 --~ H4P207 + H20

Thus we have oxidation making pyrophosphate 3//2 02 + S ~

4H3PO4

2H+ + H20 + SO~-J~..2H4%O 7 This was the initial model comparison system for oxidative phosphorylation described by Williams [5], to show that increasing acidity could make pyrophosphate. It is very closely related to Mitchell's recent coupling scheme [3]. In order to make a close parallel with oxidative phosphorylation we need to separate oxidation/reduction paths and the acid paths which we can do by controlling diffusion i.e. all the reactants are not allowed to mix freely in contrast with the above system, which is a test tube reaction, see ref. 5. We now write: I

3/2 02 + S - . ~ f

j

-.i---- S 0 2 - ~ . ~ 4

i

H20(I-~/~,-H2P20722 H+ ~ I t

I I

2 HpO2- .I-~ 4 i

~ useful energy

31 where the broken lines are diffusion control barriers which could be membranes, allowing only certain species to pass. Various fuel cells are of this design, see refs. 3 and 5.' Now this scheme makes sure that the oxidation/reduction takes place away from the condensation by allowing diffusion of protons and water only through barriers. We now wish to arrange that excess water does not reach either the generated protons or the pyrophosphate before they are transferred away from the site of formation. (Pyrophosphate is kinetically stable and so does not form or decompose in water to give phosphate in the absence of a catalyst and this is why pyrophosphate is the chosen form of biological energy storage and why a normal acid anhydride such as (CH3CO)20 could not be so used. Free diffusion of pyrophosphate, ATP, is then acceptable for, as it will only react at catalyst sites it is sufficient to immobilise the catalysts). The proton transfer could lead to a problem in that unless its diffusion is very carefully controlled it cannot be stopped from reaction at a vast number of sites in biology. Thus the biological coupling device has to be constructed carefully giving virtually separate pathways for reactants. We know that this is done in man-made electric circuits (see above) or in pipes used to carry water at high potential energy. It is also quite possible to handle electrons in this way in biology (see the cytochrome chain above) and indeed to handle ions, e.g. the pore-forming peptides such as alamethicin and gramicidin, but we do not yet know how to do this specifically for the proton although ice-like pipes have been suggested above. Some comments on the proton pathway are given in Section VIIIC. What is suggested then is that the proton could drive the Pi + ADP ~ ATP reaction in biology once it diffuses down the desired pipe, path, into the ATPase region in one of two forms: (a) the proton is not hydrated until it passes through the exact site or (b) the proton is hydrated and then goes through the exact site. In case (a) we could postulate the scheme of steps (see Figs. 3 and 4): (1) ADP a- + P~- bound at site X, access of H20 limited on binding (2) nH÷ generated by oxidation at site Y (3) Diffusion of nil* from Y to X only and relaxation of site Y to receive the next reducing equivalents (4) Change of geometry enforced by change of charge at X (5) ADP3-+ P ~ - ~ A T P 4 - + H20 reaction occurs and is favourable under the new conditions in the site X, Fig. 12 (6) Release of H20 followed by or preceded by release of ATP from X (7) Relaxation of the site X to the initial condition. (We shall discuss the implications of any conformational change later.) As a variety of points arise in the description of this series of reactions which are common to other coupling devices I shall now discuss some of these. (1) The site-binding of ADP 3- + P~- at site X must be common to all coupling devices. It can be assumed that binding of such a highly negative pair of substrate molecules will demand a highly positive binding site region. The usually binding sites of phosphates in enzymes of the dehydrogenase type are arginines, lysines and histidines but the best example is probably the diphosphoglycerate site in haemoglobin which has as many as 6 such basic groups. More or less obviously it is impossible to conceive that binding of the ADP3-+ P~- would not change the conformation of such a site. This follows from the dependence of protein conformation upon charge. It does in haemoglobin and modulates coupling. Groups such as lysine (especially), arginine, and histidine on an exposed surface of a protein are highly mobile and change their local conformational states readily. Changing charge at the centre of a protein even by one unit as in cytochrome c, here a

32

ATP ,/

ADP.P i

H20

A D P * Pi ,/ ,_. . . . . . . . . . . . . . . . . . .

(D

ATP

H20 ~. . . . . . . . . . . . . . . . . . . . . .

®

ATP H20

I ADP.Pi

®

Fig. 12. The stabilisation of ATP relative of ATP relative to ADP + Pi is shown. @ aqueous solutions. The interaction can be (~) a lowering of energy of ADP + P i but this would make for weak binding or (~) a strengthening of ATP + H20 (increased binding) in the energised state.

change in iron from Fe 3÷ to Fe 2÷, has a surface effect on the cytochrome structure which has been closely followed by NMR. Change of haemoglobin on binding phosphates causing a running conformational change which alters the haem-pocket some 2 0 - 3 0 A away. Kinases change their structures on binding substrates. We shall assume that ADP3-+ P~- binding adjusts site X considerable and that it is very probable that the adjustment runs through a large region of the coupling protein. We shall not be in a position to define these changes for many years to come, if ever, but we can be sure that some conformational change does occur. (A note on conformation changes is provided as Appendix A to this article.) (2) The generation of nH ÷. Again the points are general to all coupling schemes. The maximum redox energy associated with the nH÷ is the energy of the redox reaction i.e. the potential drop across a coupling site. It can be less through losses but it can not be more, see section IV. Thus the energy, which we shall take to be that of a two electron transfer across a drop of maximally 0.5 V, is about 95 kJ. The number of protons, n, generated directly by the redox reaction could be anything from 1 to a small number less than say 6; i.e. we write: RHp

..~

mRHv-m-2 +(m + 2) H÷ + 2 e

There can be other protons released due to changes in conformation due to the redox change, for we remember that the number of protons released in a redox reaction is a function of the pKa values of RH v and RH~n_-m_2 and the pKa values of the other molecules at the site which undergo conformational changes. Thus we have a problem with stoicheiometry in that there is no way of knowing the number of protons released. Moreover the energy of the redox reaction is also pH-dependent so that the value of 0.5 V × 2 may vary with pH. These problems require separate discussion, see Appendix B. Note again that as there are several different redox particles in mitochondria there is not a compulsory relationship between protons released and ATP formed which will apply to each of these steps or to photosynthesis. Thus as the coupling device has become an energy coupling device and not necessarily a chemical coupling device as in glycolysis there is no compulsory stoicheiometry link between numbers of protons, number of electrons and moles of ATP produced. The system is open to leakage (slippage) and this can be seen immediately as of practical importance for it can be regulated by uncouplers. Uncouplers are nucleophiles which prevent the proton from acting as it should and they can be devices for introducing new diffusion paths [3] or for blocking proton action at the ATPase (see ref. 18). (3) The non-hydrated protons diffuse through a channel toward X. Now there must be only certain restricted diffusion paths for leaks must be avoided and the protons must go

33 through coupling devices. Moreover these paths must be closed if there is no ADP + Pi (i.e. there must be compulsory coupling). Now the very facts that the X site is highly positive and that ADP + Pi binding causes a conformational change could gate proton release so that the channel is closed unless ADP + Pi are present. This is again a general point independent of which coupling path is used. The path could be a chain of acid/base groups e.g. histidines, see carbonic anhydrase C [I0], or of ice. (4) The redox carrier is reoxidised and will not absorb protons which could be those that were involved in its reaction but could arise from the surrounding media generally or assymetrically. However if it can only accept protons along certain channels which are closed in the absence of ADP + Pi then it can not be further oxidised or reduced unless (ADP + Pi ~ ATP + H20) components are present. This is compulsory coupling. (5) This statement follows from the discussion under (2). Charge changes in proteins cause conformation changes. Such a change could have a special significance here in that the conformation change could force the reaction H ÷ + ADP 3- + P?- ~ ATP4- + H20 through volume changes in the active site on binding H ÷ at that site, Fig. 12. (6) Here there are various possibilities which have been outlined in a previous paper from which we take Fig. 12. Fig. 12(a) shows that in the absence of appropriately supplied energy ATP + H20 is less stable than ADP + Pi at all pH values from +1 to +10 by approx. 40 kJ (no exact number need be given). However the very act of binding the components can alter the chemistry at the site X such as H20 + ATP is as stable as ADP + Pi. There are two ways in which this could happen: (i) H20 + ATP binding is greater than ADP+ Pi binding by approximately 40 kJ [19,20] or (ii)H20 + ATP +nil* binding is greater than ADP + Pi + nil÷ binding by approximately 40 kJ. In case (i) ATP would form at the site without energy, but it could not be released. (7) The release of products must occur in such a way that the reverse reaction can not occur. Again there are many possibilities but note that there is no way back to ADP + Pi unless protons are released back along the channel X ~ Y. But this is impossible under initial conditions since tile channel will be ready to take up new protons already formed by the next turn over of the redox reaction at X. This must be stressed for reformation of ADP + Pi from ATP is known to be coupled to reverse electron flow i.e. reverse proton flow as an intermediate. Thus we have a device which under energised conditions can not run backwards. Perhaps the comparison with the drive on a mechanical clock is worthwhile. (8) On release of ATP + H20 the site Y relaxes to its geometry at the outset and is ready to receive the next ADP + Pi. If the hydrated proton (energised) goes to the ATP site then only in small ways does this differ from (a). The proton can diffuse through hydrogen bond nets either formed from organic acids and bases or through water protons in an ice-like unit. They can also diffuse through various other disordered forms of water and organic acids and bases. The proton in these cases can be H*(H20)n but this is of little consequence for its energy can still be high i.e. AGn(H ÷) > 40 kJ required for ATP formation. The value of AGn(H÷) is dependent upon a vast number of factors. The distinction between hydrated and anhydrous protons in relation to the bioenergetics of proton-driven ATP formation [3] is quite irrelevant. The free energy of a hydrated proton, H ÷ in water, or of an anhydrous proton (e.g. bound to a carboxylate group) can be the same. There is no connection between the protons in membranes which are discussed in this article and the protons produced by the ionisation of hydrogen atoms in the gas phase, costing 400 k J, described by Mitchell [3] in his analysis of local proton energies.

34

XB. Indirect proton coupling This model requires three sites X, Y and Z, and the stages are: (1) ADP + Pi are bound at site X (2) nH+ are generated at site Y (3) nH÷ diffuses to site Z, site ¥ relaxes to accept new reducing equivalents (4) Binding at Z of nH÷ causes geometry change at X (5) At X, ADP + Pi --> ATP + H20 (and H20 moves to Z and interacts with nH ÷) (6) Release of n i l ÷ or H30 ÷ from site Z and release of ATP from site Y (7) Relaxation of site Y and Z to receive next round of reactants In order to discuss this system I shall use the scheme of Fig. 13. Protons from an

redox

~o

system + bound- H-~H.~.

H20

protein

I

I P-OH+ HO-P

H20

I I

H20

e

redox

H20

system

H O+

I

protein

ATP- ose H~O +

I

H20

L P--O--P I I I

H20

J

6 4- O ~

ATP-case

OH

redm

N20

proteln

ATP

as~

sysiem HzO P--O--P

HzO |

/

HzO OH

redox

H/O

system

(

protein

H2 0

I

H~O

I

Hz 0

I

ATP- as¢

'

OH- + H30~ZH?.O + P - - O - - P

Fig. 13. A possible scheme for the movement of protons which drives ATP formation. This indirect coupling does not demand a stoicheiometry of nH÷ to ATE It could utilise conformational rearrangements of various kinds. A channel open at both ends is shown so that the coupling device can be linked in principle to chemiosmotic gradients.

35 energy source enter the H-bond net of a channel shown here as an aqueous channel. The set of protons in the channel interacts with the ATPase see Fig. 13, and by so doing alters the energy of ADP + Pi relative to ATP + H20. This can be transmitted through-bonds i.e. a conformational change. This is step (4). The stabilisation is pictured in Fig. 13 as a stabilisation of the water of ATP + H20 and this water now enters the channel. The proton can now travel and remove the final water molecule of the channel. Relaxation liberates the ATP. The scheme is not very different from that of Boyer in which the ATP is itself made stable by the conformation change. Boyer and Slater have provided much evidence that the ATPase is very complicated and that there may be a series of intermediates involving more than one ATP (ADP + Pi) site. This is only open to experimental test for it is not an essential ingredient of coupling. We see immediately that the weakest part of the analysis of the energy transduction mechanism is that of the actual steps in which ATP is formed. At the present time we have no good knowledge about the structure of bound ADP, Pi or ATP even in kinases. This means that many elaborate schemes can be drawn. Until the experiments come forward I believe it is easier to think in terms of naive operations as in Fig. 13. Membrane energisation contains many other couplings than that to ATP formation. In the final paragraphs of the article I look at the coupling to other cation movements, but I must mention two oddities, membrane transhydrogenation and ATP transport in mitochondria both of which are energy linked although they do not require energy of necessity. Thus the energy of the membrane has here purely a control function. One of the dangers of matching observed energy requirements with numbers of protons used to make one ATP is now very apparent: there can be a deliberate use, 'waste', of energy in order to gain control and thus proton/ATP ratios will be misleading if they are presumed to be relevant to coupling only, There are many other functions apart from transhydrogenation, ATP transport and formation of ATP which these membranes are capable of coupling together. XI. Gradients of cations The cations other than the proton with which we are concerned are Na ÷, K ÷, Ca 2÷ and Mg2÷. They are very different in behaviour from one another and from the proton. The cations Na ÷ and K ÷ have a very low affinity for negative charges, anions, while Mg2+ and especially Ca 2+ can have a very high affinity for anions. The proton is quite exceptional in its affinity for monodentate anions or even neutral bases. In the case of anionic groups which form a membrane the affinity does not depend upon the affinity of isolated groups but upon that affinity plus the total fixed charge of the membrane plus any field effect. We have shown that vesicles prepared from simple neutral phospholipids such as lecithin bind calcium with at least ten times the expected affinity of the isolated phospholipid group through the presence of a residual negative charge on the membrane. When negative phospholipids or carboxylate head groups are included then the calcium binding constant can be as high as 104 to 106. Probably the binding o f N a ÷ or K ÷ does not ever exceed 102. It follows that the surface of most membranes will be bound by a relatively large number of protons, calcium and magnesium ions. Given that the normal concentration of the proton is 10-7M and that of calcium or magnesium is 10-3M the usual situation in a membrane will be that the accessible anion sites will be bound by the divalent cation. However energisation of the membrane can alter the effective charge on the membrane greatly by producing protons within it and we

36

H

L 0

I

I~4gZL ~ IMg2,x~x, ~

R

oP

h~ ~-

Mg~" 0 U

Mg7+T

N

Pig2+

L

I

R

I n _,../?)j,~"

o p

IH + ~

H

I

y

[ H+

•

L Fig. 14. A scheme showing how different membrane bound charges interact on energisation. On absorption of light the proton is retained by the sites of the thylakoid membrane which normally bind magnesium. The magnesium switches on reactions in the medium. Thus coupling of a large number of events is achieved. Calcium can function in a similar manner in systems containing mitochondria but here removal of calcium in the cytoplasm and uptake into the mitochondria can both act as switches.

can e x p e c t that the effect o f this charge will be to displace tile Mg 2+ or Ca 2÷ ions. Exactly this reaction occurs on the inner surface o f the t h y l a k o i d m e m b r a n e . The fact that before energisation this m e m b r a n e acts to retain magnesium is indicative o f the highly negatively charged state o f the t h y l a k o i d internal m e m b r a n e face, Figs. 14, 15 and 16. A parallel situation appears to exist in the case o f the sarcomplasmic r e t i c u l u m but this m e m b r a n e retains calcium. A l t h o u g h the nature o f the (proteins) which retain the

hv

a-_a-

~ H*

A"~ A-A"

*(.~) z~ *H ÷

CC+C+ c

H+ I

H*I ] H*

H*H~H~N*I.[ ÷" ,.,

hv

H÷~H*H'H*H'H*H*H+H'H'IH°H'H~ /

]

Fig. 15. Rumberg's view of energisation. Note the bound protons. This is now a very different model from chemiosmosis and introduces all the control elements of locally bound protons. The model is not one of a Gouy-Chapman charge layer, see text. (a) Initial action of light; (b) steady state (c) light off. For the movement of divalent ions see Fig. 14.

37 I~7.,,~//-~ EDTA-soluble proteins

~

Intermediary proCeins

1~

Buried

proteins

A

I @ EDTA--J

~ ~ ' ~ ]

H+

H+ H* H +

H+

H+ H +

)Ill

llt lll lllll,llll l

Fig. 16. The effect o f EDTA on m e m b r a n e organisation

[241. Note

H+

+

that the effect o f

energisation is

very similar to this as it, like EDTA, acts as a bias upon proton/M s+ (M+) competition. This figure introduces a new potential source of coupling in that some proteins are released by EDTA (energisation) which is not just a conformation change, but a change in diffusion of proteins.

magnesium cations on the thylakoid membrane face are not known the nature of calcium binding proteins is well known: they have a very high content o f glutamic and aspartic acids. From magnesium chemistry I suggest that the magnesium-retaining protein site is likely to be one histidine imidazole and two carboxylate anions. If this proves to be the case the protein magnesium sites also provide an ideal buffer of protons in the membrane for the imidazole group has a very high affinity for protons especially if these protons' free energy (+&G) is greater than that o f protons even in an aqueous phase at pH 7. We must explain not only the release of the magnesium but the fact that it is also transported out of the thylakoid inner space. We could suppose that this transport is through a simple pore for the proton could be retained by the proteins or we could propose that some o f the proton energy is used to pump the magnesium ions out. In the mitochondria it is known that the energisation o f the membrane pumps calcium ions into the inner space of the mitochondrion and that the process requires energy. In both these cases ion movement (pumping) takes place before ATP is formed. The relationship is:

~ATP energy~ i o n

pumpincl

(9)

38 As stated above ion pumping energy requires AG=RTln[M]in/[M]outand therefore changes continuously while the energy requirement for appreciable ATP formation contains both a concentration dependent term and a term due to the standard state free energy of formation of ATP. It follows that very little ATP can be ma~te before the ion gradients are rather grossly adjusted assuming Eqn. 9 for the coupling relationship. The calcium-pumping proteins of mitochondria and perhaps the magnesium-pumping proteins of thylakoids must now be coupled to energised protons in the membrane. Many molecular machines have been suggested which depend upon binding of calcium (magnesium) to sites which can be adjusted by the binding elsewhere of protons. This is usually treated as a through-bond conformation change. I have proposed an alternative pumping model which utilises the same dehydration mechanism as that proposed for ATP synthesis [2]. The idea is that calcium at one side of the membrane is bound with some residual water of hydration. On traversing the membrane the ion is partially dehydrated by the positive charges of the membrane (protons) so that the calcium binding is differentially weakened. The idea behind the model is to stress the potential functions of control of water activity in transport (and not only of ions). A quite different way of pumping ions is found in the sarcomplasmic reticulum and indeed in the usual Na+/K+ pumps. These ion movements require ATP. The general mechanism involves the formation of a phosphorylated protein, -CO-OPO~- and it is this reaction which allows the calcium ATPase to transport the calcium. A simple model follows from our NMR studies on calcium proteins. We know that the calcium proteins contract or tighten their structures on calcium binding. For a protein of low negative charge the addition of calcium could be cooperative with the binding of an anion, here phosphate. As it is a high energy anion (an anhydride) it can only be formed on the high energy (ATP) side of the membrane and calcium is driven to the other side and lost on removal of the anion: Ca 2+ +

ATP ~ ADP + Protein, P.Ca.

This is an interesting coupling as Racker has shown that it turns over even in a particle. It is very closely analogous to the proton/ATP equilibrium in the membranes discussed above. The calcium sites do not transport protons although they transport other divalent cations and are blocked by lanthanide ions. This suggest strongly that the sites for the cations are formed by a multicarboxylate binding: compare parvalbumin. This type of site does not bind protons until a pH < 4. Thus an ideal channel for Ca 2÷ (M 2÷) transport is a multi-carboxylated protein surface. This type of channel will not be suitable for the pumping of sodium or potassium ions however. Coupling between Na*/K÷ concentration gradients is not with the energised membrane but is strictly connected to a Na÷/K÷ ATPase. Thus coupling is now: Energy -~ ATP ~ Na÷/K÷ gradients The ion channel again must not act on protons and it must not be blocked by Mg2÷ or Ca ~÷. The ideal channel would contain at most one carboxylate group and would have the selectivity of the cryptate ligands. Here the specificity arises from the matching of size of cation to a preformed neutral hole. A useful way of understanding selectivity is based on the consideration of the hydration of ions and the radius ratio: ion size to hole size. Attention is drawn to the Na+/K÷ channel as it requires more than one of each of these two cations and in this respect resembles the proton-driven ATPase. However it is truly

39 an energy device linked to osmotic energies and the ATP energy can be brought into equilibrium with the [Na÷], [K ÷] concentration gradients. Peculiarly it uses a simple chemical intermedite and we may suppose that it functions in a manner not too dissimilar from that of the calcium ATPase o f the sarcoplasmic reticulum. A proton could work in this way too, when there would be a chemical high-energy intermediate. Many coupling devices for moving ions of similar charge in opposite directions demand selectivity of binding at two types of site and an interaction between the sites. The binding selectivity of Na ÷, K*, Ca 2÷ and Mg~÷ have been described above. Proton selectivity can be assured by restriction of access for the proton has a special property of movement in ice-like structures. Thus we know of selective ways of coupling energised protons in a membrane with any ion (or other chemical) movement. The fact that special channels are available for the diffusion of different ions and perhaps other chemicals means that the nature of action of these ions can be specified not only by the characteristic selectivity at the initial site of action but by the character of the access channel. This means that an ion could act in one of three ways. It could act by binding at the initial site; it could modify the channel itself by binding to proteins which form the channel; it could pass along the channel and act at an internal site which would need little or no selectivity. An uncoupler could then act in three ways too. It could block the channel, forcing the ion to find a new diffusion path. It could block the initial or final site, preventing action. It could provide a new diffusion path. The last is the Mitchell mechanism.

XII. Ion stoicheiometry In the coupling between' ion transport and ATP which uses a phosphorylated intermediate we note the following sequence of events ATP ~ Energised protein in the membrane (EP) This is a stoicheiometric reaction and the energisation is probably very much localised in a single transport protein, The protein must now bind calcium: EP + n Ca + EP" (Ca)n. The reactions are again stoicheiometric. Thus the overall process is stoicheiometric but it is not efficient except where AG(ATP) = AGca = n R Tin [Ca] in/[Ca] out. Note that if AG(ATP) = 40 kJ then for n = 2 the maximum gradient is 10 4 while for n = 1 it is 108. It pays to be able to pump two or more calcium ions at first and then to pump only one. The stoicheiometry is not fixed in the case where this is possible.If the gradient is made by energisation of the membrane and not via ATP then there is no stoicheiometric equation since the energisation which corresponds to the formation of EP is the energisation due to proton binding AG(H+)n in a situation where AG(H +) is energised by the terms A(AG °) and if' and these do not depend on n directly. Therefore the value of AG for pumping can be as great for n = 1 and n > I if ~b' and A(AG °) are changed appropriately. It is not known from experiments which of these couplings are stoicheiometric.

40 XIII. Summary I have attempted to show in this article that biological coupling is of great complexity resembling that in an electronics circuit with many different forms of feed-back control. Major features of the biological apparatus are (i) the control of diffusion (ii) the structural organisation (iii) the simultaneous use of electronic and electrolytic devices. It is important in the analysis of the coupling to discover the regions of space which can equilibrate. I have put the argument for localised reactions. Chemiosmosis, which is not so very different from these schemes, demands more general equilibration. The great uncertainties lie in the energy distribution in the system and the mechanics of ATP formation. Other couplings may be understood more easily e.g. electron/electron, proton/ proton, proton/electron, proton/cation, proton/anion. Acknowledgement The references to this article have been kept to a very small number. The understanding of energy transduction has required virtually an army of contributors who have summarised their work many times. I feel confident that the contributions of individuals is known generally or can be assessed by reading of various reviews given in the first part of my reference list, and I hesitate to state who discovered what, first, as I was not involved in this experimental work. I wish to thank Dr. S. Ferguson (Oxford) for many informative discussions. I wish to thank also Dr. P. Mitchell for a stimulating exchange of views which helped me to clarify my views in 1961. It is regrettable that subsequently the similarities in some of our views have become obscured [3,25]. ADDENDUM (July 1978) Since this paper was written evidence in favour of localised proton binding in coupling both in the redox and the ATP-synthesizing proteins of photo- and oxidative phosphorylation has been published in the Abstracts of the l l t h FEBS Meeting at Copenhagen, August 1977. Kagawa, Wakabayashi, Yoshida, Hirata and Stone (Abstract No. A.4-13, 709) have shown that the protein which allows access of protons to bacterial ATPases binds protons (pKa = 6.5). It is not clear how many protons are involved. With this pKa any attempt to build up a proton gradient would immediately energise the membrane and create a local energised proton. Evidence for chloroplast proton-binding is given by Rumberg and Heinze (Abstract No. A.4-13,714-717). Clearly bulk potential measurements need not reflect the energetics of the bound protons unless equilibria are established. The contributions of Van Dam and his coworkers (Abstract No. A.4-13, L2, 9) show that the equlibrium is not set up in mitochondria. The binding of protons to the redox proteins has been demonstrated by Wikstrom and coworkers, Abstract No. A.4-12, L2, 5, who prove apparently that energisation of the cytochrome oxidase complex occurs through proton (or ATP) binding. This is not a field effect, see Nature (1977), 266, 271-273. Further evidence that chemiosmotic mechanisms could only be at most a part of the proton-driven ATP formation is provided by Papa and coworkers, Abstract No. A.4-10, L1, 2. The lack of true stoicheiometry in proton reactions and the role of the surface potential (not essentially in equilibrium with the bulk) is stressed by Kuschmitz and Hess (Abstract No. A.4-13,708, 5-6). Given the binding of protons, even if the system should go to equilibrium, i.e. partly in keeping with the chemiosmotic assumption, the calculation of the capacity will be incorrect. The estimated binding of protons by the thytakoid membrane exceeds the chemiosmotic energy by some 10-100-fold. An interesting paper on the connection between scalar and vector quantities by F6rland, Braek and Ostrold (1978) J. Bioenergetics, submitted) shows again that the requirement for such a connection is not a membrane but a single boundary. Vectorial coupling devices do not need membranes although they may be contained within them. An interface of some kind is the minimum requirement which gives direction and control of diffusion, see Williams, R.J.P., (1978) FEBS Lett., 85, 9-19. Vectorial

41 coupling is requked in the synthesis of many biological polymers but while it requires a membrane or a surface it does not require osmotic energy. The ADP + Pi reaction is a simple condensation polymerisation as is the formation of DNA. Further indications that chemiosmotic coupling cannot explain many events in energy coupling are given in the following new references: (1) There are ways of transducing energy which do not require vesicles. At present they do not produce ATP itself. Knobloch, K. (1978) Hoppe-Seyler's Z. Physiol. Chem. 3 5 9 , 2 8 6 - 2 8 7 . (2) There are membrane changes not associated with ~pH in chloroplasts, e.g. dissociation of the inhibitor protein from the ATPase, see Fig. 16 of this article and Harris, D.A. and Crofts, A.R. (1978) Biochim. Biophys. Acta 502, 8 7 - 1 0 2 . (3) Casadio, R., Melandri, A.B. and Melandri, B.A. (1978) FEBS Lett. 87, 323-328. describe shortrange couplings between ATPase and electron-transfer chains. (4) Dilley, R.A. and Orichaska, L.J. (1978) in The Proton and Calcium Pumps, (Azzone, G.F., ed.), pp. 4 5 - 5 4 , Elsevier, Amsterdam. The authors describe intramembrane proton pools. (5) Ort, D.R. (1978) Eur. J. Biochem. 8 5 , 4 7 9 - 4 8 5 . The author studies buffer effects on photophosphorylation and explains them with a localised proton model. (6) In a series of papers Kell, D.B., Ferguson, S.J. and John, P. (1978) Biochim. Biophys. Acta 502, 111-126 find a wide variety of numbers from 3 to >10 for the apparent number of protons coupled to ATP formation. (7) Mitchell, P. (1977) FEBS Lett. 78, 1 - 2 0 , has now accepted a local proton model as possible and has referred to an equivalent two-phase system. Both coupling devices are described above and must not be confused with osmotic gradients. (8) In Bacillus alcalophilus, Guffanti, A.A., Surman, P., Blanco, R. and Krulwich, T.A. (1978) J. Biol. Chem. 2 5 3 , 7 0 8 - 7 1 5 cannot interpret the bioenergetics using a simple chemiosmotic model. (9) Morowitz, L.S. and Morowitz, H.J. (1978) Biochem. Biophys. Res. Commun. 82, 7 2 7 - 7 3 0 , claim that ATP can be made using the nitrogenase system running from NH a to N 2. If this result is confumed it would represent the formation of ATP in a particle system, which is also known to perform redox reactions involving H 2. Many bacteria form very convoluted membranes which are sometimes seen as series of concentric membranes and sometimes as deep invaginations linking almost closed vesicles (sphaeroides). It is not easy to see how such systems can reach chemiosmotic equilibrium. In the simplest sense there structures are ideally suited to discriminate between membrane regions for transport into a cell and energy capture since they provide local control over diffusion. Locally differentiated parts of membranes give differential control over metabolism. In conclusion there are energised protons in aqueous surrounding phases and in the energy-transducing membranes. Their roles and exactly how they link with ATP-production are not yet known. It is sometimes implied that localised proton coupling systems cannot be tested [3]. Some tests are: 1, The effect of buffers; 2, The effect of vesicle volume and dilution; 3, The observation of intramembrane states, especially of protonations; 4, The kinetics of energisation and of phosphorylation, i.e. rates of ATP synthesis at different energisation levels and in different chemiosmotic states. Local proton theory states that there is kinetic control not thermodynamic control of coupling; 5, Uncouplers need not be proton translocators; 6, Surface agents will affect coupling; 7, The apparent number of protons involved in coupling and calculated from chemiosmotic equations will be widely variable depending upon equilibration conditions; 8, There will be energy-transducing systems which do not require vesicles; 9, Electron flow is not controlled by thermodynamic equilibration with aqueous phases through ATP. 10. Gradients of metabolites will not be linked to ATP levels even when pumped by membrane protons. Chemiosmosis gives a single thermodynamic steady state for all membrane processes and therefore links ATP levels with all other gradients. A p p e n d i x A. T h e n a t u r e o f c o n f o r m a t i o n c h a n g e s In t h e past a c o n f o r m a t i o n c h a n g e has b e e n r e l a t e d t o a first o r d e r p h a s e c h a n g e i.e. R h o m b i c s u l p h u r crystals ~ T e t r a g o n a l S u l p h u r Crystals r a t h e r t h a n t o a l i q u i d crystal first or s e c o n d o r d e r p h a s e c h a n g e . All c o n t r i b u t i o n s to S t a t e A ~ All c o n t r i b u t i o n s to S t a t e B

42 The first equation is very closely related to the Jacob and Monod allosteric change and describes Perutz's views of two state haemoglobin systems. These are in fact special cases of the second equation in which there can be many major and minor contributing structures within state A or B which are defined by a Boltzman sum over structures. An example is the phase change of ordered to disordered/3-Brass. Visualised from the point of view of a crystallographer phase changes will always be classified as changes which lead to physical boundaries between forms and therefore they are tempted to recognise only the simple first-order phase change e.g. of sulphur or hemoglobin, fully oxygenated and deoxygenated. Other types of measurement are more helpful in recognising continuous changes in protein lattices amongst which NMR is most useful. NMR shows proteins to be dynamic bodies - more like liquid crystals open to a variety of states in rapid exchange. This includes rotational, vibrational and more extended movements of groups and segments of chains. Given this state it is bound to follow that conformation changes can arise from cooperative onsets of lattice motions. There could be very little 'structure' change to be seen by X-ray analysis of crystals as presently carried out. A model example is the onset of rotation of NH~ groups in NH4C1, or the onset of rotation of aromatic groups in a cytochrome. Such phase changes could result in small changes in ordered protein crystals until suddenly the crystals crack - a well.known observation when studying changes of conditions of protein crystals. For a further discussion of phase changes as I see then, I refer to ref. 15 and particularly the references of that article. Appendix B. The energy of a redox couple The redox potential of a redox couple which is pH dependent is given by the equation AE = AEo + ~ l n ( 1 + [H*]/Ka) where AEo is the standard free energy change for states of equal protonation when the two states are present at equal activities. The hydrogen ion concentration is given by [H÷] and Ka is the acid dissociation constant for the protonation of either of the two states. The summation sign indicates that all states of protonation must be summed properly. In general the higher the pH the more stable relatively is the oxidised form. Many of the components of the cytochrome chain undergo pH dependent redox steps. Thus (i) their energies are not obvious (ii) proton binding can control electron-transfer rates, and (iii) there is no obvious relationship between the number of electrons associated with a redox couple and the number of protons associated with that couple. A full simple analysis of this problem is given for the case of model systems by Tomkinson and Williams ((1958) J. Chem. Soc. (A) 2010-2018). Certain cytochromes c show similar effects which we have analysed in detail. The work will be published shortly. The energy storage of a redox couple is not so obvious and is not to be confused with the osmotic capacity of the chemiosmotic hypothesis. The capacity for protons of aqueous phases is usually given by buffering formulae. The capacity of the membrane is given in part by a similar treatment of acid/base dissociated equilibria but since redox states and proton binding are partly coupled in membranes by reactions such as: Q+2e+2H÷~QH~ the proton capacity of membrane depends upon the supply of electrons. This is also discussed in section under the heading of the coupling between proton and electron transfers. There are then two distinct parts of this coupling, storage of two types,

43 separated protons and electrons and bound protons and electrons, and transport. In so far as redox states of two regions of a membrane differ, whether they be on opposite sides of the membrane or between adjacent regions on the same side, there will be membrane patches of very different charge and chemical structure. Between all such charged regions potential differences will be established. Thus not only are the capacity and the transport modes of this type of system quite different from those described by chemiosmosis they are not interpretable by theories of homogeneous model surfaces, for example GouyChapman theory cannot be applicable. The heterogeneity of the surface is itself important and controls the magnitude of these local potentials. It is these potentials plus the special paths for the diffusion of electrons and protons within the membrane matrix which are an essential part of local proton diffusion theories of the connection between energy input and ATP formation (Williams (1978) FEBS Lett. in the press). It will then be seen that energy capacity much as the energy difference must be the sum of three terms i.e. ~ AG o differences + ~ potential differences + Z ApH differences. The potential differences include terms not expressed in chemiosmosis. Appendix C. Protein control of proton and water movement In the above description and in the general theories of energised proton reactions which give rise to ATP it is necessary to control (i) the diffusion of protons and (ii) the availability of water at the enzyme active sites. In many kinases it is also necessary to prevent the access of water. The first restriction (i) is required so as to prevent loss of proton energy before ATP is made and it must be the membrane proteins which prevent such leakage since they are in large excess over the lipids. The larger the capacity of the steady state the more difficult this requirement becomes. For efficient handling of the protons the only path must be that trans to the ATP-synthesizing enzyme. It is likely to be a path of hydrogen bonds. Control over such a path can be achieved by any conformational change of the proteins forming the path. This change can disorganise the H-bond network through which the proton must diffuse since it is not easy for a proton to hop large distances. Therefore very small rearrangements within proteins can control proton migration rates. We suggest that energisation may be part of the control of the path of the proton. (ii) The active sites of proteins which make anhydrides must exclude water ADP + P ~ ATP + H~O Thus in the energised state the enzyme is not a simple ATPase. No matter how ATP is made the reverse of this reaction is prevented at the site, although ATP can be released from it, unless the hydrolysis of the ATP drives reduction. This means that the enzyme does run backwards but it is not an ATPase except under artificial conditions. Most simply this can be done by using energy to prevent the formation of a new configuration in the enzyme which would allow H~O access to the ATP. A protein conformation change could readily exclude water from a reaction centre, Fig. 13. Restrictions on water diffusion are known in the field of peroxidase and haemoglobin chemistries. It must be made clear however that Fig. 13 is an artificial device which is used in an effort to clarify the problem of coupling.

44

Bibliography Extensive references to detailed experimental results have not been given in this article which is a general view of coupling. For full references the reader should consult the following: Boyer, P.D. (1977) Annu. Rev. Biochem. 46,957-966 Chance, B. (1977) Annu. Rev. Biochem. 46, 967-980 Ernster, L. (1977) Annu. Rev. Biochem. 46,981-995 Mitchell, P. (1977) Annu. Rev. Biochem. 46,996-1005 Racker, E. (1977) Annu. Rev. Biochem. 46, 1006-1014 Slater, E.C. (1977) Annu. Rev. Biochem. 46, 1015-1027 Kozlov, I.A. and Skulachev, V.P. (1977) Biochim. Biophys. Acta 463, 29-90 It is against the background of the above reports that the present article is written. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Griffith, D.E., Cain, K. and Hyams, R.L. (1977) Biochem. Soc. Trans. 5,205-208 Williams, R.J.P. (1977) Biochem. Soc. Trans. 5, 29-32 Mitchell, P. (1977) FEBS Lett. 78, 1-20 Morowitz, H.J. (1978) Adv. Biol. Med. Phys. 17, in the press Williams, R.J.P. (1961) J. Theoret. Biol. 1,1-13 Mitchell, P. (1961) Nature 191,144-148 Williams, R.J.P. (1978) Proc. Royal Society London (A review lecture) to be published Lange, Y., Ralph, E.K. and Redfield, A.G. (1975) Biochem. Biophys. Res. Comm. 62,891-894 Bjerrum, N. (1951) K. Danske Vid. Selsk. 27, 1-12 Campbell, I.D. and Lindskog, S. and White, A.I. (1975) J. Mol. Biol. 98,597-614 Kemeny, G. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3669-3671 Williams, R.J.P. (1971) Cold Spring Harbor Syrup. Quant. Biol. 36, 53-62 Hoard, J.L. (1966) in Heroes and Hemoproteins (eds. B. Chance, R.W. Estabrook and T. Yonatani) Academic Press, New York, pp. 9-24 Perutz, M.F. (1969) Proe. Roy. Soc. London B 173,113-123 Williams, R.J.P. (1975) Biochim. Biophys. Acta 416,237-286 Davison, M.T. and Garland, P.B. (1975) J. General Microbiol. 91,127-138 Olasko, S. and Moudrianakis, E.N. (1974) J. Cell. Biol. 63,936-948 Williams, R.J.P. (1962) J. Theoret. Biol. 3,209-229 Boyer, P.D. (1975) FEBS Letters 50, 91-94 Harris, D.A. and Slater, E.C. (1975) Biochim. Biophys. Acta 387,335-348 Benesch, R.E. and Rubin, H. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2465-2467 Rumberg, R. (1977) in Encyclopedia of Plant Physiology, New Series, Vol. 5, (eds. A. Trebst and M. Avron) Springer Verlag, Berlin, 1977,405-415 Witt, H.T., Schlodder, E. and Graber, P. (1976) FEBS Lett. 69,272-276 Schapendonck, A.H.C.M. and Vredenberg, W.J. (1977) Biochim. Biophys. Acta 462,613-621 Williams, R.J.P. (1978) Trends Biochem. Sci. 3, N161-NI62