Compartmentation and Communication in Living Systems. Ligand Conduction: a General Catalytic...

Eur. J. Biochem. 95, 1-20 (1979)

The Ninth Sir Hans Krebs Lecture Compartmentation and Communication in Living Systems. Ligand Conduction : a General Catalytic Principle in Chemical, Osmotic and Chemiosmotic Reaction Systems Peter MITCHELL

Glynn Research Institute, Bodmin, Cornwall

(Delivered at the 12th FEBS Meeting in Dresden: July 2, 1978)

Chemical reactions, like osmotic reactions, are transport processes when looked at in detail. Chemical catalysis by enzymes or catalytic carriers, and osmotic catalysis by porters, may be conceived as occurring by specific ligand-conduction mechanisms. In chemiosmotic reaction systems, the pathways of specific ligand conduction are spatially orientated through anisotropic enzyme and catalytic carrier complexes in which the reactions of chemical group transfer occur as vectorial diffusion processes of group translocation down gradients of group potential that represent real spatially-directed fields of chemical force. Thus, it is easier to explain biochemistry in terms of transport than it is to explain transport in terms of biochemistry.

We honour Sir Hans Krebs for being a great ex- plorer and builder of knowledge about metabolic pathways (see [l]). Perhaps the most important of these pathways - not really a pathway, more like a highway - is the Krebs carboxylic acid cycle [2], which is connected through NAD, NADP and coen- zyme Q to the respiratory chain [3,4], including David Keilin’s cytochrome system - another major metabolic highway [ 5 ] .

It has been said that the creative work of each generation of scientists is done by standing on the shoulders of the innovators of the previous generation. That being so, Sir Hans might have been crushed under the enormous weight of many of the world’s biochemists, all struggling to get a foothold. But, like David Keilin, Sir Hans had the perspicacity to provide us with secure highways of metabolism, well- metalled by solid experimental expertise, on which we could stand and further explore and survey the metabolic scene.

CLASSICAL METABOLISM

Metabolic pathways, as in the Krebs carboxylic acid cycle (Fig. 1) - which occurs in the cytoplasm of bacteria and, correspondingly, in the matrix of mitochondria [6] - were not, of course, originally meant to represent spatially defined pathways of the molecules and chemical groups in living systems. Rather, they were intended to represent the sequence of the

chemical transformations, catalysed by enzymes and catalytic carriers that could be dissolved or dispersed in homogeneous aqueous solution, according to the classical bag-of-enzymes view of metabolism. Where, then, do we stand on such pathways? Pathways convey the notion of communication from one com- partment or site to another. In what sense do metabolic pathways represent communications, and between compartments or sites of what?

Group Transfer and Chemical Reactions

The question of the nature of classical metabolic pathways was answered through knowledge of the chemically and sterically specific group-transferring activity of enzymes, and through Fritz Lipmann’s quantitative thermodynamic concept of chemical group potential [7], which evolved integrally with the pioneering knowledge of the pathways and energetics of metabolism, to which Sir Hans Krebs has contri- buted so magnificently. The sites that represent the compartments of classical metabolism are essentially alternative chemical sites dispersed in the same phase. As illustrated in Fig.2, these sites are the chemical groups that act as the donor-group species D and acceptor-group species A for a given chemical group G that is normally stably bound, and thus compart- mentalised, as the alternative metabolite species DG or AG. The transfer of the group G from D to A (thus chemically transforming DG to AG) can only

2 Ligand Conduction

pyruvate t H' 2H+* '

pyruvic acid -COA

- * c i s - a c o n i t a t e + H z O

-&15ADP+15P isocsr a t e 2H -&

(oxalosuccinate)

oxoglutarate

succlnyl CoA f

C G ~ C - +

0\2H+CGz-+ -Con

NADH deH

.I. -H,O J furnarate

oxal oa c e t a t e

KREBS CYCLE =w s% Fig. 1. Krebs' carboxylic acid cycle with communicating systems. The diagram represents the mitochondria1 cristae membrane or bacterial plasma membrane on the left, and the Krebs cycle system in the matrix or cytoplasm on the right. The components shown in the membrane are a pyruvic acid uniporter (Py), the'reversible protonmotive ATPase, and the respiratory chain system, including the NAD(P) transhydrogenase (Trans H), the NADH dehydrogenase (NADH deH) and succinate dehydrogenase (S deH), connected via ubiquinone (Q) through the cytochrome b-ct complex (&el Complex), cytochrome c (c) and cytochrome oxidase (Cyt Ox)

Fig. 2. Chemical transformation. Conversion of DG to AG by transfer of the group G from D to A in a homogeneous enzyme solution

occur when the G-retaining bond is unlatched by the ligand-conducting property of the catalytic centre domain of an enzyme or catalytic carrier of appropriate specificity. Thus, it was implicit although not entirely self-evident in the classical bag-of-enzymes view of metabolism that the metabolic pathways are actually specified spatially within the ligand-conducting catalytic centre domains of enzymes and catalytic carriers. In these ligand-conducting catalytic domains the metabolic pathways are spatially-defined pathways of chemical-group diffusion in the direction of real forces given by the corresponding vectorial group-potential gradients.

SECTION

i ADP P S

Fig. 3. Enzyme-catalysed group translocation. From Mitchell and Moyle [9, lo]. The diagram illustrates a hypothetical example of phosphoryl group (P) translocation from ATP to a substrate (S)

VECTORIAL METABOLISM GROUP TRANSLOCATION AND CHEMIOSMOTIC REACTION SYSTEMS

For enzymes and catalytic carriers that have spatially separate binding sites for donor and acceptor species, as depicted for the phosphotransferase of Fig. 3, it seemed desirable to recognise the vectorial ligand-conducting property of the catalytic domain by describing the group-transfer process as group translocation [8 - 101 - even though, in homogeneous enzyme solutions, this property would not normally be manifested outside the individual enzyme molecules, and would not lead to any macroscopic vectorial or other consequence. In 1957, I introduced the term 'chemiosmotic' [l 11 to describe this aniso- tropically-catalysed chemical-cum-osmotic type of process (Fig. 3) because the group-translocation pathway represents the field of action of a real through- space force (Greek: hooy6.~ = push) corresponding to the chemical group-potential gradient. Thus, the group-transfer process has not only a scalar chemical- transformation aspect, but also an intrinsic vectorial transport or osmotic aspect, which could be the cause of a macroscopic osmotic process if molecules of the enzyme or catalytic carrier were appropriately plugged through a membrane, or if they were organised in pairs or sequences [9,10,12], as I shall presently explain in more detail.

The explicit description of the intrinsically osmotic group-translocation property of some enzymes and catalytic carriers [8 - 1 3 ] was really only a generalisa- tion and a further development of Lundegardh's concept of vectorial electron conduction by cytochromes [13]; and it owed much to earlier exploitations of Lundegardh's concept by Conway [14], Davies and

P. Mitchell

Ogston [15], Davies and Krebs [16], Robertson [17] and others (see 118,191).

The general drift of ideas towards the notion of what I have called vectorial metabolism [20] was encouraged in the 1950’s and early 1960’s by two major biochemical mysteries : one, the mechanism of linkage between metabolism and transport; the other, the bioenergetic function of respiratory metabolism in redox chain systems, where the reactions occur, not in aqueous enzyme solution, but in tightly integrated enzyme and catalytic carrier complexes, present in a partially fluid lipid membrane. It was from this context that the general chemiosmotic concept of group translocation led on to some experimentally useful developments and conjectures, about which I propose to comment in this paper.

Ligand Conduction : a General Enzymic Catalytic Principle

The first of these developments was related to Linus Pauling’s suggestion in 1950 that enzymic catalysis may depend upon the positioning of residual bonding sites in the region of the catalytic domain of enzymes in such a way that tight bonding with the transitional complex is particularly favoured, and the free energy of the transition state correspondingly lowered [21] (and see Jenck’s Circe principle [22]). To describe the catalysis of group translocation in similar terms, we added that the different components of the enzyme reaction would have to approach the transition state along different routes, each substrate moving along its own route because of the suitability of a particular region of the enzyme molecule for residual bonding with it [lo]. Thus, the efficiency, in the sense used recently by Knowles and others [23], and indeed the actual mechanistic course of an enzyme reaction, may depend as much on the topology and articulations (or specific conformational mobility) needed to determine the intrinsically osmotic ligand- conduction aspect of group translocation, as upon the static chemical specifications necessary for tight binding and specific chemical bond labilisation. This is illustrated in the following equations, which represent the multiplicity of different chemiosmotic processes or reactions that could theoretically result from the imposition of different translocational specifications on a given chemical transformation reaction, such as the homolytic transfer of the phosphoryl group, from ATP to the substrate S, shown here [9,10,12].

(1) * c:, P

ADP

s C S ( 2 )

ATp 1 ADP + S P

ATP + S

ADP I S P

S + ATP

ADP d‘ ADP c S P

ATp-J S P A: -<Ip

3

( 3 )

ATP + S

(7)

L ADP + S P P + S

In this vectorial equation notation, the chemical (group transfer) reaction aspect is represented as progressing up or down the page, while the intrinsically osmotic (group translocation) reaction aspect is represented as progressing sideways across the enzyme or catalytic carrier complex. It is obviously easier to visualise how some of these group-translocation processes might be mechanistically specified than others. The notion of specific enzymic ligand conduction represented here [9,10] (and see [12,24,25]) is, of course, related to Koshland’s notion of induced fit [26,27], to the linked function concept of Wyman [28,29], and to Hammes’ idea of intra-enzymic stress- strain relationships [30]. It is especially relevant to recent considerations by Jencks [31] in the context of ‘the utilisation of the binding energy in coupled vectorial processes’.

Chemicomotive Aspect of Group Translocation

The second, and somewhat more specialised, development, encouraged by the concept of group translocation, is related to the idea of electrochemical fuel cells, first described by Grove in 1839 [32]. The idea of electrochemical cells and circuits was genera- lised by Guggenheim in 1933 [30] to include the chemically motivated transport of any two species of chemical particle around a suitably conducting circuit [33] (and see [12,34,35]). Guggenheim’s simple but rather abstract thermodynamic treatment effectively showed that chemical transport can be coupled rever- sibly to chemical transformation by splitting the chemical reaction spatially into two half reactions, connected internally by a specific conductor of one chemical species, and connected externally by a specific con-

4 Ligand Conduction

, I' H,

\ f \

' ,. PROTON CONDUCTOR I , ,, ,

1 --- 2H' + -H70

I

A

\ / ELECTRON C~NDUCTOR

SOURCE OF ELECTRICITY

SOURCE OF P R O T I C I T Y

PROTON CONDUCTOR

B

ELECTRON CONDUCTOR

C

H+- CONDUCTING H+- CONDUCTING WATER WATER

0

Fig. 4. Hydrogen-oxygen ,fuel cells and protonmotive redox loop ,sysfem. After Mitchell [38]. (A, B) Hydrogen-oxygen fuel cells arranged to generate electricity and proticity, respectively. (C) Suggested redox-loop arrangements of carriers generating proticity in the oxygen-terminal part of the respiratory chain. Cytochromes are represented by italic letters. The Rieske iron-sulphur protein is represented by FeS

ductor of another chemical species, needed to complete the overall reaction. When we include the leading in and out of the reactants and resultants, as in a fuel cell (Fig. 4), we see that there have to be two internal specific ligand conductors arranged in a looped configuration between the interfaces where the chemical half reactions occur [36 - 381. Obviously the external specific ligand conduction process (in Fig.4B, the flow of protons or proticity) must be the sum of the internal specific ligand conduction processes (in Fig. 4B, of hydrogen atoms one way and of electrons the opposite way).

The specification of group translocation by an enzyme or catalytic carrier complex may usefully be considered to correspond to the specification of an internal ligand-conduction reaction of a chemicomotive cell, the other internal and external circuit components of which may be determined by the topological arrangement of the group-translocating complex relative to other osmotic or diffusion-regulating systems. Thus, as the name chemiosmotic implies, the intrinsic osmotic property of a group-translocation reaction represents its chemicomotive potentiality, which may be exploited by appropriate topological organisation.

For example, the notion of the protonmotive redox loop, introduced to explain the protonmotive property of redox chain or photoredox chain complexes plugged through coupling membranes [36,37], is based on this type of development of the specific ligand-conduction group-translocation concept [38 1.

Here, as indicated by Eqn (8), the internal (trans- osmotic barrier) ligand conductors in the redox complex are conceived as being specific for hydrogen atoms that diffuse down their potential gradient one way and for electrons that diffuse down their (electrochemical) potential gradient the other way, exactly as in the fuel cell of Fig. 4B, as emphasised by Fig. 4C. The outer circuit consists of the proton-conducting aqueous phases on either side of the relatively proton- impermeable membrane.

The notion of the protonmotive hydro-dehydra- tion loop, introduced to explain the protonmotivated phosphorylation of ADP by the reversible FoFl ATPase complex plugged through the coupling membranes of bacteria, mitochondria and chloroplasts [36 - 381, depends on a similar principle to that of the redox loop. Here, I should mention first that the Fo

P. Mitchell 5

component is the part of the FOFI ATPase complex that is plugged through the lipid membrane, and its function is simply to conduct protons to the active site of the F1 component, which possesses the protonmotive ATPase activity [38 -401.

2H+

ADPOP + H20

( 9 )

As indicated by Eqn (9), which represents the protonmotivated reversal of ATP hydrolysis (ADPOP is ATP, POH is inorganic orthophosphate), the internal (trans-osmotic barrier) ligand conductors in the F1 component of the ATPase complex are conceived as being specific for the diffusion of ADP and inorganic phosphate down their potential gradients into the active site, in protonation states represented as ADPO- and PO-, and specific for the diffusion of ATP and HzO down their potential gradients out of the active site, in the protonation states represented as ADPOP and H2O [38]. The essential point is that the ligand-conduction specificity must be such that ADPO- + PO- contains 2H' ions less than ADPOP + HzO, so that the net vectorial metabolic reaction corresponds to the translocation of 2H+ across the complex plugged through the membrane. As in the redox loop system, the outer circuit consists of the proton-conducting aqueous phases on either side of the membrane.

These considerations of the chemicomotive im- plications of the group-translocation concept represent the third development, encouraged by the concept of group translocation. They show how specific ligand-conduction in enzyme and catalytic carrier complexes can provide a very simple and direct mechanism for the reversible interconversion of chemical and osmotic energy.

Reversible Interconversion of Chemical and Osmotic Energy

The reversible interconversion of chemical and osmotic energy requires, of course, that each type of energy should be conserved. Chemical or metabolic energy differs from osmotic energy in that the former depends on the state of compartmentation of given species of chemical group in alternative stable covalent

compounds which may be dissolved in the same homogeneous aqueous phase, whereas the latter depends on the state of compartmentation of given species of solute in alternative domains or phases separated by topologically closed physical osmotic barriers. The reversibility of chemiosmotic reactions, and their coupling to other chemiosmotic or osmotic reactions, therefore depends directly on the topological arrangement of the complexes in which these reactions occur.

Osmotic Coupling between Chemiosmotic Reactions

As we now know, the essentially osmotic or transport aspect of the mystery of the bioenergetic function of respiratory and photoredox chain metabolism, which guarded the secret of the general biochemical mechanism of coupling in oxidative and photosynthetic phosphorylation, acted like the face of Medusa the Gorgon - petrifying those who approached, unprepared. Like Perseus, I was lucky, because my official teacher, Jim Danielli, and my unofficial bene- factor, David Keilin, had thoughtfully provided me with a mirror that could be used to reflect on, and clear- ly see, the gorgonian transport aspect of redox chain metabolism without danger of petrifaction.

The fourth development of knowledge and ideas, encouraged by the group-translocation concept, is that of the coupling of chemical reactions through osmotic intermediary processes.

There are two main kinds of topological principle - microscopic and macroscopic - for controlling the diffusion pathway and causing energetic coupling between intrinsically chemiosmotic reactions [9,10, 121. The macroscopic chemiosmotic coupling principle is spatially extensive, and depends on the chemiosmotic reaction complexes being plugged through a topologically closed lipid membrane separating two aqueous phases, as sumtnarised for oxidative and non- cyclic photosynthetic phosphorylation in Fig. 5 A and B, respectively [36- 541. The microscopic chemiosmotic coupling principle operates at the molecular level of organisation. It depends on the pairing of catalytic protein molecules or sub-units catalysing consecutive metabolic reactions, so that the intermediary ion or metabolite is trapped in a microscopic internal phase. For example, as illustrated in Fig. 6, Jennifer Moyle and I suggested in 1958 [9,10] that this microscopic principle may be applied to the water-soluble NADP- linked isocitrate dehydrogenase, where oxalosuccinate is trapped in the microscopic internal phase between the dehydrogenation and decarboxylation reaction centres. It is thus possible to account for the observed effect of temperature and other conditions [55,56] on the tightness of coupling between the dehydrogenation and decarboxylation reactions, and on the acces- sibility of the decarboxylation centre to oxalosuccinate, present in the enzyme solution, in terms of the

6 Ligand Conduction

A

(NAD-linked deH)

\L

B

L l /ATP w m s H,O ATp

Fig. 5. Direct mucroscopic chemiosmotic mechunisnu for mitocliondriul oxidative phosphorylution ( A ) and chloroplast non-cyclic photosynthetic phosphorylution ( B ) . These diagrams are based on the results of the painstaking work of many different research groups [36- 541. Hydrogen conductors are represented by : flavin mononucleotide (FMN) in the NADH dehydrogenase (NADH deH), by ubiquinone (9) in the cytochrome b-el complex (b-cl Complex), by plastoquinone (PQ) that may possibly be involved in a cytochrome b:f complex and by flavoprotein (Fp). Electron conductors are represented by: iron-sulphur centres (FeS), by cytochrome oxidase (Cyt Ox) containing cytochromes a and u3 and redox-functional copper (a Cu a3 c u ) by cytochromes b566 and 6562 (bb) in the cytochrome b-c, complex and possibly by equivalent b cytochromes (bb) in the putative cytochrome b-f complex, by cytochrome c (c), cytochromef (f) and plastocyanin (PC) and by components of photosystems I and I1 (PS I and PS 11) which contain chlorophylls a1 and art respectively. Proton conductors are represented by the Fo and CFO components of the FoFl and CFoCFl ATPase complexes, respectively, and possibly also by other un- named components

A

decar box

NAOP' b,co* isocit + -D oxalosucc +

NADPH~H' ~ ~ f l o x o g t u t

oxalosucc

Fig.6. Microscopic chemiosmotic coupling principle. Following Mitchell and Moyle [lo]. The diagrams represent the consecutive redox (o/r) and decarboxylation reactions catalysed by the NADP- linked isocitrate dehydrogenase, involving the sequestration of the intermediary oxalosuccinate in a microscopic internal phase. A and B represent closed and open conformational states of the enzyme respectively. Further explanations in the text

degree of opening of access between the microscopic internal phase and the bulk aqueous phase, presumed to be dependent on the conformational state of the enzyme molecules [lo].

The microscopic and the macroscopic chemiosmotic coupling principles may, of course, be employed together, as in the FOFI ATPase complex, where the protonmotive ATPase component F1 communicates protonically through the membrane via the proton- conductor or proton-well component Fo [12,34,35, 421. Likewise, as illustrated in Fig. 7, a protonmotive redox loop (A) may possibly be connected through the membrane by a proton-conducting domain (B), or proton-well component (C), as, perhaps, in the NADH dehydrogenase complex. Such cases point up the fact that the physical structure of the chemiosmotic reaction complexes must be an essential part of the osmotic barrier in both the macroscopic and the microscopic types of chemiosmotically coupled topological organisation. Where the lipid membrane is penetrated by a proton-conducting channel or carrier system, the osmotic barrier is effectively looped back and passes round the protonmotive complex [12], as indicated by the broken line in Fig.7C. One should take care not to confuse the type of direct chemiosmotic mechanism, illustrated in Eqn (9) and Fig. 7B, with the indirect type of mechanism advocated by Skulachev [57], as Ragan has done in a recent review [58]. For comparison with Fig. 7, Fig. 8 illustrates the protonmotive redox loop mechanism in a more formal but somewhat more explicit idiom that has become customary, where X and Y represent hydrogen and

P. Mitchell I

B C

\ I \ . rt

BH, B*2H+ rnh !HI+ 2H' c------S CJ

Fig. 7. Development of microscopic and macroscopic couppling principles iogether in chemiosmotic systems. (A) Represents microscopic pairing between hydrogen and electron transfer proteins (FMN and FeS respectively), as, perhaps, in NADH dehydrogenase. (B) and (C) represent macroscopic arrangements of this paired system to give chemiosmotic protonmotive redox loop systems. In (C), the osmotic barrier is effectively looped back around the redox complex, as indicated by the heavy dotted line. Further explanations in the text

A 0

Fig. 8. Redox loop diagrams showing conformutionally mobile carriers. Following Mitchell [ 3 5 , 3 6 ] . Diagram (A) shows the redox system looped across the membrane. In (B), the redox system is connected through the membrane by a proton-conducting component or proton well. A and B correspond to Fig.7B and C respectively, and X and Y represent hydrogen and electron carriers respectively. Further explanations in the text

electron carriers, respectively. This type of represen- tation [12] is useful in that it emphasises that, in this condensed type of complex, interaction between the chemically reactive components, X and Y, and the processes of ligand conduction across the complex in the osmotic barrier, require specific proximity of the reactive components, and probably also some specifically articulated conformational movements within the complex. Such conformational mobility

is obviously less likely to be required for electron conduction [59- 661 and proton conduction [67] than for the conduction of other types of ligand, such as hydrogen atoms carried by ubiquinol [68], or, as may be required in the protonmotive ATPase, 0'- groups carried by the circulation of adenine nucleotides and inorganic phosphate [Eqn (9)] [12].

Returning to the chemiosmotically coupled oxidative and photosynthetic phosphorylation systems represented by Fig. 5, I should emphasise that the bulk of the coupling membrane of mitochondria, bacteria and chloroplasts has been shown to have a very low effective proton conductance (see [42]) ; and, except where the proticity-producing and proticity-consum- ing units are plugged through it, it acts passively as an insulator between the proton-conducting aqueous media on either side. Simple proton permeability measurements in my laboratory, confirmed and ex- tended elsewhere, have shown that the effective proton conductance of coupling membranes is not greater than 0.5 pmho/cmZ. The equivalent specific conductance of the aqueous media is at least 106-fold higher

During protonmotive redox activity the aqueous media are energised by being brought to different protonic potentials. One has to be careful not to be misled by the fact that most students of membrane bioenergetics say that the membrane is energised when they mean that the proton conductors on either side of it are energised.

It is a consequence of the high reactivity and high mobility of protons in aqueous physiological media that such media are good proton conductors, even though the total concentration of hydrated protons may be only some 0.1 pM [25,42,69]. Thus, just as electron-conducting metals are used for the efficient transmission of electric power, so proton-conducting aqueous media are especially suitable for the efficient transmission of protic power [24,25,42,69]. However, the protonmotive force driving the protic current is somewhat more complex than the electromotive force driving an electric current in an electric conductor of uniform composition. In proton-conducting circuits, such as those of Fig.5, the aqueous proton- conducting media on either side of the circuit generally differ in composition, so that there is a significant protonic potential difference arising, not only from the electric potential difference of the hydrated protons but also from their effective concentration difference. Therefore the total protonic potential difference Ap must be given as the sum of the electric potential difference A$ and a thermodynamic potential difference that is equal to - ZApH, where Z is the conventional factor 2.303 RTIF and ApH is the pH difference (see [36,37,42]). Thus,

~ 9 1 .

8

Outside Membrane Inside

Ligand Conduction

Outside Membrane I n s i d e

Glucose

\

/ N a*

\

I Na+

A

Glucose

Na'

Fig. 9. Coupling between chemiosmotic and purely osmotic reactions. From Mitchell [20]. Suggested systems : (A) for coupling between the cation-translocating ATPase, and a Na'-glucose symport system in the intestinal mucosa; and (B) for coupling between a protonmotive respiratory chain system and a H'-galactoside symport system in the plasma membrane of Escherichia coli. POH = inorganic orthophosphate

At 25 "C the value of 2 is about 60 mV (when the potentials are given in mV).

We now come to the last two developments encouraged by the chemicomotive group-translocation concept, which are concerned with the coupling between chemiosmotic reactions and purely osmotic ones.

Coupling between Chemiosmotic and Purely Osmotic Reactions

The development of the chemiosmotic rationale in the 1960's focused attention on the catalytic components coupling the flows of particles in the biochemical systems catalysing solute, chemical-group and electron translocation [20,70]. Particular significance was therefore attached to the suggestions by Christensen [71,72] and by Crane [73], that the transport of amino acids and sugars through the plasma membranes of animal cells might be coupled to the translocation of K + ions or Na+ ions in the opposite or in the same direction respectively by specific carriers in the membrane. Likewise, I was encouraged to follow up my own postulate that, for reasons of osmotic stabilisation, the coupling membranes of bacteria, mitochondria and chloroplasts would have to contain tightly-coupled exchange-diffusion systems for exchanging cations against H+ ions and anions against OH- ions [74]. These putative systems represented a new class of translocation catalyst corresponding to the exchange-diffusion type of system originally postulated by Ussing [75] and by Widdas [76], but differing in that they were supposed to catalyse the sym-coupled or anti-coupled translocation, not of

analogous solutes, but of chemically unrelated solutes, as exemplified by the suggested prototype symport systems for the coupled translocation of sugars with H+ or Na'., illustrated in Fig.9 1201.

To promote the precise description of the osmotic secondary bond exchange reactions of solute translocation, it was suggested that non-coupled solute translocation should be described as uniport, and that anti-coupled and sym-coupled solute translocations should be described as antiport and symport respectively [20] (and see [77]). To distinguish the catalysts of such secondary reactions from enzymes involved in the primary bond exchanges of metabolism, it was suggested that they should be called porters, thus avoiding the termination 'ase' reserved for the names of enzymes, and generally denoting covalent bond labilisation [70]. The general mechanisms of solute uniport, antiport and symport are illustrated in Fig.10 [20]. These diagrams take account of the fact that the solutes exist as the hydrates in the aqueous media (written SWL, AWL, BWL and SWR, AWR, BWR in the left and right aqueous phases respectively), and that translocation through the porter in the lipid membrane phase generally requires disengagement of the solute from (at least part of) its hydration shell by exchange of valencies with the carrier (or carrier centre) X, specific diffusion of the solute (in the pore mechanism I) or of the solute-X complex (in the mobile carrier mechanisms I1 - IV), and re-engagement of the solute with its hydration shell, so that it is released from X on the other side of the membrane. The catalysis of uniport arises from the specific diffusional mobility of S through the pore X (Fig. lOI), or from the specific diffusional mobility of both unoccupied

P. Mitchell Y

WATER LEFT (WL) I L I P I D

JI w,' i::: swL]

WATER RIGHT (W,)

UNIPORT (pore) r swR

UNIPORT (carrier)

ANTIPORl c CF, /' wRA

- 2 w ~ SYMPORT

WRB

Fig. 10. Mechanisms of uiriport, antiport and symport. After Mitchell [20]. Diagrams of porter-catalysed translocation of solutes (S, A and B) across a lipid membrane between aqueous phases in which S, A and B exist as hydrates SW, WA and WB. Left and right aqueous phases are denoted by suffixes L and R. (I) Uniport via a pore type of mechanism; (11) uniport via the carrier X ; (111) antiport via the exclusive complexes XA and XB with the carrier X ; (IV) symport via the inclusive complex XAB with the carrier X. Further explanations in the text

X and of the SX complex across the porter system (Fig. lOII), at given concentrations of S in the aqueous phases [20,70]. The catalysis of A-B symport (Fig. 10 111) likewise arises from the specific mobility of both unoccupied X and of the inclusive complex ABX across the porter system, at given concentrations of A and B in the aqueous phases; but the tightness of coupling depends on the extent to which the flux of A and B passing through the porter as the partial complexes AX and BX is minimised, compared with the fluxes of A and B passing through as the inclusive complex ABX [20,70]. In the case of A/B antiport (Fig. 10 IV), catalysis of translocation depends on the specific mobility of both AX and BX; but the tightness of coupling depends on the extent to which the flux of unoccupied X is minimised, compared with the flux of A and B passing through the porter as the AX and BX complexes [20,70].

The rate of any reaction, such as that catalysed by a symporter or antiporter, along a given diffusion pathway, is dependent upon the concentration of the species actually mobile through the degrees of freedom constituting the pathway. Since, according to the Maxwell-Boltzmann law, the space-time concentration of a given state of a system is relatively small if its potential energy is large compared with the thermal energy kT, it follows that a pathway that it may be evolutionarily desirable to close, because it corresponds to a side-reaction and causes uncoupling, can be selected against by mutations causing it to corre-

spond to states of the system having relatively high potential energy compared with states along the pre- ferred, tightly-coupled, ligand conduction pathway

My object is to emphasise that the meaning of energetic coupling in this context is nothing more than the mutual dependence of the flows of A and B due to their conduction as exclusive or inclusive complexes (AX and BX or ABX) specifically through the antiporter or symporter system, so that osmotic work can be transferred by the transmission of forces between the trains of A and B molecules undergoing the coupled ligand-conduction process [12,19,20,24,25]. As I have endeavoured to show before, precisely similar principles apply, although perhaps less obviously, to chemical group transfer reactions coupled in the active- site domains of certain bifunctional enzymes, such as the phosphorylating 3-phosphoglyceraldehyde dehydrogenase [19,24,25,77], but I do not have time to enlarge upon this interesting and important point in the present paper.

These considerations [12,24,25,70] explain why the kinetic specificity of ligand conduction in biochemical processes is inseparable from their thermodynamic reversibility and precise stoichiometry, as Jenck's Circe principle has recently emphasised in the case of the substrate-specificity and catalytic efficiency of enzymes [22].

The conceptual development of the principles of uniport, antiport and symport reactions was followed, after a delay of several years (see [77]) by the iden- tification of many such reactions [24,70,78] in animal and plant plasma membranes [79-831, and in the coupling membranes of mitochondria [84- 861 and bacteria [77,87 - 901. These porters have been identified by direct studies of the kinetics and specific inhibitor characteristics of membrane permeability, by simple measurements of swelling, and, in the case of proton-linked porters, by observations of pH changes and electric potential changes accompanying solute translocation, and by observing the uncoupling effects of proton-conducting agents, such as 2,4-di- nitrophenate, and other ionophores. I shall mainly focus attention here on the mitochondrial porters.

In the case of proton-coupled porters, involving solutes that are directly protonatable, as in the N- ethylmaleimide-sensitive phosphoric acid uniporter represented in Fig.11, it is difficult to distinguish between : (A) phosphate/hydroxyl-ion antiport, (B) phosphate-proton symport and (C) phosphoric acid uniport, which can all be functionally and stoicheiometrically equivalent, although different in mechanistic detail. Generally speaking, it has not been possible to distinguish between these stoicheiometrically equivalent mechanisms in the porters so far studied.

Fig. 12 illustrates the main mitochondrial porters for Krebs cycle carboxylic acids. Net import of suc-

~ 4 1 .

10 Ligand Conduction

Outside Ea Ins ide

HP02- PhosphatelHfdroxyl- ion 20K antiport

HPOZ- Phosphate- Proton symport

2H' 2H' Phosphoric acid

H3P0, uniport

A

B

C

C hemios rno t i c H+ protonmotive system

K+ Cation leak

Fig. 11. Mitochondria/ phosphoric acid porter. The diagram shows the equivalence of phosphate/hydroxyl-ion antiport, phosphate- proton symport and phosphoric acid uniport catalysed by the porter (P) in the mitochondrial cristae membrane

Outside Inside

A

SUCC SUCC, ,' ,' B

SUCC I C

I

I cit ci t j D

ma( or'succ

Fig. 12. Non-electrogenic mitochondrial porters for Krebs cycle acids. (A) Phosphoric acid porter ; (B) dicarboxylate porter catalysing phosphate/dicarboxylate antiport; (C) dicarboxylate porter catalysing succinate/malate antiport; (D) tricarboxylate porter catalysing citrate/(malate or succinate) antiport; (E) oxoglutarate porter catalysing (malate or succinate)/oxoglutarate antiport. Fur- ther explanations in the text

cinate involves the phosphoric acid uniporter A and the dicarboxylate porter B (acting as a phosphate/succinate antiporter) working in series. As represented in C, the dicarboxylate porter may also act as a succi-

Outslde pT Inside

ADP

ATP

V

2H'/ 2e-A

respiratory chain

2Ht/ 2e- A

Fig. 13. Putative mitochondrial ADP phosphorylation system with t H f / P quotient of 3. P represents the phosphoric acid porter, and AdN represents the ADP/ATP antiporter. The protonmotive stoicheiometry (2Ht/2e-A) means that 2H+ ions are translocated outward per pair of electrons (2e-) traversing each effective redox loop (4

nate/malate antiporter, for example, during succinate oxidation. A net entry of citrate may occur in a three-stage process involving (A) the phosphoric acid uniporter, (B) the dicarboxylate porter, catalysing phosphate/succinate antiport, and (C) the tricarboxylate porter, catalysing succinate/citrate antiport, as indicated by the broken-shafted arrows. During citrate oxidation via isocitrate, citrate/oxoglutarate antiport can be catalysed by the tricarboxylate and oxoglutarate porters through the mediation of malate or succinate, as illustrated in DE.

These systems show that porters can be biochemically and energetically coupled in series just like enzymes. Indeed, a subject of metabolic porterology, analogous to that of metabolic enzymology is now rapidly developing. It is particularly noteworthy that, as the import and export of Krebs' cycle acids through the porter systems generally occur by non-electrogenic antiport reactions, the work done in the import reaction is recovered in the coupled export reaction. Thus (provided we also take account of the non- electrogenicity of COz transport, where appropriate), the porter reactions generally involve no net expen- diture of energy other than that dissipated by the relatively small frictional effects, inevitably accompanying the specific ligand-conduction processes.

During ADP phosphorylation in mitochondria, the reversible protonmotive ATPase is directly accessible to the adenine nucleotides and inorganic phosphate only from the inner aqueous phase, as illustrated in Fig. 13. To make the ATPase accessible to external

P. Mitchell 11

Outside t&g Inside

ATP/ADP D ant ipor ter

A T P - A T P (Ca-l inked?)

?C$' k'

Chemiosmotic protonmotive system

Cation leak K+ I----P Kt

Fig. 14. Mitochondria1 electrophoretic calcium porters. (A) CaZ +

uniporter (Ca), the existence of which is now questionable; (B and C) calcium-phosphate symporter (Cap) and calcium-fi-hydroxybutyrate symporter (Cafi) for which evidence has recently been obtained (96,100,101]; (D) speculative linkage of calcium translocation with the ADP/ATP antiporter. POHbut- = /I-hydroxybutyrate. Further explanations in the text

adenine nucleotides, there is an atractyloside-sensitive and bongkrekic-acid-sensitive porter, which catalyses a strictly coupled ATPIADP antiport. According to Klingenberg and colleagues [91,92] and Vignais and colleagues [93], the ATP/ADP antiporter is electrogenic, as indicated in Fig. 13. However, since the phosphoric acid uniporter is electrically neutral, this should mean that one Hf ion would be translocated from the outer to the inner phase per cycle of the porter system. Thus, the effective proton translocation quotient +H+/P for protonmotivated ATP synthesis should be, not 2, as indicated for the reversible ATPase in Fig. 13, but 3. Assuming (for the moment) that the protonmotive stoicheiometry of the respiratory chain corresponds to a proton translocation quotient per bivalent reducing equivalent t H + / 2e- of 2 per effective redox loop A, as indicated in Fig. 13, the P/O quotient should be, not 3, as observed, but 2 for oxidation of NAD-linked substrates, and not 2, as observed, but 413 for succinate and other FAD or Q-linked substrates. There appears, therefore, to be something wrong with the scheme of Fig. 13.

Fig. 14 summarises some old and new conjectures, and some recent findings, concerning mitochondrial porters that are specific for calcium. Although the experimental evidence available was never completely conclusive, it has been thought for about 10 years

that calcium, strontium and some other divalent cations, entered mitochondria by a lanthanide-sensitive porter catalysing the electrophoretic uniport of Ca2+, as represented in A (see [94,95]). Recently, however, Jennifer Moyle and I obtained evidence, by measuring the number of protons exported via the respiratory chain system of rat liver mitochondria per calcium ion or strontium ion taken up via the lanthanide-sensitive calcium porter(s), that each bivalent ion is conducted through the membrane with only one positive charge [96]. Akerman has supported our finding by showing that the slope of the Nernst diffusion potential with concentration is the same for the outward diffusion of calcium through the mitochondrial porter(s) as it is for the outward diffusion of the monovalent K + ion via the ionophore valinomycin [97]. This evidence for the monovalency of the porter specific species is also consistent with observations on the equilibrium distribution of calcium across the membrane under the influence of the electric membrane potential (see [96]).

Evidence for a phosphate dependence of calcium uptake by mitochondria from various animal and plant species (see [96] and also [98,99]) and evidence for the bivalency for calcium of the phosphate-stimulated porter, from the sigmoidal dependence of the import rate on Ca2+ concentration (see [96]), led us to postulate the existence in mitochondria of the calcium- phosphate symporter represented in Fig. 14B [45,96]. We have obtained confirmatory evidence for this porter by showing that rat liver mitchondria accumulate phosphate in a calcium-dependent, N-ethylmaleimide- insensitive, lanthanide-sensitive process and that two calcium ions are imported per phosphate imported in that process [loo]. By relatively indirect respiratory methods, we think we have also identified a lanthanide- sensitive calcium-a-hydroxybutyrate symporter of rather broad specificity for monocarboxylates that is distinct from the calcium-phosphate symporter [loll, as indicated by Fig.14C. Our present view is that there are probably other calcium-linked porters yet to be discovered.

Some years ago, Spencer and Bygrave [lo21 suggested that the ATP/ADP antiporter might be calcium-linked. We think that some such direct or indirect linkage as that indicated by Fig. 14D is worth serious consideration for reasons that I shall explain a little later. I must, however, hasten to add that our opinion on this, i.e. that the Ca2+ uniporter A of Fig. 14 probably does not exist in mitochondria, but that calcium is probably translocated by the sympor- ters B and C , and possibly by other similar systems yet to be discovered, is not generally accepted. Indeed, evidence and arguments against our experimental findings and interpretations have been presented by Lehninger and colleagues [95,103], by Azzone and colleagues [104], and by Crompton, Hediger and


2Ca *+ 2Ca2+

2POHbut- 2 POH b u t-

4H' 2H' 2H'

T 2H'

1 2H'

T 4H'

4 POHbut- 2POHbut- 2POHbut

-H+ /2e - A :2

Fig. 15. Mechanisms of culcium j-hydroxyhutyrate import in mitochondria, (A) Involving the questionable Ca". uniporter and a respiratory chain protonmotive stoichiometry (+ H+/2e-,4) of 4; (B) involving the calcium-j-hydroxybutyrate porter and a respiratory chain protonmotive stoicheiometry of 2. POHbut ~ = P-hydroxybutyrate

Carafoli [105]. Brian Chappell has told me privately that his paper during this FEBS meeting will also describe observations from which he concludes that calcium is translocated, not with one positive charge, as we [96] and Akerman [97] have found, but with two positive charges, as was formerly supposed.

In sciences like biochemistry, where much of the experimental evidence is highly complex and tends to be soft, in order to avoid finding that one can't see the wood for the trees, it is often strategically ad- vantageous to make inferences from broad perspec- tives that depend on the apparent dispositions of many facts and do not rely too strongly on the correct view of any one fact. In this spirit, let me therefore persevere with my thesis about the calcium porters.

Fig. 15 shows schemes for the stoicheiometry of calcium p-hydroxybutyrate uptake by respiring mitochondria : (A) depending on a Ca2 '- uniporter and (B) depending on the calcium-P-hydroxybutyrate symporter that Jennifer Moyle and I think we have identified. Lehninger and colleagues have established the fact that for every bivalent reducing equivalent traversing each effective redox loop in the respiratory chain, two calcium ions and four 8-hydroxybutyrate molecules are taken up [94,95,106], as shown in both schemes A and B. But it is especially noteworthy that, according to scheme A, which is favoured by Leh- ninger and colleagues, the number of protons translocated by the respiratory chain per bivalent reducing equivalent traversing each effective redox loop, + H + / 2e-A, would have to be 4. But, according to scheme B, which I think is more likely to be correct, the respiratory chain protonmotive stoicheiometry, cHf /2e -A, would have to be 2. A knowledge of the correct protonmotive stoicheiometry of the respiratory chain is obviously very important.

respiratory chain

2Ht12e- A

Fig. 16. Putative mitochondria1 ADP phosphorylation system with +H'/P quotient of 2. Symbols as in Fig.13 and 14. Further explanations in the text

Let us return now to the unresolved question of the stoicheiometry of ADP phosphorylation in whole mitochondria. Fig. 16 summarises a speculative scheme, involving the use of both the phosphoric acid uniporter and the calcium-phosphate symporter for the import of inorganic phosphate. It also involves a coupling between the export of calcium and the export of ATP via the ATP/ADP antiporter, as suggested by Spencer and Bygrave [102]. One reason for considering this scheme is that, unlike the scheme of Fig. 13, the porter systems would have no net protonmotive effect, and the observed mitochondrial P/O ratios would be obtained, provided that the protonmotive respiratory chain had a t H + / 2 e - ratio

P. Mitchell 13

2Na'

H l i

Fig. 17. Two-sfuge Na+-motive ATPase systems. (A) Actual duplex system; (B) imaginary compact integral system. H'/Na+ represents an H'/Na+ antiporter

3Ht

2Ca2+

I ATP4'

B 50 3Na'

3;f&H*o P2-+ADP3-+H' ATP4-

o====s

Fig. 18. Putative semi-direct A TPase mechanisms. (A) Driving 2CaZ+/3H+ antiport; (B) driving 3Na+/2K+ antlport. In these mechanisms the coupling between the cation translocation and the ATPase reaction is supposed to be due to direct electrovalent interaction between the cations and anionic phosphate groups of the ATPase reactants that diffuse spontaneously down their electrochemical gradients in the virtually anhydrous enzyme active-site domain. But the cation specificity is attributed to specific ionophoric groups leading into and out of the active-site domain

of 2 per effective redox loop. Another reason is that it provides a mechanism by which the liberation of calcium through the endoplasmic reticulum membrane, and its arrival at the outer surface of the mitochondrial membrane, could boost phosphate import and ADP phosphorylation at the same time as boosting the entry of respiratory substrates having calcium-linked porters. I would like to think that this conjecture might appeal to Sir Hans Krebs in the context of his recent studies with Stubbs and Vignais [lo71 on a possible control function of the ATP/ADP antiporter (and see [ 1081) in oxidative phosphoryla-

tion. I offer it, not as a solution of the problem, but as a stimulus for more exploratory research.

As illustrated in Fig. 17A, the coupling between the H+/Na' antiporter [34,35,78] and the protonmotive ATPase in mitochondria1 [lo91 and bacterial [87,110] membranes provides a very interesting and educative example to conclude this commentary on coupling between purely osmotically coupled solute porter reactions and chemiosmotic protonmotive reactions. This duplex system has the important physiological function of helping to conserve the electric potential component A$ of the total protonic potential difference Ap, and increasing the effective differential pH buffering power, and the total energy storage capacity, of the osmotic system [24,34,35, 78,871 (and see [ l l l ] ) . However, my object here is not to dwell on its physiological importance, but to use this two-stage chemiosmotic system, which acts as an electrogenic Na+-motive ATPase, to illuminate the mechanistic question: how direct is the coupling between the chemical and the osmotic process in chemiosmotic reaction mechanisms [12,25,44,45, 112]?

Direct and Indirect Chemiosmotic Reaction Mechanisms

It is clear that the two-stage macroscopic Na+- motive ATPase mechanism of Fig. 17A is indirect in the sense that it uses the force and flow of the proton circulation by way of the aqueous phases on either side of the membrane to transform and transmit the chemical energy of ATP hydrolysis to the osmotic energy of the electrochemical potential difference of the Na' ion. This would still be the case, even if, according to our microscopic chemiosmotic coupling principle, the system could have evolved to a minia- turised compact version, contained within a single protein complex, as suggested by Fig. 17B. The precedent set by the known use of the circulation of one of the ATPase reactants, namely the proton, for energy transfer via coupled ligand-conduction processes in the macroscopic duplex Na+-motive system of Fig.l7A, seems to me to make it worthwhile to raise again the question [112] whether, as suggested in Fig. 18, the circulation of other reactants, namely the anionic adenine nucleotides and inorganic phosphate, may be coupled to cation translocation in the CaZ+-motive (A) and Na+/K+-motive (B) ATPases by what may be described as a semi-direct electro- valently coupled ligand-conduction type of mechanism. In such semi-direct mechanisms speculatively suggested in the schemes of Fig.18 (the general features of which are based on current research [113- 117]), the specificity of cation translocation would depend on cation-specific ligand binding and conducting channels, or mobile ionophoric components, in


A

2ca2+ c

ATP

H 20 P+ ADP

2Ca2+

B

3Na* c 2K'

Fig. 19. Completely indirect black box cationmofzve ATPase mechanisms. (A) Driving CaZ+ uniport; (B) driving 3 Na'/2 K C antiport. The osmotic translocation reactions are supposed to be catalysed by the translocators T, which are regarded as being spatially separate from the active-site domain of the ATPase that catalyses the chemical reaction of ATP hydrolysis

A

Fig. 20. Completely indirect black box protonmotive redox ( A ) and ATPase ( B ) mechanisms. Symbols as in Fig.8 and 19. Further explanations in the text

the enzymic polypeptide complex, as I have pointed out before [112]. But the transformation of the chemical energy of ATP hydrolysis to the osmotic work of cation translocation could depend on electrovalent coupling between the cations and the anionic ATP and phosphate groups travelling through specific ligand-conducting pathways leading into and out of the enzymic active-site region. The value of this type of consideration for the progress of research is that it explicitly recognises that, in accordance with the Curie principle (see [19]), the transformation of the chemical energy to osmotic energy must depend on a vectorial group-translocation aspect of the ATPase reaction that gives rise to the diffusion of reactants along spatially-defined ligand-conduction pathways in the ATPase complex. In the completely direct type of chemiosmotic mechanism, the osmotically translocated species, for example the proton, is itself one of the reactants, or, at least, a covalently-linked part of it. But in less direct types of chemiosmotic mechanism - as, for example, must apply to metallic ions that do not form covalent compounds - there must be some electrovalent or secondary bonding between the osmotically translocated solute(s) and

one or more of the circulating reactant species, either indirectly via an intermediary porter, as in Fig. 17, or directly as I have suggested in Fig. 18.

Since the word conformational, applied to proteins, acquired a kind of magical significance, enabling proteins to accomplish anything (conveniently without the need to specify any biochemical mechanism), it seems to me to be very unfortuante for the strategy of biochemical research on chemiosmotic reactions that, as indicated by Fig. 19, there has been a tendency amongst many biochemists to invoke completely indirect, exclusively conformational types of chemiosmotic mechanism for the metallic-cationmotive ATPases. Moreover, this fashion has spilled over into the field of protonmotive chemiosmotic systems [57,118,119], as illustrated in Fig. 20, even though the proton, being one of the most reactive of all chemical species, may readily be involved in completely direct or semi-direct chemiosmotic mechanisms (see [12,19]). The great disadvantage of the completely indirect type of formulation is that the coupling process is indicated by nothing more informative than the wiggly line between the chemical and the osmotic reactions. In particular, this black-box type of formulation is completely open with regard to stoicheiometry, and one may insert whatever numbers suit the experiments of the day. By contrast, the direct or semi-direct chemiosmotic formulations are charac- terised by definite protonmotive stoicheiometries, and in this and in other respects they do much more to promote and guide experimental research designed to test alternative conjectures than the completely indirect black-box type of formulation. The latter is so lacking in information content that one can't think how to test it, except by waiting until complete X-ray crystallographic and other topological analysis is available for both the static and the dynamic molecular structure of the chemiosmotic catalytic complex. This brings me to the closing topic of my paper: the protonmotive stoicheiometry of respiratory chain systems. As I have tried to show in this paper,

P. Mitchell 15

-- 1pg atom O/g protein .E 0

FCCP 2 ' s I .1 -- -

- _ I 1 tvalinomycin to I\,_

0 qive hiqh K' conductance and

tEGTA to chelate ; endogenous Ca2' ,'

m

8 " 6

l pg atom Olg protein

4

no valinomycin no EGTA

eiectrophoresis of endogenous calcium via

2oS + porter system

a 4

Fig. 23. Estimation of protonmotive stoicheiometry for 8-hydroxybutyrate oxidation in rat liver mitochondria. After Mitchell [122]. The upper and lower curves represent recordings from an H+-ion- sensitive and a K +-ion-sensitive electrode respectively after inducing a pulse of respiration by injecting a small quantity of air-saturated saline into an anaerobic suspension of rat liver mitochondria. FCCP carbonylcyanide p-trifluoromethoxyphenylhydrazone, EGTA = ethyleneglycol bis(8-aminoethy1)-N,N,N',N'-tetraacetic acid. Further explanations in the text

the determination of this stoicheiometry is crucial both for understanding the interrelationships between the functional activities of the respiratory chain, the ATPase and the porter systems, and for understanding the molecular mechanisms of the protonmotive respiratory chain and ATPase complexes.

PROTONMOTIVE RESPIRATORY CHAIN

Chemiosmotic Stoicheiometry

It is widely agreed that the determination of the stoicheiometry of proton translocation by respiratory chain systems is the first prerequisite for obtaining insights into the chemiosmotic energy-conserving mechanism at the molecular level of dimensions.

The proton translocation stoicheiometry is ex- pressed in terms of a +H+/O or c H f / 2 e p quotient, by analogy with the P/O ratio for oxidative phosphorylation [36,37]. The simplest, and I think most re- liable, method of measuring t H + / O or t H + / 2 e - ratios is the 02-pulse or respiratory pulse method [49,120- 1221, illustrated by Fig. 21. A lightly buf- fered suspension of mitochondria or bacteria is equi- librated anaerobically in a closed glass vessel equipped with a mechanism for vigorous stirring, and containing fast-responding hydrogen-ion-sensitive and potassium-ion-sensitive electrodes. A brief pulse of respiratory activity is induced by injecting a small volume of air-saturated saline, containing a known quantity of oxygen, and the changes of the pH and pK of the suspension medium (pH0 and pKo) are recorded. Appropriate calibrations with HC1 and KCl solutions permit the observed pHo and pKo changes to be ex-

pressed as changes of acid equivalents, AH; (acid), and changes of K f equivalents, AK;, in the outer medium. When the membrane is made freely and specifically permeable to Kf by valinomycin, and conditions are such that the membrane is otherwise of low ion conductance, the - AKof value represents the electrophoretic import of K + which electrically neu- tralises and is equal and opposite to the electric charge translocation corresponding to the respiration-driven outward proton translocation, or - A K i = AH: (charge). As the AH: (acid) and -AK$ or AH: (charge) pulses decay towards zero after the respiratory pulse (as shown in Fig.21) and this decay is specifically accelerated by proton-conducting reagents such as FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), the values of AH; (acid) and - AK$ or AH$ (charge), extrapolated to the centre of the respiratory pulse, can be taken to represent the quantities of translocated acid, t H + (acid), and of translocated electric charge, +H + (charge), cha- racteristic of the protonmotive process [42- 45,120 - 1261. As seen in Fig.21, the NAD-linked oxidation of P-hydroxybutyrate gave an +H+/O ratio of 6. In similar experiments, the ubiquinone-linked oxidation of succinate and the NADP-linked oxidation of isocitrate gave c H + / O ratios of 4 and 8 respectively [42-45,49,120,123- 1251; while, with a somewhat modified technique, the tH ' /2e- ratio of the NADP/ NAD transhydrogenase was found to be near 2 [126]. We concluded that in rat liver mitochondria, and in the mitochondrion-like bacterium Paracoccus deni- trificans, the t H + / O or c H f / 2 e - ratio per effective redox loop in the respiratory chain is 2.

A potential source of error in measurements of ApHo, and therefore of+ H+ (acid), may be introduced by the presence of solutes, such as Na' or phosphate, for which highly active proton-coupled porters are generally present in the membrane. As shown in Fig. 22 A, such porter-catalysed proton-coupled reactions may, under certain conditions, cause ApHo to collapse so rapidly after the respiratory pulse that t H + (acid) may be underestimated in spite of the usual extrapolation [123]. However, as such porter- catalysed reactions are electrically neutral, and as the H f / K f antiporter generally has a very low activity at pHo values of 7 or higher under the usual experimental conditions, the porter-catalysed reactions do not collapse ApKo, and they do not, therefore, cause underestimation of t H + (charge). This is the advantage of estimating both t H + (acid) and t H + (charge) from the ApHo and ApKo measurements [122], as I have described in outline here (Fig.21).

By using media that do not contain permeant ion species, or solute species for which there are highly active proton-linked porters, the extrapolated t H + (acid) and t H + (charge) values are found to be virtually identical, as shown in Fig. 21.


0.1

0.5 I

2.0

l B 5.0

0 2 0 40 60 Time (5)

0 2 0 40 60 Time (s)

Fig. 22. Effect of phosphate on measurements of +H' 10 for fl-hydroxybutyrate oxidation in rat liver mitochondria. From Mitchell & Moyle [123]. The numbers against the curves show the concentrations (mM) of P, added to the mitochondria1 suspensions. The arrows indicate the centre of the respiratory pulse. The experiments were done (A) at 25 "C and (B) at 5 "C. Further explanations in the text

Lehninger and colleagues (see [127]) have suggested that the endogenous inorganic phosphate of mitochondria, which equilibrates across the membrane during the anaerobic preincubation and gives a concentration of about 0.1 mM in the suspension medium in the usual 02-pulse experiments, causes a shortfall of c H + (acid) by as much as a factor of 2 by rapidly entering the mitochondria with H t ions via the phosphoric acid uniporter during the respiratory pulse, unless this porter is blocked with N-ethylmaleimide or with other -SH reactors. However, as Jennifer Moyle and I showed ten years ago (Fig.22A) [123], even the supplementation of the endogenous inorganic phosphate by a further 0.1 mM in the suspension medium does not collapse ApHo fast enough at 25 "C to cause a large shortfall of the extrapolated value of t H + (acid), a fact that has recently been confirmed by Brand, Reynafarje and Lehninger [128]. We showed, as illustrated in Fig. 22 B, that concentrations of phosphate ten times higher than the endogenous level can be tolerated if one inhibits the porters by working at 5 " C ; and, at all events, the c H + / O quotient for the NAD-linked oxidation of p-hydroxybutyrate is not raised above 6 when the phosphoric acid porter is inhibited in these experiments [123]. Moreover, as shown in Fig.21, there is not normally a significant discrepancy between c H + (acid) and -+K+ or c H + (charge), as there should be if dpHo were collapsed via an electrically-neutral porter system, such as the phosphoric acid uniporter.

As a direct test of Lehninger's idea [128] that protons are lost through the phosphoric acid porter in our usual respiratory pulse experiments, we used antimycin-treated rat liver mitochondria to set up the artificial ApH-creating system, represented by Fig. 23, employing diaminodurene as a specific external reductant reacting with the respiratory chain

system at the level of cytochrome c [49]. Added cytochrome c itself was used as reductant in control experiments (Fig. 23B). As indicated in Fig. 23A, correcting for a small respiratory leak through the antimycin-sensitive site on the ubiquinol side of cytochrome c, the estimated dH,+/O ratio was very near 2, regardless of whether the phosphoric acid porter was blocked by N-ethylmaleimide. We observed, inciden- tally (Fig. 23 A and B), that cytochrome oxidase translocated almost exactly 2 electrons inwards through the membrane per 0 atom reduced from the inner aqueous phase; that is to say, it had a +e-/O ratio near 2 but it had a c H + / O ratio of zero [49] (and see [48]), contrary to reports by Wikstrom and colleagues [129]. These observations seem to us to leave very little doubt that the c H + / O ratios corresponding to 2 per effective redox loop, measured by our 02-pulse method, are not spurious underestimates, as Leh- ninger and colleagues (see [127]) have persuasively suggested, and as other authoritative workers, including Boyer [130], Chance [131], Ernster [132], Klingenberg [91,92], David Nicholls [133], Skulachev [134], Slater [47] and Wikstrom [129], have tended to accept. On the other hand, our conclusion that the +-H+/2e- ratio is 2 per effective redox loop has been largely corroborated by respiratory pulse measurements (see [12,42,43,45]), with whole mitochondria and bacteria, for example, in the laboratories of Chap- pel1 [135], Garland [136-1391, Jones [140-1431, Packer [144] and Papa [145] (but see [146]), with sub- mitochondria1 vesicles, in the laboratories of Hinkle [147] and Papa [145], and with liposomes inlaid with respiratory chain complexes, in the laboratories of Hinkle and others (see [148]). Cox and Haddock [I211 have shown that an organic phosphate auxotroph of Escherichiu coli, which cannot transport inorganic phosphate, gives the same c H f / 2 e - ratio of 2 per

P. Mitchell 17

a ,Cu

-MalNEt +MalNEt - Mal NEt +MalNEt

AH:/O (Val) 1.98 (8) 1.98 (8) 0.00 (8) 0.00 ( 4 )

- A K i / O (Val) 2.03 (8) 2.0 1 (8) 1.98 (8) 1.95 (2)

- A H i / O (FCCP) 0.00 ( 4 ) 0.00 ( 4 ) 1.96 (10) 2.01 ( 4 )

Fig. 23. Lack of influence of N-ethylmaleimide on esfimation <$'the AH: 10 ratio in a well-controlled system. From Moyle and Mitchell I48.491. The diagrams show oxidation of diaminodurene (DADHz) and added cytochrome c (c) in rat liver mitochondria inhibited with antimycin (ant). Other symbols as in Fig.5. The numbers in the table below each diagram show the mean values of the numbers of protons and K+ ions (n) entering the medium per 0 reduced in the presence of valinomycin (Val) or in the presence of carbonylcyanidep-trifluoromethoxyphenylhydrazone (FCCP) with and without N-ethylmaleimide (MalNEt). Further explanations in the text

effective redox loop as its parent and other E. coli strains, thus confirming that phosphate translocation does not cause underestimation of the t H ' / O or c H t / 2 e - ratios in the usual 02-pulse experiments.

Since we conclude that the c H + / O or t H f / 2 e - ratios, corresponding to 2 per effective redox loop, are not normally falsified by activity of the N-ethylmaleimide-sensitive phosphoric acid porter, the interesting question remains : how does N-ethylmaleimide increase the number of protons translocated per oxygen atom reduced by the respiratory chain system under certain conditions?

When I first drew attention to this remarkable effect some years ago [125], I suggested that N-ethylmaleimide treatment favoured the involvement of the complete respiratory chain from NADPH to oxygen, because Jennifer Moyle and I had found that N- ethylmaleimide largely inactivated several NAD-linked enzymes, and also inhibited succinate dehydrogenase, but did not inhibit the NADP-linked isocitrate dehydrogenase or the NADH oxidase or NAD(P) transhydrogenase [126].

Fig.24 summarises some of our earlier and some more recent observations on the effects of N-ethylmaleimide on t H + / O and -+e-/O ratios, measured by the respiratory pulse technique, under various conditions. It is important to realise that the molecular quantities of the endogenous reductants, citrate + isocitrate, NADPH, NADH and ubiquinol, in the mitochondria are each several times as great as the quantity of oxygen usually injected (1 pg atom O/g of mitochondrial protein) to produce the respiratory pulses. As shown in A, in a sucrose/potassium sulphate medium, N-ethylmaleimide raises the stoicheiometry from 6 to 8. B shows that added citrate does not increase the stoicheiometry above 8 in the presence of N-ethylmaleimide, but added L-malate, which is a

A H i / O 0 2 4 6 8

A. Sucrose + 10 rnM K,SO,

8 . Sucrose + 10 rnM KzSO, zF$r&e

F- (- e-10)

C. Sucrose + 10 mM choline chloride + citrate

D. Sucrose + 10 rnM MgSO, + oxoglutarate

E . Sucrose + 5 rnM MgCI, frotenone

F. Sucrose + 10 rnM MgSO, + DADH, + antimycin

Fig.24. Dependence of + H '10 or j e - 1 0 ratios on Juspension medium and N-ethylmaleimide in rat liver mitochondria. Previously unpublished data of Moyle and Mitchell (1971 -1978). The inito- chondrial suspension media (pHo = 7, 25 "C) routinely contained 3.3 mM glycylglycine, and the general experimental methods were as previously described [120,123]. Empty blocks, N-ethylmaleimide absent; filled blocks, N-ethylmaleimide present. Further explanations in the text

very active NAD-linked substrate, brings it down to 6.5, despite the presence of N-ethylmaleimide. In a sucrose/choline chloride medium with added citrate (C), the stoicheiometry is 8 with or without N-ethylmaleimide. But (D) when oxoglutarate, the oxidant of the NADP-linked isocitrate dehydrogenase, is present in a sucrose/magnesium sulphate medium, the stoicheiometry is close to 6, and it is only mar- ginally increased by N-ethylmaleimide. As shown in E, in a sucrose/magnesium chloride medium, with rotenone present to inhibit the NADH dehydrogenase, the stoicheiometry without added substrate is 4, corresponding to that previously observed for ubiquinol oxidation by Garland and by Papa (see [44,145]); this is unaffected by N-ethylmaleimide under these conditions. Finally, F shows that, in a sucrose/mag-


(Isocit deH)

+ isocit ZH+oxalosucc 4 CO, ,((:; H ) e NADP OxOglut

7H'

NAD A + ~ t

FMN 2H (NADH deH)

Rotenone _ .

+ZH+

2Ht Antirnycin

FeSLOrH +b 2kH -2H'

. WHZ0 -====a

Fig. 25. Mitochondria1 respiratory chain oxiclising isocitrate. Symbols as in Fig. 5 and 6

nesium sulphate medium, the oxidation of diaminodurene, via cytochrome c and cytochrome oxidase in the presence of antimycin, gives zero proton translocation and a +e-/O ratio of 2, independently of the presence or absence of N-ethylmaleimide. Under no circumstances have we ever been able to observe stoicheiometries consistently greater than 8.

Fig. 25 summarises the general conclusions that I have drawn from these observations and from many other related observations that are too numerous to mention in the present paper. First, the observed stoicheiometry of 2H' ions translocated per bivalent reducing equivalent traversing each effective redox loop encourages one to persevere with attempts to explain the protonmotive properties of respiratory chain systems in terms of direct or semi-direct chemiosmotic mechanisms that are relatively explicit and orthodox biochemically. Such mechanisms simply depend upon vectorially organised ligand-conducting catalytic domains, in which the metabolic pathways are spatially-defined pathways of chemical-group diffusion in the direction of real forces, given by the corresponding vectorial group-potential gradients [20, 251. Second, the tightness of channelling of the ligand- conduction processes through the respiratory chain (I mean the degree to which the NADP, NAD and ubiquinone molecules that act as links in the chain, tend to equilibrate with the relatively large capacities of their respective pools) depends on the ionic strength, ionic composition and pH of the medium (and also on the presence or absence of N-ethylmaleimide) amongst other factors. This tightness of channelling of the ligand-conduction processes that influence the

observed +H+/O value might depend mainly or, perhaps, entirely on the ratios of the velocity constants of reduction and oxidation of the NADP, NAD and ubiquinone in equilibrium with their respective pools. However, as I have indicated rather crudely in Fig. 25, there are features of our experimental observations which suggest that the tightness of channelling may also depend on the binding and specific positioning of the NADP, NAD and ubiquinone (Q) in specific ligand-conducting domains that are thus partially iso- lated from the respective pools of NADP and NAD in the aqueous medium of the matrix and of ubiquinone in the lipid medium of the cristae membrane. In this context, it may be significant that the A-side and B- side specificities for reduction or oxidation of the nicotinamide ring of NADP and NAD by isocitrate dehydrogenase, the NAD(P) transhydrogenase and the NADH dehydrogenase [149,150] have the sequence A-B-A-B, as indicated in Fig. 25. Thus, hydrogen translocation could conceivably occur from the A to the B side of the nicotinamide rings without appreciable diffusional mobility of the NADP and NAD in specific ligand-conducting domains or cre- vices in the respiratory chain complexes. The recent discovery by King and colleagues of specific ubiquinone-binding proteins in the respiratory chain [59 J is likewise relevant, and is consistent with observations, for example, by Gutman [I511 on the functional compartmentation of mitochondria1 ubiquinone during simultaneous oxidation of two substrates.

At all events, the question of the enhancement of proton translocation by the - SH reactor N-ethylmaleimide under certain conditions - which is still causing confusion, and not a little turmoil, in the field of bioenergetics - is really a question of metabolism. The effects of N-ethylmaleimide can generally be mi- micked by certain, purely physical, conditions that induce an increase in the tightness of channelling of reducing equivalents through the respiratory chain system from isocitrate or NADP to oxygen. As yet, the mechanism is incompletely understood, but the experimental observations raise very interesting new questions about the functional and topological organisation of the respiratory chain complexes and Krebs' cycle enzymes (see [152]) to which we may reasonably expect to find the answers in the not too distant future.

I would like to end, as I began, by paying tribute to Sir Hans Krebs for being a great pioneer and builder of knowledge about metabolism, and for generously taking care to pave the way for further explorations of metabolic pathways and mechanisms.

I thank Dr Jennifer Moyle and Mr Roy Mitchell for helpful discussions on the subject of this paper, and also Dr Bernie Trum- power for informing me, prior to publication of his results, that his work indicated that factor OxF may be the Rieske FeS protein. I am indebted to Dr Jennifer Moyle, Mrs Stephanie Key and Mr

P. Mitchell 19

Robert Harper for help in preparing the manuscript. The general financial support of Glynn Research Ltd is gratefully acknowledged.

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P. Mitchell, Glynn Research Institute, Glynn House, Bodmin, Cornwall, Great Britain, PL30 4AU

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