Eur. J. Biochem. 166,489- 504 (1987)
i(" FEBS 1987
Review
The mechanism of the conservation of energy of biological oxidations
E. C. SLATER
Laboratory of Biochemistry, University of Amsterdam and Department of Biochemistry, University of Southampton
(Received January 5/March 24, 1987) - EJB 87 0008
In a recent review 111, it was pointed out that in tracing
the development of concepts of the mechanism of respiratorychain phosphorylation over a period ofmore than thirty years,
four distinct eras can be distinguished in succession, during
which one or at most two hypothesis dominated the thinking
of workers in this field. These eras were termed: (a) the era
of the non-phosphoryhted intermediate; (b) the era of the
'conformational' hypothesis; (c) the era of the chemiosmotic
hypothesis (delocalized protons); (d) the era of localized protons.
Implicit in this statement is that we are now in the era of
localized protons. However, a number of caveats need to be
entered. First, since there is a considerable time lag before
a current hypothesis enters the text books, these are still
practically unanimous in presenting only the chemiosmotic
hypothesis. For most of those not directly involved in bioenergetic research, and indeed for many who are, we are still in
the era of this hypothesis, even though the possibility of some
form of localized protons as the intermediate of oxidative
phosphorylation dominates current discussion of the mechanism. Secondly, the review in question dealt specifically with
the mechanism of ATP synthesis and not with other ways
in which energy is conserved, that may still be adequately
described by the chemiosmotic hypothesis. Thirdly, in that
review a hypothesis was proposed that contains features of
all four hypotheses listed [l].
In this review, 1 shall not repeat the description of the
first two hypotheses nor of the evidence in support of the
chemiosmotic hypothesis, in which AFH + (difference in
electrochemical potential of protons across the energy-transducing membrane) is the obligatory intermediate in oxidative
phosphorylation and H + is directly involved in the synthesis
of ATP, but shall take this hypothesis as the starting point.
ORIGINAL CHEMIOSMOTIC HYPOTHESIS:
DIRECT INVOLVEMENT OF PROTONS
IN ESTERIFICATION REACTION
In the original formulation of the chemiosmotic hypothesis (2, 31, hydrogen ions play the dominant mechanistic role.
Specific loops transferring hydrogen atoms from one side of
the membrane to the other and electrons in the opposite
direction were postulated in order to explain proton translocation linked with electron-transfer reactions in respiration and
photosynthesis. For example, site-1 phosphorylation was
thought to be brought about by hydrogen transfer via the
flavin of NADH dehydrogenase, followed by electron transfer
in the opposite direction through an iron-sulphur protein.
The identification of a suitable hydrogen carrier in site-2
phosphorylation gave some difficulty, but this was resolved
with the postulate of the Q cycle [4] which has been more
successful than earlier schemes as a description of the pathway
of redox equivalents in this segment of the respiratory chain
(see 151).
In the original hypothesis, protons were also considered
to be involved directly in the esterification reaction:
P;
-+
ATP
+ H20.
Until 1972, it was thought that, just as in the chemical
hypothesis, an anhydride X-I, formed between hypothetical
functional groups XH and IOH in the ATP synthase, is an
intermediate in this reaction
P;
Correspondence to E. C. Slater, Department of Biochemistry,
School of Biochemical and Physiological Sciences, Medical and Biological Sciences Building, Bassett Crescent East, Southampton,
England SO9 3TU
Abbreviations. DCCD, dicyclohexylcarbodiimmide; SI3, 5chloro-3-tert-butyl-2'-chloro-4-nitrosalicylanilide;FCCP, carbonyl
cyanide p-trifluoromethoxyphenylhydrazone;Q, ubiquinone; QH,,
ubiquinol; PQ, plastoquinone; PQH2, plastoquinol; F1.Fo, ATP
synthase.
Enzymes. ATP synthase; ubiquinol: cytochrome-c oxidoreductase
(EC 1.10.2.2); NADF3:ubiquinone oxidoreductase (EC 1.3.99.3);
cytochrome-c oxidase (EC 1.9.3.1); plastoquino1:plastocyanin oxidoreductase (EC 1.10.99.1).
+ ADP- + 2Hf
+
+
+
ADPX-I + ATP X- + I 0
X102H' -+ X-I
H2O.
+
+
+
In 1972, Mitchell [5a] suggested as an alternative that X-I,
X- and 10- might be represented by ATP, ADP and Pi,
respectively, and that the catalytic site on the enzyme is
accessible to ADP, Pi and ATP to one side and to H 2 0 and
H + to the other. This idea was elaborated further in later
papers [6 - 81, in which ATP synthesis is thought of as being
brought about by a Apw+-drivenATP/(ADP + Pi) antiporter
in the catalytic domain.
In 1974, Mitchell [9] (see also [9a]) suggested the formation
of a trigonal bipyrimidal pentavalent transitional phosphorus
intermediate:
490
ADPO- + P-OH + 2H'
ADPO- - P - OH;
+ ADPO- - P i - OH2 + ADPO-P
H20,
where P represents O=P-(O-)2.
--f
+
I
Although, in a scalar reaction, the equilibrium is far to
hydrolysis, Mitchell suggested that if the side of approach of
the ADPO- were basic and electrically negative, while the
other side of the phosphorus centre were acidic and electrically
positive, the nucleophilic-attacking capability of the unprotonated ADPO- group would be preserved, while the
leaving capacity of the -0- group would be enhanced by
conversion to an oxonium group, and there would be a poising
effect in the direction of ATP synthesis.
Boyer [lo, 111 criticised this proposal, first because the
formation of O=P-(O-), and the addition of a second proton
to give -OH: are energetically most unfavourable reactions
and, secondly, because the mechanism is not line with experience with other phosphatases which gives no evidence for the
formation of pentavalent intermediates. The rapid exchange
of ''0 between phosphate and water, which is cited by
Mitchell 191 as one of the bases for his proposal, is readily
explained by the reversible cleavage of a P - 0 bond in the P-OPO; substrate.
Mitchell has not accepted these criticisms [6, 121.
FIRST MODIFICATION
OF THE CHEMIOSMOTIC HYPOTHESIS:
A f i H + IS NOT REQUIRED
FOR THE ESTERIFICATION REACTION
It is not surprising, indeed it would be an unicum in science
if this were not the case, that in the 25 years since it
was originally proposed new findings would necessitate
modifications of the original chemiosmotic hypothesis.
The minimum modification that is now required is to
abandon any direct role of A&+ (or any other postulated
intermediate of oxidative phosphorylation) in the esterification reaction.
In 1973, Boyer [13] and Slater [14] suggested that the
energy for ATP synthesis is required not for the esterification
of ADP by phosphate per se but for the dissociation of ATP.
This postulate is now strongly supported by the following
evidence.
a) ATP binds to the enzyme catalysing ATP synthesis (ATP
synthase or F,.Fo) with a binding constant of 10l2 M-'
[15]. The dissociation of ATP, phosphate and ADP from the
enzyme is very slow (7 x
s-', 2.7 x
s-' and
s-', respectively) [15]. Binding of ATP to a second site on the
ATP synthase causes these rates of dissociaton to be increased
by about 106-fold [36].
b) The equilibrium constant of the reaction
F1 ADP(Pi) G F1 . ATP
is about 2 1151.
c) As a consequence of these kinetic constants, enzymebound ATP is synthesized from ADP and Pi added to F1
without any other source of energy [15].
d) The exchange of l80between H 2 0 and Pi, which is a
measure of the esterification reaction, is insensitive to
uncouplers [ 131.
e) During respiration, an energy-dependent dissociation
of about one third of radioactive ATP bound to F1 in submitochondrial particles takes place at a rate (k = 2 s-') more
than five orders of magnitude greater than during single-site
catalysis of ATP hydrolysis by F1 (k = 7 x
s-') [16a].
'
That energy is necessary for the dissociation of ATP from
its site of formation is now generally accepted, also by Mitchell
Pi) antiwho expresses this as an AFH+-drivenATP/(ADP
porter in the catalytic domain. The question next arises
whether the protons, driven by dfiH+ , interact directly in the
region of the ATP-binding site or bind to a site at a distance
from it. Agreement has not been reached on this point.
Mitchell [8] has proposed what he calls a 'rolling well and
turnstile' mechanism in which it is envisaged that the injection
of protons through a proton gate in the y subunit into a
hydrolytic site, formed by the juxtaposition of CI and fl subunits, causes the development of a torque by mutual net
repulsion between the subunits, which is intimately associated
with the antiport process and the abstraction of 0'- from P,
and ADP to yield ATP.
On the basis of homology with adenylate kinase, Mildvan
and co-workers [16b] have proposed that protons bind to
proton-accepting amino acid side chains involved in binding
Mg-ATP in such a way as to lead to dissociation of ATP.
Evidence in favour of the view that protons bind at a
distance from the ATP-binding site comes from Penefsky's
observation that binding of the classical inhibitors of oxidative
phosphorylation (DCCD or oligomycin) to ATP synthase
drastically lowers the binding of ATP to the first catalytic site
on F1 [17]. DCCD binds to a specific glutamic acid residue in
one of the subunits of that part of the ATP synthase (subunit
c of Fo) that is embedded in the mitochondria1 membrane
[18]. The distance between the DCCD-binding site and ATPbinding site is at least 2 nm [19]. This shows that conformational changes in subunit c are transmitted to the subunit of F1 containing the ATP-binding site (the fl subunit).
Penefsky [17] has suggested that two separate conformational changes, of the type originally postulated by
Boyer [20], lead to the dissociation of ATP from the ATP
synthase. First, cooperative interaction between catalytic sites
on separate fl subunits, whereby binding of ADP and
phosphate in the catalytic site of one subunit facilitates the
release of product ATP bound in a catalytic site in a separate
subunit; secondly, a change in conformation of subunit c in
Fo that is transmitted to the fl subunits of F1 [16a, 171.
An additional argument in favour of the view that the
primary effect of A,&+ is at a distance from the ATP-binding
site (or, as it is often termed, is 'indirect') comes from the
observation that, in alkalophilic [21, 221 and halophihc [23]
bacteria and in uncoupler-resistant mutants of Bacillus megaterium [24, 251, ATP synthesis can take place when A D H + is
very low. Membranes of an alkali-tolerant organism, Vibrio
alginolyticus, possess a respiration-dependent Na' pump [22,
261 that can be utilized for the uncoupler-insensitive synthesis
of ATP [22]. Presumably, Na+-binding sites are present in the
ATP synthase and in one of the redox enzymes in this organism, and it seems likely that binding of Na' to its binding site
on the ATP synthase causes the same type of conformational
change in the ATP-binding site as binding of protons in other
organisms. It seems unlikely that this would be brought about
by binding of Na' to groupings involved in binding of
MgATP, as suggested by Mildvan [16b] for protonmotive
systems.
It still remains open whether protons translocated by those
redox-equivalent-transferring enzymes in respiration and
photosynthesis that contain hydrogen carriers (NADH : Q
oxidoreductase, QH, :cytochrome c oxidoreductase in respiration, PQHz :plastocyanin oxidoreductase in photosynthesis) are the result of a direct loop-type mechanism or of an
indirect mechanism, as a consequence of a change of protein
+
49 1
conformation. Although the direct mechanism linked with the
Q cycle [4] is attractive, it has not been proven; Papa [27]
favours an indirect mechanism. The situation also appears to
be completely open so far as the NADH:Q oxidoreductase
is concerned. Ragan [28] has proposed a cyclic mechanism,
somewhat analogous to the Q cycle, operating in this enzyme,
in which protons are derived from reduced flavin. However,
the universality, at least, of such a mechanism is brought into
question by the isolation from Vibrio alginolyticus of an N a t activated NADH :Q oxidoreductase [26]. In this connection,
it would be interesting to know if a Q cycle is operative in this
organism in the oxidation of QH,.
In one case at least, cytochrome c oxidase, an indirect
mechanism would appear to be the more likely [29], since there
are no obvious hydrogen carriers in this enzyme, although
Mitchell has postulated an 0 loop and an 0 cycle, analogous
to the Q cycle, for this reaction [30]. Here, HzOis the hydrogen
carrier.
To conclude this section, it can be taken to be established
that A P H t is not necessary for the esterification reaction and
there is evidence suggesting that its requirement for the dissociation of ATP from the catalytic site is by virtue of an
‘indirect’ effect at a distance from this site, although the
alternative view that protons act on groupings ‘directly’ involved in MgATP binding cannot be excluded. So far as
proton translocation linked to electron transfer is concerned,
it remains to be proven whether the protons are ‘directly’
derived from hydrogenated intermediates (FMNHz, QH,,
H20) or ‘indirectly’ by dissociation of protons from amino
acid side chains as a result of a protein conformation change.
SECOND MODIFICATION
OF CHEMIOSMOTIC HYPOTHESIS:
IN OXIDATIVE PHOSPHORYLATION,
A/&+ IS NOT ONLY AN INTERMEDIATE, OBLIGATORY
OR NON-OBLIGATORY
The first modification to the chemiosmotic hypothesis,
indirect in place or direct utilization of protons, is not
considered by most observers to be fundamental (see, for
example, [31]). In the last ten years or so, however, evidence
has accumulated that seems to suggest that A/&+ is not simply
an intermediate of oxidative phosphorylation. This is based
on kinetic studies with mitochondria, submitochondrial
particles or chromatophores in which the effect of varying the
concentration of redox enzymes, ATP synthase or the putative
intermediate ( A P H + ) on the steady-state rate of ATP synthesis
(in so-called state 3) is measured.
Kinetic studies
One of the arguments persistently brought forward against
the proposition that A F H + is the obligatory intermediate of
oxidative phosphorylation is based on observations made
with chromatophores and mitochondria that a unique
relationship does not exist between the force A P H + and the
flux rates of oxidation and phophorylation, but that it is
dependent on the way in which the force and flows are varied
[l, 321. In a recent study [33], however, the same steep
dependence on the membrane potential Ay, (under conditions
in which ApH was small and virtually constant) of Jo (oxidation flux) and Jp (phosphorylation flux) was found with ratliver mitochondria whether Jo was increased from state 4 by
A
P
lkJ/mol 1
Fig. 1. Dependence of the rate of oxidative phosphorylation (J,) with
succinate on A I ~ varied
,
by addition of FCCP (@) or malonate ( 0 ) .
Reproduced with permission from [33]
adding ADP or uncoupler, or whether J p (in state 3) was
lowered by adding malonate or uncoupler (see Fig. 1).
If, as seems acceptable to the reviewer, this result is
considered more reliable than previous measurements, this
objection to the chemiosmotic hypothesis disappears. However, Zoratti et al. [34] have recently compared the dependence
of the proton flux through the ATP synthase on a driving
force, composed mainly of a potassium diffusion potential,
with the relationship between the rate of phosphorylation and
A P H + when respiration was inhibited by different concentrations of malonate. Although the two functions are in good
agreement at low A P H + , the maximum proton flux through
the ATP synthase is much slower than required to account for
the rate of oxidative phosphorylation.
If A D H + is an obligatory intermediate of oxidative
phosphorylation, formed by a primary redox pump and
utilized by a secondary ATP-synthesizing pump, one would
expect that inhibition of the primary pump would lead not
only to inhibition of ATP synthase but also to a decrease in
the magnitude of A P H + . Fig. 1 shows that this is the case, but
most importantly how small the decrease of A P H + is (80%
inhibition of the rate of ATP synthesis is associated with only
a 7% decline in A f l H + ) . Thus, an appreciable inhibition of
Jpcould be associated with an undetectably small decline in
A,&+,
which would, at first sight, appear to be inconsistent
with A P H + being an intermediate, but need not necessarily be
so (cf. [35]).
It remains, however, to be examined whether such a steep
relationship between flows and forces, which amounts to a
virtual gating effect of Aj&,+, is itself consistent with the
chemiosmotic hypothesis. We shall return to this later.
Titrations with inhibitors
Herweijer et al. [36] found that when the concentration of
active ATP synthase molecules in submitochondrial particles
is varied by irreversible inactivation by the photo-affinity label
8-azido-ATP or that of NADH :Q oxidoreductase molecules
by addition of rotenone or piericidin, the rate of oxidative
phosphorylation or of the ATP-driven reduction of NAD’
492
NADH
+ G- 0 2 -+
NAD
’ + H2O +
AfiH+
and
0
50
100
% INHIBITION ATP HYDROLYSIS
Fig. 2. Inhibition of‘ ATP-driven reduction qf‘NAD+ by succinate (‘retwsal’) as a ,ficnction of’ the degree of‘ irreversible inactivation ef ATP
synthase in .~iihnritocliondriuIparticles b,v photoinactivation with
8-azido-ATP,ineasuwd by the inactivation o f t h e ATPase activity (361.
Previous work [38] shows that residual ATPase activity is proportional
to the concentration of non-inactivated molecules of ATP synthase.
Closed and opcn symbols refcr to different ways of measuring ATPase
activity. Reproduced with permission from [36]
by succinate (see Fig. 2) is proportional to the concentrations
of residual active enzyme. This is similar to results reported
previously by several authors (e. g. [37]).
At first sight, this result is not consistent with the existence
of any ‘pool’ intermediate, whether it is ADf%+or whatever,
operating between the two enzymes. The most general
formulation of ‘pool’ kinetics is given by the sequence:
Form I
ki
ki
-+
Form 11 + Form I
in which successive enzyme systems convert the compound
acting as a pool from one form to another and to its original
form, respectively. If p equals the total concentration of the
species undergoing transformation and x that of the intermediate form 11, and if the concentrations of the two forms
are much lower than the K,,, values of the respective enzymes,
then dxldt = k , ( j - x ) - k 2 x , where k l and k2 include the
concentrations of the respective enzymes. In the steady state,
when dxldt = 0. .Y = k;p,i(k;
k2),and v (the velocity) is
given by
+
1‘ = k2.Y =
k;kzp/(k;+ k2).
(1)
If we define
as the rate of the intermediate-forming
reaction when the concentration of the intermediate is zero,
c1 = k;p, i. e. k’, = vljp. Similarly, if u 2 is the rate ofconversion
of the intermediate back to its original form when the concentration of the latter is zero. k2 = v2/p.Substituting in Eqn (1)
yields the ‘pool’ kinetics equation [39]
1‘ = L’102/(211
+
212)
.
For the chemiosmotic hypothesis, where A f i H + is the intermediate, it is perhaps difficult to envisage exactly what corresponds to Form 1. However, if we now consider p to be the
maximum ~ I f i , that
~ . can be developed when electron flow
becomes inhibited owing to back pressure by A f i H + (state 4 or
static head), and .Y = A f i H + , then ddfiH+/dt= k l (AfiH+,,, - / ( ; A & . , which leads to the identical ‘pool’-kinetics
equation.
According to thc chemiosmotic hypothesis, the ‘pool’forming and ‘pool’-destroying reactions in the coupled oxidation of NADH are
+ ADP + Pi
+
H20,
ATP
respectively, ignoring the A f i H + stoichiometry.
The proportionality between the rate of ATP synthesis and
concentration of active ATP synthase molecules is consistent
with the ‘pool’-kinetics equation only if u2 4 ul. However, the
proportionality between the rate of ATP synthesis and the
concentration of active NADH :Q oxidoreductase molecules
is consistent with this equation only if v2 % u l . Clearly, both
conditions can not be met simultaneously, which leads to the
conclusion that, on the basis of this analysis, A f i H + cannot be
the intermediate of oxidative phosphorylation. As Westerhoff
and Chen [40] have put it, ‘electron transfer and ATP synthesis
(appear to) sense each other more directly than just through
AjiHt’ .
The proportionality found between the rate of steady-state
ATP synthesis and concentration of active redox enzyme and
ATP synthase is an extreme example of the ‘double inhibitor’
approach introduced by Baum et al. [41], who argued that,
on the basis of the chemiosmotic hypothesis, an inhibitor of
one of the enzymes would be expected to have relatively less
effect on the overall reaction when the second enzyme is
already partially inhibited. To use the now popular language
of ‘control strength theory’, if the flux-control coefficient of
one enzyme is increased (by making it more ‘rate-limiting’),
that of the second enzyme would decrease. Experimentally, it
has been found by several authors [42 -441 that, contrary to
what Pietrobon and Caplan [45] have called the ‘rule of thumb’
expectation, the relative inhibition by a given concentration
of one enzyme is the same whether or not the other enzyme
is partially inhibited. Applying a model [46] in which flows
(electron transfer or ATP synthesis) are assumed to be proportional to forces (AG nAfiH+),where n is the H’ /e or H ’/
ATP stoichiometry), Pietrobon and Caplan [45] have confirmed the expectations of the ‘rule of thumb’ or the ‘pool’kinetics model.
However, in two important papers, Pietrobon and Caplan
[45,47] have pointed out that reality departs from such models
in two respects. First, particularly important in submitochondria1 particles and chromatophores, protons ‘leak’
across the membrane down the electrochemical gradient by a
process independent of ATP synthesis. Moreover, there is
reason to believe [48] that ‘slippage’, i. e. enzyme turnover
without proton pumping, occurs in the redox enzymes.
Secondly, away from thermodynamic equilibrium, the flows
are not expected to be proportional to the forces, but there is
a sigmoidal relationship between the two parameters.
The effect of a ‘leak’ is to cause a higher inhibition of the
rate of ATP synthesis by a given concentration of inhibitor,
and, in the limiting case with a high ‘leak’, the ATP synthase
can be titrated almost proportionally. Certainly, in the experiments ofHerweijer et at. [36], for example, the ‘leak’ was quite
high, and possibly it was sufficiently high to accomodate
the experimental findings, on the basis of the chemiosmotic
hypothesis.
That the relationship between forces and flows is far from
proportional has already been illustrated by the data shown
in Fig. 1. The straight line obtained experimentally is presumably part of a steep S-shaped curve, since it must pass near
the origin (note that this is far to the left of the ordinate drawn
in Fig. 1), i.e. Jp= 0 when Ay! (taken to be equal to d,CH+)
= 0, since the ATPase activity is low when substrate oxidation
AfiH+
+
493
S,3conc. (nM)
5
10
15
20
GRAMlClDlN conc. (nM1
Fig. 3. Effect of rotenone and oligomycin on titration with S13 of
ATP-driven reduction of NAD’ by succinate (‘reversal’) catalysed by
suhmitochondriulpurticles. (D) Control; ( 0 )in presence of rotenone;
(‘I)
in the presence of oligomycin. In the inset, the results with
the inhibitors are normalized to 100% in the absence of uncoupler.
Reproduced with permission from [56]
Fig. 4. Effect of rotenone on titration with gramicidin on ATP-driven
reduction of NAD’ by succinate (‘reversal’) catalysed by suhmitochondrialparticles. (m) Control; ( 0 )(+) in the presence of two
concentrations of rotenone. In the inset, the results are normalized as
in Fig. 3. Reproduced with permission from [56]
is fully inhibited, and Jp must eventually reach saturation at
high AfiH + . Indeed, as Pietrobon and Caplan [49] have pointed
out, sigmoidal flow-force relationships are a general characteristic of models of ion pumps or cotransport systems involving cyclic reaction schemes with one voltage-dependent
step. It is important, too, to note that such relationships are
also characteristic of any enzyme-catalysed reaction in which
the total concentration of substrate product is unchanged
succinate is decreased proportionally to the inhibition of the
reaction by either an inhibitor of electron transfer (rotenone)
or of ATP synthase (oligomycin). Margolis et al. [57] obtained
similar results already in 1967 with mitochondria in which
oxidative phosphorylation was uncoupled by FCCP and the
redox enzymes were inhibited by rotenone, malonate or cyanide. Herweijer et al. [56] found that, in contrast to the results
with the protonophoric uncouplers, the titre of the unambiguous pore former, gramicidin, is not affected by inhibition by
rotenone (Fig. 4)’. It is concluded that, whereas gramicidin
uncouples in submitochondrial particles and intact mitochondria preferentially by dissipation of the bulk A P H + , the
typical protonophoric type of uncouplers interfere with a
direct energy transduction between the ATPase and redox
enzymes (see later). Significantly, van der Bend et al. [59] have
shown that light-driven ATP synthesis in liposomes containing
bacteriorhodopsin and ATP synthase, a system expected to
follow a chemiosmotic mechanism, the amount of protonophore required to uncouple ATP synthesis is independent
of the degree of inhibition of the ATP synthase. These
conclusions have also been challenged. Some of the objections
seem to based on a misconception. O’Shea and Thelan [60],
for example, claimed that the results obtained by Herweijer
et al. [56] would be expected, even on the basis of the
chemiosmotic model. However, as van de Bend and Herweijer
[61] have pointed out, the treatment of O’Shea and Thelan
leading to this conclusion implies that the rate of ATP synthesis is dependent on the concentration of active ATPase
molecules and independent of A D H + ,which is, in fact, identical
+
POI.
It is possible, on the basis of a six-state model of such
a pump, to simulate inbibition curves in which the rate of
phosphorylation is virtually proportional to the concentration
of residual ATP synthase molecules even in systems with low
‘leaks’.With a sufficiently high ‘leak’, identical inhibition titration curves may be obtained whether or not the other enzyme
is inhibited. Moreover, the experimentally observed insensitivity of ADH+ to inhibition of either primary or secondary pump can be simulated on the basis of this model [51].
From these analyses of Pietrobon and Caplan, it is clear
that it is difficult, if not impossible, unequivocally to draw
the conclusion from double-inhibitor [41] and related [36]
experiments that AgH+is not an obligatory intermediate of
oxidative phosphorylation (see also 152, 531). It is possible
that, by accurately measuring the magnitude of the ‘leak’
and determining the precise flow-force relationship, a more
rigorous comparison between the experimental findings and
those predicted by the chemiosmotic hypothesis will become
possible, but this has still to be achieved.
Effect of inbibitors on titration by uncouplers
A second type of experiment, in which the effect of inhibitor on the effectiveness of uncouplers of oxidative
phosphorylation are examined, was introduced by Hitchens
and ~ ~ [54,
1 551,
1 using chomatophores. ~h~ results of a
similar type of experiment using submitochondria1 particles,
carried out by Herweijer et al. [56], are illustrated in Figs 3
and 4. With uncouPlers ofthe type O f S i 3 or2,4-dinitroPhenol,
the amount of uncoupler required for complete inhibition (Or
90% inhibition) of the ATP-driven reduction of NAD’ by
The lack of effect of inhibitors on the gramicidin titre is found
only with inhibitors of the secondary pump: the titre is decreased
when the primary pump, the ATP synthase in this case, is inhibited
[561. Hitchens and Kell [55] found that, in chromatophores,
gra m idin, just like protonophores, uncouples with proportionally
higher efficiency when the secondary pump is inhibited. A possible
explanation [56] of the different behaviour of gramicidin in submitochondria1 particles and chromatophores is that, due to the small
diameter of the latter and the resulting curvature of the membrane,
gramicidin dimers cannot be formed and that gramicidin in its monomeric form possibly acts as a carrier-type ionophore [%].
494
with the conclusion of Herweijer et al. and in conflict with the
chemiosmotic hypothesis. Thus, O’Shea and Thelan are in
reality in complete agreement with Herweijer et al. although
they do not seem to realize it.
Pietrebon and Caplan [45,62] agree with van de Bend and
Herweijer [61], but challenge the conclusion of Herweijer et
al. [56]. They point out that, since inhibition of the A f i H t utilizing reactions leads to an increased steady-state level of
AF,, +, the rate of dissipation of A f i H + through the membrane
is increased so that a lower concentration of protonophore is
sufficient to bring about the same degree of uncoupling. In
other words, the relative inhibition of the rate of the energyconsuming reaction by a given concentration of uncoupler is
greater in the presence of an inhibitor of the secondary pump
that it is in its absence. Pietrobon and Caplan illustrate their
conclusion with simulated curves based on a linear model of
chemiosmotic coupling.
Although Pietrobon and Caplan’s [45, 621 conclusion is
undoubtedly correct in a qualitative sense, the model does
not account for the most important quantitative findings of
Herweijer and co-workers [36, 561 and others [54, 55, 571,
namely that both the rate of the coupled reaction and the
amount of uncoupler required for a given degree of
uncoupling are proportional to the concentrations of both the
primary and secondary pumps. Also the model predicts that
the amount of uncoupler required to abolish ATP synthesis
‘completely’ (or by 90% say) would be unaffected by inhibition of the secondary pump, whereas experiment shows that
the amount declines proportionally with the activity of the
secondary pump. Finally, Herweijer [56, 631 has shown, by
entering parameters from her own experimental data into the
model of Pietrobon and Caplan [45, 621, that the calculated
increase of A$,, a t 90% inhibition of the secondary pump is
only 1%, compared with 75% calculated with the parameters
used by Pietrobon and Caplan. With any reasonably realistic
parameters, the simulations show that, although there is a
higher uncoupling efficiency when the secondary pump is
inhibited, the calculated effect is negligibly small compared
with that found experimentally. The higher uncoupling
efficiency of gramicidin when the primary pump (the ATP
synthase) is inhibited can, however, be simulated by this
model, which is in conformity with the conclusion that
gramicidin acts ‘purely’ by a chemiosmotic mechanism.
The more realistic model used in a later paper by Pietrobon
and Caplan [47], based as it is on the sigmoidal relationship
between flow and force, fails also to simulate the experimental
situation with protonophores. This is illustrated in Fig. 5 for
the three simulations made by these authors. The simulations
are for uncoupling of oxidative phosphorylation in the absence (Fig. 5A) and presence (Fig. 5B) of inhibitors of the
secondary pump. The experimental points, taken from Fig. 2,
relate in Fig. 5 B to inhibition by oligomycin and rotenone of
the primary and secondary pump, respectively, in the ATPdriven reduction of NAD ’by succinate. The simulation based
on the linear model 1621is also included in Fig. 5. Only simulation C approaches the experimentally observed curve in the
absence of an inhibitor, and in no case is the eperimentally
observed effect of an inhibitor on uncoupling by protonophores simulated. In particular, in all the simulations,
100% uncoupling is approached at the same concentration of
uncoupler, independent of the presence of an inhibitor of the
secondary pump, this in contrast to the experimental results.
In Fig. 3, it is shown that the effect on the titration with
uncoupler is the same whether it is the primary or secondary
pump that is inhibited. It is possible, on basis of the
0
20
10
30
Sl3(nM1
Fig. 5. The effect of concentration of tincoupler on oxidative phosphorylation rate (.I,,),according to simulations A , B and c‘ of ( 4 7 1
(full lines) and of’ the linear model in (621 tvith L, = 1 (dottrd line),
cornpared with the experimental results o j Herweijer et al. (561. (A)
Fully active ATP synthase. Open circles arc experimental results for
the ATP-driven reduction of NAD’ by succinate. (B) The case in
which 50% of the ATP syntbase (secondary pump) molecules have
been removed. Open circles are experimental results for inhibition
by 39% with oligomycin; crosses are experimental results for 38%
inhibition with rotenone. J , (0) refers to the rate in the absence of
inhibitor and with fully active ATP synthase. The simulations
published by Pietrobon and Caplan [47, 621 have been normalized,
so far as the abscissa is concerned, to the experimentally measured
concentration of S I 3 that gives J p / J p(0) = 0.1 with fully active ATP
synthase
chemiosmotic model, to simulate the proportional effect of
inhibition of the primary pump on the amount of uncoupler
required, but not the identical effect of inhibition of the secondary pump.
Thus the results of the uncoupler-inhibitor experiments
are inconsistent with A & + functioning only as an intermediate of oxidative phosphorylation. Whether or not they are
consistent with a variant of the chemiosmotic hypothesis,
namely that A f i H + functions in two ways (primarily as the only
intermediate of oxidative phosphorylation and secondarily as
an allosteric activator) is not completely clear in the absence
of a further kinetic analysis. It seems unlikely that such effects
could be playing a role in the experiments [56] in which the
concentration of active molecules of NADH :Q oxidoreductase was lowered , since no allosteric control by A f i H + on this
enzyme has been demonstrated.
The sigmoidal relationship between the rate of ATP synthesis and A i l H + has been taken by some (e.g. [64, 651) to be
495
evidence of an allosteric effect of A F H + . Indeed, especially in
chloroplasts, a physical basis for such an allosteric effect exists, since it is known that Aj2H + causes the dissociation from
its inhibitory site of an inhibitor of the ATP synthase [66].
However, Graber [67] has shown that the effect of eliminating
any such allosteric effect by prior activation of the ATPase is
purely to shift the relationship between rate of ATP synthesis
and AfiH+ to lower values of the latter, while retaining the
sigmoidal relationship (see also Mills [65]). In any case, as
already mentioned, Pietrobon and Caplan [49] have shown
that the sigmoidal relationship is an inherent property of the
chemiosmotic model, in the absence of any such allosteric
effect.
In the next section, evidence will be brought forward
favouring the view that, whether or not it can act as an
allosteric activator, A j i H + is not an obligatory intermediate of
oxidative phosphorylation.
IN DISAGREEMENT WITH THE CHEMIOSMOTIC
HYPOTHESIS, BULK-PHASE dj&+
IS NOT AN OBLIGATORY INTERMEDIATE
IN OXIDATIVE PHOSPHORYLATION
The results of two types of experiment have been presented
as evidence in favour of this standpoint: (a) measurements of
the AG,/AfiH+ ratio in the quasi-equilibrium state 4 (or static
head); (b) comparative biochemical studies with bacteria
adapted to different environments or with mutants.
Measurements of the AG,/ADH+ ratio
in the quasi-equilibrium state 4 (or static head)
According to the chemiosmotic hypothesis, the ATPforming reaction is: nAFH+ + ADP + Pi
ATP. When
respiring mitochondria are allowed to reach the situation in
which net synthesis of ATP ceases (state 4 or static head), this
reaction has reached equilibrium, in which case nAFH+
= AG,, where AG, = AGO Riln ([ATP]/[ADP][P,]). Here
AGOis the Gibbs free-energy change in the hydrolysis of ATP,
and AC, is often called the ‘phosphate potential’.
A number of reports have appeared (reviewed in [32])
indicating that, against the expectations of this hypothesis,
AGo/AfiH+(= n) is not constant, but increases when uncoupler
or respiratory inhibitor is added. Since the equilibrium
between Ar(iH+ and ATP synthesis cannot be affected allosterically, this so-called ‘anomaly 2’ [32] has been considered
the strongest evidence against the chemiosmotic hypothesis.
However, Woelders et al. [68]have recently published evidence
suggesting that Ac,/AjiH+ is constant over a wide range of
(down to about one-half of the state-4 level) and
ascribed the previously published apparent discrepancies to
experimental artefacts. Even more recently, however,
Petronelli et al. [69], after examination of their measurements
for possible artefacts, have reiterated their previous standpoint that the AG, declines proportionally less than the input
force A P H + with a consequent inrease of the AG,/dPH+ ratio
to values above 4, which these authors consider to be the
maximum accepted H+/ATP ratio (synthesis plus transport).
Until this experimental discrepancy between the Amsterdam [68] and Padua [69] groups is resolved, it would not seem
justified to maintain the inconstancy of the AG,: A F H + ratio
as an anomaly [32] to the chemiosmotic hypothesis.
+
Comparative biochemical studies with bacteria
As already mentioned, alkalophilic and halophilic bacteria
are capable of maintaining ATP synthesis at low values of
A j I H + . It is likely that, in the case of one type of alkalinetolerant organism (Vibrio alginolyticus), an electrochemical
gradient of Na+ replaces A P H + [22]. Possibly, an electrochemical gradient of C1- has the same function in halobacteria. In these cases, then, it appears to be necessary at least
to extend the chemiosmotic hypothesis to encompass
electrochemical gradients of ions other than protons.
Of particular significance is the finding of Cox and coworkers [70] that membranes of an Escherichia coli mutant, in
which a leucine residue in the c subunit (the very hydrophobic
subunit of F,) is replaced by phenylalanine, have normal
ATPase and oxidative phosphorylation activity with greatly
impaired proton permeability. Energization of these membranes, as measured by quenching of Atebrin fluorescence
[71], is retained when the ATPase is stripped from the membrane, this in in contrast to membranes from the wild type,
when stripping of the membranes causes loss of quenching,
owing to increased proton permeability.
ALTERNATIVE HYPOTHESES
To summarize the previous two sections, it should be noted
that a number of the arguments brought forward against the
concept that A f i H i is the only intermediate of oxidative
phosphorylation have been weakened or falsified in the last
year or two. However, to falsify a hypothesis, only one unambiguous experimental result unambiguously in conflict with
the hypothesis is necessary. The following results, in increasing
order of the weight that should be given to them, fall into this
category.
a) The existence of bacteria in which Na+ gradients replace
A F H + as the source of energy for ATP synthesis precludes a
universal role for A J H + in this reaction.
b) The proportionality between the amount of protonophoric uncoupler required to produce a certain degree of
uncoupling and the concentrations of redox enzymes and the
ATP synthase, is incompatible with A j i H + simply acting as an
intermediate of oxidative phosphorylation in mitochondria.
c) The existence of an E. coli mutant, the membranes
of which exhibit normal oxidative phosphorylation but with
greatly impaired proton permeability, demonstrate that
oxidative phosphorylation can proceed in the absence of
AFH+.
These and similar considerations have led many to
abandon the chemiosmotic hypothesis in the sense that they
propose that A F H + is not an obligatory intermediate in
electron-transfer-linked phosphorylation. In order to accomodate the clearly established findings that an artificially
imposed A f i H t can be used to make ATP, A $ H + has been
placed on a side-line. All proposals can be summarized by the
scheme
Electron transfer
=$
0 s ATP
11
AFH+
This scheme is essentially the same as that designated
scheme B in my review in 1967 in this journal [72] (see also
[73]). It is also the same as scheme C of Westerhoff et al.
[32] and to scheme I of Padan and Rottenberg [74], but, in
contradiction to statements in our two previous papers [l,561,
is not the same as the ‘parallel coupling’ model favoured by
the latter authors.
It is in the nature of the closed box that the various
hypotheses differ (see [32]). These can be classified under
496
three headings: (a) localized protons within the membrane; (b)
localized dji,, + ; (c) energized protein conformations.
Before considering these hypotheses further, however, it is
perhaps worthwile noting that to some the black box appears
to be equated with A,&+ by definition. An example is the
claim that recent studies on methanogenesis from methanol
and H2 in Metliunosurcium harkrrii have provided the ‘first
proof of a chemiosmotic mechanism of ATP synthesis in
methanogenic bacteria’ [75]. In fact, the experiments clearly
demonstrate that in these bacteria ATP is synthesised by
electron-transfer chain phosphorylation, and not by substrate-linked phosphorylation. They also show that A j H + is
increased somewhat by addition of methanol and is dissipated
by uncoupler, coincident with inhibition of ATP synthesis, but
they do not ‘show clearly a sequence of events: methanogenesis
generation of membrane potential + ATP synthesis’, as claimed by these authors, since this implies a separation in time o f generation of membrane potential and ATP
synthesis that was not observed. In fact, none of the experiments address the question whether o r not A j H t is the
sole intermediate of ATP synthesis, as required by the
chemiosmotic hypothesis, o r is on a side-path.
electrostatic interaction between the protons and fixed negative charges that are generated in localized areas on the surface
of energized membranes (cf. [72, 89a. 89bl. Malpress gives
equal importance to the translocated, but localized, protons
and these fixed negative charges, and proposes that the primary mediating force in ATP synthesis is the electrostatic
potential of protons in selective areas of the diffuse double
layer.
In both Kell’s [ X l , 821 and Dilley’s [X3] hypotheses, a
rather rigid structure of the membrane with specific channels
connecting different parts of the membrane are implied.
Although I a m unable to deny this possibility, 1find if difficult
to reconcile this with my picture of the structure of energytransducing membranes, except in a modification which will
be considered below.
--$
Localized w x w i f m i t i e protons
The hypothesis of the involvement of membrane protons
is as old as the chemiosmotic hypothesis, having been proposed by Williams in the same year [76]. The specific proposal
was that protons produced in a hydrophobic environment by
the reduction of the ferric ion atoms of cytochrome by substrate hydrogen ;ItoliiS are utilized to combine with H 2 0
liberated by the esterification of A D P by Pi to drive the reaction towards ATP synthesis [77]. Williams maintains that the
removal of protons from the membrane into the bulk medium,
a s envisaged by Mitchell, would mean a loss of energy [7X XO]. In his hypothesis, the protons generated a t high equivalent
aqueous activity inside the membrane diffuse only slowly
through the membrane phase to the outside. They pass
through a series of hydrogen bonds, for example with oxygen
atoms, to reach the ATP synthase. The driving force is given
by pulling water from the polyphosphate-condensation site
into the diffusion channel. The proton channel is thought of
as being composed of fixed water molecules and/or to be
associated with proton-binding groups on proteins [78, 791.
Kell [81, 821 postulated a neural network of channels
conducting protons from their site of origin near the electrontransferring enzymes to the ATP synthase. A similar idea was
put forward by Dilley [83]. A recent study by Prats et al.
[84] (see also [85]) implicates the polar head groups of the
phospholipids at the surface of the membrane as a pathway
for rapid proton conduction, much more rapid than in the
bulk phase.
Like everyone else in the 1950’s and 1960’s (e.g. [86]),
Williams envisaged that the energy-requiring reaction in ATP
synthesis is the esterification reaction [76, 771. Now, however,
he prefers a mechanism in which protons bind to the ATP
synthase and drive two conformational changes. One releases
ATP from F,, the other opens the Fo channel to the inside of
the mitochondria allowing downhill diffusion [79, 871.
Williams’ hypothesis has been frequently invoked by those
having difficulty in accepting the obligatory role of bulk protons, sometimes in a modified form in the sense that the
protons are placed close to the membrane, but on the aqueous
side of the interface (e.g. [XS]). This is also the case in the
‘coulombic hypothesis‘ of Malpress [89], who proposes an
Loculized ADII+
Westerhoff and colleagues [32] havc proposed, under the
name ‘mosaic chemiosmosis’, the existence of a large number
of independent local proton domains, each of which would
be supplied with protons by only one or ii few electron-transfer
chains and each of which would be connected to only one or
a few ATP synthase molecules. A small number of protons
pumped into the domain would be sufficient to generate a
local proton electrochemical potential sufficiently high to
drive ATP synthesis which would then depend rather directly
on the rate of turnover of electron-transfer chains. Westerhoff
and Chen [40] have shown that a system in which the freeenergy coupling intermediate (e. g. the proton) occurs only in
small numbers of molecules per coupling unit exhibits a
number of peculiar properties: (a) the reactions involving the
intermediates to not follow the conventional kinetic rate laws
in terms of the average concentration or chemical potential
of the intermediate; (b) the variation of the output reaction
rate with the average intermediate concentration is not
unequivocal but depends on whether the input reaction or the
leak is varied to alter that concentration; (c) when the apparent free energy contained in the average concentration of
the intermediate is compared with the average free energy
recovered in the output reaction, apparent violations of the
second law of thermodynamics may occur. These phenomena
closely parallel the experimental findings that are in conflict
with the traditional interpretation of chemiosmotic coupling
and on the basis of which Westerhoff based his model [32].
It should be noted that implicit in this model is that the
proton box does not contain a large number of rapidly exchangeable protons on buffer groups. 1 find this difficult to
envisage since the ‘box’ must contain protein molecules with
many dissociable protons. I have three other problems with
this hypothesis.
(a) It requires a specific association of a small number of
electron-transferring proteins and ATP synthase in the energytransducing membrane. This is at variance with the evidence
that these proteins can diffuse in the plane of the energytransducing membrane a t least as rapidly as electron transfer
[901.
b) Since, according to ‘mosaic chemiosmosis’, each microcompartment operates in accord with the Mitchell hypothesis
(translocation of protons from one aqueous phase to another),
the differences between protonophoric uncouplers and
gramicidin found by Herweijer et al. [56] are unexpected.
c) If the constancy of the AG,/AfiH+ ratio as A g H t is
lowered by adding uncoupler, with or without respiratory
inhibitor, reported by Woelders et al. [66] is confirmed (see,
497
however, [69] and [91]), an additional argument can be
brought against this hypothesis, since this constancy would
not be expected if there is a barrier between the interior bulk
phase reflected in Afi,, + and the micro-compartment where
the micro A,&+ is in equilibrium with the enzyme systems
concerned.
in the summer of 1985 [l]. In the meantime, Boyer independently presented a collision hypothesis to a symposium,
the proceedings of which have been published [92].
The proposal of a direct interaction between the redox
proteins and the ATP synthase may be written formally:
Energized protein conformation
The following considerations led us to propose what we
have called a ‘collision hypothesis’ [l], in which, as in Boyer’s
[20] ‘conformation’ hypothesis, the closed box in the above
scheme represents an energized protein conformation.
a) The increasing number of reports (not all of which, as
we have seen, have stood up to further examination but a
number have done so) indicating the difficulty of maintaining
the bulk-phase ADH+ as an obligatory intermediate of
electron-transfer-linked phosphorylation, which had led many
to postulate that the energy is, in some way, localized within
the membrane.
b) The picture [90] of energy-transducing membranes that
is emerging, in which a comparatively small number of large
multi-subunit proteins diffuse as separate entities in or on a
phospholipid bilayer that also contains quinone, leads
logically to the idea that, if the conserved energy is localized,
it is done so on these proteins.
c) The observations of Herweijer et al. [36] that, whether
it is the ATP synthase or the electron-transfer protein that is
inactivated, the rate of ATP synthesis is directly proportional
to the product of the concentrations of the remaining active
redox enzyme and ATP synthase molecules. On the other
hand, the rate of the 32P,-ATP exchange reaction is proportional to the square of the concentration of the residual
ATP synthase [36]. To a chemist, this is what is to be expected
if the rate-determining step in ATP-synthesis is the rate of
collision between a redox enzyme and the ATP synthase and,
in the exchange reaction, between two molecules of ATP
synthase, one energized and the other non-energized. As already indicated above, double-inhibitor titrations have lost
their force now that they can also be explained on the basis
of a purely chemiosmotic hypothesis. However, while one
would not like to make too much of it, the quadratic relationship between the rate of the 32P,-ATPexchange and the concentration of active ATP synthase molecules remains
suggestive.
d) The observation that the amount of protonophoric
uncoupler required to prevent the ATP-driven reduction of
NAD’ by succinate is proportional to the concentration of
the redox enzyme and the ATP synthase [56].
e) As discussed above, it can now be taken as proven that
the energy transmitted to the ATP synthase is used to induce
a conformational change in the latter enzyme in such a way
as to bring about the energy-requiring dissociation of alreadysynthesized ATP. It is highly likely that the energy transmission within the ATP synthase occurs by subunit-subunit
interactions. It seemed not unlikely, then, that collision between an energized redox enzyme and the synthase is the way
in which energy is transferred between the two systems,
Our first attempts to publish this hypothesis in early 1984
did not pass the reviewers. It was presented to a symposium
in the spring of that year and the manuscript given to the
organizers at the time of the symposium, but the published
proceedings of the symposium have not appeared at the time
of writing this review. A somewhat extended version of the
originally submitted paper duly appeared in another journal
EATp
-
h+
+ ADP + Pi $ EArp + ATP
in which the reactions at the right-hand side of the first two
lines are seen as equilibrium reactions. The first reaction describes a redox reaction catalysed by a redox enzyme, in which
the Gibbs free energy made available by the redox reaction is
conserved by bringing the redox enzyme (E,) into a ‘highenergy’ conformational state. This is denoted by E, h + to
indicate that the energy can be utilized to create an
electrochemical gradient of protons between the bulk phase
on the two sides of the membrane. The source of the protons
is not specified in this simple formulation. Energy it transduced as the result of direct collision between the ‘energized’
redox enzyme and a molecule of ATP synthase (EATp), with the
formation of an energized form the the synthase (EATp h’),
which can be utilized either to synthesize ATP in the third
reaction or to create O f i H + . Just as in the chemiosmotic hypothesis, the redox enzyme and ATP synthase are considered
to be proton pumps that can deliver protons to the bulk
phases. The main route of energy, however, is the transfer of
energy as a result of collision between the two molecules.
Under conditions in which the equilibria between the
energized proteins and the bulk-phase A f i H t is set up more
slowly than the rate of electron transfer and phosphorylation,
there will be no simple relation between ADH+ and the rate of
ATP synthesis. However, in state 4 when the reactions shown
above reach equilibrium, a nearly constant AG,/ApH+ ratio is
to be expected.
The collision hypothesis postulates that the energy is transduced as the result of direct collision between the interacting
molecules. Zoratti et al. [93] also considered protein-protein
interaction as one possible explanation of their experimental
results. The direct-collision hypothesis differs from those involving localized protons or localized AfiH+, which require a
‘pool’ of protons, albeit maybe a small one [32, 941,
functioning between the two enzymes. It differs also in that,
in our model, the direct interaction is not permanently
localized. The transduction of energy is localized in the
encounter complex, but this can be anywhere in the membrane, and the separated energized and non-energized proteins diffuse randomly in the membrane.
The proportionality of the uncoupling titre with the degree
of inhibition of the secondary pump in the experiments of
Herweijer et al. [56] suggests that protonophoric uncouplers
interfere with a direct interaction between active redox
enzymes and the ATP-synthase (cf. [93]). In consequence,
the concentration of uncoupler needed is related only to the
concentration of the interacting enzymes concerned in the
energy transduction. In contrast, the degree of stimulation of
ATP hydrolysis by protonophoric uncouplers or gramicidin
is not affected by the concentration of active ATP synthase
molecules.
The contrasting behaviour of protonophoric uncouplers
and gramicidin is illustrated, not only by the different effects
of inhibition by rotenone on the uncoupler titration of the
ATP-driven reduction of NAD’ by succinate (see Figs 3 and
4), but also by a comparison of the relative concentrations
-
-
498
g ramicidin
S13 (1)
S13 (2)
ATP' ADP+ P,
f3g. 6. Diugrammulic, wpresc~nttrtionofc.ollision hypothesis us upplied
the ATP-driveti reduction of NAD' bv Q H 2 (not shown) and the
proposed fti'ii modes of uction of the uncoupler S13 and the single mode
c!/uc.rion ofgrcimicidin. S 13( I ) indicates a direct effect of uncoupler
on thc proposcd proton transfer. At 20-fold higher concentrations,
S13 is ablc to translocate protons by a chemiosmotic mechanism and,
thereby, inducc the hydrolysis of ATP in the absence of reductant.
Gramicidin, by creating a pore in the membrane through which protons can diffuse. has the same cffcct as S13 a t the higher concentrationr. Diagram reproduced with pcrmission from [56]
to
required to stimulate ATP hydrolysis and to inhibit NAD'
reduction. The two processes are affected similarly by
gramicidin, but with the protophoric uncoupler S I 3the concentration required for maximal stimulation of ATP hydrolysis is much higher than that for inhibition of NAD' reduction
(for example half-maximum stimulation of ATP hydrolysis at
1.49 nmol/mg protein and 50% inhibition of NAD' reduction at 0.077 nmol/mg protein) [%I. The maximum ATPase
activity reached is the same with both types of uncoupler.
These findings also strongly suggest that stimulation of ATP
hydrolysis is brought about by a different mechanism from
inhibition of N A D reduction, and, specifically, that interference with the direct interaction between NADH: Q reductase and ATP synthase occurs at concentrations of S I 3
that have little effect on the rate of transfer of protons over
the membrane.
A schematic representation of the collision hypothesis, as
applied to the ATP-driven reduction of NAD' by succinate,
is represented in Fig. 6. In this scheme, the interference by
protonophoric uncouplers with the direct energy transfer is
indicated by S I3( 1 ). and the conventional uncoupling pathway by S13(2). Higher concentrations of uncoupler are required for the latter pathway, probably because the lipophilic
uncoupler is present mainly in the lipid phase. Gramicidin,
on the other hand, is not able to react with protons in the
membrane and can function only as a pore, allowing the free
passage of protons from one bulk phase to the other. Thus,
uncoupling by gramicidin occurs via dissipation of A,&+
which. since this is in equilibrium with the energized redox
enzymes and ATP synthase, results in a lowering of their
concentration.
Rate of ' c d i s i o t i hc.tnecw redox enzymes and ATP synthase
In order to maintain a collision hypothesis of oxidative
phosphorylation. it is necessary to demonstrate that the
collision rate is at least as rapid as the rate of phosphorylation.
The lateral diffusion coefficient ( D L ) of ubiquinone,
QH2:cytochrome c' reductase, cytochrome c and cytochrome
c' oxidase in intact, ultra-large, spherical inner membranes of
rat-liver mitochondria has been measured by fluorescence
recovery after photo-bleaching of suitably fluorescently
labelled diffusing species [90, 951. The diffusion coefficients
of ubiquinone and the two larger proteins were found to be
independent of ionic strength, but that of cytochrome c' is
increased by increasing the ionic strength. The diffusion of
ubiquinone is about as fast as that of the phospholipid [96]
and that of the large proteins about ten times slower. The
recovery of fluorescence was more than 90% and monophasic,
indicating that the molecules diffuse as single components
in a single pool. It is concluded that, at physiological ionic
strength, cytochrome c diffuses largely in a three-dimensional
phase within the inter-inembrane space 1961, but the other
proteins and the ubiquinone diffuse in the plane of the membrane.
Using these values of D ,and other data on the concentration (nmol/mg protein) of the reacting species in the membrane, the amount of inner-membrane protein per mitochondria, the diameter of each reacting enzyme molecule in the
surface of the membrane and the redox state of the reacting
enzyme during steady-state electron flow, Hackenbrock and
colleagues [90,95] calculated the diffusion-controlled collision
frequency for each pair of reacting molecules involved in
the oxidation of succinate by oxygen. It was shown that the
collision frequency in every case exceeds the experimentally
determined maximum turnover number. Thus, electron transfer is coupled to (preceded by) lateral diffusion. This is the
basis of the random-collision model of electron transfer, in
which the membrane is envisaged structurally as in a fluid
rather than solid state. The redox components are seen to
be randomly dispersed rather than organized in a macromolecular or chain-like assembly. Ubiquinone (cf. [97]),
cytochrome c (cf. [98]) and the integral membrane proteins
are common-pool, mobile electron carriers.
Productive collision between randomly spaced redox
partners require long-range diffusion of each partner, the rate
of which is limited by obstructive multicollisions with other
membrane proteins. Since the collision efficiency was found
to be almost completely independent of temperature,
Hackenbrock et al. [96] conclude that electron transfer is
not only diffusion-coupled, but is diffusion-controlled. In a
diffusion-controlled reaction, the collision efficiency will be
virtually independent of temperature since any effect of temperature on the diffusion process will be approximately the
same for the electron-transfer process.
In view of the importance of these conclusions for our
understanding of the interactions between the membrane
constituents, some space will be devoted to a discussion of
possible errors. A considerable reductance seems to exist in
some quarters to accept a random-collision model of electron
transfer. This seems partly to be based on a misconception,
namely that energy-transducing membranes are so heavily
packed with proteins that there is little room for them to
move. It is true that it has long been known that such membranes consist of about 70% protein and only about 30%
lipid. However, one of the greatest advances in our knowledge
of membrane structure, culminating in the fluid-mosaic model
of Singer and Nicholson [99], has been the realisation that a
large part of a protein embedded in a membrane lies outside
the phospholipid bilayer.
Calculations of the packing in the membrane require data
for the concentration of each protein (e.g. nmoljmg total
protein) in the membrane, the total amount of protein in a
single mitochondria, the area of surface of inner membrane
in a single mitochondrion and the radius of the cross-section
in the membrane of the protein in question.
For rat-liver mitochondria, two sets of data have been
used in the literature, by Gupte et al. [95] and Schwerzmann
499
Table 1. Occupancy of inner membrane of rat-liver mitochondria by
muin redox enzymes and ATP synthase
Parameter
Gupte et al.
[951
Schwerzmann
[1001
Surface area (cm2/mg protein)
304"
560
Radius (nm) of:
NADH :Q reductase
succinate:Q reductase
QH,:cytochrome c reductase
cytochrome c
cytochrome c oxidase
ATP synthase
4.0
0.7
2.5
1.5
2.5
n.d.b
4.0
0.7
5.0
1.o
4.4
4.5
Concentration (nmol/mg protein) of:
NADH :Q reductase
0.014
succinatc: Q reductase
0.027
QH2:cytochrome c reductase
0.041
cytochrome c
0.122
cytochrome c oxidase
0.095
ATP synthase
n.d.
0.037
0.074
0.078
0.236
0.222
0.222
Concentration (molecules/cm2)of:
NADH:Q reductase
succinate :Q reductase
QHz:cytochrome c reductase
cytochrome c
cytochrome c oxidase
ATP synthase
2.77 x loiG
5.35 x 10'0
8.12 x 10"
2.42 x 10"
1.88 x
n.d.b
4.28 x 10"
8.56 x 30"
9.01 x 10'0
2.75 x 10"
2.61 x 10"
2.57 x 10"
Area occupied (YO)by:
NADH :Q reductase
succinate:Q reductase
QH2:cytochrome c reductase
cytochrome c
cytochrome c oxidase
ATP synthase
3.4"
0.1
1.6'
1.7'
3.7"
n.d.b
2.2
0.1
7.1
0.9
15.9
16.3
a Calculated from diameter of liver mitochondrion (1.5 pm [loll)
and number of mitochondria per mg protein (4.3 x lo9 [102]).
Not determined.
Calculated by the author.
et al. [loo], respectively (see Table 1). The occupancy of the
five redox proteins shown in Table 1 amounts to 8.4%
calculated from the data of Gupte et al. [95] and 26.2% according to that of Schwerzmann et al. [IOO], which is an important
discrepancy. Together with the ATP synthase, the occupancy
calculated by Schwerzmann et al. [loo] is 42.5%, which, since
there are other membrane proteins (for example the ATPADP translocator) present, seems uncomfortably high for the
collision hypothesis. However, there is reason to believe that
at least the occupancy calculated for the ATP synthase is too
high. First, the radius for that part of the ATP synthase
embedded in the membrane, taken to be the same as the radius
of the F1 moiety, which is generally assumed to project from
the membrane, might perhaps be on the high side, although
the assumption is not unreasonable given the lack of detailed
structural information on this enzyme. More important is that
the concentration assumed for this enzyme (0.222 nmol/mg
protein) is certainly too high. From the amount of oligomycin
required for complete inhibition of the ATPase activity [103],
a concentration of 0.1 nmol/mg protein may be calculated,
which yields an occupancy of 7.3%. Also the occupancy of
the QH2:cytochrome c reductase seems high, since it is based
on a radius of the monomer of 5.0 nm, compared with half
that value assumed by Gupte et al. [95], which makes a
difference of a factor of 4 in the calculation of the occupancy.
I suggest that, since there is good reason to suppose that this
enzyme is a dimer in the membrane, occupancy should be
based on the concentration and dimension of the dimer. From
the data of Leonard et al. [104], the dimer has a diameter of
about 13 nm, which yields an occupancy of 5.6%. Applying
these two corrections would bring the occupancy of the five
enzymes down to 32.0%.
From the density of the different proteins in the membrane, Schwerzmann et al. [loo] calculated the nearestneighbour distance between them to be about 7 - 20 nm, small
enough in their view to cause the formation of microaggregates. These distances become a little greater if the
occupancy values proposed in the above paragraph are used,
for example 12.5 nm instead of 9.1 nm between dimeric
QH2: cytochrome c reductase and ATP synthase.
In the previous review [I], data were calculated for ratheart mitochondria, based largely on early calculations of the
author for the concentrations of the enzymes. The concentrations (molecules per cm2) and occupancy of the redox
components were quite close to those used for rat-liver
mitochondria by Hackenbrock and co-workers. Although
these calculations are critically dependent on the assumed area
of inner-membrane surface in heart mitochondria, determined
from the diameter of fully swollen mitochondria as measured
by phase-contrast microscopy in 1953 [105], it is not
unreasonable that the concentration of the respiratory
enzymes in the inner membranes of the two types of
mitochondria should be similar, despite the well-known much
higher concentrations in heart when expressed as nmol/mg
protein. This is due to the larger amounts of inner membranes
in these mitochondria [106].
According to measurements by Cherry and colleagues
[lo71 of rotary diffusion, which is directly related to lateral
diffusion, a considerable fraction of cytochrome c oxidase is
relatively immobile, presumably owing to the formation of
aggregates with other cytochrome c oxidase molecules or with
other proteins. This was not observed by Hackenbrock and
colleagues, who found that the recovery of fluorescence was
unicomponent. A possible reason for the discrepancy is the
use of 60% (w/v) sucrose to isolate the mitochondria by
Cherry and co-workers. Sucrose in these hypertonic concentrations is known to bring about irreversible inactivation of
electron-transfer reactions. Indeed, we considered it to behave
as a fixative [loti].
I see no reason to doubt the direct measurements of the
lateral diffusion coefficients in the mitochondrial membrane
made by Hackenbrock and co-workers. It is true that, for
technical reasons, many of the measurements were made on
fused swollen mitochondria, in which possible constraints to
diffusion in intact mitochondria as the result of the juxtaposition of the extra-membrane moieties of the proteins in
adjacent membranes in the cristae [lo91 would be removed.
However, the same values of the diffusion coefficient were
obtained with intact megamitochondria isolated from
cuprizone-fed mice [96]. Such mitochondria have normal
oxidative phosphorylation functions [llO - 1121. In any case,
the density of the proteins in the mitochondrial membrane
would not be affected by fusion. If the aggregation of the
proteins proposed by Cherry and Schwerzmann were to occur,
it would be present in the membranes used by Hackenbrock.
Since only one population of diffusing proteins was detected,
it would appear that any aggregate is dissociating more
rapidly than the rate of diffusion and that the presence of
such aggregates does not invalidate the collision hypothesis
of electron transfer.
500
Tabk 2. Calculution o/ the frc>qurvicyof collision between redox enzymes and ATP synthase in rut-liver mitochondria und of c.ollisionlturnover
rirtio
The following assumptions were made. (a) The D Lfor NADH:Q reductase is equal to the mean of the values obtained by Gupte et al. [95]
for cytochromc c' rcductase and cytochrome c oxidase, namely 4.0 x lo-'' cm2 s - ' , and that for ATP synthasc equals 8.4 x 10- l o cm2 sC1
(C. R. Hackcnbrock, personal communication). (b) The concentration of 'energized' redox enzyme (E, h + ) and of non-energized ATP
synthasc (EATp),during steady-state ATP synthesis, is equal to one-half of the total concentration of thc respective enzymes. (c) The
concentrations of NADH:Q reductasc and cytochrome c oxidase are the same as those assumed by Schwcrzmann et al. [loo]. (d) The
concentration of ATP synthase is 0.1 nmol/mg protein [103]. (e) QH2:cytochrome c reductase reacts as a dimer with a radius of 6.5 nm (see
text). The radii of N A I 1 I i : Q reductase, cytochrome c oxidase and ATP synthase are 4.0 nm [ Y S . IOO], 4.4 nm [loo] and 4.5 nm [IOO],
respectively. (f)The rate of ATP synthesis in sites 1, 2 and 3 were calculated from the rates of oxidation of pyruvatc + malate (35.9 x 10" e
s c m - '), succinate ( 1 2 5 . 4 ~10" e s - cm 2, and ascorbate (249.5 x 10" c s - cm -'),respectively. as determined in [IOO]. I t was
that P/2c = 1 for cach sitc
-
Pdramerer
E,
Value for site
- h(molecules
i (molecule\ cni
cm
')
')
EArp
Radius of rcdctivc drcd (cm)
Collision frcqucncy ( \ cm '1
Turnober (s c m ')
Collisions/ turnover
1
2
2.14 x 10"
5.79 x 10'O".
8.5 x
0.3s x 10'3
0.18 1013
1.9
2.25 x
5.79 x
8.5
0.46 x
0.63 x
0.7
3
1010".
lo1"",
10-~'
1013
1OI3
1.3x101'"
5.79 x lo1"".
8.9 x to-'
8.9 x 1 0 1 3
1.25 x 1013
7.1
[loo].
See text.
If this hypothesis is correct, it follows that collisions between redox enzymes and the ATP synthase occur. The question is whether such collisions are relevant or not for the
mechanism of oxidative phosphorylation. First it is necessary
to examine whether the collision is sufficiently rapid to
account for measured rates of phosphorylation. In making
this calculation. 1 havc used the data of Schwerzmann et al.
[loo] for the rate of electron flow and for the density in
the membrane of the enzymes, except for modifications with
respect to the QH 1 cytochrome c' reductase and ATP synthase
referred to above. I t was assumed that one half of the ATP
synthase is in the energized state (EATp h') and similarly
for the redox enzymes. The results of this calculation are given
in Table 2.
Although the calculation of the collision frequency is
straightforward, the calculation of the collision efficiency (on
the assumption that collision does lead to energy transfer) is
more difficult. In the first place, the real values for both the
P/2e ratios for the three phosphorylation sites and the H + / e
and H ' iATP ratios for the redox enzymes and ATP synthase,
rcspectivcly, are still not established. Secondly, the collision
hypothesis is not yet sufficiently refined as to specify how
many collisions are necessary to transfer 'energy' (protons'?)
from E, h + to E,,, If only one proton were transferred per
collision and two protons are required for the synthesis of
one molecule of ATP, two collisions would be required to
synthesise a molecule of ATP, which is rather higher than the
ratio calculated in Table 2, especially for site 2. However, it
is also possible to conceive mechanisms in which the two
transferable protons are transferred simultaneously. Indeed,
the implications for both the chemiosmotic and collision hypothesis of the higher H + / e and H+/ATP ratios now being
reported need to be examined more deeply than is possible in
this review. It is also possible that energy is transferred from
E, h i to E A I Pdirectly without actual transfer of protons
[I]. I n view of these uncertainties and also of the arbitrary
assumptions made concerning the fractions of the redox
enzymes and A'TP synthase that are in the energized state, I
conclude that the collision frequencies calculated in Table 2
-
-
-
are not seriously inconsistent with hypothesis that the rate of
chemical interaction between the redox enzymes and the ATP
synthase, if there is one, is diffusion-coupled. Indeed, the small
number of collisions per turnover would suggest that it might
be diffusion-controlled.
Nature ojenergy transfer consequmt on collision
What is the nature of the energy transfer formulated in
the reaction
E,hht
+ EATp$Er + E A r p - h t ?
The simplest proposal is to supposc that electrostatic
attraction between a positively charged group in E, h ' and
a negatively charged group on EATp results in the former losing
one or more protons and the latter gaining them. It is inherent
in the collision hypothesis that the gain of protons leads to the
conformational change in EATp required for the dissociation of
the ATP formed at the catalytic site, which, as discussed above,
is the energy-requiring step in oxidative phosphorylation.
Specific proposals have been made for the way in which
binding of protons to the ATP synthase might lead to the
conformational change at the ATP-binding sites causing the
dissociation of ATP. In view of Penefsky's [I71 finding that
the binding parameters of a catalytic site on the [j subunit of
F1 are affected by binding of DCCD to a specific glutamic
acid residue in the c subunit, it is attractive to postulate that
this group is involved in binding a proton. Since there are
many copies of this subunit in a molecule of ATP synthase, it
is not difficult to visualize a mechanism in which binding of
more than one proton is required for the synthesis of ATP.
Cross [113], Mitchell [8] and Cox et al. (1141 have presented
models in which the three /l
subunits or (m b) subunits rotate
relative to the three smaller subunits of F1. Cox et al. 11141
proposed that the minor subunits are attached to a dimer of
the b subunit of Fo and that the whole complex rotates, the
driving force being supplied by an interaction between Lys23 in the membrane-embedded portion of the b subunit in E.
coli Fo and Asp-61 (which replaces the corresponding
-
+
50 1
glutamic acid in mitochondrial F,) in the c subunit. However,
this hypothesis has had to be modified in a more recent paper
[I151 by these authors in which it is shown that replacement
of Lys-23 by threonine has no effect on oxidative phosphorylation. In the new modification, the proposed interaction is by way of hydrogen bonds between Asp-61 in the c
subunit and lysine and arginine residues flanking a leucine in
a transmembrane cc-helix segment of the a subunit, whereby
a proton pore is created. (Note, however, that the lysine is not
present in mitochondrial Fo).It is proposed that the rotation
of the inner complex consisting of the a, b, y, 6 and F subunits
relative to the outer components consisting of cc, fi and c
subunits allows successive interactions of the helix with the
conserved acidic residue of the c subunits. The proposal of
Williams [I161 is quite similar, except that it is the binding of
protons to carboxylate groups in the c subunit of the synthase
that is thought to set off a running conformational change,
via the other subunits in the synthase, to the catalytic site.
The essential point is that binding of protons to the c subunit
in that part of the ATP synthase that is embedded in the
membrane (F,) is transmitted to a site on the fl subunit far
away from the proton-binding site. Williams draws the analogy with the action of CaZ+ions on troponin C and considers
the c subunit as a proton-modulin.
The simplest proposal mentioned above requires that the
proton-donating and proton-accepting groups on the two
proteins approach one another very closely. Alternatively, just
as in electron transfer, it is possible to envisage specific proton
channels within the proteins being involved in the proton
transfer. Indeed, such channels are frequently invoked as part
of the mechanism of proton translocation (pumping) across
membranes.
The best studied proton pump is bacteriorhodopsin. A
conformational change in this protein, triggered by a lightinduced change of configuration of the 13,14 double and 14,
15 single bonds in the side-chain of the prosthetic group,
retinal [116a], induces two changes in protonation in that part
of the protein that is deeply embedded in the membrane. One
is in the Schiffs base formed by the aldehyde group of the
retinal and a lysine residue, the second in some aspartic acid
residues. The result is that protons are taken up from the
cytoplasmic side of the membrane and ejected to the exterior.
Since this process is not sensitive to the pH outside the membrane, it is unlikely that a non-specific aqueous channel, open
to the exterior, is involved. Engelhardt et al. [I171 have also
brought forward evidence against proposals that the conduction of protons from the site of ejection in the membrane to
the bulk phase proceeds via a hydrogen-bond network. Rather
it seems that protons are transferred in distinct steps from one
aspartic acid residue to another, the process being governed
by successive changes in the pK values of the residues.
In this connection, it seems significant that the structure
of halorhodopsin, a second retinal-containing protein present
in halobacteria and whose function is to catalyse a lightinduced transport of chloride anions across the membrane, in
the opposite direction from that of protons driven by
bacteriorhodopsin, resembles very closely that of bacteriorhodopsin [l 181.In this case, the light-induced configurational
change in the retinal side-chain induces a conformation
change in a specific chloride-binding site. Although the reversible binding of protons to their side-chains is an inherent
property of proteins, the specific binding of anions is not, but
requires a specifically constructed anion-binding site. Since it
is somewhat unlikely that more than one such site is present
in halorhodopsin, a chloride channel penetrating far into the
membrane or even a transfer of chloride in discrete steps
seems unlikely.
This comparison of bacteriorhodopsin and halorhodopsin, together with the existence of alkaline-tolerant
micro-organisms in which an Na' gradient seems to replace
a proton gradient, makes one wonder if there is anything so
special about protons in energy transduction. The common
feature of the energy-conserving step in all energy-transducing
membranes is a charge separation in a membrane-spanning
protein, which can itself lead to a membrane potential, and
to the creation of fixed negative charges on the surface of the
membrane [71, 89, 89a, 89b].
Indeed, it is clear that for certain mitochondrial processes,
a membrane potential is required and cannot be replaced by
a proton concentration gradient. These include the uptake by
mitochondria of Ca2+,required for the activity of a number
of mitochondrial matrix enzymes, the import into the mitochondria of proteins or their precursors [119] and, most
importantly, the exchange of ADP for ATP [120].
Heterogeneity in membranes
At first sight, the random-collision hypothesis of electron
transfer and phosphorylation is difficult to reconcile with the
clear evidence of heterogeneity in some energy-transducing
membranes. This is not necessarily so, since, as Kaprelyants
[I211 has pointed out, rapid lateral diffusion can co-exist with
the segregation of the same proteins to defined localities in the
membrane. The lateral non-homogeneity reflects the ability of
the proteins to form dynamic protein-protein assemblies. At
the same time (milliseconds time scale), the membrane
contains the same proteins in multienzyme assemblies and as
freely diffusing proteins. The observed value of DL,as a result
of the time averaging of the diffusion coefficients for
molecules differing in mobility, would, if this were the case,
be lower than that expected for unconstrained proteins.
Two examples of membrane heterogeneity were treated in
our previous review [l]:the thylakoid membrane in chloroplasts and the membranes of halobacteria.
In chloroplasts, the stacked (grana) regions are enriched
in photosystem I1 and the light-harvesting complex, wheras
the non-stacked (stroma) regions are enriched in photosystem
I, plastoquinol :plastocyanin reductase and the ATP synthase
[122]. Electron transfer between the photosystem I1 and the
plastocyanin reductase can be taken care of by the lateral
diffusion of plastoquinol and plastoquinone [97]. The lightharvesting complex in a phosphorylated form migrates from
the stacked into the non-stacked regions, whereby a fraction
of the light energy absorbed by the complex pool is transferred
to photosystem I. When the complex is dephosphorylated, it
migrates back into the stacked region [123].
If the electrochemical gradient generated by photosystem
I1 is involved in ATP synthesis, the separation between this
photosystem and ATP synthase implies that the electrochemical gradient must be continuous throughout the entire
lumen of the photosynthetic membrane and, in the case of
cyclic phosphorylation, ferredoxin. However, many authors
(e. g. [124]) have found localised behaviour in chloroplasts.
Specifically, De Kouchkovsky and co-workers [125] have reported that protons generated by photosystem I1 have
hindered access to the ATP synthase, but this is denied in a
recent paper by Davenport and McCarty [126], who have
reported that phosphorylation by isolated thylakoids depends
on the bulk-phase APH+ and is independent of the source of
the protons. The discrepancy appears to be resolved by Beard
502
and Lilley [I271 who have shown that suspension of
chloroplasts in KCI shifts the energy-coupling mechanism
from localised to bulk-phase delocalised proton gradients. It
remains to be seen which type of chloroplast preparation
represents the situation in vivo. Even if, in the green plant,
protons liberated from water as a result of the light-driven
charge separation in photosystem I1 contribute to ATP
synthesised in the phosphorylation in a purely chemiosmotic
mechanism, collision between the plastoquinol :plastocyanin
reductase and the ATP synthase, as suggested for the analogous reaction in mitochondria, may also play a role.
It is clear that bacteriorhodopsin is Concentrated in purple
patches in halobacteria membranes, well separated from the
ATP synthase. However, the main function of the light-driven
proton efflux catalysed by bacteriorhodopsin might be to
provide an electrochemical gradient of protons for the operation of a Na+/H antiport [128], whose function is to pump
sodium out of the cell in a medium of high NaCl concentration. ATP synthesis in this organism proceeds at low values
of d c H +which makes it problematical whether ATP is synthesised by a chemiosmotic mechanism. It cannot be excluded
that in the intact cell a sufficient number of bacteriorhodopsin
molecules diffuse to the ATP synthase. It is true that in
artificial vesicles composed of bacteriorhodopsin and ATP
synthase, ATP synthesis is driven by a purely chemiosmotic
mechanism [59], but the concentration of the proteins in the
phospholipid is far lower in the artificial vesicles than in the
intact bacteria.
+
CONCLUDING REMARKS
This review has, of necessity, concentrated on one aspect
of the mechanism of the conservation of energy of redox
reactions, the nature of the intermediate between the redox
reaction and ATP synthesis. In a review in the first volume of
this journal [72]in 3967, entitled An evaluation of the Mitchell
hypothesis of’ ik>miosmoticcmipling in oxidative and photosJ’nthrtiipho.vphor!.lution,I concluded that “the chemiosmotic
theory, in its present form, is untenable”.
This conclusion was partly based on a misunderstanding
of the changes in the theory that had been made since 1961
(see [73]). Ten years later [129], 1 stated in a passage referred
t o by Mitchell in his Nobel Prize Lecture [7] as a classical
Popperian view:
“There is now wide, if not perhaps completely universal,
agreement that the fundamental energy-conserving processes
are the separation of electrical charges, that is the creation of
an electrical field, and the net movement of hydrogen ions
across the membrane, as first proposed by Mitchell. The areas
of disagreement and uncertainty can all be accomodated
within (or, perhaps, just outside) the general framework of
the Mitchell hypothesis. I for one am now prepared to accept
(you my prefer the word concede) this statement, and I hope
that most workers will now use this framework as the starting
point for the presentation of their work, aimed at filling in
the gaps or even, if they wish, to disprove the theory. What
has, I believe, now become redundant are experiments
directed only to converting the unbelievers. This is really an
over-kill opera tion. ”
Now, again 10 years later, 1 would no longer maintain this
position. Although there is no doubt that ATP can be made, in
vitro, by a purely chemiosmotic mechanism, the quantitative
importance of this mechanism for the synthesis of ATP in vivo
has still not been established. The use of the chemiosmotic
hypothesis as a universal framework might now be unduly
restrictive and I would support those who are now re-examining the possibility of a more direct interaction between
redox enzymes and the ATP synthase. It might be particularly
fruitful to keep this possibility in mind as the detailed threedimensional structure of the energy-transducing enzymes, so
brilliantly opened up by Michel [I 301, unfolds.
I would like to thank Drs C. R. Hackenbrock, K . Schwerzmann,
B. Hess and D. Pietrobon for fruitful discussion and for providing
information about their work before publication.
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