Bioscience Reports, Vol. 11, No. 6, 1991
Glynn and the Conceptual Development of
the Chemiosmotic Theory: A Retrospective
and Prospective View
Bruce H. Weber
The origin and evolution of the chemiosmotic theory is described particularly in relation to Peter
Mitchell's application of it to model oxidative phosphorylation. Much of the deployment, development and evaluation of the theory occurred at the independent laboratory of the Glynn Research
Foundation; the value and future of such an institution is discussed. The role of models mediating
between theories and phenomena is analyzed with regard to the growth of knowledge of chemiosmotic
systems.
KEY WORDS: chemiosmotic theory; vectorial chemistry; energy transduction; Q-cycle; Glynn;
history of biochemistry; Peter Mitchell; logic of discovery and justification; models; phenomena;
evolution of knowledge.
INTRODUCTION
Although the origin of the chemiosmotic theory lies in Peter Mitchell's work at
the Universities of Cambridge and Edinburgh, the practical application, detailed
development and dissemination of the theory is inextricably tied with the Glynn
Research Institute. Twenty-five years ago, Mitchell began a double experiment:
to test and extend chemiosmotic concepts and to see if first-rate science, which
would have a disproportionately large impact, could be done in a small,
financially and administratively independent laboratory. A t a time when the
biological sciences are gearing up for the h u m a n g e n o m e project, it is worthwhile
to pause and reflect on the kind of achievement produced at Glynn and to analyze
the types of problems that can be addressed by such an institution and the
necessary conditions for its success.
To account for the synthesis of A T P in mitochondria and chloroplasts,
Mitchell (1961e) p r o p o s e d a specific hypothetical model, based upon general
chemiosmotic principles, which did not invoke chemical intermediates that were
Department of Chemistry and Biochemistry, California State University, Fullerton, California,
U.S.A.
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0144-8463/91/1200-0577506.50/0O 1991 Plenum Publishing Corporation
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common to the redox or photoredox chain system and the A D P phosphorylation
system.
The fundamental principles of the chemiosmotic theory were largely formulated during the 1950s, while Mitchell was studying bacterial phosphate transport
and developing the concept of specific vectorial ligand conduction. During this
time Mitchell explored his conceptual commitment to the notion that osmotic
phenomena and the chemistry of metabolic processes share a fundamental
causality and connection in vectorial chemistry. A balance of vectorial forces
colinear with flow was assumed to relate the structural and dynamic elements in
living systems, with compartmentation and communication as organizational
correlatives descriptive of this notion. This commitment guided Mitchell in his
deliberate choice to study phosphate transport as a common link between
metabolism and transport. The theoretical structure that emerged was based upon
the concept of specific vectorial ligand conduction, catalysed by membranelocated enzymes and catalytic carriers, in which ligands (i.e. electrons, chemical
groups, molecules or ions) are driven by the resultant of primary and secondary
chemical forces, down their classical electrochemical potential gradients, along
the conduction pathways. Thus, the "active transport" of a given ligand was
conceived as the passive diffusion of a derivative, and the driving of one ligand by
another was seen to depend upon the ligands being directly or indirectly bound
together (i.e. mechanically coupled) in a compound or complex that was
translocationally mobile in a conduction pathway.
In developing his model of oxidative phosphorylation, Mitchell extended
precedents concerning vectorial electron transfer set by Lundegardh, Davies and
Ogston, Conway and others, and added crucial new concepts, based on general
chemi0smotic theory (see Mitchell 1961e). He postulated that the respiratory
chain must be situated in the mitochondrial cristae membrane, and must
translocate protons through the membrane, thus producing a protonmotive force
(in analogy to an electronmotive force) composed of two components, an electric
membrane potential and a trans-membrane pH difference. To this, Mitchell
added three other basic postulates: that there must be a reversible protonmotive
ATPase, plugged through the cristae membrane, that would use the protons,
driven by the protonmotive force, to dehydrate inorganic phosphate + A D P and
produce ATP; that the membrane must contain proton-coupled anion and cation
exchange-diffusion systems to maintain the electric component of the protonmotive force, and to maintain pH and osmotic stability of the mitochondrial matrix;
and that the membrane must be relatively impermeable to protons, hydroxide
ions and other hydrophilic solutes .under physiological conditions. This chemiosmotic system for mitochondrial oxidative phosphorylation represented a special
case of a more general chemiosmotic system for protonic osmotic energy and
force transfer, for example, in bacteria (see Mitchell, 1961a, 1962, 1963, 1970a,b).
New scientific theories and knowledge evolve out of the context of previous and
current work; innovation and novelty arise in response to what has gone before,
and are recognised as such against the context of the relevant research traditions
(Depew and Weber, 1985; Richards, 1987; Basalla, 1988).
Glynn and Chemiosmotic Theory
MITCHELL'S P R E - G L Y N N D E V E L O P M E N T
CONCEPTS
579
OF C H E M I O S M O T I C
Fundamental Chemiosmotic Principles
Peter Mitchell has provided a detailed description of the d e v e l o p m e n t of his
thought and how that came to bear on the p r o b l e m of oxidative phosphorylation
(Mitchell, 1981a). Mitchell was an undergraduate at Cambridge University,
England, at the beginning of World W a r II. H e remained there, first to join a
research group working on antidotes to poison gases, and then to do graduate
research on the mechanism of action of penicillin (which led to his Ph.D. in
1950), and finally to w o r k on p h o s p h a t e transport in bacteria, until his departure
for the University of Edinburgh in 1955. While at Cambridge he was influenced
by a combination of the m e m b r a n e studies of Jim Danielli, the solution
enzymology of Malcolm Dixon, and most importantly by the work and personality of his unofficial m e n t o r David Keilin, who focused on understanding
cellular respiration catalysed by the insoluble enzymes and catalytic carriers of the
cytochrome system present in the particulate cell fraction. Mitchell has described
these influences as follows (interview, 5 July, 1979):
Malcolm Dixon was a classical metabolic enzymologist. He hardly believed in
enzymes that weren't soluble, whereas Jim Danielli was working with surface films on
Langmuir troughs, and was interested in membrane transport at the time. I think that the
thing that influenced me most was noticing that the transport people, with whom I became
associated because of Danielli, not only failed to understand the metabolism people, but
they were positively scornful about their attitude. Conversely, the metabolism people not
only did not understand the transport people, but they were scornful about them. So I
think one of the strong feelings I can recognize or recollect is that of sadness that this
should be the state of affairs. [I]t was pretty clear [to me] that there was a transport aspect
of metabolism. Even simple things such as how NAD could couple two redox reactions,
one going forwards and the other going backwards. When you come to think of it--and
the way Malcolm Dixon drew it--you have two enzymes, and the N A D is shuttling
between. It immediately becomes obvious that it is a sort of a transport process. So, by the
late 1940s I had become fairly determined to find time at some stage to look at both
transport and metabolism, and actively try to produce some sort of rationale that would
integrate them. David Keilin stood between the transport people and the metabolism
people-- because he had the insoluble cytochrome system--and so he was much more
open-minded . . . . Danielli was very interested in how metabolism drives transport. But he
had the feeling that people like Goldacre, who wanted to achieve some sort of elastic~
largely conformational, type of mechanism, were on the right track. Keilin was interested
in transport phenomena through red blood cell membranes, but he was more of a
physiologist. But his attitude to me strongly suggested that he had a feeling that what I was
trying to do was in principle a good idea.
Besides setting the overall goal of integrating transport and metabolism
during his Cambridge years, Mitchell developed a vectorial approach to biochemical processes. As he has written (Mitchell, 1981a), G u g g e n h e i m ' s b o o k Thermo-
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dynamics According to the Method of Willard Gibbs (Guggenheim, 1933)
appealed to his imagination. H e has stated (interview, 23 May, 1980) that when
studying Guggenheim while thinking about enzyme kinetics and transport:
I realized that there is a fundamental difference between free energies, which are the
scalar products of two vectors (generally a force and a displacement), and entropic
energies (e.g. in the form of TdS), which are the scalar products of two scalars which did
not have any vectorial content. It think that I may have understood then that the
fundamental difference between heat energy, on the one hand, and the other forms of
energy that could do useful work, on the other hand, were to do with whether, in the
process of the abstraction required to obtain the energies from the appropriate scalar
products, you were losing some of the information; and that what we would have to do in
biochemistry to get the directionality of biology back again would be to put the vectors
back into our mechanistic formality. That, I think, may have been the underlying notion
that led me to the concept of vectorial chemistry.
Mitchell's inclination towards thinking in vectorial terms was probably
affected by his ideas in the 1940s about what he called fluctids, statids, and
fluctoids that he defined to classify different kinds of things (Mitchell, personal
communication, 5 December, 1990, and 20 February, 1991). About 1945
Mitchell's original speculations about the interplay of static (statid) and dynamic
(fluctid) components and forces in systems dynamics (fluctoid), such as in a living
cell, led one of his teachers to suggest that he should read about Heracleitos, who
taught that "everything flows". Actually, Mitchell's idea was that one could
discriminate between different types of things because only some things are
flow-things. However, he was delighted to discover from Burnet (1945) that
Heracleitos was reputed to have said, " Y o u can't step into the same river twice
because, although the name of the river remains the same, the water has flowed
on. But although this is obvious in the case of a river, it is not so obvious in the
case of a man". The use of scalar energies was seen by Mitchell as throwing away
literally vital information that was needed for understanding biological processes.
Mitchell's first version of his Ph.D. thesis, which included observations on
some effects of penicillin on bacteria, examined organisational effects of transport
in explicitly vectorial chemical terms, and employed concepts of diffusional fields
in a non-equilibrium thermodynamic context (Mitchell, interview, 4 October,
1982). Mitchell's committee found this presentation to be mystifying and had him
remove it and bolster the experimental section.
Through the efforts of Ernest Gale, Mitchell gave a paper at the 1949
meeting of the Society for General Microbiology on " T h e Osmotic Barrier of
Bacteria" (Mitchell, 1949), which appears to contain the first usage of "osmotic
barrier" for transport across biological membranes. The key analogy was that
diffusion of hydrated solutes across a topologically closed osmotic barrier between
two aqueous domains was similar to the progress of chemical groups through the
activation energy barrier in chemical reactions. Flow of metabolites or ions
through the barrier was seen as driven by an energy gradient corresponding to a
force with vectorial character.
With this conceptual background, Mitchell became involved in studying
phosphate metabolism and transport in Staphylococcus aureus, demonstrating the
Glynn and ChemiosmoticTheory
581
presence of a membranous osmotic barrier and blockage of transport by specific
inhibitors including D N P (Mitchell, 1953, 1954a). In summarizing this work,
Mitchell (1954b) treated bacterial transport and oxidative phosphorylation as
conceptually related p h e n o m e n a and stated, "In complex biochemical systems,
such as those carrying out oxidative p h o s p h o r y l a t i o n . . , the osmotic and enzymic
specificities appear to be equally important and may be practically synonymous".
However, Mitchell did not immediately follow up on the implications of this
concept. In 1955 he moved his laboratory to Edinburgh where, in addition to
other responsibilities and projects, he continued work on Staphylococcus and
other bacteria, assisted by Jennifer Moyle with whom he had previously
collaborated at Cambridge (Mitchell and Moyle, 1956a, 1957, 1958a, 1959). H e
further developed theoretical concepts for transport p h e n o m e n a (Mitchell and
Moyle, 1956b; Mitchell, 1956, 1957, 1959). H e (Mitchell, 1956; Mitchell and
Moyle, 1956b) proposed a detailed analogy between enzyme action and proteinmediated " p e r m e a t i o n " mechanisms in which the secondary chemistry of
solvation change was regarded as a fundamental mechanistic feature. The term
translocation was introduced to refer to the change of accessibility of hydrated
solute molecules from one side of the osmotic barrier to the other.
In a paper read to the Biochemical Society in 1957, but not published until
1959, Mitchell (1959) summarized his arguments for considering the catalysts
involved in m e m b r a n e transport as being normal enzymes that express vectorial
behaviour because of being e m b e d d e d in a membrane, rather than possessing some
unique structural property as suggested by Danielli (1954). Mitchell introduced
the term "chemi-osmotic" to describe enzyme-catalysed group-transfer processes
having both chemical transformation and osmotic translocation attributes (Mitchell, 1959, p. 90):
To simplify the analysis of the systems responsible for this spontaneous organising
activity we normally divide them arbitrarily into: (i) the endergonic systems more directly
responsible for the accumulation of the nutrients of the medium and the rearrangement of
the constituent atoms; and (ii) the exergonic systems which drive the systems of group (i),
being tightly coupled to them by mechanisms which involve the exchange of covalent
bonds between pairs of molecules. The endergonic systems of the cell bring together
certain groups of atoms and molecules which are generally present in relatively dilute
solution in the medium, and it has consequently been customary to divide the work done
by these systems into: (a) osmotic work, such as is done in transporting a solute through a
natural membrane against an electrochemical gradient; and (b) chemical work, such as is
done in linking carbon atoms together covalently. In this paper I have suggested that in
general, active transport depends upon the formation of covalent bonds between the
solutes that are taken up from the medium and components of the plasma-membrane, and
that these bonds are formed by exchange with the covalencies arising from the exergonic
systems of metabolism. According to this view, both osmotic work and chemical work is
represented by the spontaneous exchange (channelled by enzymic and carrier specificities)
of the covalencies from the substances arising in the exergonic systems of group (ii) with
those of endergonic group (i). If this view is correct, we should regard the plasmamembrane of bacteria not simply as an osmotic barrier and an osmotic link between the
media on either side of it, but we should consider it as a chemical link, allowing (as in the
well-known group-transfer reactions of classical biochemistry) the exchange of one
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covalently linked group for another, provided that the necessary specificity requirements
are met . . . . In view of certain similarities between mitochondrial membranes and the
plasma- membranes of bacteria, I would venture to suggest that the function of the
membranes of mitochondria and of the endoplasmic reticulum may be, like that of the
plasma-membrane, to act as chemi-osmotic links between the media that they separate.
This general chemiosmotic concept was developed in more detail in two key
papers (Mitchell and Moyle, 1958b, 1958c). The basic notion was that enzymecatalyzed group transfer was in effect a microscopic group-translocation process,
because the movement of the chemical group undergoing transfer along the
reaction coordinate between donor and acceptor groups in the catalytic domain of
the enzyme would be directed in space relative to a coordinate system based in
the enzyme. Thus, if the group-donor and group-acceptor substrate species were
to gain access from the aqueous medium to the catalytic domain by different
routes, the overall primary and secondary chemical process of chemical group
transfer would have a vector component relative to the enzyme molecule. This
group translocating action would not be detected at the macroscopic level in such
an enzyme in aqueous solution, using laboratory coordinates, because of the
isotropic tumbling of the enzyme molecules. However, if such an enzyme were
plugged through a topologically closed lipid membrane of low permeability, there
could then be a macroscopic consequence of group translocation because, given
the side-specific accessibility of the enzyme catalytic domain to group-donor
and/or group-acceptor substrate species, concentration differences of these
species could develop between the aqueous phases on either side of the
membrane. Further, if two Such enzymes with a common group-donor or
group-acceptor substrate species were plugged through the same topologically
closed membrane, the chemical action catalysed by the one enzyme could be
osmotically coupled to that catalysed by the other through the circulation of the
common substrate. This was described as macroscopic chemiosmotic coupling.
Jennifer Moyle's part in this development of ideas arose from her studies of
isocitrate dehydrogenase in Malcolm Dixon's laboratory. Like malate enzyme,
isocitrate dehydrogenase catalyses a dehydrogenation followed by a decarboxylation. In the light of the concept of enzyme-catalysed group translocation, Mitchell
and Moyle formed the conjecture that in the oxidative decarboxylation of
isocitrate, the intermediate compound, oxalosuccinate, might be confined to a
microscopic internal domain, from which it could not escape into the external
aqueous medium, thus coupling the decarboxylation to the dehydrogenation
through a microscopic osmotic mechanism. The results of some experimental tests
done by Jennifer Moyle appeared to be in accordance with this conjecture. In this
case, unlike the case of macroscopic chemiosmotic coupling (requiring enzymes to
be plugged through a membrane) the osmotic coupling action would presumably
be observed in the dual-function enzyme in aqueous solution, but should be
released by exposing the enzyme to treatments that opened up the microscopic
internal domain, and allowed the intermediate (e.g. oxalosuccinate in isocitrate
dehydrogenase) to equilibrate with the outer medium. Mitchell and Moyle
described this type of coupling as micro-chemiosmotic.
At this stage in Mitchell's thinking, the concept of enzyme-catalysed group
Glynn and ChemiosmoticTheory
583
translocation was applied only to homolytic actions. In other words, the
involvement of anionic or cationic groups in enzyme-catalysed group translocation had not been explicitly considered. However, in the course of supervising
work by B. P. Stephen that involved the possibly reversible action of a glucose
6-phosphate phosphatase present in the region between the cell wall and plasma
membrane of Escherichia coli (for which Mitchell coined the term periplasm), he
began to consider how the hydrolytic poise of the phosphate ester would be
affected if the catalytic site of the phosphatase were inaccessible to water as
such, but the activity of water there depended on hydrogen ions equilibrating
with that site only from one side of the plasma membrane and on hydroxide ions
equilibrating with it only from the other side. Obviously, the water activity could
be depressed, and phosphorylation promoted, if the electrochemical potentials
(i.e. pH values and electric potentials) of the hydrogen ions in the aqueous media
on either side of the membrane were appropriately different, so that hydrogen
ions would be drawn away towards the medium of low protonic potential, and
hydroxide ions would be drawn away to the medium of high protonic potential
(Mitchell, 1961b). This idea prompted more general considerations of enzymecatalysed group translocations involving heterolytic chemical actions and,
amongst other things, provided the starting point for the formulation of reversible
protonmotive ATPase mechanisms.
The general theory of vectorial metabolism amounted to a broad generalisation and development of the proposal of vectorial electron transfer in redox chain
systems (Davies and Ogston, 1950; Lundegardh, 1940, 1954, cited in Mitchell and
Moyle, 1958c), although it was actually derived from general principles, and not
by expanding directly on the earlier specific proposals. The maturation of the
general theory of chemiosmotic systems encouraged Mitchell to think more
seriously about applying it to the so-far intractable problem of the coupling
mechanism in oxidative and photosynthetic phosphorylation. Mitchell recalls that
around 1959 he became convinced that the membrane of bacteria and mitochondria could be impermeable to protons (Mitchell, interview, 4 October, 1982); and
his laboratory began performing proton conductance measurements in 1960. At
the same time B. P. Stephen was writing up her Ph.D. dissertation on the studies
in Mitchell's laboratory on glucose 6-phosphatase. During the summer of 1960
Mitchell prepared papers for delivery at the Prague Symposium and the
International Union of Biochemistry Symposium in Stockholm. A colleague of
Mitchell, Jack Dainty, recalls this period (personal communication, 18 August,
1987 and 28 November, 1987):
Peter would often come (at one period, every day) and discuss his ideas with me; in a
sense he was "using" me to sound out and sharpen his ideas. I think he expected from me,
who am trained in the physical sciences, a check on whether any of his notions were
physically nonsensical. This was particularly so with respect to the thermodynamic aspects
of his model(s) . . . . I remember that he seemed obsessed by physical models of vectorial
transport coupled to ATP synthesis. He would build them from plywood (-1 cm thick)
and each model would consist of several pieces articulated together and able to swivel in
various ways. He would produce a new plywood model every week or so at one stage and
he would try them all out on me~emonstrating exactly what he thought was happening
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with the ADP, Pi, ATP, H20, H +, and so on. He was very, very dedicated and obsessed
with them. I believe that his discussions with me about the thermodynamics of the model
and the role of gradients began well before the Prague Symposium (which I attended).
Mitchell has characterized this period (interview, 4 October, 1982) as one in
which "the way ideas developed wasn't sort of 'snap-snap', but was [rather a]
gradual clearing of mist".
At the Prague Symposium on Membrane Transport and Metabolism, 22-27
August, 1960, for which the formal papers were precirculated, Mitchell presented
a paper entitled "Biological Transport Phenomena and the Spatially Anisotropic
Characteristics of Enzyme Systems Causing a Vector Component of Metabolism", which the organisers appropriately placed first in the symposium and in the
resulting volume (Mitchell, 1961a). In this paper Mitchell made an explicit
attempt to link transport phenomena with enzymology using the chemiosmotic
concepts he had developed (Mitchell, 1954b, 1956, 1957; Mitchell and Moyle,
1956b, 1958b, 1958c; Mitchell, 1959) to provide a general account of the
mechanisms by which enzymes and catalysts of facilitated diffusion, anisotropically arranged in a membrane, produce a spatially organized diffusion of ligands,
thus providing the fundamental vectorial component of the primary and secondary chemical processes of metabolism underlying transport. Independently,
Crane (Crane, Miller and Bihler, 1961) proposed a model of the secondary
chemical process of the co-transport of glucose with sodium ion, building upon
ideas of exchange diffusion and counter-transport of Ussing (1947) and Widdas
(1952), in which it was implicit that the trans-membrane force and energy of the
electrochemical gradient of one solute could be used to drive the translocation of
another solute. By Crane's account (Crane, 1983) Mitchell alone fully understood
the implications of his proposal. Crane went on to apply his model of co-transport
to problems in mammalian transport, and provided the basis for a rich research
programme (Alvarado and van Os, 1986).
As can be seen from his precirculated contribution at the Prague Symposium,
Mitchell had already reached the general conclusion that the driving of the
transport of one ligand by another required only that the two ligands be (literally)
coupled together, either directly or through an intermediary component, and that
their compound or complex be free to diffuse through a common catalytic
translocation pathway traversing the osmotic barrier. It would be irrelevant
whether the coupling of the ligands involved primary and/or secondary valencies.
Mitchell continued to develop his general theory of vectorial metabolism and
chemiosmotic systems, and began to apply it explicitly to the problem of oxidative
and photosynthetic phosphorylation, as first outlined two weeks after the Prague
meeting, in the latter part of a paper on transport principles related to his
laboratory's work on glucose transport, at the International Union of Biochemistry Symposium in Stockholm (Mitchell, 1961b). In the autumn of 1960, a visitor
to Mitchell's laboratory in Edinburgh, Guy Greville, reported back to his
colleagues at Cambridge that Mitchell was trying to measure the pH of
mitochondrial suspensions and getting quite excited (Chappell, interview, 19
May, 1980).
Glynn and ChemiosmoticTheory
585
In order to appreciate that excitement, it is necessary to place the problem of
oxidative phosphorylation in its context in 1960.
Initial Proposals for the Mechanism of Oxidative Phosphorylation
Once there was some understanding about the basic phenomenon of
mitochondrial oxidative phosphorylation by the late 1940s, it was natural that
attempts would be made to postulate and test for the mechanism by which the
redox reactions of the electron transport chain were coupled to the synthesis of
ATP. The expectation was that the linkage of the processes ought to be
analogous to other coupled processes in metabolism and involve chemical
intermediates (Lipmann, 1941, 1946).
The first detailed proposal that had an impact on the field was that of
Edward Charles "Bill" Slater (Slater, 1953), that followed the preliminary
suggestion of Lipmann (1946) in which putative chemical intermediates were
assumed to be phosphorylated compounds produced by the redox reactions of the
electron carriers. Slater worked on his Ph.D. research (1946-49) in the laboratory
of David Keilin on the succinate oxidase system of Hartree-Keilin particles
(submitochondrial particles). After finishing his thesis research and before leaving
to spend a postdoctoral year with Ochoa at the Rockefeller Institute, Slater used
the first Beckman spectrophotometer at the Molteno Institute to see if the
Hartree-Keilin preparations would catalyze the oxidation of NADH by oxygen,
since Friedkin and Lehninger (1949) had demonstrated this with whole mitochondria; and he demonstrated for the first time the oxidation of NADH by fumarate.
Slater's interest in oxidative phosphorylation was stimulated by Ogston and
Smithies' (1948) review. While in Ochoa's laboratory, Slater developed a
spectrophotometric method to measure the production of ATP and demonstrated
phosphorylation with ferricytochrome c as the acceptor in the absence of oxygen,
but probably more importantly he followed Racker's demonstration of the
intermediate for glyceraldehyde-3-phosphate dehydrogenase that was proceeding
two floors below. Racker and Krimsky (1952) showed that an enzyme-bound
thioester intermediate forms prior to attack by inorganic phosphate, producing
the acyl phosphate intermediate 1,3-diphosphoglycerate that subsequently transfers the phosphoryl group to ADP under the action of 3-phosphoglycerate kinase.
Upon his return to the Molteno Institute, Slater's interests shifted more toward
elucidating the mechanism of oxidative phosphorylation. He has described the
background to his 1953 paper (interview, 6 July, 1979):
The stimulus for my paper in 1953 came while I was attending a Society of
Experimental Biology Symposium in Bangor, Wales, early in 1953. Ernest Gale gave a
paper, in which he reported that dinitrophenol stops growth and substrate assimilation in
Streptococcus faecalis. As I knew that DNP was an uncoupler in oxidative phosphorylation, I asked Gale why does DNP have this effect in an organism that does not possess
oxidative phosphorylation? I received the crushing answer, "Well, you enzymologists
might think that everything that goes on in cells is just as with your isolated enzymes, but I
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assure you, a bacterial cell is different". It was my dissatisfaction with this answer that
stimulated me to think about it, and I got the idea that DNP might destroy an intermediate
in oxidative phosphorylati ,n. And since these reactions would be reversible, you could
form the intermediate from ATP, as well as from the respiratory chain. So it was really to
explain why DNP was damaging cells when ATP only came from glycolysis that made me
think of the ATP formation in oxidative phosphorylation as being a reversible process.
The sort of intermediate I had in mind was analogous to that proposed by Ef Racker for
glyceraldehyde-3-phosphate dehydrogenase. And after I wrote up the 1953 paper, I
thought that it was trivial and I very much hesitated sending it to Nature. I very nearly
didn't send it in.
At about the same time Lehninger proposed a similar mechanism in his
Harvey Lecture that was not published until later (Lehninger, 1954), although
Lehninger had X - P as the primary intermediate, harking back to Lipmann
(1946). What was novel in Slater's proposal, and not always appreciated by
chemists in the 1950s, was the inclusion of a non-phosphorylated intermediate as
the primary event with phosphorylated intermediates forming later. Lardy's
introduction of oligomycin as an inhibitor of phosphorylation (Lardy, Johnson
and McMurray, 1958) allowed the demonstration that phosphorylation followed
the formation of "squiggle" (Huijing and Slater, 1961).
Although it was widely and commonly taken for granted that this nonphosphorylated intermediate had to be a chemical compound, the possibility was
not excluded that the so-called "squiggle" could represent another type of
potential energy source. It is relevant that, following work by Davies and Ogston
(1950), Davies and Krebs (1952) considered the use of a trans-membrane proton
current as a possible means of coupling between two electron-transfer systems,
one of which was coupled to phosphorylation through a chemical intermediate.
Thus, they did not overcome the problem of the "squiggle" that was believed to
be a chemical intermediate, but they invoked a trans-membrane proton current as
an extra "squiggle". Two decades later, after it had become apparent that the
"squiggle" of oxidative and photosynthetic phosphorylation was extremely
difficult to identify as a covalent chemical compound, Boyer (1965) suggested that
it might represent a protein coupling factor in an energy-rich conformational
state. A detailed analysis of the conceptual structure of the chemical intermediate
hypothesi s and its development over time has been provided by Rowen (1986).
Through the 'fifties and 'sixties most research on mitochondrial oxidative
phosphorylation was informed, guided, and interpreted in terms of Slater's
hypothesis or some subsequent variant, assuming that the coupling mechanism
involved covalent chemical intermediates (see for example Racker, 1965); indeed,
some workers pursued this approach well into the 'seventies. Between 1954 and
1984 there were 378 citations (less self-citations) to Slater (1953) with 310 before
1974. The fraction of citations that assumed the correctness of Slater's hypothesis
dropped from 73% + 6% over the period of 1961 through 1966, to 18% • 17%
over the period of 1971 to 1976, with only a handful of such "positive" citations
subsequently. This change was due, on the one hand, to not finding any of the
postulated intermediates and, on the other hand, to the availability of alternative
hypotheses (Mitchell, 1961e; Boyer, 1965) that came to dominate research in this
field.
Glynn and ChemiosmoticTheory
587
Application of Chelniosmotic Principles to Oxidative Phosphorylation
By 15 February 1961 Mitchell submitted an abstract for the 29-30 March
1961 Biochemical Society meeting (Mitchell, 1961c) in which he published for the
first time the basic assumptions for the application of chemiosmotic principles to
oxidative and photosynthetic phosphorylation: 1) the flow of electrons and
hydrogen atoms through the redox chain (driven by an electron source and sink
or by photons) occurs vectorially; 2) this results in a vectorial translocation of
protons between the aqueous phases separated by the membrane; 3) the
membrane is impermeable to protons, and so a pH difference and a membrane
potential develop; 4) the membrane contains a reversible anisotropic ATPase that
5) enables the return flow of protons to drive the dehydration reaction of
phosphate and ADP. Leakiness of the membrane would cause uncoupling.
Based upon an initial assumption of one proton/ATP Mitchell calculated that
a pH difference of 2 and an electric membrane potential of 300 mV would give
equipoise of [ATP]/[ADP]. About the same time (prior to 15 February) Mitchell
completed experiments and a few months after submitted an abstract (Mitchell,
1961d) on membrane conductance. Working with suspensions of Micrococcus
lysodeikticus and rat-liver mitochondria Mitchell showed, by studying the
time-course of pH changes in response to a simple acid pulse, that the low rate of
proton diffusion through the membranes was increased manyfold by a variety of
uncouplers of oxidative phosphorylation. This result was consistent with the
chemiosmotic model of oxidative phosphorylation and provided an elegant
explanation for the uncoupling due to chemically very different acid/base
compounds; all that was required for uncoupling was that both the acidic and the
basic form be lipid soluble.
At about this time the first issue of the Journal of Theoretical Biology was
published with "Possible Functions of Chains of Catalysts" by R. J. P. Williams
(1961) as the lead article. Williams suggested that the electron carriers could
produce anhydrous protons in dislocated phases between catalytic protein
molecules, in which the proton concentration could be great enough to drive the
dehydration of phosphate and ADP. On 24 February 1961 Mitchell wrote to
Williams requesting clarification on the chemistry Williams was proposing and
began a correspondence that lasted sporadically over the next 17 years (an
analysis of this correspondence will be published elsewhere).
It is clear that all the key components of Mitchell's hypothesis, which is quite
distinct from Williams', were in place well before Mitchell opened the correspondence; nor did Mitchell have access to Williams' manuscript as a referee prior to
publication (James Danielli, interview, 2 March, 1982). As with Crane's
development of the gradient-coupled transport concept, Williams' proposal that
redox-chain derived protons drive ATP synthesis represents independent and
virtually simultaneous work. In any event, Williams did not pursue his model
experimentally and, less self-citations, there are no citations of his paper until
1966 and only 10 by 1973. Later in the 'seventies and 'eighties the number of
citations did increase to a dozen or so a year, especially as interest in "localized"
proton coupling increased. A further relevant factor in assessing the impact of
new ideas is their conceptual context. Crane's ion-gradient proposal and Davies'
588
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and Williams' proposed roles for protons in ATP synthesis correlate only with
components of the larger conceptual structure of the chemiosmotic theorizing of
Mitchell in the late 'fifties and early 'sixties. The earlier proposals were at a
phenomenological level whereas the models advanced in 1960 moved toward
specific mechanisms, but only the chemiosmotic model had a set of articulated
principles that provided falsifiable causal explanations that could guide research
on the specific problem of oxidative phosphorylation and hold out the promise of
a conceptual unification of energy-transducing mechanisms and of integrating
metabolism and transport. This issue will be explored more fully in the last
section.
On 28 April 1961 Mitchell submitted "Coupling of Phosphorylation to
Electron and Hydrogen Transfer by a Chemi-osmotic Type of Mechanism" in
which he expanded on the basic principles of the model previously presented
at meetings (Mitchell, 1961b, 1961c) and placed those principles within the
context of his earlier work on the general concept of chemiosmotic ideas and the
transport literature (which has been summarized above) and made specific
testable, falsifiable predictions from the model (Mitchell, 1961e). He justified the
presentation of the chemiosmotic model as an alternative to the orthodox
chemical intermediate model because of the lack of success in the isolation of any
intermediates and difficulties of that orthodox model in explaining the observed
facts of oxidative phosphorylation.
In summer 1961 Mitchell read a paper at the Oxford meeting of the Society
for General Microbiology (Mitchell, 1962) in which he presented data on internal
ethanol oxidation by external ferricyanide for suspensions of M. lysodeikticus that
showed that a stoichiometric amount of H § was liberated into the medium from
internal N A D H + H § This observation was consistent with the expectations of
the chemiosmotic model. Further he showed that the cytochrome oxidase
appeared to be anisotropically arranged in the membrane and that the electrons
had to move inwards through the membrane to reach the active site of the
cytochrome oxidase. Also, these experiments revealed that the polarity Mitchell
had indicated for mitochondria in the Nature paper was reversed and that
mitochondria, and bacteria, pump protons outwards and not inwards.
During the verbal presentation of this paper, Mitchell made reference to
Liebig having thought that chemical momenta were important in biological
processes. On 13 October 1961 Hans Krebs, who had attended the meeting,
wrote to Mitchell indicating that he was bothered by Mitchell's vectorial concepts
linking metabolism and transport and specifically concepts such as momenta,
" . . . my view is that the classical approach, as practised by, say, Lehninger and
D. E. Green, still seems to me the most likely one to solve the problem. Your
return to Liebig appears to me to be a retrograde step". Krebs also disclaimed
that his and Davies' earlier suggestion of an osmotic link in oxidative phosphorylation (Davies and Krebs, 1952) was anything but an hypothesis that had
little impact. Despite criticism, such as Krebs', Mitchell continued to hold to his
belief in vectorial chemistry, drawing support in part from the work of Glasstone,
Laidler and Eyring (1941) in which chemical reactions were represented as a
trajectory in multidimensional phase space. Subsequently, Mitchell wrote an
Glynn and ChemiosmoticTheory
589
extended paper on the general concepts of chemiosmotic coupling and vectorial
metabolism (Mitchell, 1963) that was presented at the March 1962 Biochemistry
Society meeting. At that meeting G. D. Greville reported that he and K. E.
Bicknell had repeated Mitchell's earlier experiments on the effect of uncouplers
on the p H of mitochondrial suspensions. A promising start was evidently made in
exploring and revising the chemiosmotic model which had grown out of Mitchell's
thought in the 'fifties, and the process of the "clearing of the mists" was
continuing (Weber, 1986b).
In early 1963 Mitchell became seriously ill with acute gastric ulcers and it
would not be until 1965 that he would resume publishing on his chemiosmotic
model. Even so, there were 30 citations, less self-citations, of Mitchell's 1961
Nature paper (Mitchell, 1961e), many from researchers in the transport field, but
also some from the oxidative phosphorylation community. One of the key figures
in that community, Albert Lehninger published two major reviews in 1962
(Lenhninger, 1962; Lehninger and Wadkins, 1962) in which he gave a sympathetic summary of Mitchell's proposal. Mitchell's hypothesis also attracted the
interest of Slater whom Mitchell knew from their Cambridge days and with whom
he maintained a lively correspondence. On 4 January 1962 Slater wrote a long
letter to Mitchell providing detailed comments on Mitchell's Nature paper. After
defending the adequacy of his chemical intermediate hypothesis to account for
experimental observations, he makes some laudatory comments about Mitchell's
model and concludes:
To summarize, I find your theory most stimulating. It has many attractive features,
especially the stoichiometry. There are a number of "facts" about oxidative phosphorylation which I think it might have difficulty in explaining, but I should prefer to write again
about these. One of its great virtues to my mind is that it is the first theory described in
concrete terms in which there is mechanism for all three phosphorylation steps in the
respiratory chain. Many people, including Lipmann, have from time to time wondered if
that might be the case, but I could never picture such a theory.
I am not saying that I believe in your theory. But I do plan to think pretty hard about
experiments which might be able to make a clear distinction between the chemiosmotic
theory and the orthodox chemical.
In response Mitchell wrote to Slater on 14 February 1962 and made clear that
he did not imply that the facts of oxidative phosphorylation could not be
explained by the orthodox conception of chemical intermediates, only that is was
difficult to do so, and therefore that it was worthwhile to suggest a new
alternative approach. H e concluded with:
I have no preconceived ideas at present as to which of the alternative conceptions is
the more likely to fit all the facts, and so I look forward to being able to discuss the whole
question with you in an entirely open-minded way. We have-done a number of
experiments here aimed to disproving the feasibility of the chemi-osmotic conception, but
so far without success.
During this time notes of meetings of the Slater group indicate that they were
discussing the chemiosmotic model in critical detail (a more comprehensive
Weber
590
analysis of the initial responses of the various oxidative phosphorylation
laboratories will be given elsewhere). By 1964 Slater was corresponding with
Mitchell about his presentation of the chemiosmotic theory in a chapter of
oxidative phosphorylation for Comprehensive Biochemistry (Slater, 1966).
Thus there was a surprisingly reasonable response to the chemiosmotic
model during the early 'sixties by several of the leaders of the oxidative
phosphorylation field even if it was considered as an elegant but outside
alternative. Overall, however, most of the field's reaction was unfavourable
(Mitchell, 1981a), and later on both Slater and Lehninger became critics of
aspects of the chemiosmotic model. In any event, the initial experimental
evaluation in Mitchell's laboratory was cut short due to his illness and did not
resume until after the founding of the Glynn Research Institute.
THE F O U N D I N G OF THE GLYNN R E S E A R C H INSTITUTE
Mitchell has written a brief account of the founding of the research
organisation at Glynn (Mitchell, 1980). While in Edinburgh Mitchell had, with
the help of craftsmen, restored an old manse "Carrington Hill" in the nearby
village of Carrington for his family's residence. In October 1961 Mitchell saw an
advertisement for a cottage, Glynn Mill, which he inspected and purchased as a
vacation home. While there the estate agent, Mr Weekes, took him to look at
Glynn House, and he was saddened to see that this beautiful building was in such
an advanced state of decay that it would soon be completely ruined if nobody
took on the responsibility of restoring it. He happened to remark that it would be
an ideal setting for a small research institute. Upon his return to Edinburgh, Mr.
Weekes contacted Mitchell to interest him in the possible purchase and
restoration of Glynn House. Further contacts in January 1962 led nowhere as the
owner Mr. Claude Selleck, a speculative builder, was asking too high a price. But
Mr. Selleck died unexpectedly in April 1962 and his widow wanted to get rid of
the house. Mr. Weeks wrote and asked Mitchell if he would like to make an
offer. Mitchell responded that he felt that the house had no value, but that the
site might be worth s
Weekes' response was that the offer was accepted but
that it should be reduced to s
because the widow wanted to avoid Estate
Duty.
By summer 1962 Mitchell spent his holidays attempting to dry the house out
and halt the spread of dry rot by putting up sheets of polyethylene and blowing
air in through the front door with a large grain-drying fan. He hoped to halt the
dilapidations, and get the house onto the market so that it could enjoy a new
lease of life. He had no intention then to use it as a research institute. Shortly
after this time Mitchell's health deteriorated and he was facing an operation to
remove four-fifths of his stomach. He decided not to submit to surgery, and
accepted leave of absence to try to recover by relaxing at Glynn Mill. He soon
realized that he would have to resign his post at Edinburgh and began to think
seriously about the possibility of converting Glynn House into a research
institute. As Mitchell has described to me (Mitchell, from interviews 5 July, 1979
Glynn and ChemiosmoticTheory
591
and 5 October, 1982):
I wrote to Dr. Moyle after I'd come down to have a protracted leave from Edinburgh,
at Glynn Mill, and said to her, if I got involved in a scheme of restoring the house, which
would probably take a couple of years, and then making an independent foundation,
would she join me? Rather surprisingly, she wrote back and said "Yes". If she had said
no, I'm almost sure I wouldn't have undertaken it.
We spent two years restoring the house with about twenty men and no architect. We
did all the surveying and all the necessary drawings and looking after the workers . . . . I've
never had to control a gang of twenty real men before; two of them were jailbirds. Really,
it was one of the most enjoyable periods of my life . . . . Another beneficial thing that
happened was that I became involved in farming the land around Glynn House. As a
result of a fortuitous series of events, I had to spend several months milking eight cows by
hand, morning and evening. That proved to be an excellent therapy for healing my gastric
ulcers.
The restoration and reconstruction of Glynn House and its rehabilitation as a
residence and a m o d e r n scientific research institute continued well into autumn
1964. The funding of the works cost s
paid out of Mitchell's pocket, and
the original e n d o w m e n t of the institute required s
which was provided by
Mitchell and his brother, transferring their shares in George Wimpey & Co., run
by their uncle Sir G o d f r e y Mitchell.
The M e m o r a n d u m and Articles of Association of Glynn Research Limited,
the Company formed to administer the work of the Research Institute, were
signed on 1 April, 1964 and the Company was Incorporated on 21 April, 1964
" T o promote and carry on scientific research, in particular in the field of
fundamental biology, with a view to the development and spread of scientific
k n o w l e d g e . . . " . The Company was accepted for registration as a U.K. Charity.
Peter Mitchell and Jennifer Moyle were the two share holders and members of
the Council of Management. The Company proceeded to lease the major part of
Glynn House from Peter Mitchell at a nominal rent. During the summer of 1964
Mitchell and Moyle finished and equipped the laboratories, and by autumn
started experimentation on a total equipment budget of s
Mitchell thus
launched his experiment to see if a small, independent research institute could
provide a " . . . quiet haven for untrammelled scientific work and thought"
(Mitchell, letter to E. C. Slater, 16 April, 1964), which could have a disproportionately large impact on the development of a specific field of science. In
comparing the above monetary costs with contemporary ones, it is necessary to
bear in mind that, according to the U.K. Retail Prices Index, the purchasing
power of s in 1964/65 was equivalent to about s in 1990/91.
GLYNN A N D THE EVOLUTION OF CHEMIOSMOTIC KNOWLEDGE
The Early Years
During the final phases of constructing the laboratories at Glynn, Mitchell
exchanged a number of letters with Slater. Initially the correspondence involved
592
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Slater making sure that he had correctly represented Mitchell's chemiosmotic
model in his review for Comprehensive Biochemistry (Slater, 1966). Mitchell
suggested revisions to more accurately portray his position, especially with regard
to the mechanism of A T P synthesis. In particular Slater had questioned whether
the assumption that the proton involved in the ATPase had to come from the
opposite side of the membrane. In a letter dated 13 May, 1964, Mitchell wrote to
Slater:
I prefer to assume the ATPase to be accessible to H § only from the right-hand
[cytoplasmic] side, because that seems to me to be the simplest assumption . . . . It is, of
course, important to note that although the assumption which you have questioned is not
necessary, as my theory could not be sufficiently defined without this or some similar
assumption, the assumption about accessibility to H § cannot be regarded as a superfluous
excrescence of the kind to be shaved off by the razor of Mr. Occam.
Mitchell went on to describe his plans for research at Glynn:
No doubt it will take us quite some time to build up to about 12 personnel which we
consider to be the maximum size for the efficient and happy administration of this lab., but
meanwhile we are quite confident that we shall succeed in getting down to some research
and useful writing in this peaceful and, incidentally, very beautiful place.
The correspondence continued on the question of the magnitude of the p H
difference across the m e m b r a n e and about the mechanistic role of phosphorylium. Also, on 25 May 1964 Andr6 Jagendorf wrote to Mitchell to tell him
of experimental evidence from his laboratory indicating that energized chloroplasts had a pool of high energy, non-phosphorylated "intermediate" that
appeared to be a p H gradient as Mitchell had predicted. The kinetics of the p H
rise were correlated to the kinetics of the "intermediate". While not proving the
chemiosmotic model to be correct for chloroplast phosphorylation, it was
gratifyingly consistent. On 14 D e c e m b e r 1964 Slater wrote to Mitchell in part
about the implications of Jagendorf's results expressing the concern that the
tenfold excess of H § to A T P produced suggested that the chemical intermediate
was driving the transport of protons. A little later in the month Slater invited
Mitchell to present his chemiosmotic model at the first Bari meeting in late April
1965. The Bari meeting itself was an experiment in a smaller more focused
meeting rather than the large scientific congresses that had become c o m m o n after
World War II (Slater, 1985).
Thus, during the time that Mitchell and Moyle were deciding what focus to
give to the Glynn Research Institute, Mitchell was being drawn more specifically
into thinking about his chemiosmotic model of oxidative phosphorylation. The
correspondence with Slater had emphasised that the theory, as presented in 1961,
needed to be revised and described in more detail, and the Bari Conference
would give him an opportunity to present the model to the oxphos community.
Also, it became clear that it would be appropriate to begin research in the
laboratory by extending the preliminary experiments with mitochondria and
bacteria that Mitchell and Moyle had started back at the University of Edinburgh.
Glynn and ChemiosmoticTheory
593
Thus began what might be characterized as the Glynn style of doing science. This
style includes intensive theoretical analysis combined with carefully conceived and
executed experiments. Results of both are presented at major meetings coupled
with Mitchell visiting laboratories (such as he did in Slater's laboratory in early
May 1965 on his way back from Bari); but more importantly having scientists
come for a few days to a week or so for undistracted and focused discussions at
Glynn. The first such visitor was R o b e r t Crane in 1964. In 1965 seven scientists
visited including Andr6 Jagendorf, Brian Chappell and E. C. Slater. Such
interactions often led to the visitor pursuing new experiments based upon the
discussions at Glynn, as was the case for Jagendorf's p H overshoot experiments.
During the first twenty years of Glynn, over 150 of the scientists involved in
various aspects of bioenergetics visited Glynn, many several times. Virtually all
the main participants in oxidative phosphorylation research have come to Glynn,
including some of Mitchell's most vocal critics. This type of venue for scientific
discourse is certainly less stressed then the typical scientific meeting, and when
there is a reasonable framework of conceptual affinity, effective communication
has resulted. It is not so clear what impact such interactions have had on critics,
but at least the differences should become more clearly defined. In his statement
as the Chairman and Director of Research in the Second Annual General
Meeting of Glynn Research Institute, 13 June 1966 5Mitchell stated:
In a small research organisation it is not, of course, possible to work on a wide range
of research problems. Sound economic and intellectual considerations show that it is
necessary to concentrate upon a limited range of experimental techniques (thus minimising
expenditure on specialised equipment), and to focus attention upon a limited area of the
intellectual field in which the research workers have competent knowledge and experience.
The fact that the research problem is selected within a limited experimental and
intellectual field does not, however, necessarily mean that the solution of the problem will
have a correspondingly limited application. In practice, according to the best traditions of
research strategy, one attempts to select small-scale problems, the solutions to which will
have large-scale applications. Using these and other less easily defined principles of
selection, the research activities of our Company have been focused upon "the mechanism
of oxidative and photosynthetic phosphorylation".
In the next year's report Mitchell pointed out that several million dollars had
been expended in the United States alone funding research based upon the
orthodox chemical intermediate hypothesis by a number of first-rate laboratories,
whereas the work at Glynn was being done with minimal expenditure. Even so,
Mitchell reported that the Institute was operating at a deficit. The theme of the
financial problems of doing research in even such a small setting are referred to
throughout the subsequent reports. By 1970, the goal is expressed of completing
research on oxidative phosphorylation by 1972. The deadline for moving research
from bioenergetics to some other area kept being moved back and bioenergetics
has continued as the primary focus for the first twenty-five years. An evaluation
of the effectiveness of the Glynn experiment will be undertaken in the final
section.
The intense experimental and conceptual efforts of the first few years of
594
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research at Glynn were crucial for the subsequent degree of acceptance of the
chemiosmotic model of oxidative phosphorylation and the degree of its ultimate
impact on bioenergetics. The next decade of work would show the power of the
chemiosmotic research programme to evolve conceptually and to not only
accommodate but to generate new data.
The paper presented at the Bari meeting in late April 1965 (Mitchell, 1966a)
gave the most extended treatment of the chemiosmotic model to that date and,
while reiterating the basic principles of the 1961 Nature paper, corrected details
of the original model, especially with regard to the polarity of the protonmotive
force across the membrane. Mitchell pointed out that the chemiosmotic model
not only could account for the synthesis of ATP, as did the chemical intermediate
model, but that it explained the action of uncouplers of disparate structure,
accounted for transport of ionic metabolites across the mitochondrial inner
membrane, and could also explain simply the observed osmotic swelling and
shrinking of mitochondria. Of course the chemical intermediate model had been
elaborated to explain such osmotic phenomena, but at the expense of postulating
further chemical intermediates in addition to those supposed to be involved in
ATP synthesis (Lardy, Connelly and Johnson, 1964). In this presentation Mitchell
first illustrated his concept of a redox loop that would stoichiometrically conduct
protons across the membrane via a type of ligand conduction. The response to
Mitchell's talk was muted, just two questions were asked and Slater commented
that in trying to explain both mitochondrial ion transport and ATP synthesis as
different aspects of the same process, Mitchell was trying to have his cake and eat
it. But it should be remembered that, because of the protonmotive stoichiometry
that Mitchell was assuming at the time, a membrane potential as large as 420 mV
or a pH gradient of 7.0 was required by the model to reach an equipoise of ATP
and ADP, which values seemed beyond what was likely physiologically.
Jagendorf arranged for Mitchell to be invited to the Gordon conference
scheduled for late August, 1965. Mitchell met Slater in Amsterdam and they
joined Chance in Boston to go together to the meeting. According to Slater's
notes on the sessions, Mitchell was the "hit of the conference". The impact of the
presentation of the chemiosmotic model was enhanced by Jagendorf's report of
pH-gradient induced ATP synthesis in dark chloroplasts. Jagendorf had written to
Mitchell on 5 August about this work:
My work with ATP induction by pH changes has been swimming right along . . . . We
have been able to show that it is ATP that is formed; that its formation is inh~bited by
typical uncouplers; and that the stored material decays in 2 seconds at pH 8 (just as does
light-driven XE) and also ATP formation is finished in 4 seconds at pH 8. Nicely enough,
it's a reversible phenomenon . . . rul[ing] out a one-shot, acid denaturation artifact. We
can rule out an oxidative process at pH 8, because we get even more ATP-forming under
strictly anaerobic conditions than under aerobic conditions. The maximum yield observed
so far has been . . . a grand total of 120 ATP per cytochrome f. Hardly likely to be an
electron carrier, at that rate.
This work (Jagendorf and Uribe, 1966), which Robinson (1986) has argued,
contra Gilbert and Mulkay (1984a), represents a key experiment, coupled with
Glynn and ChemiosmoticTheory
595
the earlier reports from Jagendorf's laboratory on light-induced pH changes for
chloroplasts (Hind and Jagendorf, 1963; Jagendorf and Hind, 1963; Neumann
and Jagendorf, 1964) plus Mitchell's preliminary reports of his experimental
studies at Glynn moved the chemiosmotic model forward as a serious contender
to the orthodox chemical intermediate model.
Mitchell and Moyle (1965a) reported the results of their oxygen-pulse
experiments. Using more sophisticated methodology they were able to confirm
the earlier measurements of low proton permeability of the mitochondrial
membrane. More importantly, they were able not only to confirm that the
respiratory chain could translocate protons as could the ATPase, but they were
also able to obtain the first stoichiometric data on the number of protons
involved. They reported that oxidation of fl-hydroxybutyrate gave <---H+/O = 6,
oxidation of succinate a ratio of 4, and for ATP hydrolysis ~---H+/P = 2, which
was consistent with the expected P/O ratios of 3 and 2 respectively for
fl-hydroxybutyrate and succinate. These proton stoichiometries, which differed
from those originally assumed by Mitchell, meant that the total protonmotive
force needed only to be 210 mV with a maximum membrane potential of 210 mV
or a pH difference of 3.5, or more likely some combination of the two at lesser
values. This correction was borne out nicely in Jagendorf's experiments where the
pH jump involved was 4 pH units; a pH difference of lesser magnitude, say 3, did
not drive the synthesis of ATP.
Mitchell appended a note about the stoichiometries, and the more physiological values of A~p and ApH that result, to the published version of his Bari paper
(Mitchell, 1966a). While supportive of the chemiosmotic model, such results
could be explained by postulating that proton translocation was a secondary
process driven by the intermediates of ATP synthesis or the intermediates
postulated to be involved in osmotic phenomena of mitochondria. Mitchell and
Moyle (1965b) addressed this issue in experiments on submitochondrial particles
which gave proton translocation with reversed polarity, consistent with the
observations of Lee and Ernster (1966) that the submitochondrial particles were
intact, but inverted, vesicles formed from the inner membrane. Also they showed
that reversal of electron transport caused uptake of protons as expected in the
chemiosmotic model but not expected in the chemical model.
Work in Brian Chappell's laboratory at about this time on mitochondrial
transport (Chappell and Crofts, 1965, 1966) confirmed that the mitochondrial
inner membrane had a low permeability to monovalent cations and that
uncouplers equilibrated the pH difference across the membrane; the interpretation of their data was most intelligibly couched in terms of the chemiosmotic
theory. Chappell visited Mitchell at Glynn in early August 1965, after Jagendorf's
and just before Slater's interactions at Glynn. Reid, Moyle and Mitchell (1966)
used valinomycin, introduced by Pressman (1965) and used by Chappell's
laboratory, as an additional probe to confirm basic stoichiometries and reported
preliminary results of experiments that qualitatively, if not quantitatively,
confirmed that a pH gradient could cause ATP synthesis in mitochondria in a
fashion similar to chloroplasts. At the same time Mitchell had been reading
widely in the literature of oxidative phosphorylation, guided by the review by
596
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Ernster and Lee (1964), after his return from the Bari meeting. Mitchell reports
(interview, 24 May 1980): "My reading was interspersed with days spent thinking,
drawing doodles, trying to integrate the knowledge that was available from the
literature with the conjectures that one was trying to produce".
When the editor of Biological Reviews requested a review article on the
chemiosmotic model for oxidative phosphorylation, Mitchell was quite ready to
comply. The review Mitchell produced was too long for publication in the journal
and the editor required that it be significantly shortened. Mitchell continues:
So I then shortened it. But, although I felt that several of the parts that I took away
from it really weren't vital, it would have been a pity not to publish the review complete in
its more extensive form... We found a small printer in Plymouth who was willing to use
our typed manuscript... He agreed to produce the first grey book, including binding and
putting on a grey cover for four shillings a copy. So we were able to sell it for ten shillings
a copy with a small margin of profit.
The first grey book (Mitchell, 1966b), Chemiosmotic Coupling in Oxidative
and Photosynthetic Phosphorylation, which was cited in the Biological Reviews
article (Mitchell, 1966c) had a reasonable circulation and the Glynn Research
Institute was still sending out several copies a month in the early 'eighties. The
grey book and the review article provided an extensive statement of the rationale
of the chemiosmotic theory and the chemiosmotic model of oxidative phosphorylation. For the first time the energy coupling was shown as a full circuit of
proton flow. Mitchell reviewed the evidence supporting the four basic postulates
of the chemiosmotic model, which could be fitted to the chemical intermediate
model only by additional assumptions.
At the Federation of European Biochemical Societies meeting in Warsaw in
1966, Mitchell and Moyle (1967a) presented results from experiments with
valinomycin and gramicidin that demonstrated that the greater part of the
protonmotive force in mitochondria was as membrane potential rather than as a
pH gradient. He also invoked "backlash", probably due to Ca ++ as suggested by
ChappeU, to explain the slack between the flow of redox equivalents through the
electron-transport chain and the initiation of respiratory control. Backlash had to
be minimized in order to get accurate stoichiometries. At this meeting Slater and
Chance became more critical of the chemiosmotic model. Tager, VeldsemaCurrie and Slater (1966) were not able to repeat the stoichiometry experiments
but Mitchell and Moyle (1967b) responded providing evidence, from work done
in 1966, defending their experiments. Experimental difficulties and differences of
procedures between laboratories in determining proton stoichiometries was to
remain a major problem for the next twenty years. Additional work in 1966 by
Mitchell and Moyle (1967c), employing pulsed acid-base titrations, provided
evidence of H + distributed over a three-phase system and further confirmed the
low membrane permeability to H + and O H - .
On April 19, 1967 Mitchell read a paper at the Federation of American
Societies for Experimental Biology meeting in Chicago (Mitchell, 1967a) in which
he not only reviewed the mounting evidence in support of the chemiosmotic
model but for the first time extended the notion of a proton circuit coupled with
redox and hydrodehydration reactions to an analogy with fuel cells. Unfortun-
Glynn and Cherniosmotic Theory
597
ately Mitchell's p a p e r was the last before lunch and due to shortened time had to
be read rapidly, so the full impact of the fuel cell analogy was not realized in the
audience. A t this meeting Chance continued his challenging, begun in Warsaw,
that biological m e m b r a n e s could be i m p e r m e a b l e to protons. T h e sessions were
held in the Blackstone T h e a t r e and E. Racker, who was chair, m a d e h u m o r o u s
reference to the fact that the current play on the boards was The O d d Couple.
The next m o n t h Mitchell and Moyle (1967d) published a very thorough study
confirming their p r o t o n stoichiometries, detailing the backlash effects, and
showing that pulses of acidity due to pulses in respiration were not consistent with
an intervening primary formation of a chemical intermediate. Chance, and also
Pressman, had argued that the putative chemical intermediates might be coupled
to translocation of cations and that translocation of protons might be a
consequence. H o w e v e r , in this p a p e r Mitchell and Moyle d e m o n s t r a t e d that
uncouplers did not change the initial ~---H+/O but did change the m e m b r a n e
conductance; a result that would be difficult to reconcile with the notion that the
proton translocation was a derivative consequence of the primary formation of
intermediate.
As a result of an earlier visit by Mitchell to R a c k e r ' s laboratory, in 1967
Peter Hinkle came to Glynn for a post-doctoral year. Hinkle (interview, 4 July
1979) has described what research was like at Glynn at that time:
I spent the first month when I first got there just talking with Mitchell and reading.
His library was very interesting. The library in the laboratory consists of his personal
books plus all the journals since about 1963. I'd just take books home and generally get
more familiar with physical chemistry of electrochemical gradients. Then we designed a
rather elaborate experiment involving the effect of membrane potentials on equilibrium
between cytochrome c and cytochrome a. The laboratory had a very nice routine. The
technicians made rat liver mitochondria two or three days a week, which was a very slow
pace for an American lab . . . . One of the first weeks I was working I just stayed and kept
working in the evening, which I was used to doing, and Mitchell came in and looked rather
shocked. I don't remember exactly what he said, but generally he implied that the
mitochondria were no longer any good and that I was wasting my time and should go
home and go to bed. And so I didn't work much in the evening anymore.
The group consisted of Mitchell, Jennifer Moyle, Peter Scholes, who was a post-doc at
the time, working on bacteria, and about three technicians. We would have tea twice a day
at around eleven or ten-thirty and around three, which would last for about half an
hour . . . . Mitchell would come out of his study and [the discussion] would always be some
topic related to the philosophy of science, or the fact that he did not like big institutions
but liked small groups of people working together, or of course about something that he or
we had read. Actually, I enjoyed it tremendously because having this period twice a
d a y . . , when it is carried out on a small scale like this, is particularly intense, so you are
always involved in a rather interesting conversation with a group, whereas in a larger
group you would not have as much speaking yourself.
Jennifer Moyle (interview, 5 July 1979) has described the work at Glynn
during this time:
When we first went to Cornwall, Peter and I both worked in the laboratory. We had
just one technician. But it soon became obvious that Peter needed more time for his
598
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theoretical work, and that it would be a happy compromise for the experimental work at
the laboratory to be mostly done by me and by any postdocs that we had with us, and that
he would spend most of his time on theoretical angles and writing papers, writing the two
grey books, and of course going to meetings. And that is how it developed. So, in fact,
every day we talk for several hours about the latest experimental work, which very often
triggers off a new bit of theory on his part. Then he'll come back and say, "well, if what I
was thinking could be true, then this should happen." Then I'll do the experiment the next
day, and then we'll discuss again. So it's very much talk and experiments, theory going
along with practice.
The on-going theoretical work was reviewed in Comprehensive Biochemistry
and in Advances in Enzymology that placed the chemiosmotic model of oxidative
phosphorylation within the broader conceptual context of the chemiosmotic
theory (Mitchell, 1967b, 1967c), which was itself much more explicitly articulated
and analyzed in physical chemical terms. The following year Mitchell (1968)
published the second grey book, Chemiosmotic Coupling and Energy
Transduction, that provided a more sophisticated treatment of the fuel cell
analogy for chemiosmotic systems, expanded on the thermodynamic treatment of
gradients by Guggenheim (1933), and discussed the mechanism of action of
ionophores.
Mitchell and Moyle (1968) responded to a series of articles by Chance (see
references in Mitchell, Moyle and Smith, 1968) which purported to demonstrate
that mitochondrial membranes were permeable to protons by spectroscopic
measurement of the internal pH with the dye bromothymol blue. Mitchell and
Moyle, in colloborative work with Lucille Smith, were able to demonstrate that
mitochondria in state 3 or 5 had one-third of the dye in the inner phase, but when
converted to state 4 the dye was expelled due to the formation of the membrane
potential upon the onset of respiration. Mitchell and Moyle (1969) published a
detailed study using valinomycin to estimate how much of the protonmotive
force was membrane potential under various experimental conditions. For
example, for a Ap = 230mV, they showed A~p = 200mV and ApH = 0.5, and
that through manipulation of the experimental system the relative contributions
of each could be altered.
In the first four years Glynn established itself as a focused and productive
laboratory that had an unusual balance of theory and experiment. Twenty-four
papers and two books were published during this period, with ten more papers
the following year, with an almost equal emphasis on theoretical and experimental work. By the end of 1970, there had been sixty-three consultations by visiting
scientists, and Mitchell had given twenty-five presentations at meetings. Although
situated at Glynn, forty miles from the nearest university, it was possible not only
to be active in research but to be in close touch with the community of scientists
engaged in similar work. Not only had the general chemiosmotic theory and the
model of oxidative and photosynthetic phosphorylation based on that theory been
considerably developed, but a sizable body of evidence was produced that
demonstrated that energy-coupling membranes are impermeable to H § that the
redox reactions of the respiratory chain generate a protonmotive force with a
characteristic stoichiometry, that the ATPase also has a characteristic proton
Glynn and ChemiosmoticTheory
599
stoichiometry, that an artificially induced proton gradient of sufficient magnitude
is thermodynamically competent to drive ATP synthesis, and that uncouplers act
to increase the H § permeability of membranes but do not decrease the initial
<---H+/O. Further, it was possible to estimate the membrane potential and show
that it was in accord with theory. In contrast, the majority of laboratories that
continued to pursue the orthodox chemical intermediate model had no success in
isolating any of the putative intermediates, nor was any evidence found for direct
interaction of the redox chain and the ATPase, and the number of ad hoc
additions to the theory was making it a less elegant and economical explanatory
model than the chemiosmotic one.
In 1969 a major review of the literature (Greville, 1969) not only was
supportive of the chemiosmotic model but presented it in a manner more easily
understood by a larger number of biochemists. Harold followed with a lucidly
written review (Harold, 1972) that was sensitive to the wider implications of
chemiosmotic theory. The supportive and more popular reviews by Deamer
(1969) and by Hinkle and McCarty (1978) explained the rationale to a broader
community of scientists. The majority of remaining doubters of the concept of
proton coupling as a phenomenon-were persuaded by the 1973 experiment of
Stoeckenius and Racker in which the purple-membrane bacteriorhodopsin, which
was known to translocate protons when activated by light, isolated from
halophilic bacteria by Stoeckenius, was reconstituted in an artificial bilayer with
horse heart FoF1 isolated by Racker. The membrane was energized by light and
ATP production was detected (Racker and Stoeckenius, 1974).
After 1973, only a few workers continued to base their research programme
on the chemical intermediate hypothesis. Although there would be a dispute
about the location of the proton path (Kell, 1979) as to whether bulk-phase or
membrane-surface protons are energy coupling as opposed to protons that move
intramembranely (Williams, 1961, 1989), the phenomena of chemiosmotic
coupling were generally accepted. In the seventies and eighties the field as a
whole shifted from ascertaining whether the phenomena predicted by the
chemiosmotic model occurred to experimental probes of the mechanisms underlying the phenomena.
Mitchell has described the concept of energy transduction through proton
coupling across a proton-impermeable membrane as the "physiological" level of
the chemiosmotic model, and I will argue in the next section that it can be also
considered as the phenomenological aspect. Behind this level for Mitchell is the
"chemical" level that deals with the mechanism of the model and its causal
explanations, which I shall later refer to as the aspect of the model that flows
from the more general chemiosmotic theory. What Mitchell terms the "metabolic" level would be the general chemiosmotic theory by the considerations that I
shall advance.
Analysis of the citations in the literature of the "physiological" level shows
not just an explosion of the number of citations (as documented by Robinson,
1984) but a rise in the number of positive citations from about 40% in 1965 to
over 68% by 1977, and after 1977 well over 80% positive citations are observed.
Negative citations are constant over much of the period at about 20% with a
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gradual decline to under 10%. Citations expressing ambiguity fluctuate over a
range of 15-45%. There was a trough of positive citations and a peak of
ambiguous citations around 1970, maybe due to concerns about measurements of
membrane potentials, but the elegant work of Skulachev, published in 1971 and
subsequently, provided a reliable method of determining the membrane potentials and there was a rapid rise of positive and decrease of ambiguous citations
thereafter. In striking contrast to this overall picture of acceptance of the
physiological level (or phenomenological aspect) is the response in the literature
to the chemical level (or mechanistic aspect). In the late sixties about 50% of the
citations were positive, and after a drop in the early seventies surged to 66%
positive citations after 1973, but as arguments about stoichiometries became more
intense the positive citations declined to 35% by 1978, further decreasing to 20%
by 1983. It should be remembered that even though there were negative citations
over stoichionaetries and mechanistic detail, the discourse in the field itself was
being defined by the work from the Glynn laboratory; in the absence of Mitchell
and Moyle's work, the <---H+/O, if observed at all, woOld have been regarded as
an interesting consequence of a side reaction of the m~iin reaction pathway
involving chemical intermediates. Further, citation analysis doeS:not of course
reflect the scientific validity of an idea but r~ther the attitude of the field as a
whole at a particular time. Mitchell has responded to this challenge to the
chemical level by not only refinements of the mo;del but also by enriching the
theory, as discussed below.
THE EVOLUTION OF THE CHEMIOSMOTIC MODEL A N D THEORY
SINCE 1970
The past twenty years have seen a continuation of the intense experimental
and theoretical collaborative activity that was established during the first five
years, with an average of 6 papers a year published, 6 lectures a year presented,
and over a dozen consulting visits per year at Glynn. They have been years also
characterized by increased concerns over funding, direction and type of research,
and the future role of such places as Glynn. There is not room here for a detailed
account of the richness of activity for this period. Rather I will focus on how the
controversies over stoichiometry and mechanism have led to advances not just in
the chemiosmotic model but in the general theory, with special regard to the
development of the Q cycle and the mobile carrier concept, the development of
the notion of a type of rotational catalysis and its application to the ATPase, and
the more recent development of the mobile barrier concept.
In the first grey book, Mitchell (1966b) arranged the respiratory chain
diagrammatically into loops, and based on the evidence then available, had two
loops (loops 2 and 3), one between FMN and Q and the other between Q and
cytochrome c~. That ~---H§ = 4 for loops 2 and 3 and that cytochrome oxidase
was electronmotive but not protonmotive appeared to be supported by experimental evidence from Glynn (Hinkle and Mitchell, 1970; Mitchell and Moyle, 1970).
Loop 3 was criticized by Slater (1971) because in going from cytochrome bK to
Glynn and ChemiosmoticTheory
601
cytochrome cl no H-carrier was available. Mitchell (1972) wrestled with the
anomalous redox poises of the cytochromes and suggested that they might be due
to the membrane potential c o m p o n e n t of the protonmotive force. Mitchell was
increasingly worried about the inadequacy of the linear loop to explain what was
happening in the cytochrome c reductase span of the respiratory chain.
Correspondence with Chance focused Mitchell on kinetic anomalies of the
cytochromes b in 1974-5.
During October 1974 Mitchell spent some time in Tsoo King's laboratory
during which time King impressed upon Mitchell the need for diffusional mobility
to facilitate interactions between the redox complexes of the respiratory chain. In
late 1974 (18 December) Mitchell gave the Biochemistry Society C I B A Medal
Lecture. In preparation for that lecture Mitchell made a thorough review of the
literature and became even more acutely aware of the problems with the linear
loop model. In early 1975 Mitchell continued to reconsider the available evidence
as he describes (interview, 24 May 1980):
As I did so, I became more and more unhappy about my ability to account for our
observations on the stoichiometric translocation of protons. Also, I became more and
more uncomfortable about my inability to explain the kinetics of the respiratory-pulse type
of experiment, especially the poises of the cytochromes b, which had always been regarded
by Chance and other people as anomalous. The more one looked at them the more it was
clear that one couldn't think how to place the ubiquinone-ubiquinol couple in the chain.
Well, ! had been wrestling with that, and feeling more and more that this might well be a
point where we could succeed in falsifying the chemiosmotie hypothesis. Then, while I was
just toying with thoughts about that system, I think, not intentionally intending to get
anywhere, on the 20th of May, 1975, I wasn't sleeping well. I had in my mind's eye loops 2
and 3, and I put in my mind's eye, as I bad been doing for some little time before that,
ubiquinone couples,... I was more inclined to put the Q/QH couple in loop 3 and the
QHE/QH couple in loop 2. Being in bed with my eyes shut, it was easy to visualize the
thing, as it were, in front of me, and suddenly as I was visualizing it I noticed what hadn't
been noticed before. In these two loops, drawn like that, QH was going one way across
the membrane in one loop and the other way across the membrane in the other loop. I
suddenly said to myself, "Well, that's funny. Why does it have to go across them at all
then? If it is going both ways, it is as though, from the formal view, it's not going across at
all". Then, of course, in my mind's eye, I could see that you could allow the QH to go
along the surface of the osmotic barrier, formally speaking, on each side, and you would
then have a cyclic arrangement of the two couples. And that, of course, was the Q cycle.
Under those circumstances, the cytochromes b would conduct electrons across the centre
of a cyclic system instead of between the two loops. So it was immediately obvious, as
soon as that thought occurred to me, that the redox behaviour of the b cytochromes would
thereby be completely different because, assuming that it was QH2 that was the reductant
in what would have been loop 3, when the QHz was oxidized to QH on the outer side of
the membrane by cytochrome Cl, the QH would have to be further oxidized by a
cytochrome b. Therefore, the cytochrome b would go reduced, which would explain the
observation, so far inexplicable in a context where you were also trying to explain the
proton translocation induced by cytochrome c oxidation . . . . Well, of course, I didn't
sleep for the rest of that night. In fact I got dressed and started doodling and drawing
diagrams.
602
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As Mitchell was scheduled to present a paper at the Fasano meetings later
that year, he sent preprints of his paper (Mitchell, 1975a) to the participants.
This, coupled with a working mechanical model to illustrate his talk, resulted in
considerable discussion about the Q-cycle concept. The Q cycle was first
published in F E B S Letters (Mitchell, 1975b), subsequently generalized (Mitchell,
1975c), and placed solidly in the conceptual context of the chemiosmotic theory
(Mitchell, 1976). The Q cycle represented a triumph of the chemiosmotic theory
that viewed chemical transformation and osmotic translocation as different
aspects of vectorial chemistry in specific ligand conduction processes. The vitality
of the theory was demonstrated by its ability to absorb "counterfactuals". As
shown especially in studies by Trumpower (see Bowyer and Trumpower, 1981), it
resolved a number of problems and reorganized thinking about the respiratory
chain, especially about the cytochrome c reductase (Slater, 1981, 1983). Alternative models were proposed that involved protein conformational changes,
participation of polypeptide chains and "membrane Bohr effects" to account for
the data (Wikstr6m and Krab, 1980; Papa et al., 1982).
After several years of strong opposition, Crofts described evidence in favour
of the Q cycle, based on the kinetic behaviour of cytochrome b and associated
components (Crofts, Meinhardt and Bowyer, 1982). Mitchell and Moyle (1985)
and Mitchell (1987c) reviewed the controversies in the literature and found the
weight of evidence favouring the Q cycle. Simulations of the Q cycle seem best to
account for experimental data (Palmer et al., 1985). Recent papers from Glynn
(Rich, Heathcote and Moss, 1987; Moss and Rich, 1987) have provided further
evidence for the operation of the Q cycle in the cytochrome bf complex of
photosynthetic systems and for some refinements in the model.
In the late 'seventies controversy broke out over the protonic stoichiometries,
which Mitchell and Moyle had previously documented; and the considerably
higher values that were apparently being found were thought to have the
potential to falsify the chemical-level chemiosmotic models favoured by Mitchell.
Lehninger first presented higher stoichiometries at the 1975 Fasano meeting; the
ensuing correspondence between Mitchell and Lehninger has been analyzed for
rhetorical style rather than scientific content by Mulkay (1985). Lehninger's group
(Brand, Reynafarje and Lehninger, 1976; Alexandre, Reynafarje and Lehninger,
1978) published ~--H§ = 4 for each of the coupling sites and ~---H§ = 3 for
the ATPase. While these data posed no challenge for the cytochrome c reductase,
for which the chemiosmotic model also predicted a stoichiometry of 4 via the Q
cycle, it was at variance with the *--H+/O = 2 for the NADH-dehydrogenase and
the ~---H+/P = 2 for the ATPase as determined in the Glynn laboratories. Most
dramatic was the discrepancy for cytochrome oxidase. Mitchell had argued as far
back as the first grey book (Mitchell, 1966b) that this was electronmotive but not
protonmotive. He and Moyle (Moyle and Mitchell, 1978a) had presented
evidence denying the ejection of protons, claiming that proton ejection observed
in the presence of antimycin was the result of an electron-transfer leak, a
phenomenon requiring a correction factor when calculating the stoichiometry.
Wikstr6m (1977) and Wikstr6m and Krab (1978, 1979) found ~---H§ = 2 for the
cytochrome oxidase and offered an indirectly-coupled model to account for the
Glynn and ChemiosmoticTheory
603
stoichiometry. Brand, Lehninger and Reynafarje (1977) claimed that the oxygenpulse method that the Glynn group used underestimated the true proton
stoichiometries by one-third to one-half. This error they said was due to rapid
phosphate transport, a factor that the Baltimore group avoided by use of NEM.
Moyle and Mitchell (1978b) defended their earlier interpretation (Moyle and
Mitchell, 1973) that NEM affected the stoichiometries with succinate dehydrogenase and NAD-linked enzymes but not with the NADP-isocitrate
dehydrogenase or transhydrogenase and that the higher stoichiometries being
claimed were thus an artifact.
Because of the experimental complexities, this controversy continued on into
the eighties (Hinkle, 1981), by which time the focus was primarily on the
cytochrome oxidase. In an effort to facilitate communication amongst the scientists
involved in experimental work on the cytochrome oxidase system, Mitchell
arranged for a conference to be held at Glynn 22-24 March, 1983. Rather than
structuring the meeting around formal lectures an "octavian discussion" was held.
The twenty participants from eight countries gathered around an octagonal table
with not more than eight self-selected participants sitting at the table; only those
at the table were allowed to speak. There was a continual exchange of observers
and speakers with a steady-state of six speakers at any given time. This format
was judged by the participants to be especially conducive to a nonconfrontational exploration of the issues.
The problem continued to be investigated at Glynn and the issue was
resolved by 1985. Mitchell, Mitchell, Moody, Baum and Wrigglesworth (1985)
accepted the stoichiometry of Wikstr6m and Casey (1985), and West, Mitchell,
Moody, and Mitchell (1986) showed that the use of myxothiazol with antimycin
obviates the need of the correction for "leaks" and that under these conditions
they obtained the same stoichiometry as originally observed by Wikstr6m (1977).
Also, Mitchell, West, Moody and Mitchell (1986) and West (1986) re-examined
the NEM effect and showed that, among other factors, the use of Li + rather than
K + buffers by Lehninger's group allowed a Li+/H § exchange via a Na+/H +
porter to counter the valinomycin-induced H + uptake, thus allowing NEM to give
a higher and more accurate stoichiometry. Thus the NADH-dehydrogenase has a
stoichiometry of at least 3, probably 4~--H§
the cytochrome c reductase
stays unchanged at 4, and the cytochrome oxidase is 2, with the ATPase at
3 "~'--H+/P.
Mitchell has gone on to show that these results, although not anticipated in
the earlier model, can be explained by the chemiosmotic theory. Incorporation of
these results leads to an evolUtionary advance not just by suggesting novel
testable models but also by advancing the theory with regard to the concept of
mobile barriers, which is implicit (Mitchell, 1957) in the chemiosmotic rationale.
Mitchell, Mitchell, Moody, West, Baum and Wrigglesworth (1985) used the
concept of vectorial ligand conduction to propose protonmotive O loop and O
cycle mechanisms for the cytochrome oxidase. This proposal was made more
specific (Mitchell, 1987a) with regard to the role of Cu in the cytochrome oxidase
and expanded (Mitchell, 1988) to the CuB "zoop" mechanism. Here Cu acts as a
hydrogen translocator (Cu§247
for example) by a mechanism in
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which the electronation or de-electronation of the Cu centre from one side is
coupled to protonation or de-protonation from the opposite side, which is
different from what happens in protonmotive Q systems (Mitchell, 1991).
As with the development of the Q cycle, Mitchell has adhered to the basic
core principles or themata in applying chemiosmotic theory to the available
experimental information to generate a novel, testable model. He has provided a
similar application of theory to the model of the ATPase mechanism (Mitchell,
1981a, 1981b, 1984, 1985a, 1987b) that preserves the direct role for protons in the
active sites in F1 and accounts for the third proton as participating in an antiport
of adenine nucleotides and phosphate to and from the active sites in F1 by a
mobile barrier type of process (Mitchell, 1990). Mitchell's model, in which
protons are conducted through F0, and are involved in the overall process of
ADP phosphorylation in F1, differs from Boyer's model, in which the protons are
supposed to interact only with a proton-pumping system in F0, and coupling with
ADP phosphorylation in F1 is attributed to conformational energy transfer
between F0 and F1 (Boyer, 1974, 1975; Kayalar, Rosing and Boyer, 1977; Boyer,
1987). This issue has yet to be resolved experimentally, although a majority in the
field currently favour some version of an indirect mechanism as judged by citation
analysis. The elaboration of the mobile barrier mechanism (Mitchell, 1987b,
1990) opens new avenues of research on transport processes and points toward an
even greater unification of osmotic, chemical, and chemiosmotic mechanisms by
specifically addressing how chemical action over distances of 0.1 nm are coupled
to osmotic action over distances of 4 nm. In this work Mitchell is exploring the
implications of the vectorial character of chemical forces (see for example
Mitchell, 1977 and Mitchell 1981a) and the fundamental correlation of chemical
and osmotic forces (Mitchell, 1985b, 1991).
There is no question about the scientific success of the experiment Mitchell
undertook when he and Moyle established the Glynn Research Institute, or about
the continuing vitality of the chemiosmotic concepts. In contrast to a strictly
private laboratory of a scientist working alone, such as that of Lundegardh, the
experiment of Glynn as a place and style of scientific research has to be evaluated
in terms of its ability to continue to function and evolve on its own. In June 1983
Jennifer Moyle retired. In 1985 Glynn House was purchased by The Glynn
Research Foundation Ltd from Mitchell, and the position of Director of Research
was split from that of Chairman of the Council. In January 1987, Peter Rich
became the Director of Molecular Research, while Mitchell remained as
Chairman and Director of the Foundation, and temporarily took on the position
of Director of Behavioural Research. Peter Rich has continued with the
chemiosmotic research programme (Rich, 1988a, b; Rich, West and Mitchell,
1988; Rich, Moody and Mitchell, 1989; Moody and Rich, 1989, 1990; Rich, Jeal,
Madgwick and Moody, 1990; Rich and Harper, 1990), and is extending it through
the introduction of further novelties (Rich, 1991).
To function as at present with a total staff at Glynn of about a dozen
individuals requires currently about s
per year. At best, interest on the
present endowment can bring in less than a quarter of that amount, so the
remainder has to be made up from contributions from benefactors and research
Glynn and ChemiosmoticTheory
605
grants. This on-going process of raising funds for research is not unlike that which
has to be done at larger institutions; however, all scientists should hope that
places like Glynn can survive and provide a venue for independent research and
reflection.
Implications and Explorations
This historical analysis has two aims beyond the obvious one of developing a
narrative of the growth of conceptual and experimental knowledge of biological
energy-transducing mechanisms. As science crosses the "complexity barrier"
(Pagels, 1988) and addresses the problems of the "sciences of the artificial"
(Simon, 1969), attempting to deal robustly with more complexly-organized and
self-organizing systems, it is important to have a realistic understainding of the
evolution of scientific knowledge, of the distinctions between successful strategies
in physics, chemistry and biology, and of the interplay of various types of
reduction and interfield connection in research strategies. Having a narrative of
the past helps provide a context for creative understanding of new problems
(MacIntyre, 1980). More specifically, it allows us to delineate the conceptual,
economic, material and cultural conditions that foster a successful research
programme at institutes such as Glynn and illuminates an alternative way of doing
science.
Mitchell has described his process of discovery (Mitchell, 1980; 1982) in
terms of the philosophy of science of Karl Popper (Popper, 1959, 1962, 1972).
This provides a good starting point, but the analysis can be enriched by Lakatos'
(1970) extension of Popper's ideas. Lakatos describes research 'programmes' as a
privileged set of core concepts and background assumptions that provide the
selective, interpretive and explanatory principles from which theories are
constructed and revised. These principles could be likened to the concept of
scientific themata (Holton, 1988). Theory is applied to the real world through
auxiliary assumptions. Another philosopher of science, Nancy Cartwright (1983),
has distinguished between phenomenological laws and foundational laws in
physics; the former are idealized generalizations from experimental experience
whereas the latter are intended to subsume phenomenological laws under formal
explanation. Cartwright (1983) and Hacking (1983) argue that in periods of
scientific progress the contexts of discovery and justification significantly overlap
in ways that do not correlate with the conventional distinction of theory and
experiment. Both Cartwright and Hacking emphasize the intermediary role
between theory and data provided by the models that scientists develop to depict
and explore phenomena. Finally, Bogen and Woodward (1988) and Woodward
(1989) have emphasized the distinction between data and phenomena. The
phenomena are the conditions and processes responsible for the production of
data. Scientists study phenomena by means of data that are "idiosyncratic" to
specific experimental conditions. How the phenomena are conceived in the
model, within the context of the theory, will constrain the types of experiments
performed and the evaluation of what data are valid or relevant.
The conventional dichotomy of theory and data provides a less robust
Weber
analytical vocabulary than the view in which modelling provides theoretical access
to formalized phenomena causally responsible for data (Hofmann, 1990). With
these conceptual tools we can make sense of the growth of knowledge about
chemiosmotic systems and explore the specific way in which models are used in
the biochemical sciences.
As shown in Fig. 1, the core principles would appear to include the concept
that chemical and osmotic processes in living systems should be given equal
ontological and explanatory status rather than assuming that the osmotic
processes are secondary products of primary chemical processes. Further, in
chemiosmotic actions, metabolic and transport processes are fundamentally
vectorial and are coupled. Finally, the balance of vectorial forces colinear with
flows relate structural and dynamic elements in living systems. Mitchell (1962)
stated this core assumption succinctly (p. 35):
We must not lose sight of the fact that transport processes in biology are integral with
the activities of growth and survival. When we separate transport from morphogenesis,
growth, and movement in living organisms, the separation is not real, but abstract. Thus, it
emerges that the activities of biological transport represent the elusive directiveness of the
phenomena of life. Scalar (or directionless) biochemistry is a subject of dead things, like
crystals. To describe the flame-like properties of living things, we have to represent the
metabolic processes as projections in space as well as in time. This requires the recognition
and development of a new subject: the subject of vectorial chemistry.
These principles employed together allow the development of the theory as it
was applied to specific phenomena by the inclusion of information about the
specific phenomena. During the 1950s Mitchell had developed the concept of
ligand conduction and had shown its application to the description not only of
transport processes but to the mechanism of action of enzymes. An important
component of the theory was that the chemical force for chemiosmotic actions
was transmitted and transduced by direct atomic (orbital) contact. Finally, the
thermodynamics of chemical potentials and gradients were used to describe the
macroscopic consequences of microscopic vectorial chemistry (Fig. 1).
In order to apply the core principles and theoretical structure to a particular,
real case it was necessary to add auxiliary information and assumptions. For
example to deal with phenomena of metabolic transport there needed to be
added specific information about what was transported, its directionality, other
metabolites co-transported, amount and form of energy requirements, membrane
organization and basic properties, and the membrane components involved. To
model a phenomenon such as oxidative phosphorylation required knowledge of
the structure and function of the redox chain, information on the ATP
synthesizing components, the P/O values for various substrates under different
experimental conditions, effects of uncouplers and inhibitors, the requirement for
a topologically intact membrane, and so forth. In constructing the specific model
it was necessary to assume that the connection between redox reactions involving
hydrogen atoms (proton plus electron translocated across the membrane) and
hydrodehydration reactions involving protonation was a transmembrane protonmotive chemical potential and that the membrane was impermeable to protons.
Glynn and ChemiosmoticTheory
667
The model provided a specific molecular picture with attendant equations that
explained various aspects of the phenomenon of oxidative phosphorylation. The
chemiosmotic model focused attention on phenomena not anticipated by the
orthodox chemical intermediate model, such as the generation and the utilization
of the protonmotive force, the anisotropy of the mitochondrial membrane and the
impermeability of such energy-coupling membranes to protons. The model was
connected through the phenomena to the specific experimental data and the new
data then affected the model. As evidence for the phenomena predicted by the
model accrued, the auxiliary assumptions rather became auxiliary information
(see Fig. 1). As the auxiliary information was refined, revised, and/or added to,
the model was also revised. Those of Mitchell's critics who complain that the
theory should have got the model right in all aspects de novo essentially ignore
how knowledge grows in the face of the complexity of nature. The development
of the mobile carrier concept in the Q cycle demonstrates how the core is
preserved and enriched at the theoretical level and how the model can be
improved in a testable way in the light of problematic new information about the
phenomena. A similar process is at work in the elaboration of the mobile barrier
concept. The controversies over stoichiometry can be located at the level of data
and the idiosyncratic nature of the experiments; with the resolution of that
controversy, the new information is being incorporated into the model via the
O-loop and Cu-zoop proposals within the principles of the chemiosmotic theory.
Mitchell's characterization of the three levels of the chemiosmotic theory are
consistent with the above analysis. The "metabolic" level involves the core
principles of vectorial chemistry and osmochemistry that apply generally. The
"chemical" level would encompass the way the model connects to the theory and
the mechanistic aspects of the model. The "physiological" level corresponds to
the phenomena predicted by the model that have been experimentally confirmed.
Just about everyone in the field has accepted the physiological level for some
time, although there are still disputes about localized protons and whether these
behave as predicted in the chemiosmotic model. As noted above, there has been
more controversy over the chemical level, with many making the improper
assumption that any stoichiometry higher than Mitchell originally predicted
falsifies directly coupled chemiosmotic mechanisms and supports some form of
conformational coupling mechanism.
The advantage of pursuing the chemiosmotic model in which the conformational changes are viewed as driven by the vectorial chemistry is that it makes
specific, testable predictions. This was the great advantage of the chemiosmotic
model as originally presented (Mitchell, 1961e) over less direct and less specific
models. Also, attempts to develop a narrative of the growth of knowledge about
bioenergetics can founder if attention is not paid to the complex relationship of
these levels as happened to Gilbert and Mulkay (1984a, 1984b). They interviewed
34 scientists and reviewed the key literature and the public correspondence
among bioenergeticists. Mulkay found that the sociological complexity of
bioenergetics and the multiple voices of each actor were such that he and Gilbert
felt that they could not tell the story without distorting the reality. Instead they
provide inter alia a discourse analysis and an analysis of the scientists'
608
Weber
CONCEPTUAL STRUCTURE OF THE CHEMIOSMOTIC PROGRAMME
coRE
t.
Chemical and osmotic processes given equal
explanatory weight, i.e., one not reduced to the other
Metabolic and transport processes are fundamentally
vectorial and are aspects of a single underlying process
Balance of vectorial forces cclinear with flow relates
structural and dynamical elements in livin~ systems
Auxiliary information ' I
about transport - e.g.
j
[enzyme- ke spec f c t es
~'-! ~
""
I Auxiliary information
about metabolism - e.g.
1 9r~ translocatlon
,
THEORy
ligand conduction
anisotropic membranes and membrane-bound
enzymes
vectorial chemistry
chemical force
thermodynamics of gradients
1.
2.
3.
4.
5.
Auxiliary information
phenomenon to be
modeled - oxidative
phosphorylation
1lb.~
W
\
%
system and their kinetic
thermodynamics and
structure
Phe~ena
Mechanism
1. redox chain produce
A~H+ by vectorial release of H+
1. redox loop translocates
via H atom in bond
2. H+ directly protonates 0 2of Pi in ATPase active site
2.
3.
Membrane impermeable to H+
Anisotropic ATPase reversed
by ~ H + to make ATP, H+ catalyzed dehydration
Consequences of theory applied to model
4,~H+ = F~,~ + 2.3 RTz~pH
e
r
i
m
e
n
t
s
N'Nz "chemical level"
/
J
[
Experimentally Accessed Phenomena
respiratory chain redox coupled to ATP synthesis by ,5~H+
membrane impermeable to H+
uncouplers increase H+ conductance of membrane
~
>"metabolic level"
y
MODEL
I
J
|
1
]
Fig. 1. A conceptual analysis of the components of the chemiosmotic theory in relation to the core
principles, auxiliary information, the specific model of oxidative phosphorylation, phenomena, and
data. See text for details.
reconstruction of their enterprise. This sociological analysis was extended by
Mulkay (1985) to an analysis of the discourse as recorded in the private
correspondence between Mitchell and Lehninger in 1975-6 about stoichiometries.
Valuable as these contributions are, they need to be reexamined in light of the
multiple levels of theorizing, modelling, phenomena and data, and their complex
interrelationships.
Crofts (1979), among others (Harold, 1986; Allchin, 1991) has argued that
Glynn and Chemiosmotic Theory
609
the development of the chemiosmotic theory of oxidative phosphorylation
represents a Kuhnian paradigm change. It is true that most articles on oxidative
phosphorylation in the fifties and sixties interpreted experimental data in terms of
chemical intermediates, whereas the papers of the seventies and eighties focus
interpretation in terms of membrane potentials and pH gradients. However, if we
look at the broader range of topics now encompassed by bioenergetics the
paradigm change would be more properly located in the development of vectorial
chemistry. At this level and at this time it is premature to determine if a paradigm
change is occurring. To the extent that the lucidly written text by Harold (1986)
becomes standard in training the next generation of bioenergeticists, it will have a
role in determining such a paradigm shift. The ultimate arbiter is the success of
the problem-solving research tradition (Laudan, 1977). For the chemiosmotic
tradition the questions will be do the core chemiosmotic concepts continue to
provide robust principles for interpreting phenomena and for guiding further
research through the development of testable models.
What the core of the chemiosmotic principles allows is the ability to model
apparently disparate phenomena, such as oxidative phosphorylation, photosynthesis, transport, and biomechanical systems, within a single theoretical
structure. This could be represented as an "interfield connection" (Darden and
Maull, 1977; Weber, 1986a), however, Mitchell was not seeking to just bring
concepts from transport studies to the problem of oxidative phosphorylation, but
rather to develop a fundamental theory in which transport and metabolism were
treated as aspects of an underlying vectorial dynamic process. We normally think
of biochemistry as a reductionist discipline (Mathews and van Holde, 1990) and
certainly Mitchell did and does aspire to molecular descriptions and a grounding
in physical law (Rosenberg, 1985). But for that reduction it was necessary to first
develop the concepts of vectorial chemistry and to be guided by the biological as
much as by the chemical (Mitchell, 1962), in accord with the suggestion of Simon
(1969) for a general strategy when faced by complex systems. Further, Mitchell
did not follow the methodological reductionist strategy that informed the
orthodox chemical intermediate model of oxidative phosphorylation and which
had been so successful in guiding research into the scalar chemical transformations of metabolism. Nor did he assume that the secondary osmotic barrier was
less fundamental than the primary chemical energy barrier. But with the full
articulation of the chemiosmotic theory and vectorial chemical principles, it is
now possible to achieve a reduction where is was not possible before.
True to his Popperian principles, Mitchell has emphasized the "logic of
justification" in his own accounts of his research and has bracketed the "logic of
discovery" under the rubric of creative conjecture. Yet, increasingly, philosophers of science have questioned whether it is not possible to perceive some of
the contours of the discovery process (Schaffner, 1974; Kelly, 1987; HoyningenHuene, 1987; Kleiner, 1988). Popper's cutting of the Gordian knot of induction
probably was necessary to break with the tradition of "Baconian Induction" as
presented by Hershell (1830). It is interesting to note that Brewster (1831) in his
landmark biography of Newton rejected a purely "Baconian" description of
Newton's method of discovery and emphasized the interplay of theoretical
610
Weber
conception and inductive inference. Hacking (1983) has reinterpreted Bacon as
having advanced a theory of learning by refutation through an alliance of
experimental and theoretical skills. In Mitchell's case there was an early
commitment to the concept of vectorial chemistry as an interpretive and
explanatory principle that sustained his early empirical and theoretical studies of
transport and enzyme mechanisms. Theory was not allowed to become untethered from available information and experiments were not piled up in the
hope that some pattern would emerge. As the theory and the models became
more articulated they were better guides to experimentation and more subject to
the rigors of empirical falsification. As Cartwright (1983) and Hacking (1983)
have both emphasized, during times of creativity in science the distinction
between the logic of discovery and justification becomes harder to draw. For
example, the development of the Q cycle could be viewed as an example of
discovery rather than of reconjecture during justification, It was and is the intense
coupling of discovery and justification, of theory and experiment, that has
characterized the development of the chemiosmotic research programme and the
work at Glynn.
The past twenty-five years of research at Glynn have demonstrated that it is
possible to accomplish high-quality experimental and theoretical research that has
a disproportionate effect on a scientific field at a small institution with limited
funding. It is especially important to keep in mind the model of research at Glynn
at a time when biological research is moving, with the rise of the human genome
project, toward the model of "big science" that has characterized physics research
in the latter half of the twentieth century. The small size and restricted resources
demanded that the research at Glynn be sharply focused and that through
interaction with other groups a broader range of experimentation be encouraged.
The isolation encouraged maintenance of this focus, but by frequent attendance
at meetings and visits to other laboratories, and more importantly by inviting
colleagues to spend time at Glynn, it was possible tO keep open vital lines of
communication. The pace and rhythm of research at Glynn is less frenetic than
that found in most university laboratories, which helps to reinforce a focus on the
essentials of a problem.
The most distinctive feature of the biochemical research at Glynn is the high
ratio of theory to experiment. Biochemists, and more generally biologists, usually
conceive of their science as fundamentally experimental with theory relegated to
a role of generalization or phenomenological equation (Huszagh and Infante,
1989). What has been unusual in the work at Glynn is the extent to which the
experimental programmes have been conceived within a context of testing a
specific model that was the product of the well developed theoretical framework.
Further, once begun, the experimental work has been conducted in an on-going
dialogue with the development of the theory. Not only has the model become
more articulated by the interaction of theory and experiment, but the theory itself
has beome more fully developed within the overall core principles or themata.
The future of Glynn and similar such experiments rests most importantly in
maintaining this balance of theory and experiment.
Glynn and Chemiosmotic Theory
611
ACKNOWLEDGEMENTS
This w o r k has b e e n s u p p o r t e d b y g r a n t s f r o m t h e N a t i o n a l S c i e n c e
Foundation, the American Philosophical Society, Wellcome Research Travel
Grants, and by Hughes Faculty Grants, California State University, Fullerton. I
wish to t h a n k D o u g l a s A l l c h i n , H a r o l d B a u m , D a v i d D e p e w , F r a n k l i n H a r o l d ,
James Hofmann, Peter Mitchell, Jennifer Moyle, Joseph Robinson, Kramer
R o h l f l e i s c h , a n d E. C. S l a t e r f o r r e a d i n g an e a r l i e r v e r s i o n o f this m a n u s c r i p t a n d
for p r o v i d i n g h e l p f u l c o m m e n t s a n d s u g g e s t i o n s .
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