Bioscience Reports, Vol. 16, No. 3, 1996
Successful Theory Development in Biology:
A Consideration of the Theories of
Oxidative Phosphorylation Proposed by
Davies and Krebs, Williams and Mitchell
John N. Prebble I
The chemiosmotic theory is normally attributed to Peter Mitchell's formulation published in Nature in
1961. However, the essential elements of the theory were published 9 years earlier by Davies and
Krebs. Why, then, was this earlier formulation overlooked? The success of Mitchell's theory is
examined in comparison with those of Davies and Krebs and of Williams.
KEY WORDS:
formulation.
Chemiosmotic theory; proton translocation; history of biochemistry; theory
INTRODUCTION
The process of oxidative phosphorylation involves the synthesis of ATP by a
membrane-bound ATPase coupled through an energy transfer mechanism to the
transport of electrons from oxidation of metabolic intermediates to oxygen. In the
early days of oxidative phosphorylation the coupling was assumed to be through a
chemical intermediate, a view based on the understanding of conventional
substrate phosphorylation. An alternative to this approach proposed independently by Davies and Krebs, Williams and Mitchell was that coupling occurred via
a proton gradient. Two of the proposals, those of Davies and Krebs and Mitchell
were almost identical while that of Williams had much in common with the other
two. However, only one--that of Mitchell--attracted substantial attention although not immediately; that of Davies and Krebs was almost totally overlooked
while William's proposals received scant attention for the first decade of their
existence. The proposals of Mitchell became the benchmark for studies in the
field and Mitchell was awarded the Nobel Prize for Chemistry in 1978 in relation
to his work on bioenergetics. Since Mitchell's theory was proposed last, it is of
interest to understand why this theory dominated the field.
Primacy in Science is normally regarded as a desirable and even essential
1 Royal Holloway University of London, Egham, Surrey TW20 0EX, England,
207
0144-8463/96/0600-0207509.50/09 1996PlenumPublishingCorporation
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attribute for successful scientists. It is the first person to achieve a particular
understanding who is normally regarded as meriting the credit for a particular
development. The fact that Mitchell's proposals were the last to be formulated,
but yet within a decade achieved the greatest attention, raises the question of
what constitutes successful theory generation in the biological sciences. The
reasons why the hypothesis, proposed by Mitchell, was after a few years accepted
as a subject for serious debate and investigation and was hence widely quoted,
will be analysed. It should, however, be remembered that the whole issue of a
proton gradient as an intermediate in the process of oxidative phosphorylation
proved a difficult view for the biochemical community to accept. The field was
enmeshed in a series of controversies some of which have been analysed from
both a philosophical (1) and sociological (2) point of view. Those working in the
field were predisposed by background training and collective view to accept a
chemical explanation of oxidative phosphorylation and to find gradients and
physico-chemical approaches problematic.
The three theories can readily be distinguished when simple extrinsic criteria
are considered. It should be borne in mind that these theories lacked experimental support when they were first propounded; consequently they could only be
assessed in terms of their innate probability and attractiveness. However, the key
issue was whether they should be considered seriously at all. The relevant criteria
for this issue are as follows.
1. Mode of presentation. It is suggested that the mode of presentation is of
importance in formulating a theory which needs to attract a broad range of
interest in the field. Thus a presentation of the theory on its own in a high
profile journal might be taken to be significant in communicating a new and
difficult idea to the scientific community.
2. A Coherent Statement. A clearly presented theory complete and well developed in a single presentation might be seen to be the most powerful form of
communication.
3. Timeliness. This is relevant in two distinct ways. Firstly, for a theory to be of
interest it is necessary to present it when the scientific community recognises it
has a problem to solve to which existing solutions are no longer seen as
adequate and is therefore open to new viewpoints. (3) Secondly, the extent to
which a theory can be successfully developed will depend upon an adequate
appreciation by the scientific community of the principles on which it is based.
4. Suggestion for Experimentation. Perhaps the most critical element in successful
theory presentation is that the proposals suggest experimentation for which
methods are available or could reasonably be developed and that the theory is
therefore capable of being tested.
5. Advocacy. Probably of equal importance with 4 for a theory proposed with
little or no experimental backing there is a need for a worker or group of
workers (usually the orginal proposers) to advocate the study of the theory.
Theories placed in the literature without subsequent publicity are liable to be
overlooked.
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209
MITCHELL
In assessing the three theories according to the above criteria, the proposal of
Mitchell (the chemiosmotic hypothesis) will be considered first. This was widely
accepted as a serious proposition meriting debate after an initial delay of some
five of more years.
Mitchell's hypothesis was put forward in a full paper to Nature in the
summer of 1961 (4). The statement was substantial and in his letter to the editor
submitting the paper he apologised for its length (5). He set out three principles
on which his theory was based, namely the anisotropic ATPase, the proton
pumping respiratory chain and the impermeability of the mitochondrial membrane to protons. Although significant details of the theory were modified with
time, the fundamental principles were not altered.
Many workers found the 1961 paper difficult to understand and a later
revised statement prepared by Mitchell in 1966 was the basis for the much wider
understanding of the theory. In 1966, Mitchell wrote a substantial review (6) and
at the same time the private institute which he had set up at Bodmin in Cornwall,
the Glynn Research Institute, published a monograph on the Chemiosmotic
Theory (7). It is this latter privately published statement known as the First Grey
Book, which was highly influential on a wide range of workers in the field of
oxidative phosphorylation in the late sixties and seventies. Although most
references to the Chemiosmotic Theory have continued to refer to the 1961.
Nature article (8), the understanding of the theory came from the later
descriptions. Indeed, the 1961 paper was subsequently identified as a "citation
classic" by the Institute of Scientific Information because of its high citation rate
(9). The revised version in 1966 benefited from a number of developments in the
field over the intervening 5 years. The presentation became clearer following the
elucidation of some issues in oxidative phosphorylation.
The question of timeliness is complex. Those workers who had been
concentrating on cation movements in biological systems had developed theories
for proton transport across membranes (10); The Chemiosmotic Theory could be
seen as a logical development from those views as well as from Mitchell's earlier
work (11). Alternatively, those working in the traditional field of oxidative
phosphorylation were still seeking a chemical intermediate for the process and
their thinking was dominated by a fundamentally chemical approach to biochemical issues, an approach which had justified itself in studies hitherto (12). For this
reason, Mitchell's theory was initially regarded as irrelevant, although by the late
1960's it had become a matter for serious debate.
A particular virtue of Mitchell's theory was that it did suggest experimentation. The proposal that protons were pumped across a membrane implied the
possibility of measuring a pH change associated with respiration. Equally, the fact
that protons were seen as driving the ATPase suggested the possibility that ATP
might be synthesised in response to a pH gradient. Further, it would be possible,
if such measurements could be made, to carry out thermodynamic calculations.
While the technical requirements of this approach were significant at the time, it
was not unrealistic to believe that sensible measurements could be made. The
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fact that within a very few years chloroplast ATP synthesis was demonstrated in
response to a pH gradient in Jagendorf's laboratory (13, 14) greatly enhanced the
position of the theory in biochemical thinking.
Probably the most critical factor in the early success of the chemiosmotic
theory was its advocacy by Peter Mitchell himself. Mitchell had prepared the way
for the publication of the hypothesis initially at a conference in Prague (15)
regarded as the birth of modern membrane transport (16, 17). Later in the same
year, 1960, in Stockholm when the leaders of the oxidative phosphorylation field
were gathered for a conference on Biological Structure and Function, Mitchell
had private discussions with them on the issue of whether, in view of the failure to
find a chemical intermediate, it was now time to consider other possibilities (5).
Those discussions led to further correspondence with some of his fellow scientists
at that meeting. At the same time, in a different section of the conference,
Mitchell put forward views on which the chemiosmotic theory was based,
although they were being applied to the function of a membrane-bound enzyme
not related to oxidative phosphorylation (18).
Several papers were published in 1961/62 in support of the theory but serious
work began after the creation of the Glynn Research Institute in 1964/65. This
work led to extensive correspondence with many workers in the field. It was thus
impossible for others to ignore the existence of the Chemiosmotic Theory. Slater
outlined the theory (19) with Mitchell's help (20) in 1966 while in 1967 a major
review on oxidative phosphorylation (21) gave prominence to the Chemiosmotic
Theory, Mitchell's advice having been sought on this section of the review (22).
Thus Mitchell had not only prepared the ground for his theory but advocated it
strongly in the ensuing years.
DAVIES AND KREBS
The proposal put forward by Davies and Krebs (23) at a symposium of the
Biochemical Society in 1951, although rudimentary involves the same principles
as the hypothesis put forward by Mitchell ten years later. Davies and Krebs,
whose proposals differ in several respects from those put forward only two years
earlier by Davies and Ogston (24), had arrived at their theoretical position
through a consideration of hydrochloric acid secretion in the stomach. Following
from a substantial group of papers in the field of gastric secretion by Davies (25),
Davies and Krebs presented a theoretical explanation for the movement of
protons into the stomach. Measurements had led to the conclusion that 12
protons per molecule of oxygen taken up in respiration were secreted. Two
complementary mechanisms were proposed. Protons derived from the oxidation
of organic substrates could account for four of the twelve protons observed while
the remainder were ultimately derived from water using pyrophosphate bond
energy. Moreover Davies and Krebs suggested the reverse process, namely that
"pyrophosphate bonds can be synthesised, in principle . . . . from the energy
stored in concentration differences of H + ions and by subsequent exchange of
Theory Development in Biology
211
any other cations". Thus, in essence, they proposed a proton translocating
ATPase although in the absence of any understanding of the enzyme or of
structure the concept was inevitably primitive. Further, they anticipated that such
a mechanism could be the basis for oxidative phosphorylation. They wrote, "It is
feasible that ionic concentration differences form the link between the free energy
of (respiration) on the one hand and (ATP synthesis) on the other". (The words
in parentheses are substituted for equations.) Thus, two of the core features of
Mitchell's theory were anticipated. In addition, the third element was covered by
a statement "the operation of a mechanism depending on concentration
differences necessarily postulates specific structures which prevent wasteful
mixing of ions". The authors concluded, "Osmotic energy may in fact be a link in
oxidative phosphorylation".
The proposal seems to have been almost completely ignored although Slater
corresponded with Davies about the proposals and made a very brief reference to
the paper when reviewing oxidative phosphorylation (26). There are several
reasons for this lack of interest. While it is difficult to see that Davies and Krebs
could have developed the theory much more at the time, it was nevertheless
extremely rudimentary in its presentation, causing Slater to seek clarification from
Davies. The correspondence suggests that the implications of the proposals had
not been developed by the authors and appears to reach a conclusion at variance
with the quotations above. In particular there is no consideration of membrane
potentials. Major limitations arose because the first high resolution electronmicrographs of mitochondria showing their membranes were not published until some
months later (27, 28), and therefore the location of these processes in mitochondrial particles was not fully appreciated although location of the respiratory chain
in mitochondrial membranes had been deduced by Cleland and Slater (29). The
understanding of the nature of the ATPase, little enough understood when
Mitchell put forward his proposals in 1961, was almost non-existent in 1951. The
chemical theory formulated by Slater (30) which stimulated the study of the
mechanism of oxidative phosphorylation and which emphasised the possibility of
"high energy" chemical intermediates had also not been published although an
earlier version had been proposed by Lipmann (31).
In addition to the difficulties of presentation and timeliness, the paper was
published in an obscure place so far as oxidative phosphorylation was concerned.
The Symposium was entitled "Metabolism and Function in Nervous Tissue".
Thus it was unlikely to attract the attention of those interested in the field of
oxidative phosphorylation since the question of mechanism had not yet become a
very serious issue and because studies in that field were reported in the general
biochemical literature and in Nature rather than in specialised symposia.
Although Davies continued to show interest in ion transport in gastric
secretion, in the nervous system and later in mitochondria, he did not press his
theoretical views about the mechanism of oxidative phosphorylation. Further, the
form in which the theory was couched did not immediately suggest experimentation in the way that Mitchell's proposals did, primarily because of the lack of
adequate understanding of the mitochondrial biochemistry at the time. As Krebs
subsequently wrote to Mitchell about his proposal, "After all, it was no more
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than an idea--hardly a hypothesis--and did not lead directly to any useful
experiments (32).
WILLIAMS
The theory of Williams also involves a proton gradient. However, this theory
does not require the same transmembrane orientation of the gradient as the other
two proposals. The protons are essentially restricted to the membrane itself and
the details of the mechanism are significantly different. Any gradient observed
across the membrane would be only a reflection of the effective gradient within
the membrane. The immediate consequence of this modification of the concept of
proton driven phosphorylation is that experimental evidence for the theory is
difficult to obtain. Indeed, up until relatively recently it had been difficult to see
how such a gradient could be demonstrated to exist.
The first indication of Williams' thoughts along these lines appeared in a
couple of sentences at the end of a paragraph within a major article devoted to
other issues (33). Williams had consulted Hans Krebs about these theoretical
views but Krebs had felt that hypotheses should not be published without
evidence and has dissuaded Williams from publication (34). Krebs did not refer
Williams to the much earlier paper by Davies and himself. However, when the
new Journal of Theoretical Biology was launched in 1961, Williams was invited to
contribute and provided a formal and substantial statement of the thedry (35).
This was supplemented by a second statement in the same journal a year later
(36). The first paper was published some three months before Mitchell submitted
his paper to Nature and in advance of the submission of Mitchell's abstract for a
meeting of the Biochemical Society (37).
The Journal of Theoretical Biology was designed to appeal to those scientists
who wished to develop the theoretical side of the subject and tended not to be
popular with the strong experimentalists, such as the major workers in the field of
oxidative phosphorylation. Thus, the location of the paper in the literature was
not one to draw wide attention to the new theory. Nevertheless, the account given
by Williams was extensive and detailed, even if not readily susceptible to
experimental verification.
Following the publication of the theory, Williams, whose prime interests
were in metallo-enzymes and inorganic chemistry, devoted much of his time to
the writing of a major text in inorganic chemistry, returning to his theory after a
space of a few years. By this time, the first indications that protons might in fact
have significance in the process of oxidative phosphorylation had been obtained
and linked to the proposals of Mitchell. Indeed, as already indicated, it was
difficult to see how, at the time, a proton gradient in a membrane could be
detected or other relevant evidence obtained in support of the proposals of
Williams. The significance of Williams' proposals were taken up more actively in
the 1970's in association with the proposals and advocacy of Mitchell. It is
interesting to note that Williams' theory was not included in one of the first
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213
reviews to take Mitchell's proposals seriously. This review considered the major
mechanisms of respiratory chain phosphorylation stating:
"Three mechanisms of respiratory chain phosphorylation are currently under
consideration. The basic difference among these proposals is the nature of
the primary energy conservation step. According to the "chemical" hypothesis the energy of oxido-reduction is conserved as an energy-rich compound
whereas in the "chemiosmotic" hypothesis advanced by Mitchell oxidoreduction energy is utilised for the establishment of an electrochemical
potential across the mitochondrial membrane. Finally, the more recent
"conformational coupling" hypothesis provided by B o y e r . . . " (17)o
Thus, some six years after publication, Williams' theory which predated
Mitchell's remained in oblivion and failed to command substantial interest from
the biochemical oxidative phosphorylation community. Although presented in full
it lacked advocacy, failed to suggest experimentation and indeed only now in the
1990's do its implications begin to resonate with the understanding of the
respiratory chain (38).
CONCLUSION
What was the influence of the earlier paper of Davies and Krebs on Williams
and Mitchell? There is also the question of the effect of William's paper on
Mitchell. In the case of Davies and Krebs, it seems unlikely that Mitchell, a
microbiologist working on bacterial permeation at the time with no particular
interest in mammalian and neurochemical issues ever saw the paper in the
symposium on "Metabolism and function in nervous tissue". The title of the
paper "The Biochemical aspects of the transport of ions by nervous tissue" was
not one which would have necessarily attracted Mitchell's attention. After
publication of his major paper, Mitchell's attention was drawn to the early paper.
In a letter to Krebs (39), Mitchell apologised for failing to refer to the earlier
work of which he was unaware although he later dismissed the paper as a version
of the chemical theory (40). Similarly, Williams, a chemist, was also unlikely to
have shown any interest in this symposium and indeed became interested in
biochemical issues only later. Thus both Williams and Mitchell claimed to have no
knowledge of the earlier paper. Significantly so far as Mitchell's work was
concerned, Robertson's review (10), which could be regarded as the basis for
Mitchell's theory, did not quote the paper by Davies and Krebs although it did
cover much of Davies' work on acid secretion in the stomach.
By contrast, Mitchell was very aware of the paper by Williams and eleven
letters passed between the two workers on the subject of William's proposals
before Mitchell submitted his key paper to Nature (41). Williams had expressed
his views on the relationship of his own work to Mitchell's proposals (34). It is,
however, quite possible that Mitchell, having discussed Williams' theory by
correspondence, regarded it as a version of the chemical theory. Nevertheless it
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is puzzling that Mitchell rarely cited Williams papers; in particular the publications in 1961 and 1966 which provide the initial formulations of the chemiosmotic
theory make no reference to Williams' theory. The correspondence between the
two, while initially friendly, eventually lacked cordiality and it is unlikely that the
extent of any influences of Williams on Mitchell can readily be determined.
Scientists in viewing their own history attach much more importance to
primacy. It is assumed that the credit for scientific development attaches to the
first worker to propose a particular view. However, there are several examples in
the biological sciences where this is not true. Although the discovery of
cytochromes is normally attributed to Keilin, he himself drew attention to the
earlier work of MacMunn. In the proton-based theories of oxidative phosphorylation, where Mitchell's proposals have dominated the field, it is clear that factors
other than being first in the field play a significant role in the attribution of credit
for initiating new developments and ideas. Advocacy, timeliness, potential for
experimentation and presentation are major factors.
ACKNOWLEDGEMENTS
I am grateful to Dr Peter Rich, Director of the Glynn Research Laboratory,
for access to the Mitchell archives, to Prof Slater for copies of correspondence
and to Dr Helen Davies, Dr Rich, Professor Slater, Professor Weber and
Professor Williams for valuable discussions.
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