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J. Biol. Chem., Vol. 277, Issue 42, 39045-39061, October 18, 2002
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From the Molecular Biology Institute, UCLA, Los Angeles, California 90095-1570
These reflections present a perspective of how I and my
graduate students and postdoctoral fellows, over a span of many years, arrived at the concept that ATP is made by an unusual rotational catalysis of the ATP synthase. A recent sketch of the structure of this
remarkable enzyme is given in Fig. 1.
Such a depiction is the culmination of the efforts of many
investigators.1 The two
portions of the enzyme are the membrane-imbedded F0 and the
attached F1 that has three catalytic sites, principally on the large
INTRODUCTION
subunits. ATP is formed when protons pass through the
F0, driving the rotation of the ring-shaped cluster of
c subunits and the attached 
and 
subunits. Other
subunits attached to outer portions of the F0 and
F1 served as a stator. The internal rotary movement of the
subunit is coupled to sequential changes in the conformation of the
catalytic sites. During ATP synthesis these conformational changes
promote the binding of ADP and Pi, the formation of tightly
bound ATP, and the release of ATP.
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Fig. 1.
The ATP synthase. The enzyme from
E. coli has an F1 portion with subunits
designated as 
33
. When separated
it acts as an ATPase. The F0 portion subunits are
designated as ab2c9-12. The passage of
protons, at the interface of the a subunit and the ring of c subunits,
causes a rotation of the c and attached and subunits relative
to the rest of the enzyme. The asymmetric subunit
(yellow and light green)
extends through the center of the 33
cluster. The b2 and subunits serve as a
stator. The rotation of the subunit results in sequential
conformational changes of the catalytic sites that promote ADP and
Pi binding, ATP formation, and ATP release. The
mitochondrial and chloroplast enzymes are similar, except the
F0 portion has more subunits. The three catalytic
sites are principally on the subunits at an interface with the subunits. The subunits also have three non-catalytic sites that
bind nucleotides. The figure is from Ref. 112 (copyright 2001, National
Academy of Sciences, U. S. A.).
Revealing the mechanism of the ATP synthase became a major research
goal in the latter part of my long career. This paper recalls how my
career developed as related to the remarkable progress in biochemical
knowledge. It presents the background and results of fruitful, as well
as mistaken, approaches that were explored.
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The Early Years |
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Born and educated through college in Utah, at the age of 21 I entered graduate school in the Department of Biochemistry at the University of Wisconsin in the fall of 1939. The biochemical research and teaching there were excellent. Not until years later did I appreciate all that is necessary to create such a fine scientific environment.
I had had no previous courses or research experience in biochemistry and was uncertain about my career choice. By the end of my first year of graduate study the fascination of biochemical understanding and the addictive effect of experimental attempts to uncover new knowledge had firmly launched me toward a career in biochemical research. The Department of Biochemistry at Wisconsin was at the forefront of research in nutrition and metabolism. Recent achievements included the identification of nicotinic acid as a vitamin, the irradiation of milk to produce vitamin D, the discovery of a vitamin K antagonist (dicoumarin), and the discovery of lipoic acid as a growth factor for bacteria. At that time incoming graduate students were assigned to a mentor professor. Both Henry Lardy, from South Dakota, and I joined the group of Professor Paul Phillips whose major interest was in dairy cattle nutrition. Evidence had been obtained that vitamin C might help prevent reproductive difficulties in cattle, and one of my assignments was to find if vitamin C might ameliorate the reproductive failure that occurred in rats with vitamin E deficiency. No benefits of vitamin C were noted, but the rats also showed the striking muscular dystrophy characteristic of vitamin E deficiency. Exploration, together with Henry Lardy, of the possible cause of this dystrophy led me into study of ATP-related enzymes. Henry is still active in an exceptionally distinguished career that has included major contributions to the understanding of oxidative phosphorylation.
The milieu at Wisconsin (meetings where students and staff discussed recent research papers, frequent research seminars, and class instructions) introduced me to the wonder of enzyme catalysis. A prominent event was a symposium on respiratory enzymes at which the outstanding biochemists Meyerhof, Cori, Ochoa, Lipmann, Kalckar and others contributed (1). From this and other sources I learned that ATP and phosphorylations were central to the capture and use of energy derived from foodstuffs.
Perhaps defective formation of ATP might underlie the muscle dystrophy
in my vitamin E-deficient rats. One approach was to measure the ability
of muscle extracts to make phosphocreatine during glycolysis. No
definitive defect from vitamin E deficiency was found, but in the
course of these experiments, I noted a stimulation of the transfer of
phosphate from 3-phosphoglycerate to creatine by K+ ions.
This was traced to a requirement of K+ for transfer of the
phosphoryl group from 2-phosphoenolpyruvate to ADP. The discovery of
the K+ activation of pyruvate kinase was the first
demonstration of a K+ requirement for an enzyme reaction.
The two Journal of Biological Chemistry publications
reporting this were the best of several from my graduate studies (2,
3). An understanding of the K+ activation was attained at
the University of Wisconsin some 50 years later from the x-ray
structure of pyruvate kinase (4). The K+, coordinated to
four protein ligands, to an oxygen of the -phosphate of ATP, and to
a water oxygen, apparently provides a requisite positive charge.
Oxidative phosphorylation was discovered only 7 years before I started
graduate studies. As noted in an interesting Prefatory chapter by
Englehardt in Annual Reviews of
Biochemistry (5), ATP was discovered by Lohmann in 1927, and
oxidative phosphorylation was first demonstrated by Engelhardt and
Liubimova in 1932. These salient contributions at that time seemed far
from recent to me, and discoveries such as that of cell-free
fermentation by Buchner made about 40 years earlier were relegated to
the distant past
science after the escape from the Middle Ages. Now,
from my present perspective, research of 30 years ago still seems
fairly recent and vibrant. Time seems to go much faster, but it is I
who has changed while a unit of time has retained its constant value.
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An Introduction to Properties of Proteins |
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Some 20 amino acids linked in peptide bonds can yield proteins with a truly remarkable diversity of structural properties and the ability for specific combination and catalysis. The versatility of proteins is arguably the most important property of matter that has made life possible. Little was known about protein structure when I was a graduate student. As stated in a 1946 textbook of biochemistry (6): "Since the protein molecule is often built up of hundreds, even thousands, of these amino acids, the problem of protein structure is one of almost insuperable difficulty." In the following years, to be an observer as the wondrous properties of proteins have been revealed is one of the finest rewards provided by my profession.
My appreciation of protein structure and function arose in 1943 when I
joined a small group at Stanford University that was supervised by
Murray Luck, founder of the Annual Review of Biochemistry. Our nation was at war, and Luck's group was asked if they could find
how concentrated solutions of human serum albumin, used primarily for
the treatment of shock in wounded soldiers, could be heated to
inactivate pathogens without denaturing the albumin. The group found
that low concentrations of long chain fatty acids or other non-polar
anions such as acetyltryptophan would satisfactorily stabilize the
albumin. Albumin preparations used militarily and commercially are
still stabilized with small concentrations of N-acetyltryptophan. As part of these studies, I noted that
when albumin solutions were exposed to urea or guanidine hydrochloride, the large viscosity increase accompanying denaturation could be reversed by fatty acid addition; a specific combination was markedly influencing the folding of the protein (7). My interest in protein
structure was firmly initiated.
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Early Studies at Minnesota |
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In my 17 years at the University of Minnesota, I studied a
wide variety of biochemical problems, including such items as the chemistry of -tocopherol oxidation products, possible formation of
antibodies by a refolding of denatured -globulins, sulfhydryl groups
and enzyme catalysis, and the free energy of hydrolysis of ATP. Mostly
my interests have concerned enzymes, and over the years names of some
25 different enzymes have appeared in titles of my publications.
An unsettled problem from my graduate studies at Wisconsin was clarified by the demonstration that mitochondria from the muscles of vitamin E-deficient rats performed oxidative phosphorylation as well as those from as normal muscle (8). Studies in my laboratory (9) and those of my graduate colleague Henry Lardy (10) independently reported that during oxidative phosphorylation oxygen uptake was decreased by the lack of phosphate acceptors. Such respiratory control was the basis for the later development, by Britton Chance and others, of the extensive use of an oxygen electrode to replace the cumbersome Warburg manometric method for measuring rates of oxygen uptake during oxidative phosphorylation.
More importantly, stimulated by the pioneering studies of Mildred Cohn (11), we initiated studies using the heavy oxygen isotope, 18O, for probing phosphorylation reactions. As noted in later sections, insights into ATP synthase catalysis by my group were crucially dependent upon the use of 18O. The 18O isotope and mass spectrometer facilities were made available by physics professor Alfred Nier (a benefit of a research university and a cooperative faculty). Over the years we and others have modified and improved techniques for 18O measurements. Yet studies with 18O remain more laborious than many approaches and have not been widely used. The lack of familiarity with the 18O measurements probably added to the reluctance of the field to accept our concepts, as they were later developed in the 1970s.
In our early studies with 18O we demonstrated that in the glyceraldehyde-3-phosphate dehydrogenase reaction an oxygen from inorganic phosphate appears in the carboxyl group of the 3-phosphoglycerate formed (12). This was explained by a phosphorolysis of an acyl enzyme intermediate demonstrated by studies of Racker's group (13) and mine (14). The phosphorylation accompanying this oxidative step of glycolysis was a prominent basis for the widely adopted paradigm that a phosphorylated intermediate was likely formed during the oxidative phosphorylation of the respiratory chain.
In related experiments my group showed that the enzymic catalyses for formation of phosphocreatine from 3-phosphoglycerate occurred with the retention of all 3 oxygens of the phosphoryl group. Thus such phosphoryl transfers do not involve any steps giving exchange of Pi oxygens with water (12). Also we found that syntheses coupled to ATP cleavage, such as formation of glutamine from glutamate and ammonia, occur with transfer of an oxygen from the substrate to Pi (15). No water oxygen is incorporated into the Pi.
Our initial studies of oxidative phosphorylation with 18O
revealed an important characteristic of the oxidative phosphorylation process. We incubated mitochondria with Pi labeled with
both 18O and 32P and unlabeled ATP in the
presence or absence of substrates or of oxidation inhibitors. We were
surprised to discover that, in addition to the strikingly rapid
exchange of Pi oxygens with water, a quite rapid
Pi 
ATP exchange was occurring (16). The reactions of
oxidative phosphorylation appeared to be dynamically reversible. The
reversibility continued even when electron carriers were inhibited or
nearly fully reduced. This gave evidence for formation of some type of
energized compound or state, independent of oxidation-reduction reactions that allowed the ready reversal of the reaction sequence. We
thought this likely was some type of chemical intermediate; the idea of
an electrochemical gradient across a coupling membrane was far from our thoughts.
Possibilities arose of pursuing interesting aspects of enzyme catalysis not related to ATP formation. For example, in 1955 while on a Guggenheim fellowship for study in Sweden with Nobelist Hugo Theorell, I noted a previously overlooked shift in the fluorescence of NADH upon binding by a dehydrogenase (17). This gave a new basis for measuring combinations of NADH with enzymes. However, the problem was not as interesting as the studies of oxidative phosphorylation that I was also pursuing in the laboratories of Olov Lindberg and Lars Ernster at the Wenner Gren Institute. In an experiment conducted in part in Sweden 18O was used to demonstrate that the terminal bridge oxygen in ATP formed by oxidative phosphorylation came from ADP, not Pi. This and some other research were reported at an International Union of Biochemistry symposium in Japan (18). At that time I was a bit pessimistic about gaining a satisfactory insight into how oxidative phosphorylation occurs. In my contribution I stated: "Our basic knowledge of the chemistry involved does not appear adequate for the task, and the problem is likely to be with us for some time. Researchers who undertake indirect approaches to the problem should do so with recognition that their experiments cannot give final answers, and may not even point the way to final solutions." In retrospect, the pessimism seems appropriate.
During the next several years we undertook experiments looking for intermediates in oxidative phosphorylation, particularly by making use of 32P as a tracer. We learned that radioactively induced reactions of phosphorus compounds with highly labeled 32Pi could give rise to radioactive impurities that stick to mitochondrial components but that did not behave like intermediates. Most of my publications during this period were from some worthwhile investigations with other enzymes; one needs to keep research funding available. One of my favorite sayings is that most of what you accomplish in research is the coal that you mine while looking for diamonds.
Some of our studies concerned patterns of isotope exchanges at
equilibrium with glutamine synthetase using 18O,
32P, and 14C. It soon became apparent that
covalent bond cleaving and formation may not be rate-limiting in
enzyme-catalyzed exchanges. Somewhat surprisingly, adequate rate
equations governing exchange reactions of enzymes were mostly lacking.
I spent a fair effort in a pioneering development of appropriate
relationships (19). To some reviewers these relationships were
unexpected, and there is an interesting story not told here about what
I needed to do to get the publication accepted. Various applications
were made by my group. For example, data with glutamine synthetase
revealed that the binding of ATP and glutamate was random, and such
subtleties as a spatial selectivity of transfer of only one oxygen of
the glutamate -carboxyl group to phosphate when glutamate and
glutamine are readily interconverted at the catalytic site (20). The
understanding obtained was useful for later measurements of isotope
exchanges that helped in the discovery of compulsory sequential
participation of catalytic sites of ATP synthase.
An observation of later interest was that myosin and actomyosin can
catalyze an exchange of phosphate oxygens with water oxygens. This can
occur with Pi in the medium without added ATP (21) or with
the Pi formed from ATP before it is released to the medium (22). We did not pursue such observations until about a decade later
when we belatedly recognized their potential relationship to the
mechanism of oxidative phosphorylation.
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The Phosphohistidine Story |
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In 1961 it seemed that our searches with 32P had hit pay dirt. We reported that under oxidative phosphorylation conditions a brief exposure to 32Pi and solubilization of the mitochondria with concentrated urea and detergent gave a non-dialyzable 32P-labeled substance. The rate of its formation from Pi or ATP, the disappearance in a cold Pi chase, and the effect of inhibitors and reaction conditions were consistent with its being an intermediate in oxidative phosphorylation. Our interest was heightened when my capable associates identified the substance as a phosphorylated histidine residue in a protein (23). This was the first recognition of a phosphohistidine in biochemical systems. The ability to form the bound phosphohistidine in soluble preparations from mitochondria encouraged the possibility that we could characterize details of the formation process. As the research developed, I became overly enthusiastic in regarding the phosphorylated protein as an intermediate of oxidative phosphorylation (24). In retrospect, I should have been more cautious. It was at this stage that my laboratory group moved to UCLA where we joined the Biochemistry Division of the Chemistry Department.
Our continued studies showed that dialyzable substances from
mitochondria could modulate the bound phosphohistidine formation, and
this led to the recognition that CoA and succinate were particularly effective. We had overlooked the substrate level phosphorylation accompanying the citric acid cycle. We became aware that a Ph.D. thesis
at Illinois by Upper (25) had reported evidence of formation of a
phosphoenzyme with the Escherichia coli succinyl-CoA
synthetase and that such formation had been suggested earlier from
catalysis of an ADP ATP exchange by the synthetase (26). Our
further studies showed that the phosphorylated protein we had detected was indeed an intermediate in the formation of nucleoside triphosphate (ATP or GTP depending on enzyme source) from Pi by
succinyl-CoA synthetase (27, 28). Our bound phosphohistidine was
clearly not an intermediate in oxidative phosphorylation. In Olympic
analogy, we were reaching for a gold but were fortunate to have
obtained a bronze.
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Another Decade with Little Essential Progress |
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At this stage I felt that perhaps I could do more for science by accepting an opportunity to become the initial Director of the Molecular Biology Institute at UCLA. Fortunately this did not prevent reasonable continuation of laboratory studies, although I was not encouraged about the progress we and others were making toward elucidation of the major problem of how cells captured energy from oxidations to make ATP. At that time I of course did not know that a decade later we would be fortunate in developing a new concept for oxidative and photosynthetic phosphorylation.
Meanwhile my group pursued some worthwhile studies with other enzymes
and continued a few probes of ATP synthesis that were useful but did
not yield or point to breakthroughs. In a more sensitive search for the
labeling of unidentified components with 32P, a small
amount of rapidly labeled lipid fraction was detected (28). However,
this labeling was found to continually increase with time, not an
expected characteristic of an intermediate. The independence of oxygen
exchanges from oxidation-reduction reactions was more firmly
established (29). A claim that a localized AMP might be the initial
phosphoryl acceptor was refuted and ADP as the initial phosphoryl
acceptor more firmly established (30). A sensitive search for possible
substances that might transitorily bind an oxygen from Pi
on its way to water was negative (31). An exploration of the source of
phosphate oxygens in E. coli and Bacillus
subtilis showed that only a few oxygens that entered with
the Pi remained (32). Most of them came from water and substrates, undoubtedly by exchange patterns we had been investigating. Other studies gave a welcome observation that laid the base for the
later extensive use of chloroplasts by my group; under appropriate conditions chloroplasts catalyzed rapid Pi ATP,
Pi HOH, and ATP HOH exchanges. Like oxidative
phosphorylation, photophosphorylation was dynamically reversible and
its mechanism could be probed by oxygen exchange measurements (33).
From later developments the lack of the exchanges noted previously was
likely because of the unusual and particularly strong Mg-ADP inhibition
of chloroplast ATPase activity that can occur in the dark but is
readily reversed by protonmotive force.
In an attempt to gain more insight about energy coupling we also conducted some studies on active transport by E. coli. We obtained convincing evidence that a common energized state or intermediate could drive transport or ATP synthesis (34), a view that had been independently developed by Harold (35) and others. However, unlike Harold, we were reluctant at that time to regard that the energized state was a protonmotive force. We were not alone in this reluctance. The field was active, and frequently reviewed. The 1967 (36), 1969 (37), 1971 (38), and 1974 (39) reviews in the Annual Review of Biochemistry on electron transport and phosphorylation gave brief and generally negative assessments of Mitchell's proposal that protonmotive force drove ATP synthesis. My hesitation in accepting this proposal came from the lack of a satisfying explanation as to how proton migration could drive ATP formation.
The mechanism of the ATP synthase remained unclear. As noted in the
reviews mentioned above, there were a plethora of hypothetical compounds and reactions suggested for participation in ATP formation. A
possibility consistent with our various experiments was that an
energized state, not involving oxidation-reduction reactions, was used
to drive a reaction in which an oxygen from Pi formed water
as ADP was phosphorylated to yield ATP. We and others wondered if in
some manner energy captured in conformational changes of proteins was
involved. Remarkable advances in recognizing the versatility of protein
structure were occurring. The x-ray structure of hemoglobin and other
proteins and the allosteric properties of enzymes suggested the energy
requirements for ATP formation might be accommodated in conformational
changes of proteins. But we still had no clear idea about how the
conformational changes might function.
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A New Concept |
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In 1972, the first part of what I later called the binding change mechanism of ATP synthesis came from further considerations of past data, not new experimental findings. While attending a seminar that I did not understand, some puzzling aspects of oxygen exchange measurements were occupying my mind. Our thoughts had been that the major use of captured energy was to make the covalent structure of ATP. The realization struck me that past data could be explained if the major use of energy was not to form the ATP but to release a tightly bound ATP from the enzyme. Reversible formation of bound ATP at a catalytic site could explain why the exchange of Pi oxygens was less sensitive to uncouplers than net oxidative phosphorylation. For me it was a rare moment of insight, like suddenly reaching a summit on a mountain climb and seeing a beautiful valley spread below. All enzymes have the capacity for ready reaction reversal at catalytic sites and to bind both products and reactants. The reversal of the hydrolysis of ATP by the ATP synthase is no more remarkable than the reversal of simple hydrolyses by many enzymes, except that with the ATP synthase the product ATP is tightly bound. An additional step or steps must intervene for ATP release. This could logically be an energy-requiring conformational change of the catalytic site.
Richard Cross had joined our laboratory as a postdoctoral fellow. At UCLA he further documented the uncoupler-insensitive oxygen exchange and other aspects. We submitted a paper, "On a New Concept for Energy Coupling in Oxidative Phosphorylation Based on a Molecular Explanation of the Oxygen Exchange Reactions," to the Journal of Biological Chemistry for consideration. The publication was declined; at that stage our evidence was not strongly convincing. However, the concept remained appealing. I had recently been elected to the National Academy, and the paper was published in Proceedings of the National Academy of Sciences as the first paper I sponsored for the journal (40). A follow-up paper gave additional details (41).
The presence of multiple binding sites for ADP and ATP on the isolated F1-ATPase and the ATP synthase had been recognized by Slater's group and others. During catalytic turnover some of these nucleotides exchanged with medium nucleotides, and Slater and associates had also suggested the possibility that energy-requiring release of bound ATP might occur in oxidative phosphorylation (42).
The validity of the concept of the role of a tightly bound ATP was
strengthened by our finding that myosin ATPase would spontaneously form
a tightly bound ATP from medium Pi (43). The estimated 
G0 of the binding of ATP from our and other
data was 12-13 kcal/mol. A corresponding tight binding was anticipated
for the ATP synthase. In related experiments, Bagshaw and Trentham had
recently shown that the apparent G for the hydrolysis of
the bound ATP to bound ADP and Pi was only about 1.3
kcal/mol (44); the equilibrium was not far from unity. In a subsequent
cooperative study with these investigators an exchange of phosphate
oxygens of bound ATP with water was demonstrated to accompany the ATP
hydrolysis by myosin (45). The ability to form a bound ATP from
Pi by the reversal of ATP hydrolysis readily accounts for
the capacity of myosin to catalyze a Pi HOH exchange we
had observed years earlier (21). Later observations characterized how
the combination of myosin with actin promotes the release of the
tightly bound ATP, a conformational transition analogous to that
proposed for the ATP synthase (46).
At this time I contributed a chapter on "Conformational Coupling in
Biological Energy Transductions" in which the possibility that
changes with ATP synthase were driven by protonmotive force was
recognized. However, my preferred view was still that the conformational changes were driven by some type of interaction with
oxidation-reduction enzymes (47).
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Recognition of the Role of Protonmotive Force |
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Peter Mitchell introduced his concept of energy-linked proton translocation in 1961 (48), and in ensuing years he and others continued to present evidence and win converts. By the early 1970s even holdouts like myself were beginning to see the light. It seems probable that the role of protonmotive force would not have been recognized for a long time without Mitchell's contributions.
If proton translocation were coupled to ATP synthesis, I felt it would be accomplished indirectly by protein-linked conformational changes. In contrast, Mitchell proposed that the translocated protons reached the catalytic site and participated directly in the removal of a water molecule. I found his 1974 proposal in FEBS Letters (49) unattractive and called attention to some deficiencies in a FEBS Letters contribution (50). Without informing me, the journal allowed Mitchell to present a rebuttal following my paper (51). This seemed inappropriate, and the journal agreed to publish my subsequent paper presenting a model of how, through conformational coupling, proton translocation could drive ATP synthesis (52). The suggestions made still seem applicable.
Over the years Peter and I had extensive correspondence and shared a
mutual respect. Although we were looking at essentially the same
mechanism we tended to present different pictures of our views. Too
often in science there is rancor between those who disagree. An
important lesson that I have learned is that more will be accomplished
if one can maintain cordial relations in an exchange of interpretations.
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Other Developments |
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By the mid-seventies other investigators had provided much
welcomed information about the ATP synthase that was quite relevant to
mechanism studies. Hatefi and others in David Green's laboratory had
shown that the mitochondrial inner membrane could be fractionated to
yield separate complexes of the respiratory chain components and the
ATP synthase. They (and particularly
Racker2 and associates) had
separated and characterized the F1-ATPase. The knobs
visible in electron micrographs of mitochondrial membranes were
identified with the F1-ATPase, connected by a stalk to the membrane portion of the synthase. A similar ATPase had been found in a
wide variety of organisms. The ATPase was known to have two or three
copies of major and subunits and single copies of other smaller
subunits. The unusual subunit stoichiometry and observations in a
number of laboratories that modification of one subunit per enzyme
essentially stopped catalysis raised intriguing questions about
mechanism. The portion of the synthase imbedded in the membrane,
F0, was recognized as being involved in proton transport.
The addition of F1-ATPase to F0 preparations could restore oxidative phosphorylation or photophosphorylation. Either
proton gradients or membrane potential sufficed to drive ATP formation.
Beechey had shown that a buried carboxyl group on a small hydrophobic
subunit of F0, present in multiple copies, readily reacted
with dicyclohexylcarbodiimide
(DCCD)3 and that this blocked
oxidative phosphorylation.
Although information about the ATPase was becoming extensive, how
proton translocation could be coupled to ATP formation remained poorly
understood. We were encouraged some by the concept that energy-linked
binding changes were involved. Fortunately, at this time we obtained
evidence for an unusual catalytic site cooperativity displayed by the
ATP synthase and the isolated F1-ATPase. There was a
feeling in my research group that some important secrets about the ATP
synthase were being revealed. This created an ambience that stimulated
research efforts. Such occasions are an all too infrequent reward of
basic research. They help soften the disappointments of the many
experiments that yield little or no helpful information.
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Alternating Site Participation |
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Many enzymes have more that one catalytic site, suggesting the possibility of a catalytic cooperativity between sites such that catalytic events at one site are promoted by substrate binding at another site. With most multicatalytic site enzymes, limited or no cooperativity has been observed. In contrast, we found that the ATP synthase showed a nearly complete dependence of continued catalytic steps at one site on the presence of substrate(s) at a second site. This was the first enzyme for which such a striking behavior had been discovered, adding to our interest in the phenomenon.
Our discovery arose from researches by Jan Rosing, a postdoctoral
fellow with exceptional experimental skills from Slater's group, and
Celik Kayalar, a gifted graduate student. They were symbiotically
productive. We devised methods for estimating oxygen exchanges by
submitochondrial particles that accompany: (a) the binding,
exchange, and return to the medium of Pi; (b)
the binding, exchange, and return to the medium of ATP; (c)
the binding of Pi, intermediate exchange, and the release
of ATP formed; and (d) the binding of ATP, intermediate
exchange, and the release of the Pi formed. These
measurements with 18O were accompanied by measurement of
the Pi ATP exchange with 32Pi.
The exchange patterns gave evidence that besides promoting ATP release,
energy input also increased competent Pi binding. More
importantly, the measurements yielded exchange patterns that Kayalar
proposed could be explained if the binding of a substrate at one site
was necessary for the release of a product from another site.
Whether two or three catalytic sites per enzyme were present was not known at that time. We proposed alternating behavior of two sites, although it was recognized that the results would also be compatible with sequential participation of three sites (53, 54). During net ATP formation or hydrolysis, sites were considered to proceed sequentially through the steps of binding, interconversion of reactants, and release so that at any one time each catalytic site was at a different stage of the catalysis. The concept seemed attractive, but more evaluation was needed.
David Hackney, a talented postdoctoral fellow from Dan Koshland's laboratory, had joined our group and initiated his excellent experimental and theoretical studies of the oxygen exchanges. We were proposing that Pi and ADP can bind and reversibly form bound ATP but that ATP cannot be released until Pi and ADP bind to an additional site. If dynamic reversal of ATP formation at a catalytic site continued in the absence of net reaction, then reductions in the concentration of Pi or ADP should increase the amount of intermediate oxygen exchange per ATP made. We were encouraged by a report from a former postdoctoral fellow of our group, Robert Mitchell, that he and his colleagues observed increased intermediate oxygen exchange accompanying ATP hydrolysis by submitochondrial particles when ATP concentration was lowered (55). Support for the possibility also came from the observation of Wimmer and Rose (56) that when ATP was exposed to chloroplasts in the light, the ATP showed nearly complete exchange of its oxygens before being released. This is as expected if low ADP concentration in the medium prevented the release of the ATP and many reversals occurred before its release.
Hackney observed that during net oxidative phosphorylation as either ADP or Pi concentration was decreased, there was a marked increase in water oxygen incorporation into each ATP formed (57). Additional observations made it unlikely that some type of enzyme heterogeneity or hysteresis could explain the exchange patterns. It deserves emphasis that these experiments were performed with submitochondrial particles during net ATP synthesis, giving them relevance to the actual oxidative phosphorylation process.
An interesting possibility was that catalytic site cooperativity might
also be found with the isolated F1-ATPase. Several years
earlier, Ef Racker brought some of his purified F1-ATPase to our laboratory to find if his enzyme would catalyze an intermediate Pi HOH exchange. We tested this at millimolar
concentrations of ATP and found that the Pi formed
contained only close to the one water oxygen necessary for the
hydrolysis. Now, however, with our evidence for cooperativity, it was
evident that if reversible ATP formation could occur in the absence of
protonmotive force and if participation of alternating sites was
necessary, then the extent of intermediate Pi HOH
exchange with each Pi released should increase as ATP
concentrations are lowered. This was found to be so (58) and as ATP
concentrations were lowered the number of reversals before the
Pi was released approached a limit of over 300 (59).
Tightly bound ATP at a single site was undergoing reversible hydrolysis
waiting for ATP to bind to another site and promote ADP and
Pi release.
The reaction rates and equilibrium characterizing the slow catalysis at
a single site were determined in a widely recognized study by Cross
together with Grubmeyer and Penefsky (60). They termed this "uni-site
catalysis," and their results added considerably to the acceptance by
others of alternating site participation. In these studies the
Kd for ATP binding to one site of the
F1-ATPase was shown to be near 10
12
M (61), indicative of the need for energy input for ATP
release and akin to the affinity of ATP for myosin.
The capacity to make bound ATP from medium Pi and ADP/ATP ratio near unity on the enzyme was nicely demonstrated with the chloroplast F1-ATPase by Feldman and Sigman (62), a contribution that warrants wider recognition. In a slow reaction, needing relatively high Pi concentration, a tightly bound ADP became phosphorylated. Other findings made it probable that this was at the same site as the ADP that was rapidly released in the acid-base transition of thylakoid membranes and thus that this site was likely where covalent bond formation occurred during photophosphorylation.
In addition, results of various investigators established that chemical
modification of only one catalytic site effectively stopped catalysis
and that each of the three catalytic sites had a different capacity for
derivatization. Such behavior agreed with the concept that during
catalysis all three catalytic sites were in different conformations and
proceeded sequentially through the conformations.
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The Basis of 18O Exchange |
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Our studies with 18O are interpreted on the basis
that the exchange results from a reversal of the formation of bound ATP
from bound ADP and Pi. As covered in the Appendix of a
review there is strong support for this interpretation (63). This
includes demonstrations that the Pi oxygen exchanges
catalyzed by the sarcoplasmic reticulum ATPase (64, 65) and
pyrophosphatase (66, 67), as well as that of myosin ATPase as mentioned
above, result from reversible formation of a phosphorylated enzyme or
enzyme-bound pyrophosphate or ATP, respectively.
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Probes of Initial Reaction Rates |
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Other evaluations of our postulates were needed. Rapid mixing and quenching techniques yielded essential information. One objective was to find if a tightly bound ADP on the chloroplast ATP synthase might react with medium Pi to form ATP in the first turnover of the enzyme. We used rapid mixing in an acid-base transition of chloroplast thylakoid membranes, as introduced by Jagendorf and colleagues, to start ATP synthesis in a few milliseconds. We found that the tightly bound ADP was not directly phosphorylated but was rapidly released to the medium and that the first ATP formed came from medium Pi and ADP (68). As substantiated in later experiments, the tightly bound ADP in such chloroplast membranes prior to release is tightly bound at a catalytic site without Pi.
The demonstration that exposure to protonmotive force caused the
release of a tightly bound ADP from a catalytic site without phosphorylation had important implications for later developments. The
tightly bound ADP in the presence of Mg2+ causes potent
inhibition of ATPase activity of the ATP synthase and
F1-ATPase. Thus such inhibition in the intact synthase is readily and quickly overcome by protonmotive force. When a step of
rotational catalysis occurs, the binding site with the tight ADP is
opened as if it had an ATP present, while another site is binding ADP
and Pi. The properties of the tightly bound ADP also aided
interpretation of Walker's 1994 x-ray structure of the major portion
of the F1-ATPase, in which one subunit has a tightly
bound ADP and Mg2+ present (69).
Our rapid mixing experiments verified that medium ADP was rapidly bound
and phosphorylated as if no phosphorylated intermediates were involved.
They provided evidence that during photophosphorylation, in addition to
a transitorily bound ATP, about one bound Pi and one bound
ADP per enzyme are present and committed to ATP synthesis (70). Such
results harmonize with the alternating site model with more than one
catalytic site having bound reactants, as required if a tight site is
already filled and substrates must initially bind at another site.
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Research Conferences and Binding Change Mechanism |
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Research conferences are important to scientific progress because concepts can be freely discussed, and the publication of proceedings often allows inclusion of material not suited for the usual journals. For example, in my contribution to a 1979 conference honoring Ef Racker, I summarized our concepts and considered how to name our suggested mechanism. A name seemed desirable for ease of discussion and to identify the concept in the field. My contribution entitled "The Binding Change Mechanism for ATP Synthesis" was the first publication in which this nomenclature was used (71).
The binding change mechanism at that time included the following
concepts. The first compound made from Pi is ATP itself (no intermediates); a principal requirement of energy is not for the formation but for the release of ATP; energy input also promotes the
competent binding of Pi and the sequential participation of catalytic sites so that binding of substrate(s) at one site is necessary for release of product(s) from another site. Two years later,
another and even more novel concept of the binding change mechanism was
developed, namely the proposal of rotational catalysis. The suggestion
that rotation of internal subunit(s) drives the binding changes for
catalysis was first published in reports from 1981 and 1983 conferences
at the University of Wisconsin (72, 73). How this concept came about is
outlined next.
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The Proposal of Rotational Catalysis |
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In the 1970s highly enriched 18O was available, mass spectrometry techniques for 18O analysis had improved, and Mildred Cohn had introduced an NMR method for measuring 18O in phosphate compounds. David Hackney developed theoretical aspects of 18O measurements relevant to observed distributions of 18O isotopomers of Pi with 0 to 4 18O atoms per Pi or 0 to 3 18O atoms per ATP molecule. Measurement of the presence of 18O in ATP formed by photophosphorylation showed a pronounced increase in 18O loss at lower ADP and Pi concentrations (74). More importantly, the distribution of 18O isotopomers corresponded to that statistically expected if all the ATP were produced by the same catalytic pathway. This eliminated the possibility that substrate modulation arose from heterogeneity of the enzyme used and made modulation by control sites unlikely. We now regarded the catalytic site cooperativity of ATP synthase to be reasonably well established.
Companion studies with the F1-ATPase showed that when highly 18O-labeled ATP was hydrolyzed by F1-ATPase at different ATP concentrations, the distribution of 18O isotopomers was as expected for a single catalytic pathway (58). At appropriate labeling and substrate concentration ranges, the distribution patterns provided a sensitive test for more than one catalytic pathway. A statistically homoge
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INTRODUCTION
The Early Years