Tools and Applications of Biochemical Engineering Science

3Molecular Mechanism of ATP Synthesis in the F1 and F0 Portions

3.2Latest Experimental Evidence not in Agreement with any Version

of the Binding Change Mechanism . . . . . . . . . . . . . . . . . . 73

3.3Further Specific Difficulties Associated with the Binding Change

3.4.1Some Novel Predictions of the Torsional Mechanism

3.4.3Resolution of Difficulties Achieved and Experimental Evidence

4Further Insights into the Molecular Mechanism in the F0 Portion

5Thermodynamic Analysis of Molecular Mechanisms

1

Introduction and Brief History

From knowledge of metabolic pathways and the extent of the world’s biomass, it is estimated that the energy currency of the cell, the molecule adenosine triphosphate (ATP) and the adenosine diphosphate (ADP) and inorganic phosphate (Pi) from which it is formed, participate in more chemical reactions than any other compound on the surface of the earth with the exception of water. ATP was co-discovered by Fiske and SubbaRow and independently by Lohmann in 1929 [1, 2]. Over 60 years ago, the vital cellular process of oxidative phosphorylation was demonstrated by Belitzer and Kalckar. It was recognized that this was the major pathway by which our bodies captured energy from foodstuffs and used it to carry out a variety of essential cellular functions, but how it took place was largely unknown. In 1941, Lipmann postulated the key role of ATP in cellular metabolism and suggested that it possessed a “high group transfer potential” [3]. Lehninger identified the site of this reaction as the mitochondrion in 1948.

The synthesis of ATP is catalyzed by the enzyme ATP synthase (or F1F0-ATP synthase); the F1 portion of this enzyme was first isolated by Racker and coworkers in 1960 [4]. ATP synthase is present in abundance in the membranes of animal mitochondria, plant chloroplasts, bacteria and other organisms. ATP synthesized by our ATP synthase is transported out of mitochondria and used for the function of muscle, brain, nerve, liver and other tissues, for active transport, and for synthesizing myriad compounds needed by the cell. Since the pool of adenosine phosphates in the body is limited, the use of ATP must be continually compensated by its synthesis, and an active person synthesizes his own body weight of ATP every day. The synthesis of ATP is the most prevalent chemical reaction in the body [5]. This is indeed a very important reaction. How exactly does it occur?

Historically, a first step towards resolution of this problem was taken by Slater in 1953 through his formulation of the chemical hypothesis [6]. Un-

fortunately, no chemical intermediate between oxidation and phosphorylation could be isolated despite frenzied efforts by several groups. Between 1961 and 1966, Mitchell formulated his then radical chemiosmotic hypothesis of oxidative phosphorylation [7, 8], while Williams proposed localized models coupling oxidation and ATP synthesis [9, 10]. Boyer presented the salvage conformational hypothesis in 1965 in which oxidation was directly coupled to phosphorylation via protein-protein conformational interactions [11]. In 1973, Boyer and colleagues proposed a precursor [12] to what later became the binding change mechanism of ATP synthesis, which he reviewed in great detail in 1993 [13]. Cross modified the mechanism and presented it in a review in 1996 [14], but still referred to it as the binding change mechanism.

Exactly a decade ago, after a long, rigorous and thorough education in both the engineering and biological sciences at IIT Kanpur, Princeton University, MIT, GBF/TU Braunschweig (under the inspiring guidance of Prof. Dr. W.-D. Deckwer), I decided, as a young Assistant Professor at IIT Delhi, to “work on something really challenging.” Basic studies in bioenergetics had intrigued scientists for a long time; yet the molecular mechanism of biological energy transduction remained an enigma. It appeared that a completely fresh and original hypothesis was needed that could explain the wealth of existing data and better withstand further experimental challenge. In a series of papers during 1998– 2001, Nath and co-workers proposed the torsional mechanism of energy transduction and ATP synthesis [16–20, 56].

In this review, I shall attempt to critically examine, as best as I can in the limited space available, some of the major candidate molecular mechanisms of ATP synthesis.

2

The ATP Synthase

2.1

Subunit Composition

ATP is synthesized by the enzyme ATP synthase (or F1F0 ATPase), which transforms energy from a transmembrane gradient of protons, or, in some cases, Na+ ions into the chemical energy of ATP. This enzyme, the smallest known molecular machine, consists of two major parts: a membrane-extrinsic, hydrophilic F1 containing three a, three b, and one copy each of g, d, and e subunits, and a membrane-embedded, hydrophobic F0 composed of one a, two b, and twelve c subunits (Fig. 1). The molecular masses of the a, b, g, d, and e subunits in E. coli measure about 55, 50, 31, 19, and 15 kDa, while the a, b, and c subunits have molecular masses of 30, 17, and 8 kDa, respectively [21–30]. The F0 and F1 domains are linked by two slender stalks [21–30]. The central stalk is formed by the e subunit and part of the g subunit, while the peripheral stalk is constituted by the hydrophilic portions of the two b subunits of F0 and the d subunit of F1. The ion channel is formed by the interacting regions of the a and c subunits in F0, while the catalytic binding sites are predominantly located in the b subunits of F1 at the a-b interface [21, 22, 28].

Fig. 1. Schematic diagram of the Escherichia coli ATP synthase enzyme

2.2

Molecular Machine Characteristics

ATP synthesis takes place by conformational changes at the catalytic binding sites. Recent structural [21, 27], biochemical [13, 28, 31], spectroscopic [32, 33] and microscopic [34, 35] studies indicate that these conformational changes arise from rotation of the g-e subunit in a static barrel of a3b3 subunits in ATP synthase, making it the world’s smallest molecular machine with a rotor radius of ≈1 nm (Fig. 1).

2.3 Structure

2.3.1

The Walker Structure

The X-ray crystal structure of bovine heart mitochondrial F1 was solved by the group of Walker to 2.8 Å resolution in 1994, and represents the largest asymmetric structure ever solved [21]. The elongated a and b subunits are arranged alternately in the form of a hexagon.A 9-nm long a-helix of the g subunit (comprising of residues 209–272 at the carboxy terminal) extends from top to bottom of the central cavity of the hexagon. The lower part of the carboxy terminal helix forms a coiled coil with a a-helix consisting of residues 1–45 at the amino terminus of the g subunit. A short a-helix consisting of residues 73–90 of the g subunit projects from the bottom. The d and e subunits, and 145 residues of the g subunit (~50%), were not sufficiently ordered to diffract to high resolution and were not located in the electron density map.

3.2

Latest Experimental Evidence not in Agreement with any Version of the Binding Change Mechanism

Recently, in a significant development, Senior and colleagues designed an optical probe by inserting a tryptophan residue to directly monitor, for the first time, the occupancy of the catalytic sites by nucleotides [28, 38]. Their tryptophan fluorescence experiments in the hydrolysis mode established definitively that Vmax activity is attained by F1-ATPase only when all three catalytic sites are occupied by bound nucleotide, i.e., an enzyme species with all three b subunits occupied is the only catalytically competent species [38]. Due to the inviolability of microscopic reversibility, this result holds for ATP synthesis also, and agrees with an earlier assessment based on enzyme kinetic and radioactive nucleotide binding data [39]. This experimental evidence is in complete disagreement with the fundamental tenets of the binding change mechanism. Further, the tryptophan fluorescence experiments showed that Pi binding can not be spontaneous, as suggested in diagrams illustrating the binding change mechanism (e.g., Figs. 2 and 3). The recent X-ray structure of mitochondrial F1-ATPase in the transition state [39a] shows nucleotide bound to all three catalytic sites, which can never be explained by the binding charge mechanism.

Direct visualization of F1-ATPase by sophisticated single molecule spectroscopy techniques such as epifluorescence/confocal microscopy clearly and unequivocally demonstrates unidirectional and discrete motion of the g subunit in steps of 120° [34, 35, 40]. In various disciplines, from quantum chemistry to chemical/biochemical and mechanical engineering, such motion is considered irreversible. It is very difficult to conceive how a unidirectional, discrete motion can take place by a reversible mode of catalysis (E.ATP E.ADP.Pi), i.e., how can a subunit of a single enzyme molecule oscillate back and forth in the presence of a driving force in one direction? This irreversibility of operation in a single molecule mode contradicts the fundamental tenet of the binding change mechanism that ATP synthesis occurs reversibly (and spontaneously) in a catalytic site of the enzyme. In my view, it is imperative to relate the chemical kinetics (the arrows representing an elementary step in the kinetic scheme) to the mechanical aspects (structure and dynamics of the molecular machine). It is interesting to note that our approach to the problem, in contrast, has incorporated irreversibility from the very outset [16–20, 41, 42, 56].

3.3

Further Specific Difficulties Associated with the Binding Change Mechanism

In addition to the very serious recent evidence in total disharmony with the binding change mechanism discussed above (Sect. 3.2), we pointed out several other specific difficulties in a paper published in January 2000, and stated that it was most important “to recognize and understand the imperative need to consider novel ideas with an open mind, and to overcome the limitations imposed on our own thinking by currently accepted mechanisms, by paradigms that are no longer applicable” [18]. For instance, since the loose conformation, L (bTP), con-

tains bound (MgADP+Pi), and the open site, O (bE), contains no bound nucleotide or Pi (Fig. 2), we found it difficult to conceive how both the binding steps could take place in the same catalytic site conformation (bTP), especially keeping in mind that the binding of Pi is not spontaneous and requires energy [18]. The spontaneity of inorganic phosphate binding was emphasized as a serious defect. In the latest version [37], in addition to competent binding of ADP and Pi to O, even ATP is synthesized in a single O Æ T binding change, which is even more problematic. We considered it highly unlikely that nucleotide could bind to the catalytic site in the O conformation as proposed by Boyer [13, 22, 37], because of the absence of a proper nucleotide binding site [18]. This is even more difficult to reconcile in the hydrolysis mode, as pointed out by other workers [27]. Moreover, in this mode, in Boyer’s bi-site mechanism, MgATP binds to the low affinity O site, but the medium affinity L site is left unfilled, which is a problem, as discussed [43]. Further, negative binding cooperativity and positive catalytic cooperativity need not occur simultaneously, as discussed by us [19], and as shown by recent experimental evidence [44].We have also shown from first principles that there is absence of site-site cooperativity in ATP synthase under physiological steady state operating conditions [19]; in fact, there is no need for cooperativity [18]. Moreover, the molecular basis of cooperativity in ATP synthase remains unelucidated, despite almost three decades of experimental effort! Finally, our kinetic analysis necessitates that in the steady state physiological mode of ATP synthesis, product ATP release must precede substrate ADP binding [19], in agreement with the latest experimental evidence by direct optical probes [38], while, according to all versions of the binding change mechanism from 1973 to 2000, ADP binding must precede ATP release or be simultaneous with it. In the latest modification [37], the L site has no real role to play. It appears to be there just to accommodate the existence of three catalytic sites. Further, in the synthesis mode, T contains bound ATP while in the hydrolysis mode, L has bound ATP. If, in the synthsis mode, release of ATP takes place from O, then, at any instant, there exist two bound ATP molecular in F1 . This contradicts experimental data [bottom of p. 255 in 37]. If, in the synthesis mode, ATP is released from L, why is it that in the hydrolysis mode [39a] the ATP, entering in O, is not released from L itself? A logical explanation of these numerous discrepancies is that the basic tenets of the binding charge mechanism are incorrect.

Cross’s modification of the binding change mechanism (Fig. 3) is also inadequate; in addition to the difficulties detailed above, it is beset with other serious problems. For instance, the open conformation (O) causes release of ATP, therefore, how can it cause binding of ADP, as envisaged? Moreover, it is hypothesized that ATP forms from tightly bound (ADP+Pi) in T only after ATP release has taken place from O (Fig. 3; steps 1, 2); since these conformational changes are caused by rotation of g-e, they should logically occur simultaneously at the different catalytic sites. How substrate (MgADP) binding enhances product (MgATP) release at a different catalytic site, which in turn promotes ATP synthesis at a third site, has never been proposed, i.e., how is this signal communicated, and, above all, how is the energy transmitted from one b subunit to another through the intervening a subunits? Further, in the nonequilibrium mode of functioning, tightly bound (ADP+Pi) should not be present in T; instead,