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ATP synthase FAQ

This list of Frequently Asked Questions (FAQ) on the ATP synthase is written with the assumption that the reader has some background knowledge in biochemistry, enzymology, and physical chemistry.
This is NOT a review article or something of that sort, there are no references or credits, and no detailed description of the experiments underlying each piece of information. If you are interested in getting into details, just write me an e-mail and I will be happy to discuss any of the questions below. I hope you will help me to extend this list.
Recommended reading is added for some sections under "Recommended reading"-sign.

Correct name

According to the IUBMB Enzyme Nomenclature, the enzyme is called "ATP phosphohydrolase (H+-transporting)". However, the name "ATP synthase" reflects the primary function of the enzyme more clearly, and nowadays is the most wide-spread one.
The other name that was commonly used in the past is "H+-ATPase", sometimes a more precise "FOF1  H+-ATPase". After the discovery of many other types of ATP-driven proton pumps these old names are less used.
The other names that were used for ATP synthase are:

F1-ATPase
FoF1-ATPase
F-type ATPase
H+-transporting ATPase
mitochondrial  ATPase
coupling factors (F0, F1 and CF1)
chloroplast ATPase
bacterial Ca2+/Mg2+ ATPase
Complex V (five)

Differences between F-,  A-, V-, P-, and E-ATPases

 
F-, A-, and V-type ATPases are multisubunit complexes, similar in terms of overall architecture, and most probably have the same catalytic mechanism. They couple transmembrane proton (or Na+ in some F-ATPases) transport, presumably achived by the rotation of a certain subunits complex relative to the rest of the enzyme, with ATP hydrolysis (or synthesis in A- and F-ATPases).
The common features for them are: "mushroom" shape, hexameric hydrophilic catalytic domain of  Alpha 3 Beta 3 - type with Gamma subunit inside it. The catalytic act performed by those enzymes does not include a phosphorylated enzyme intermediate. 
The proton-translocating portion of those enzymes is composed of a ring-shaped subunit oligomer (c-subunit oligomer in case of F-type ATPases); each subunit bears a critically important carboxyl group approximately in the middle of one of it's transmembrane helices. It is presumed that this carboxyl is directly involved in proton translocation.
 
P-type ATPases are quite a different family of ion-translocating ATP-driven pumps. Most of them are also multisubunit membrane proteins;  one large subunit performs both  ATP hydrolysis and ion pumping. There are many different subfamilies of P-type ATPases, usually classified according to the ion they transport. H+, Na+, K+, Mg2+, Ca2+, Ag+ and Ag2+, Zn2+, Co2+, Pb2+, Ni2+, Cd2+, Cu+ and Cu2+ pumping P-ATPase are described.
During ATP hydrolysis by a P-ATPase at a certain stage of catalytic cycle the phosphate is transferred to one of the Asp residues of the enzyme. There is no evidence (neither structural nor functional) for rotary catalysis in P-type ATPases. Typical examples of such enzymes are yeast plasma membrane H+ ATPase, K+/Na+ membrane ATPase, Ca2+ membrane ATPase.

Recommended reading 1) Pedersen, P. L., and Carafoli, E. (1987) Ion motive ATPases. I. Ubiquity, properties, and significance to cell function. Trends Biochem. Sci. 4: 146-150.
2) P-type ATPase Database (By Kristian B. Alexsen, Swiss Institute of Bioinformatics)
3) Kawasaki-Nishi S, Nishi T, Forgac M. (2003 ) Proton translocation driven by ATP hydrolysis in V-ATPases.
FEBS Lett. 545(1): 76-85.
4) Perzov N, Padler-Karavani V, Nelson H, Nelson N. (2001) Features of V-ATPases that distinguish them from F-ATPases. FEBS Lett. 504(3): 223-8.

The architecture and subunit composition of ATP synthase

ATP synthase is a large mushroom-shaped asymmetric protein complex. The simplest bacterial enzyme (see the cartoon below) is composed of 8 subunit types, of which 5 form the catalytic hydrophilic F1-portion (the "cap" of the mushroom). These subunits are named by Greek letters (Alpha, Beta, Gamma, Delta and Epsilon) in accordance with their molecular weight. The proton translocating FO portion is composed of subunits of 3 types named a, b and c.

ATP synthase illustrative pic

The catalytic portion of ATP synthase (F1) is formed by Alpha 3 Beta 3 hexamer with Gamma subunit inside it and Epsilon attached to the Gamma. Subunit Delta is bound to the "top" of the hexamer and to subunits b. The  hydrophobic transmembrane segment of subunit b is in contact with subunit a. Subunits Gamma and Epsilon of the catalytic domain are bound to the ring-shaped oligomer of c-subunits. Proton translocation take place at the interface of subunits a and c.

The stoichiometry of the subunits is:

F1
FO
   Alpha
 3     
   a 
1
   Beta
 3    b
 2
   Gamma
 1
    c
 10-14(?)
   Delta
 1

   Epsilon
 1


Chloroplast ATP synthase and the enzyme from some photosynthetic bacteria have 2 different, although similar, b-type subunits  in the proton translocating FO portion, namely  b and b', one copy of each.
High homology is found for most of the ATP synthase subunits from different bacteria and chloroplasts.

Mitochondrial enzyme is much more complex; 17 different types of subunits are described at the moment. Some of these subunits have a high homology to bacterial and chloroplast counterparts, especially subunits Alpha, Beta and Gamma in the F1 portion and subunits a and c in the FO portion. Many subunits are unique for the mitochondrial enzyme (see Subunit Nomenclature Table for details). However, the catalytic and proton translocating "core" of the enzyme is still highly homological to that of bacterial and chloroplast ATP synthase. The overall topology of the enzyme is also quite similar.

The reaction catalyzed

ATP synthase catalyzes ATP synthesis/hydrolysis coupled to transmembrane proton transfer. In case of synthesis the energy input comes from protonic flux through FO downhill the transmembrane electrochemical proton potential difference (Delta mu H+). In case of hydrolysis the enzyme functions as an ATP-driven proton pump and generates Delta mu H+.
The equation of the reaction catalysed is

ADP3- + Pi2- + nH+P  <=>   ATP4- + H2O + (n-1)H+N              ( pH > 7.2 )
 
The "P" and "N" indices denote the positively and the negatively charged sides of the coupling membrane.
The pH value is important: the pK value for   Pi2- + H+ <=> Pi-   is 7.2, while the corresponding pK values for phosphate in ADP and ATP are close to 6.9.
This means that in the pH  interval of 6.9-7.2 the prevailing reaction will not include trapping of protons:

ADP3- + Pi- + nH+P  <=>   ATP4- + H2O + nH+N              ( pH  6.9-7.2 )

However, below pH = 6.9, the prevailing reaction is again accompanied by proton trapping:

ADP2- + Pi- + nH+P  <=>   ATP3- + H2O + (n-1)H+N              ( pH < 6.9 )

Thermodynamics of the ATP synthesis/hydrolysis

Traditionally the thermodynamics of ATP synthesis/hydrolysis is described for the hydrolysis reaction:

ATP4- + H2O   <=> ADP3- + Pi2- + H+              ( pH > 7.2 )

"Physical Chemistry" (P.W.Atkins, 2nd edition) gives a value of -30 kJ mol-1 (-7.16 kcal/mol) at 37oC as a "biological" standard Gibbs free energy change (Delta Go´) for this reaction. This is a reasonable estimate, for figures from -28 to -36 kJ mol-1 can be found in literature, the most popular being -30.6 kJ mol-1 (-7.3 kcal/mol).
The standard Gibbs free energy change, Delta Go, is the total amount of energy which is either used up or released during a chemical reaction under standard conditions when the chemical activities of all the reactants is equal to 1. In case of reactions in aqueous solutions the activities are usually substituted by concentrations (i.e. 1 M); the activity of water itself is taken as 1. "Biological" standard Gibbs free energy change, Delta Go´, is a similar parameter, but is defined at pH 7, i.e. the concentration of H+ is not 1 M, but 10-7M. It is more practical and convenient, for most biological reactions take place at physiological pH.

A very important, and sometimes ignored point, is that Delta Go´ is not the amount of energy available to drive other, endothermic reactions in the cell, because the conditions in the cell are not standard (see the definition above). The actual Gibbs energy change is

          Delta G  =  Delta Go' + 2.3 RT log [CADP CPi (CH+ / 10-7) /  CATP ],

where CADP, CPi, CH+, and CATP are the actual concentrations of the corresponding reactants, R is the molar gas constant (8.314  J mol-1K-1), and T is the temperature in Kelvins. To make this point clear, let us consider the following example with the arbitrary values that are close to the real intracellular concentrations:

CATP 2 x 10-3 M-1
CADP  2 x 10-4 M-1
CPi  10-2 M-1
CH+ 5 x 10-8  M-1(pH approx. 7.3)

The Gibbs energy change under such conditions (temperature 310oK, or 37oC) will be

Delta G  =  Delta Go' + 2.3 RT log ( CADP CPi CH+  /  CATP ) =  -30 - 19.6 = - 49.6 kJ mol-1

 This figure, calculated from the actual concentrations of the reaction components, reflects the energy available as a driving force for any other process coupled to ATP hydrolysis under given conditions.
It follows that the same 49.6 kJ mol-1 must be provided by the proton transport across the membrane down the electrochemical gradient to maintain such a high ATP/ADP ratio. If we assume that 3 protons are transported per each ATP molecule synthesized, a transmembrane H+ electrochemical gradient of  49.6 / 3 =  16.5  kJ mol-1 (i.e., protonmotive force of 171 mV) is necessary.

The conclusion from the example above is:
The energy provided by ATP hydrolysis is not fixed (as well as the energy necessary to synthesize ATP). In first approximation it depends on the concentrations of ADP, ATP, Pi and on the pH. This energy increases logarithmycally upon decrease in ADP and Pi concentration and upon increase in ATP concentration and pH. The graphs below illustrate this point, showing change in the upon the change in the concentration of one reactant (x axis), assuming that the concentrations of other reactants are kept constant at values used in the example above (red dots indicate the calculated in this example).

Graphs of Delta G dependence on C(ATP), C(ADP) and pH.

To close up this section, I would like to note that although the thermodynamics of the ATP synthesis described here might seem rather complex, it is actually much more complex. One point neglected here was the different ADP and ATP protonation states (see above), the other is that the actual substrates in the reaction catalysed by ATP synthase are not pure nucleotides, but their magnesium complexes. However, as the magnesium concentration in the living cell is relatively high and the pH is usually above 7.2, so the description given is still applicable for thermodynamic estimates.
Recommended reading 1)  Nicholls, D. G. and S. J. Ferguson. Bioenergetics 2, London:Academic Press, 1992.
2) Any edition of "Physical Chemistry" by P. Atkins

Driving force for ATP synthesis by ATP synthase.

ATP synthesis catalysed by ATP synthase is powered by the transmembrane electrochemical proton potential difference, Delta mu H+ composed of two components: the chemical and the electrical one. The more protons are on one side of a membane relative to the other, the higher is the driving force for a proton to cross the membrane. As proton is a charged particle, it's movement is also influenced by electrical field: transmembrane electrical potential difference will drive protons from positively charged side to the negatively charged one.

  Picture illustrating the protonmotive force

A water mill is a good analogy: the difference between the water levels before and after the dam provides potential energy; downhill water flow rotates the wheel; the rotation is used to perform some work (ATP synthesis in our case).


          ATP synthase picture 


Quantitatively Delta mu H+ is measured in  Joules per mole (J mol-1) and is defined as:

Delta mu H+  =  -FDelta Psi  + 2.3 RT (pHP - pHN),

where the "P" and "N" indices denote the positively and the negatively charged sides of the coupling membrane; F is Faraday constant (96 485  C mol-1); R is the molar gas constant (8.314  J mol-1K-1), T is the temperature in Kelvins, and is the transmembrane electrical potential difference in volts.  The value of Delta mu H+ tells, how much energy is required (or is released, depending on the direction of the transmembrane proton flow) to move 1 mol of protons across the membrane.
It is often more convenient to use not Delta mu H+, but protonmotive force (pmf):

  pmf  = - Delta mu H+ / F  =  Delta Psi - 2.3 RT/F (pHP - pHN)

At room temperature (25oC) the protommotive force (in millivolts, as well as Delta Psi) is:

  pmf   =  Delta Psi - 59 (pHP - pHN)

In the abcence of transmembrane pH difference pmf equals the transmembrane electrical potential difference and can be directly measured by several experimental techniques (i.e. permeate ion distribution, potential-sensitive dyes, electrochromic carotenoid bandshift, etc.). Each pH unit of the transmembrane pH gradient corresponds to 59 mV of  pmf.
For most biological membranes engaged in ATP synthesis the pmf value lies between 120 and 200 mV (Delta mu H+ between 11.6 and 19.3 kJ mol-1)
Recommended reading 1)  Nicholls, D. G. and S. J. Ferguson. Bioenergetics 2, London:Academic Press, 1992.
2)  A Lecture on Electrochemical potential by Prof. A.R. Crofts
3)Cramer, W.A. and D.B. Knaff. Energy Transduction in Biological Membranes: A Textbook of Bioenergetics, Springer-Verlag New York/Berlin/London

Rotary catalysis

The catalytic mechanism of the ATP synthase most probably involves rotation of Gamma subunit together with subunit Epsilon and c-subunit oligomer relative to the rest of the enzyme. Such rotation was experimentally shown for ATP hydrolysis uncoupled to proton translocation. Moreover, recent experiments revealed, that if Gamma subunit is mechanically forced into rotation, ATP synthesis takes place even without proton-translocating FO-portion.
 It seems most probable that such rotation takes place in vivo. However, there is no direct experimental evidence for such rotary mechanism in the intact enzyme under physiological conditions.
  The proposed mechanism is the following:
  1. Driven by the protonmotive force, protons are transferred through the FO portion of the enzyme. This transfer drives the rotation of the c-subunit oligomer ring relative to the a and b subunits (see here for details).
  2. The rotation  is passed to Gamma and Epsilon subunits that are bound to the c-subunit oligomer ring. The rotation of asymmetric Gamma subunit mechanically causes conformational changes in Alpha 3 Beta 3 -hexamer. Each 120 degrees of the Gamma subunit rotation forces one of 3 catalytic sites located at Alpha-Beta interface into an opened conformation. Freshly synthesized ATP molecule is released, and phosphate and ADP are bound instead. High affinity of the opened site to phosphate impairs rebinding of ATP and favours ADP binding.
  3. Rotation goes further, Gamma subunit turns another 120 degrees forcing the next site into the opened conformation, and the ADP and phosphate bound to the previous opened site are occluded and ATP synthesis takes place. The ATP molecule formed is released when the Gamma subunit makes one 360 degrees turn and once again opens the site.
Recommended reading 1)  W. Junge, H. Lill, and S. Engelbrecht. (1997) ATP synthase: an electrochemical transducer with rotatory mechanics. Trends Biochem.Sci. 22 (11):420-423, .
2)  H. Wang and G. Oster. (1998) Energy transduction in the F1 motor of ATP synthase. Nature 396 (6708):279-282.
3) Weber, J., and Senior, A. E. (2003) ATP synthesis driven by proton transport in F1FO-ATP synthase.
FEBS Lett.  545(1): 61-70.
4) Nice movies at http://nature.berkeley.edu/~hongwang/Project/ATP_synthase/

Proton/ATP ratio

From the early experiments with mitochondria the H+/ATP ratio for ATP synthesis was estimated as 3. However, for chloroplast enzyme the figure of 4 was found more probable. From the thermodynamic considerations less then 3 protons pro ATP is hardly feasible, for the energy required for ATP synthesis under physiological conditions is about 50 kJ mol-1 (~520 meV), so at physiological protonmotive force values in the range of 120-200 mV at least 3 protons should be transferred to get the energy necessary.

There is no convincing evidence or arguments that this ratio should be a whole number.

This ratio is expected to depend on the number of c-subunits in the FO: as there are 3 catalytic sites on the enzyme and
it is most possible that ATP synthesis is driven by a rotary mechanism,

H+/ATP = (number of c-subunits) / 3

But here the problem is that the experimentally determined number of the c-subunits is 10 for a mitochondrial enzyme,14 for a chloroplast enzyme and 11 for a bacterial one, suggesting ratios of 3.33, 4.67 and  3.67, respectively. It is also possible that  c-subunit stoichiometry varies depending on the situation in the cell.

ATP synthase location

ATP synthase is found in bacteria, mitochondria and chloroplasts. In bacteria it is located in the cell membrane with the bulky hydrophilic catalytic F1 portion sticking into cytoplasm. The orientation is quite easy to remember, for the bacterium need ATP to be synthesised inside the cell, not outside. With the proton flow it is less easy; I found it helpful to think that protons always go “along” with ATP: during ATP synthesis they enter the bacterial cell (more ATP inside, more protons inside), and during ATP hydrolysis they leave the cell and go into the outer medium (less ATP inside, less protons inside).
In mitochondria ATP synthase is located in the inner membrane, the hydrophilic catalytic F1 portion is sticking into matrix. In a way a mitochondrion is a bacterium “swallowed”  by the eucariotic cell: then the inner mitochondrial membrane corresponds to the bacterial cell membrane.
In chloroplasts the enzyme is located in the thylakoid membrane; F1 portion is sticking into the stroma.



How many catalytic site has the enzyme?

The answer is three. Not five. However, the total amount of the nucleotide-binding sites is six, three of them being non-catalytic. Each site is located on the interface between subunits Alpha and Beta. Larger part of each catalytic site is composed from aminoacid residues of the respective Beta-subunit, while each non- catalytic site is situated mostly on the respective Alpha subunit. The role of the non-catalytic sites is obscure, they are not necessary for the catalysis. One possibility is that they facilitate the enzyme assembly in the cell.
There is also evidence that the Epsilon subunit binds adenine nucleotides.
 Picture illustrating the position of catalytic and non-catalytic sites

How fast is ATP synthase?

For simplicity let us leave aside the more "biochemical", but less understandable values of "micromoles of ATP per minute per mg protein" and discuss the number of ATP molecules synthesized (or hydrolyzed) by one ATP synthase in one second.
Maximal rates over 100 s-1 were reported for bacterial, mitochondrial and chloroplast enzymes for ATP synthesis. ATP hydrolysis rates is a less clear issue, for the coupled enzyme in small membrane vesicles (most commonly used experimental system) quickly builds up relatively high protonmotive force that acts as a back pressure and stops the hydrolysis. For uncoupled or solubilized enzyme rates over 100 s-1 were also reported.
In the living cell the enzyme most probably operates below the maximal possible rate, making tens of ATP molecules per second.
Recommended reading 1) C. Etzold, G. Deckers-Hebestreit, and K. Altendorf. (1997) Turnover number of Escherichia coli FOF1- ATP synthase for ATP synthesis in membrane vesicles. Eur.J.Biochem. 243 (1-2):336-343.
2) R. L. Cross, C. Grubmeyer, and H. S. Penefsky. (1982) Mechanism of ATP hydrolysis by beef heart mitochondrial ATPase. Rate enhancements resulting from cooperative interactions between multiple catalytic sites. J.Biol.Chem. 257:12101-12105.
3) U. Junesch and P. Gräber. (1985) The rate of ATP synthesis as a function of Delta pH in normal and dithiothreitol-modified chloroplasts. Biochim.Biophys.Acta 809:429-434.

Proton translocation through FO

Although the Fo portion of the ATP synthase is often referred to as “proton(ic) channel”, it is NOT a channel. It differs significantly from "real" proton channels (e.g. gramicidin, M2 from influenza virus, etc.). The most important distinction is that when being in conducting state, a membrane channel does not require conformational changes for proton translocation, while FO portion of ATP synthase does. The transfer rate is also too slow for a channel: at voltage of 100 mV textbooks give a rate of about 106 ions per second for an ion channel, more than 100-fold higher than the maximal corresponding values reported for FO portion. So the latter is a typical example of a proton transporter (the ability to operate as a pump is further confirming it - no channel can do that).
However, the term "proton channels" is often used for certain regions in the membrane proteins that are involved in proton translocation (e.g. proton channels in the cytochrome oxidase, or proton entrance channel in bacteriorhodopsin). As they never cross the entire  membrane, they are sometimes called "proton half-channels".
The proton-translocating region of ATP synthase is formed by subunit a and c-subunit oligomer. There are two certain aminoacid residues that are critically important for proton translocation. The first is an acidic residue (mostly Glu, in some organisms Asp) in the middle of the second transmembrane alpha-helix of subunit c. The second is an Arg at the last but one transmembrane helix of subunit a. Almost all mutations in those two residues result in a complete loss of activity. Several other important hydrophillic aminoacid residues are located on subunit a, but their substitution  leads only to a partial loss of activity.
The currently favored hypothesis of proton transport through ATP synthase is based on the stochastic rotary mechanism. It is presumed, that the conserved acidic residue on the c-subunit can be deprotonated (i.e. negatively charged) only when facing the protein-protein interface between a and c subunits, because it is energetically unfavorable to expose a charge into hydrophobic lipid bilayer.
Proton enters through one half-channel, binds to the unprotonated, negatively charged carboxyl group of the c-subunit conserved Glu (or Asp). The latter becomes electrically neutral and can now enter the hydrophobic lipid phase. As soon as it does, another c-subunit with protonated Glu (Asp) comes from the lipid phase into protein-protein interface area from the other side and releases it's proton through the other half-channel. Carrying now a negative charge, it cannot go back, but can go one position forward and accept another proton from the first half-channel. The cycle is completed. Click here for an animated cartoon illustrating the mechanism above, or download a much nicer (and therefore much larger) movie from Prof. Junge's webpage!

Other questions are welcome!

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Constructed and maintained by Boris A. Feniouk
 Summer 2002