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Why protons flow back to the matrix through ATP synthase?

Why protons flow back to the matrix through ATP synthase?


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I am reading oxidative phosphorylation and I can't understand why the protons that are pumped out must go again into the matrix and finally produce ATP.

Suppose initially that the inside (Matrix-M) and outside of the membrane (Intermembrane space-IMS) are neutral (for sake of simplicity). The exergonicity of the redox reactions powers the proton pumping. As the pumping goes on an electrochemical gradient arises because of the concentration gradient the pumping produces and due to the electrical gradient which is produced due to the unequal distribution of protons.

After the equilibrium of the redox reaction an electrochemical gradient has established. I can't understand why the protons have to flow back into the matrix. The redox reaction equilibrium means that the ratio $ce{H_{M}}/ce{H_{IMS}}$ has a specific value. Do they flow back because of the electrochemical gradient? I was thinking that the flow back is coupled to the pumping, is that correct? I mean we have the equilibrium: $$ce{H_{M}} ightleftharpoons ce{H_{IMS}}$$ and by pumping we increase $ce{H_{IMS}}$ so protons must flow back according to Le Chatelier principle. If that is the case then what is the purpose of ATP synthase? Why the protons must flow back through ATP synthase and not from any other region? Are respiratory chain and ATP synthase seperated and the only way for protons to flow back is throught ATP synthase?

I have read Biochemistry textbooks and still can't understand the importance of the so called proton motive force.


5.5: Uncoupling Electron Transport from ATP Synthesis

  • Contributed by E. V. Wong
  • Axolotl Academica Publishing (Biology) at Axolotl Academica Publishing

So, that is oxidative phosphorylation. It productively utilizes the energy of the proton gradient across the inner mitochondrial membrane (created by oxidation-powered pumps) to drive ATP formation at an approximate rate of 3 protons to 1 ATP. The system is normally highly self-regulated due to impermeability of the inner mitochondrial membrane to H + . If the ATP is not used up quickly, then its concentration slows the action of ATP synthases, which slow the movement of protons out of the intermembrane space. This buildup of protons will eventually be enough that the free energy needed to transfer a proton into the intermembrane space (from the electron transport chain) will not be sufficient to overcome the concentration gradient. Electron transport is slowed, and working backwards, the chain reaction slows respiration rates in general. As the cell/organism requires more energy and uses up the ATP more quickly, protons flow more quickly and the electron transport chain is disinhibited. Thus there is a direct association between respiration rate and physiological energy need.

Interestingly, there is an exception to this tight coupling of the electron transport chain and formation of ATP. The purpose of brown fat (aka brown adipose tissue), which is most often found in newborn and hibernating mammals, is to generate non-shivering (non-movement-based) heat to keep the animal warm. This is accomplished by uncoupling the electron transport chain from the ATP synthesis. This uncoupling is a hormonally controlled process based on the presence of a mitochondrial proton channel called thermogenin. The hormone norepinephrine increases production of free fatty acids, which open the thermogenin channel. This allows protons to ow from the intermembrane space back into the matrix without having to go through ATP synthase. Because of this, the electron transport chain can keep chugging away, ATP levels do not build up, there is no reduction in respiration rate, and the excess energy not being used in ATP production is released as heat.

In fact, 2,4-dinitrophenol, which is used in a variety of research and industrial applications today, was at one time used as dieting drug (in the 1930&rsquos) because through a different mechanism, it too uncoupled electron transport from ATP synthesis. Its mechanism of action derived from its ability to carry and release protons as it freely diffused through the mitochondrial membrane (since it is a small hydrophobic molecule). As this continues, cells catabolize more and more stores of carbohydrates and fats, which is the reason for the interest by dieters. Unfortunately for some of those dieters, this pharmacological means of uncoupling the electron transport chain from the ATP synthesis had no regulation other than the amount of DNP taken. In cases of overdose, respiration rates could rise dramatically while producing little ATP and a great deal of heat. In fact, overdose illness and death are generally due to the spike in body temperature rather than lowered ATP availability. Unfortunately, there are still some dieters and bodybuilders who self-medicate with DNP despite the dangers.


This occurs during Oxidative phosphorylation and the "proton leak" process is also called Uncoupling of Oxidative Phosphorylation

The mitochondrial electron-transport chain

Electron transport, causes Complexes I, III, and IV to transport protons across the inner mitochondrial membrane from the matrix, a region of low [ $ce$ ] and negative electrical potential, to the intermembrane space (which is in contact with the cytosol), a region of high [ $ce$ ] and positive electrical potential.

Three of the four electron-transport complexes, Complexes I, III, and IV, are involved in proton translocation. Two mechanisms have been put across that would couple the free energy of electron transport with the active transport of protons: the redox loop mechanism and the proton pump mechanism.

Uncoupling of Oxidative Phosphorylation

Electron transport (the oxidation of NADH and FADH2 by $ce$ ) and oxidative phosphorylation (the synthesis of ATP) are normally tightly coupled due to the impermeability of the inner mitochondrial membrane to the passage of protons. Thus the only way for $ce$ to re-enter the matrix is through the $ce$ portion of the proton-translocating ATP synthase.

In the resting state, when oxidative phosphorylation is minimal, the proton-motive force across the inner mitochondrial membrane builds up to the extent that the free energy to pump additional protons is greater than the electron-transport chain can muster, thereby inhibiting further electron transport.

However, many compounds,including 2,4-dinitrophenol (DNP) and carbonylcyanide-ptrifluoromethoxyphenylhydrazone (FCCP), have been found to “uncouple” these processes. In a pH gradient, they bind protons on the acidic side of the membrane, diffuse through, and release them on the alkaline side, thereby dissipating the gradient.

The chemiosmotic hypothesis has provided a rationale for understanding the mechanism by which these uncouplers act.

Uncouplers act by dissipating the proton gradient across the inner mitochondrial membrane created by the electron-transport system.

As uncouplers, they function by carrying protons across the inner membrane. Their tendency is to acquire protons on the cytosolic surface of the membrane (where the proton concentration is high) and carry them to the matrix side, thereby destroying the proton gradient that couples electron transport and the ATP synthase.

In mitochondria treated with uncouplers, electron transport continues and protons are driven out through the inner membrane. However, they leak back in so rapidly via the uncouplers that ATP synthesis does not occur. Instead, the energy released in electron transport is dissipated as heat.

The presence in the inner mitochondrial membrane of an agent that renders it permeable to $ce$ uncouples oxidative phosphorylation from electron transport by providing a route for the dissipation of the proton-motive force that does not require ATP synthesis. Uncoupling therefore allows electron transport to proceed unchecked even when ATP synthesis is inhibited.

Uncoupling of oxidative phosphorylation: The proton-transporting ionophores DNP and FCCP uncouple oxidative phosphorylation from electron transport by discharging the electrochemical proton gradient generated by electron transport


4.6: ATP Synthase

  • Contributed by John W. Kimball
  • Professor (retired) at Tufts University & Harvard

ATP synthase is a huge molecular complex (>500,000 daltons) embedded in the inner membrane of mitochondria. Its function is to convert the energy of protons (H + ) moving down their concentration gradient into the synthesis of ATP. 3 to 4 protons moving through this machine is enough to convert a molecule of ADP and P i (inorganic phosphate) into a molecule of ATP. One ATP synthase complex can generate >100 molecules of ATP each second.

Figure (PageIndex<1>): ATP Synthase

ATP synthase can be separated into 2 parts:

  • F o - the portion embedded in the inner mitochondrial membrane
  • F1-ATPase &mdash the portion projecting into the matrix of the mitochondrion

This is why the intact ATP synthase is also called the F o F 1-ATPase.

When the F 1-ATPase is isolated in vitro, it catalyzes the hydrolysis of ATP to ADP and P i (which is why it is called the F 1-ATPase). While it is doing so, the central portion of F o attached to the stalk rotates rapidly in a counter-clockwise direction (as viewed from above).

In the intact mitochondrion, the protons that have accumulated in the intermembrane space enter the F o complex and exit from it into the matrix. The energy they give up as they travel down their concentration gradient rotates F o and its stalk (at

6000 rpm) in a clockwise direction. As it does so, it induces repeating conformational changes in the head proteins that enable them to convert ADP and P i into ATP. (In the figure, two of the three dimers that make up the head proteins have been pulled aside to reveal the stalk inserted in their center.)

In both these cases, the machine is converting chemical energy from the hydrolysis of ATP in the in vitro case and the flow of protons down their concentration gradient in the intact mitochondrion into mechanical energy &mdash the turning of the motor. But this remarkable device can be made to do the reverse, converting mechanical energy (turning of the motor) into chemical energy.

A group of Japanese scientists interested in nano-machines have succeeded in attaching magnetic beads to the stalks of the F 1-ATPase isolated in vitro. Then using a rotating magnetic field they were able to make the stalks rotate. When rotated in a clockwise direction, the F 1-ATPase synthesized ATP from ADP and P i in the surrounding medium &mdash at a rate of about 5 molecules per second! (When rotating the stalks in the counter-clockwise direction, or not rotating them at all, ATP was hydrolyzed into ADP and P i .)

Their achievement was reported in Itoh, H., et al., Nature, 29 January 2004.


Biochemistry. 5th edition.

Figure

Mitochondria, Stained Green, Form a Network Inside a Fibroblast Cell (Left). Mitochondria oxidize carbon fuels to form cellular energy. This transformation requires electron transfer through several large protein complexes (above), some of which pump (more. )

The NADH and FADH2 formed in glycolysis, fatty acid oxidation, and the citric acid cycle are energy-rich molecules because each contains a pair of electrons having a high transfer potential. When these electrons are used to reduce molecular oxygen to water, a large amount of free energy is liberated, which can be used to generate ATP. Oxidative phosphorylation is the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH 2 to O 2 by a series of electron carriers. This process, which takes place in mitochondria, is the major source of ATP in aerobic organisms (Figure 18.1). For example, oxidative phosphorylation generates 26 of the 30 molecules of ATP that are formed when glucose is completely oxidized to CO2 and H2O.

Figure 18.1

Electron Micrograph of a Mitochondrion. [Courtesy of Dr. George Palade.]

Oxidative phosphorylation is conceptually simple and mechanistically complex. Indeed, the unraveling of the mechanism of oxidative phosphorylation has been one of the most challenging problems of biochemistry. The flow of electrons from NADH or FADH2 to O2 through protein complexes located in the mitochondrial inner membrane leads to the pumping of protons out of the mitochondrial matrix. The resulting uneven distribution of protons generates a pH gradient and a transmembrane electrical potential that creates a proton-motive force. ATP is synthesized when protons flow back to the mitochondrial matrix through an enzyme complex. Thus, the oxidation of fuels and the phosphorylation of ADP are coupled by a proton gradient across the inner mitochondrial membrane (Figure 18.2).

Figure 18.2

Essence of Oxidative Phosphorylation. Oxidation and ATP synthesis are coupled by transmembrane proton fluxes.

Oxidative phosphorylation is the culmination of a series of energy transformations that are called cellular respiration or simply respiration in their entirety. First, carbon fuels are oxidized in the citric acid cycle to yield electrons with high transfer potential. Then, this electron-motive force is converted into a proton-motive force and, finally, the proton-motive force is converted into phosphoryl transfer potential. The conversion of electron-motive force into proton-motive force is carried out by three electron-driven proton pumps—NADH-Q oxidoreductase, Q-cytochrome c oxidoreductase, and cytochrome c oxidase. These large transmembrane complexes contain multiple oxidation-reduction centers, including quinones, flavins, iron-sulfur clusters, hemes, and copper ions. The final phase of oxidative phosphorylation is carried out by ATP synthase, an ATP-synthesizing assembly that is driven by the flow of protons back into the mitochondrial matrix. Components of this remarkable enzyme rotate as part of its catalytic mechanism. Oxidative phosphorylation vividly shows that proton gradients are an interconvertible currency of free energy in biological systems.

Respiration—

An ATP-generating process in which an inorganic compound (such as molecular oxygen) serves as the ultimate electron acceptor. The electron donor can be either an organic compound or an inorganic one.

  • 18.1. Oxidative Phosphorylation in Eukaryotes Takes Place in Mitochondria
  • 18.2. Oxidative Phosphorylation Depends on Electron Transfer
  • 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle
  • 18.4. A Proton Gradient Powers the Synthesis of ATP
  • 18.5. Many Shuttles Allow Movement Across the Mitochondrial Membranes
  • 18.6. The Regulation of Cellular Respiration Is Governed Primarily by the Need for ATP
  • Summary
  • Problems
  • Selected Readings

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.


7.2: The Electron Transport Chain (ETC)

  • Contributed by Gerald Bergtrom
  • Professor Emeritus (Biosciences) at University of Wisconsin-Milwaukee

All cells use an electron transport chain (ETC) to oxidize substrates in exergonic reactions. The electron flow from reduced substrates through an ETC is like the movement of electrons between the poles of a battery. In the case of the battery, the electron flow releases free energy to power a motor, light, cell phone, etc. In the mitochondrial ETC, electrons flow when the reduced electron (NADH, FADH2) are oxidized. In plants and other photosynthetic organisms, an ETC serves to oxidize NADPH (a phosphorylated version of the electron carrier NADH). In both cases, free energy released when the redox reactions of an ETC are coupled to the active transport of protons (H+ ions) across a membrane. The result is a chemical gradient of H+ ions as well as a pH gradient. Since protons are charged, the proton gradient is also an electrical gradient. In a kind of shorthand, we say that the free energy once in reduced substrates is now in an electrochemical gradient. That gradient free energy is captured in ATP synthesis reactions coupled to the flow (diffusion) of protons back across the membrane in the process called oxidative phosphorylation. In aerobic respiration, electrons are ultimately transferred from components at the end of the ETC to a final electron acceptor molecular oxygen, O2, making water. In photosynthesis, electron transfer reduces CO2 to sugars.

The Chemiosmotic Mechanismexplained how the creation of an electrochemical gradient and how gradient free energy ends up in ATP. For this insight, Peter Mitchell won the Nobel Prize in Chemistry in 1978. You can read Mitchell&rsquos original proposal of the chemiosmosis model of mitochondrial ATP synthesis in Mitchell P (1961) Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic type of mechanism. Nature 191:144-148. Here we focus on the details of respiration as it occurs in the mitochondria of eukaryotic cells. The end products of electron transport are NAD+, FAD, water and protons. The protons end up outside the mitochondrial matrix because they are pumped across the cristal membrane using the free energy of electron transport.

Electron transportand oxidative phosphorylationare summarized in the illustration below.

Roman numbered protein complexes along with Coenzyme Q (just &ldquoQ&rdquo in the drawing) and cytochrome C (Cyt c) constitute the ETC), the sequence of reactions that oxidize NADH or FADH2 to NAD+ and FAD (respectively). The electrons from these reduced electron carriers are transferred from one ETC complex to the next. At the end of chain, electrons, protons and oxygen unite in complex IV to make water. As you might expect, under standard conditions in a closed system, electron transport is downhill, with an overall release of free energy (negative DGo) at equilibrium.

In the illustration above, we can see three sites in the respiratory ETC that function as H+ pumps. At these sites, the negative change in free energy of electron transfer is large and coupled to the action of a pump. The result is that protons accumulate outside the matrix of the mitochondrion. Because the outer mitochondrial membrane is freely permeable to protons, the electrochemical gradient is in effect between the cytoplasm and the mitochondrial matrix. Proton flow back into the mitochondrial matrix through lollipop- shaped ATP synthase complexes releases the gradient free energy that is harnessed as chemical energy.


The Electrochemical Proton Gradient

Coordinately with electron transport, the three respiratory protein complexes described above pump protons from the matrix into the intermembrane space, resulting in an electrochemical gradient across the inner membrane. As electrons move through these multi-protein complexes, they are frequently paired with a proton (H+) to neutralize their charge as they move from one side of the membrane to the other. Allosteric changes in the protein complexes can also result in the pumping of protons across the membrane. The pumping of protons generates a gradient across the inner membrane of one pH unit difference (a ten-fold difference in hydrogen ion concentration) between the matrix and the intermembrane space. In addition, a membrane potential is generated due to a positive charge in the intermembrane space and the net negative charge in the matrix. The electrochemical gradient across the inner membrane is comprised of both the pH gradient and membrane potential and is the force, often referred to as the proton motive force (PMF), which will drive protons back across the inner membrane into the matrix and, in doing so, drive ATP synthesis.


Why protons flow back to the matrix through ATP synthase? - Biology

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 kind 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 (feniouk [at] atpsynthase.info) and I will be glad to discuss any of the questions below.
Recommended reading is added for some sections under ""-sign.

Table of Content

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 most wide-spread.
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:

Physiological role of ATP synthase

To make a long story short, the primary function of ATP synthase in most organisms is ATP synthesis. Hence the name. However, in some cases the reverse reaction, i.e. transmembrane proton pumping powered by ATP hydrolysis is more important. A typical example: anaerobic bacteria produce ATP by fermentation, and ATP synthase uses ATP to generate protonmotive force necessary for ion transport and flagella motility.
Many bacteria can live both from fermentation and respiration or photosynthesis. In such case ATP synthase functions in both ways.
An important issue is to control ATP-driven proton pumping activity of ATP synthase in order to avoid wasteful ATP hydrolysis under conditions when no protonmotive force can be generated (e.g. leaky damaged membrane, uncoupler present, etc.). In such case ATP hydrolysis becomes a problem, because it can quickly exchaust the intecellular ATP pool. To avoid this situation, all ATP synthases are equipped with regulatory mechanisms that suppress the ATPase activity if no protonmotive force is present. The degree of ATP hydrolysis inhibition depend on the organism. In plants (in chloroplasts), where it is necessary to preserve ATP pool through the whole night, the inhibition is very strong: the enzyme hardly has any ATPase activity. In contrast, in anaerobic bacteria where ATP synhase is the main generator of protonmotive force, such inhibition is very weak. Mitochondrial ATP synthase is somewhere inbetween.

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

  • "F-type ATPase" is just another name for ATP synthase letter "F" comes from "phosphorylation F actor". F-ATPases are present in bacteria, mitochondria and chloroplasts. Their major function in most cases is ATP synthesis at the expense of the transmembrane electrochemical proton potential difference. In some bacteria, however, the primary function of the enzyme is reversed: it hydrolyzes ATP to generate this potential difference. In vitro F-type ATPases can operate in both directions depending on the experimental conditions.
    A few Na + -bacterial F-type ATPases are also found.
  • A-type ATPases were found in A rchaea, their function is similar to that of F-type ATP synthase, but structurally they are very similar to V-type ATPases (see below).

F-, A-, and V-type ATPases are multisubunit complexes, similar in terms of overall architecture, and most probably have the same core catalytic mechanism. They couple transmembrane proton (or Na + in some F-ATPases) transport, achieved 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 its second transmembrane helix. This carboxyl group 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 f 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 + , Mg 2+ , Ca 2+ , Ag + and Ag 2+ , Zn 2+ , Co 2+ , Pb 2+ , Ni 2+ , Cd 2+ , Cu + and Cu 2+ 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, Ca 2+ membrane ATPase.

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 .


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
10-15(?)
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 p ortion, 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 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 (). In case of hydrolysis the enzyme functions as an ATP-driven proton pump and generates .
The equation of the reaction catalyzed is

ADP 3- + P i 2- + nH + P <=> ATP 4- + H 2 O + (n-1)H + N ( pH > 7.2 )

The " P " and " N " indices denote the p ositively and the n egatively charged sides of the coupling membrane.
The pH value is important: the pK value for Pi 2- + H + <=> P i - 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:

ADP 3- + P i - + nH + P <=> ATP 4- + H 2 O + nH + N ( pH 6.9-7.2 )

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

ADP 2- + P i - + nH + P <=> ATP 3- + H 2 O + (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:

ATP 4- + H 2 O <=> ADP 3- + P i 2- + H + ( pH > 7.2 )

" Physical Chemistry " (P.W.Atkins, 2nd edition) gives a value of -30 kJ mol -1 (-7.16 kcal/mol) at 37 o C as a "biological" standard Gibbs free energy change ( o ´) 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, o , 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, o ´, is a similar parameter, but is defined at pH 7, i.e. the concentration of H + is not 1 M, but 10 -7 M. It is more practical and convenient, for most biological reactions take place at physiological pH.

A very important, and sometimes ignored point, is that o ´ 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

/>= />o ' + 2.3 RT log [ C ADP C P i (C H + / 10 -7 ) / C ATP ],

where C ADP, C Pi, C H + , and C ATP are the actual concentrations of the corresponding reactants, R is the molar gas constant (8.314 J mol -1 K -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:

C ATP 2 x 10 -3 M -1
C ADP 2 x 10 -4 M -1
C P i 10 -2 M -1
C H + 5 x 10 -8 M -1 (pH approx. 7.3)

The Gibbs energy change under such conditions (temperature 310 o K, or 37 o C) will be

/>= />o ' + 2.3 RT log ( C ADP C P i C H + / C ATP ) = -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 logarithmically upon decrease in ADP and Pi concentration and upon increase in ATP or H + concentration (= decreases linearly with increase in 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).

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 catalyzed 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.

Driving force for ATP synthesis catalyzed by ATP synthase.

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

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).

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

where the " P " and " N " indices denote the p ositively and the n egatively 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 -1 K -1 ), T is the temperature in Kelvins, and is the transmembrane electrical potential difference in volts. The value of 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 , but protonmotive force ( pmf ):

At room temperature (25 o C) the protonmotive force (in millivolts, as well as ) is:

In the absence 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 ( between 11.6 and 19.3 kJ mol -1 ).

Rotary catalysis

  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.

Inhibitors of ATP synthase

ATP synthase activity is specifically inhibited by several compounds (both organic and inorganic). Most of these inhibitors are very toxic, so great care and appropriate safety precautions are essential when working with them (it is not very surprising that we get unhappy when OUR ATP synthase is blocked!). Most inhibitors are specific for either proton-translocating FO-portion, or hydrophilic F1-portion, so the section below is divided accordingly.

Inhibitors of FO

Oligomycin

Oligomycin is the inhibitor that gave the name "FO" to the membrane-embedded portion of ATP synthase. The subscript letter "O" in FO(not zero!) comes from Oligomycin sensitivity of this hydrophobic phosphorylation Factor in mitochondria.
Oligomycin binds on the interface of subunit a and c-ring oligomer and blocks the rotary proton translocation in FO. If the enzyme is well-coupled, the activity of F1 is also blocked. Because of the latter phenomenon, a subunit of mitochondrial F1-portion that connects F1 with FO was named Oligomycin-Sensitivity Conferring Protein (OSCP). This subunit is essential for good coupling between F1 and FO and makes the ATPase activity of F1 sensitive to FO inhibitor oligomycin, hence the name.
Oligomycin is specific for mitochondrial ATP synthase and in micromolar concentrations effectively blocks proton transport through FO. This inhibitor also works in some bacterial enzymes that show high similarity to mitochondrial ATP synthase, e.g. enzyme from purple bacterium Rhodobacter capsulatus. But ATP synthase from chloroplasts and from most bacteria (including Escherichia coli) has low sensitivity to oligomycin.
It should also be noted that oligomycin in high concentrations also affects the activity of mitochondrial F1.

DCCD (abbreviation for Dicyclohexylcarbodiimide also known as DCC, as N,N'-dicyclohexylcarbodiimide, as Bis(cyclohexyl)carbodiimide, and as 1,3-dicyclohexylcarbodiimide) is a small organic molecule that can covalently modify protonated carboxyl groups. When added to ATP synthase at pH above 8, DCCD almost exclusively reacts with the carboxyl group of the conserved acidic amino acid residue of subunit c (that is why subunit c is sometimes called "DCCD-binding protein"). that has elevated pK and can therefore be protonated at such a high pH. Modification of the carboxyl group in a single c-subunit is enough to render the whole c-ring oligomer inactive. Because DCCD covalently binds to c-subunit, this inhibition is irreversible.
The carboxyl group of the conserved amino acid residue in subunit c-subunit is present in all ATP synthases known so far. So DCCD is a universal inhibitor that can FO function in bacterial, mitochondrial and chloroplast enzymes. Moreover, V- and A-type proton-transporting ATPases are also sensitive to DCCD for the same reason. Sodium-transporting ATP synthases are also effectively inhibited by DCCD.
At lower pH (

Venturicidin

The macrolide antibiotic venturicidin (also known as Aabomycin) isolated from a Streptomyces sp. was originally described as an antifungal agent. Later it was found that venturicidin is a potent inhibitor of ATP synthase that specifically blocks proton translocation through FO. Like oligomycin, it binds on the interface of subunit a and c-ring oligomer. However, venturicidin specificity is not limited to mitochondrial ATP synthase, and it is effectively inhibiting bacterial and chloroplast enzymes. Na + -translocating ATP synthases are also strongly inhibited with venturicidin.
If the coupling between FO and F1 is good, venturicidin also blocks the activity of F1. So this inhibitor is a good choice for quick test of the coupling efficiency. Its important advantages over DCCD are quick effect and ease of use. Unlike DCCD, venturicidin can be stored as a concentrated stock solution for a long time without loss of inhibitory power.
The affinity of FO to venturicidin is very high. In Rhodobacter capsulatus ATP synthase half-maximal inhibition was observed at 2-5 nM venturicidin concentration.

Inhibitors of F1

Azide

Azide selectively inhibits ATPase activity of ATP synthase, leaving its ATP synthesis activity unaffected. It is demonstrated in mitochondrial F1 that azide binds together with MgADP (interacting with its beta-phosphate) in a catalytic site, and presumably prevents ADP release from this site. However, rotation of subunit gamma forced by sufficiently high pmf or by external force can expell the occluded ADP from the catalytic site, bringing the enzyme to active ATP synthesis.

Tentoxin

Tentoxin is a phytotoxin produced by fungi of the Alternaria species. It specifically inhibits the ATPase activity of some chloroplast ATP synthases it has no effect on bacterial and mitochondrial enzyme. Moreover, some chloroplast ATP synthases are also tentoxin-resistant.
Tentoxin binds at the cleft between Alpha and Beta subunits close to the N-terminal beta-barrel crown of F1. At small concentration (about 1-10uM) tentoxin inhibits ATP hydrolysis, while at higher concentrations the inhibition is relieved. The binding site of tentoxin was determined by X-ray analysis of chloroplast F1 crystallized in the presence of the inhibitor.

Efrapeptin

Efrapeptin (also known as A 23871 or A23871) is a common name for a group of small peptides antibiotics that can bind inside F1 with high affinity and inhibit both ATP synthesis and hydrolysis. The binding site of efrapeptin was determined by X-ray analysis of the bovine mitochondrial F1 crystallized in the presence of the inhibitor. It is likely that efrapeptin fixes subunit Gamma inside F1 and block the rotation of this subunit.
Efrapeptins are potent inhibitors for mitochondrial ATP synthase and for some bacterial enzymes. The inhibitory effect was first noticed in chromatophores of purple bacterium Rhodospirillum rubrum. Chloroplast ATP synthase is only mildly sensitive to efrapeptin.

Fluoro-aluminate (AlF4)

Fluoro-aluminate based inhibitors mimic the transitional state of ATP gamma-phosphate. They bind together with ADP in catalytic sites and freeze the enzyme in a conformation that presumably reflects an intermediate step of ATP hydrolysissynthesis.

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 than 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 numbers of the c -subunits in ATP synthases from different organisms are 10, 11, 14, and 15, suggesting ratios of 3.33, 3.67, 4.67 and 5, 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 synthesized 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 eukaryotic 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 does the enzyme have?

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.

1) C. Etzold, G. Deckers-Hebestreit, and K. Altendorf. (1997) Turnover number of Escherichia coli F O F 1 - 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 10 6 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 amino acid 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 hydrophilic amino acid 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 its 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!

What is Beta DELSEED sequence?

Beta DELSEED region is a part of subunit Beta that has amino acid sequence of -Asp-Glu-Leu-Ser-Glu-Glu-Asp- (hence the name: in single-letter amino acid code it is DELSEED). This fragment is highly conserved in all ATP synthases. However, its role is not completely clear. In bacterial ATP synthase from thermophilic Bacillus PS3 it was demonstrated that this region is essential neither for ATP hydrolysis nor for ATP-driven rotation of subunit Gamma in Alpha3-Beta3 complex, but plays a role in the inhibitory action of subunit Epsilon. It is likely that in Bacillus PS3 the negatively charged Asp and Glu residues interact with positively charged Lys and Arg in the C-terminal domain of Epsilon, and block hydrolysis.
It is probable that the same mechanism works in ATP synthase from other bacteria and in chloroplast enzyme. In mitochondrial ATP synthase such mechanism is unlikely, because subunit Delta (mitochondrial homologue of bacterial epsilon) lacks the important positive charges in its C-terminal domain.


ATP Synthase: A Molecular Motor

ATP synthase is a huge molecular complex (>500,000 daltons) embedded in the inner membrane of mitochondria. Its function is to convert the energy of protons (H + ) moving down their concentration gradient into the synthesis of ATP. 3 to 4 protons moving through this machine is enough to convert a molecule of ADP and Pi (inorganic phosphate) into a molecule of ATP. One ATP synthase complex can generate >100 molecules of ATP each second.

ATP synthase can be separated into 2 parts:

  • Fo - the portion embedded in the inner mitochondrial membrane and
  • F1-ATPase &mdash the portion projecting into the matrix of the mitochondrion.

This is why the intact ATP synthase is also called the FoF1-ATPase.

When the F1-ATPase is isolated in vitro, it catalyzes the hydrolysis of ATP to ADP and Pi (which is why it is called the F1-ATPase). While it is doing so, the central portion of Fo attached to the stalk rotates rapidly in a counter-clockwise direction (as viewed from above).

In the intact mitochondrion, the protons that have accumulated in the intermembrane space enter the Fo complex and exit from it into the matrix. The energy they give up as they travel down their concentration gradient rotates Fo and its stalk (at

6000 rpm) in a clockwise direction. As it does so, it induces repeating conformational changes in the head proteins that enable them to convert ADP and Pi into ATP. (In the figure, two of the three dimers that make up the head proteins have been pulled aside to reveal the stalk inserted in their center.)

In both these cases, the machine is converting chemical energy

  • from the hydrolysis of ATP in the in vitro case and
  • the flow of protons down their concentration gradient in the intact mitochondrion

into mechanical energy &mdash the turning of the motor.

But this remarkable device can be made to do the reverse, converting mechanical energy (turning of the motor) into chemical energy.

A group of Japanese scientists interested in nano-machines have succeeded in attaching magnetic beads to the stalks of the F1-ATPase isolated in vitro.

Then using a rotating magnetic field they were able to make the stalks rotate. When rotated in a clockwise direction, the F1-ATPase synthesized ATP from ADP and Pi in the surrounding medium &mdash at a rate of about 5 molecules per second! (When rotating the stalks in the counter-clockwise direction, or not rotating them at all, ATP was hydrolyzed into ADP and Pi.)

Their achievement was reported in Itoh, H., et al., Nature, 29 January 2004.


Can ATP synthase work without a proton gradient?

ATP synthesis is driven by energy of proton concentration gradient that moves the hydrogen ions (protons) from lumen of thylakoid membrane towards the stroma. Hence, ATP synthesis requires presence of more hydrogen ions inside the membrane.

Beside above, how many protons does ATP synthase use? Since the gamma-subunit catalyses the formation of 1 ATP every 120 degrees, a full 360 degree rotation would yield 3 ATPs. This is where things go awry in my mind. Within 360 degrees, 10 protons and 3 ATPs are made. Thus, that makes (10/3) protons per ATP, or 3.33 protons/ATP.

Accordingly, how does a proton gradient drive synthesis of ATP?

The proton gradient produced by proton pumping during the electron transport chain is used to synthesize ATP. Protons flow down their concentration gradient into the matrix through the membrane protein ATP synthase, causing it to spin (like a water wheel) and catalyze conversion of ADP to ATP.

What happens if ATP synthase is inhibited?

9.13 The ATP Synthase Inhibitor Protein IF Damage to the electron transport chain, increased proton leakage, or severe hypoxia can lower &Deltap such that the ATP synthase reverses in the cell and starts to hydrolyse cytoplasmic ATP generated by glycolysis.