ATP SYNTHASE STRUCTURE ATP SYNTHESIS.pptx

Atp synthase structure

1. ATP Synthesis Mechanism of ATP Synthesis Substrate level phosphorylation Chemiosmotic mechanism (oxidative and photophosphorylation) ATP Synthase Boyer’s conformational model Racker’s experiment Jagendorf’s experiment Role of uncouplers 03-03-2024 ATP SYNTHASE CHARU KALRA

2. Mechanism of ATP Synthesis Oxidative phosphorylation- It is the chemiosmotic synthesis of ATP associated with the transfer of electrons through the electron transport chain (from NADH / FADH 2 to O 2 by a series of electron carriers) and the accompanying consumption of oxygen. ATP is formed as a result of the transfer of electrons This process, which takes place in mitochondria , is the major source of ATP in aerobic organisms. Photophosphorylation - The light- dependent chemiosmotic synthesis of ATPin the chloroplast in the presence of light. ATP is formed as a result of the transfer of electrons which takes place in chloroplast , is the major source of ATP in photosynthetic organisms Substrate level phosphorylation- ATP synthesis by the transfer from a high- energy compound (phosphate group) ADP, without involvement of any electron transport is called substrate- level phosphorylation. 03-03-2024 ATP SYNTHASE CHARU KALRA

3. Photophosphorylation Light- dependent ATPsynthesis- coupled reaction This process was discovered by Daniel Arnon and co- workers in 1950s Electron flow without accompanying phosphorylation is said to be uncoupled. Photophosphorylation works via the chemiosmotic mechanism, first proposed in the 1960s by Peter Mitchell. The total energy available for ATP synthesis, called the proton motive force (∆p), is the sum of a proton chemical potential and a transmembrane electric potential, ∆p = ∆E − 59(pHi − pHο) A transmembrane pH difference of 1 pH unit is equivalent to a membrane potential of 59 mV. 03-03-2024 ATP SYNTHASE CHARU KALRA

4. Substrate- level phosphorylation ATP synthesis by the transfer from a high- energy compound (phosphate group) ADP, without involvement of any electron transport is called substrate- level phosphorylation. In simple terms, it is the production of ATP by the transfer of a phosphoryl group from the substrate of a reaction to ADP. Substrate- level phosphorylation differs from the other two ways of ATP synthesis i.e., oxidative- phosphorylation and photo- phosphorylation, in that the ATP synthesis is not coupled to any electron transport. Oxidative- phosphorylation and photo- phosphorylation involves ATP synthesis coupled with electron transport. 03-03-2024 ATP SYNTHASE CHARU KALRA

5. 2 Steps in Glycolysis where substrate level phosphorylation occurs 03-03-2024 ATP SYNTHASE CHARU KALRA

6. In the first reaction, energy is harvested in the form of ATP. The enzyme phosphoglycerate kinase catalyzes the transfer of the phosphoryl group of 1,3- bisphosphoglycerate to ADP. This is the first substrate- level phosphorylation of glycolysis , and it produces ATP and 3- phosphoglycerate. It is a coupled reaction in which the high energy bond is hydrolyzed and the energy released is used to drive the synthesis of ATP. The final substrate- level phosphorylation in the pathway is catalyzed by pyruvate kinase where Phosphoenolpyruvate is coverted to Pyruvate. Phosphoenolpyruvate serves as a donor of the phosphoryl group that is transferred to ADP to produce ATP. This is another coupled reaction in which hydrolysis of the phosphoester bond in phosphoenolpyruvate provides energy for the formation of the phospho-anhydride bond of ATP. 03-03-2024 ATP SYNTHASE CHARU KALRA

7. ATP synthase The ATP is synthesized by a large (400 kDa) enzyme complex known by several names: ATP synthase , ATP ase (afterthe reverse reaction of ATP hydrolysis), and CFo–C1 (Boyer,1997). This enzyme consists of two parts: a hydrophobic membrane- bound portion called CFo and a portion that sticks out into the stroma called CF1. CF1 is the portion of the complex that synthesizes ATP. CF1 is made up of several peptides, including three copies of each of the αand βpeptides arranged alternately much like the sections of an orange. Whereas the catalytic sites are located largely on the βpolypeptide, many of the other peptides are thought to have primarily regulatory functions. CFo appears to form a channel across the membrane through which protons can pass. The molecular structure of the mitochondrial ATP synthase has been determined by X- ray crystallography (Stock et al. 1999). Although there are significant differences between the chloroplast and mitochondrial enzymes, they have the same overall architecture and probably nearly identical catalytic sites. In fact, there are remarkable similarities in the way electron flow is coupled to proton translocation in chloroplasts, mitochondria, and purple bacteria . Another remarkable aspect of the mechanism of the ATP synthase is that the internal stalk and probably much of the CFo portion of the enzyme rotate during catalysis (Yasuda et al. 2001). The enzyme is actually a tiny molecular mo t o 03-03-2024 ATP SYNTHASE CHARU KALRA

8. Structure of ATP synthase 03-03-2024 ATP SYNTHASE CHARU KALRA

9. ATP synthase- A tiny molecular motor ATPase or CFo–CF1 CF1 is the portion of the complex that synthesizes ATP. CFo appears to form a channel across the membrane through which protons can pass. It is a large multi-subunit complex, CF1, attached on the stromal side of the membrane to an integral membrane portion, known as CFo. CF1 consists of five different polypeptides, with a stoichiometry of α3, β3, γ, δ, ε. Cfo contains probably four different polypeptides, with a stoichiometry of a, b, b‘, c12. 03-03-2024 ATP SYNTHASE CHARU KALRA

10. Model of the F o F 1 - ATPase, showing the attachment of the catalytic complex to the membrane via the β subunit and the δ subunit. When the reaction runs in reverse (ATP synthesis), protons diffuse through the F o complex down their electrochemical gradient. The movement of protons through the channel drives the rotation of the entire F o complex within the membrane. The Y subunit, which is attached to the F o complex, then turns within the catalytic complex, causing the conformational changes that are required for ATP synthesis. It is assumed that the catalytic complex itself does not rotate, but is anchored to the membrane. The δ subunit is located on the outside of the β subunit and serves as the site of attachment of the β subunit, which anchors the catalytic complex to the membrane and prevents it from spinning. In mechanical terms, the F 1 complex and its membrane anchor act as a stationary housing, or “stator,” while Y subunit (and possibly the F o complex) serves as the “rotor.” (From Junge et al. 1997.) 03-03-2024 ATP SYNTHASE CHARU KALRA

11. Uncouplers / Protonophores Uncouplers are amphiphilic compounds(which are soluble both in water and lipids). They are agents with conjugated double bonds which allow them to diffuse across the membrane in both the protonated form and the unprotonated form, and thus dissipate the electrochemical proton gradient. Uncouplers which transfer protons across the membrane are known as protonophores . They disrupts phosphorylation by dissociating the reactions of ATP synthesis from the electron transport chain. They directly bypasses the ATP synthase by allowing passive proton influx, without affecting electron flow, but ATP synthesis does not occur. The result is that the cell or mitochondrion expends energy to generate a proton motive force, but the proton motive force is dissipated before the ATP synthase can recapture this energy and use it to make ATP. Uncouplers increases the proton permeability of the inner mitochondrial membrane and dissipates the proton gradient. Uncouplers are capable of transporting protons through mitochondrial and chloroplast membranes. Both mammalian and plant mitochondria contain uncoupling protein (UCP). This protein facilitates the movement of protons across the inner membrane and therefore partially uncouples electron transport and decreases the ATP yield of respiration. Electron flow without accompanying phosphorylation is said to be uncoupled. Uncoupling proteins (UCPs) occur in the inner mitochondrial membrane and dissipate the proton gradient across this membrane that is normally u sed for ATP synthesis. 03-03-2024 ATP SYNTHASE CHARU KALRA

12. Role of uncoupling agents Addition of uncouplers results in continuation of electron transport and proton pumping, without generation of any proton gradient. ATP synthesis does not occur without affecting uptake of oxygen. In the absence of proton gradient, however, protons are transported in reverse direction through ATP synthase at the expense of ATP. Protonated DNP (a weak acid) diffuses from high proton concentration side of the membrane to low proton concentration side where it gets dissociated to generate protons resulting in dissipation of proton gradient. Membrane is permeable to both protonated and anionic forms of these. E.g. FCCP (trifluoromethoxy carbonyl cyanide phenylhydrazone), a very efficient mitochondrial uncoupler. Other exampes of uncouplers- Carbonyl cyanide phenylhydrazone (CCP) 2,4-dinitrophenol (DNP), Carbonyl cyanide m - chlorophenyl hydrazine (CCCP) 03-03-2024 ATP SYNTHASE CHARU KALRA

13. Dr. Roshni Rajamohan The mechanism of action of uncouplers (B) In the presence of an electrochemical proton gradient across the membrane, FCCP will become protonated and thus pick up a proton on the positive side of the membrane, move across the membrane in the neutral form, and lose the proton on the negative side. This is driven by the electrochemical proton gradient. In the negative form, FCCP will be driven back out across the membrane by the membrane potential. The net result is the movement of one proton from the positive to the negative side of the membrane, which results in the dissipation of the electrochemical proton gradient, and the movement of one electrical charge from the negative to the positive side of the membrane, which results in the dissipation of the electrical gradient. The shaded area represents the system of conjugated double bonds (u- orbital system). (Modified from Nicholls and Ferguson 1992.) (A) The protonation / deprotonation of FCCP which is a weak acid. 03-03-2024 ATP SYNTHASE CHARU KALRA

14. CHEMIOSMOTIC THEORY Peter Mitchell in 1961 proposed CHEMIOSMOTIC THEORY Nobel Prize for Chemistry in 1978 Membrane potential with high negative charges and positive charges operating on the opposite surfaces of the membranes can generate energy rich bond between ADP and Pi to synthesize ATPs . The basic principle of chemiosmosis is that ion concentration differences and electric- potential differences across membranes are a source of free energy that can be utilized by the cell Early evidence supporting a chemi- osmotic mechanism of photosynthetic ATP formation was provided by an experiment carried out by André Jagendorf and co- workers. 03-03-2024 ATP SYNTHASE CHARU KALRA

15. Ja g endor f ’ s Experiment Andre T. Jogendorf & Earnest Uribe Placed chloroplasts extracted from cells in darkness, thereby eliminating light absorption & electron transfer as a source of energy for photosynthesis. In the dark, thylakoids were first incubated in a medium of pH 4 until both the exterior and interior of the vesicles had ph4. Then, the thylakoid vesicles were quickly transferred to a medium with pH 8. At this point, there was a pH gradient, with the interior of the thylakoid (pH 4) having a higher H + concentration than the exterior (pH 8). When ADP was added, ATP was made, even in the dark. This is convincing evidence linking a pH gradient to ATP synthesis. 03-03-2024 ATP SYNTHASE CHARU KALRA

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17. Efraim Racker Efraim Racker- identified and purified Factor- 1 (F1), the first part of the ATP synthase enzyme to be characterised. F1 is only a part of a larger ATP synthase complex. It is a peripheral membrane protein attached to component Fo, which is integral to the membrane. Racker was able to confirm Mitchell's hypothesis that contrary Peter D. to popular opinion, ATP synthesis was not coupled to respiration through a high- energy intermediate but instead by a transmembrane proton gradient. Dr. Roshni Rajamohan 03-03-2024 ATP SYNTHASE CHARU KALRA

18. Can a proton gradient drive the synthesis of ATP? Peter Mitchell’s hypothesis that a proton gradient can drive the synthesis of ATP was proposed before experimental evidence supported it and was therefore met with skepticism. In the 1970s, biochemist Efraim Racker and his collaborator Walther Stoeckenius tested the hypothesis. RACKER’S EXPERIMENT Racker and Stoeckenius built an artificial system consisting of a membrane, a bacterial proton pump activated by light, and ATP synthase. They measured the concentration of protons in the external medium and the amount of ATP produced in the presence and absence of light. RESULTS In the presence of light, the concentration of protons increased inside the vesicles, suggesting that protons were taken up by the vesicles. In the dark, the concentration of protons returned to the starting level. ATP was generated in the light, but not in the dark. INTERPRETATION In the presence of light, the proton pump was activated and protons were pumped to one side of the membrane, leading to the formation of a proton gradient. The proton gradient, in RACKER’S EXPERIMENT CONCLUSION: A membrane, proton gradient, and ATP synthase are sufficient to synthesize ATP. This result provided experimental evidence for Mitchell’s hypothesis. 03-03-2024 ATP SYNTHASE CHARU KALRA

19. Boyer’s conformational model (Binding change model) Our understanding of how ATP is synthesized was advanced by Paul Boyer (1997) at the University of California, Los Angeles, who proposed the binding- change mechanism for catalysis by F- ATPases. From the detailed understanding of how the ATPases function, he proposed that the binding- change mechanism for ATP synthesis contains three important components: The major energy- requiring step is not the synthesis of ATP from ADP and P i , but the release of ATP from the enzyme. Substrate is bound and products are released at three separate but interacting catalytic sites, corresponding to the three catalytic subunits (β subunits). Each catalytic site can exist in one of three conformations: tight, loose, or open. The binding changes are coupled to proton transport by rotation of the γ subunit. That is, the flow of protons down their electrochemical gradient through the F o complex causes the γ subunit to rotate. Rotation of the γ subunit then brings about the conformational changes in the catalytic complex that allow the release of ATP from the enzyme, and the reaction is driven forward. The reverse occurs when the enzyme functions as an ATP- driven proton pump Dr. Roshni Rajamohan 03-03-2024 ATP SYNTHASE CHARU KALRA

20. Binding change model In this model, the F1 subunit exists in three states: an O - open - state with very low affinity for substrates and has no catalytic activity; a L - loose - state with low affinity for substrates and also no catalytic activity, and a T - tight - tight state with high affinity for substrates and with catalytic activity. The F1 subunit consist of three α and three β subunits, which can cycle between three conformations, bind substrate, and have catalytic activity. The collapse of the proton gradient (i.e. the proton- motive force) causes the γ subunit to rotate like a crankshaft relative to the F1 subunit, forcing the β subunit to change conformation from the T to the O (releasing ATP) and then the L (binding ADP and Pi). The γ subunit does not appear to undergo any significant conformational change on ATP hydrolysis as evidenced by tritium exchange studies of amide protons. Top view 03-03-2024 ATP SYNTHASE CHARU KALRA

21. The binding- change mechanism as seen from the top of the F 1 complex. There are three catalytic sites in three different conformations: loose, open, and tight. (For clarity, only the three β subunits are shown.) Substrate (ADP + P i ) initially binds to the open site and is converted to ATP at the tight site. In step 1, rotation of the γ subunit causes a conformational change, resulting in a change in the formation of the sites. As a result, ATP is released from the enzyme. In step 2, substrate again binds to the open site, and another ATP is synthesized at the tight site. (After Duncan et al. 1995.) http://6e.plantphys.net/topic12.04.html 03-03-2024 ATP SYNTHASE CHARU KALRA

22. The model of Boyer's binding change mechanism . The α and β subunits configure three nucleotide binding sites: O, which provides the early binding site for ADP and inorganic phosphate, L, to which the ADP and inorganic phosphate bind after migrating from O, and T, which tightly binds ATP. Energy ensuing from the movement of protons from the chloroplast lumen to the stroma drives the rotation of the Y subunit of CF 1 , and the interconversion of the binding sites and the release of an ATP molecule. 03-03-2024 ATP SYNTHASE CHARU KALRA

23. Rotary motion of the Y subunit To visualize the rotation of the enzyme, Masasuke Yoshida and his colleagues at the Tokyo Institute of Technology attached an actin filament labeled with a fluorescent dye to the base of the γ subunit using another protein as a "glue." They then attached the F 1 complex upside down to a glass surface. If the γ subunit rotates with respect to the catalytic complex, the actin filament should swing around with it. Since the filament is very long compared to the ATP synthase (about 1 μm), its rotation should be visible in a fluorescence microscope. In other words, the fluorescently tagged actin filament, which is large enough to visualize in a light microscope, reports the rotation of the γ subunit. When ATP was added to the modified enzyme, the actin filaments were seen to swing around in a circle at as much as 4 revolutions per second in a fluorescence microscope . Demonstration of the rotary motion of the γ subunit made it possible to put together a model of how the ATP synthase works 03-03-2024 ATP SYNTHASE CHARU KALRA

24. The γ subunit is inserted like a shaft through the center of the catalytic complex, which consists of three α subunits and three β subunits arranged alternately in a doughnut- like structure. The interface between the γ subunit and the α and β subunits is highly hydrophobic. The hydrophobicity of the interface minimizes the interactions between the subunits, consistent with the rotation of the γ subunit within the hole formed by the catalytic complex. In other words, the γ subunit looks like a molecular bearing lubricated by a hydrophobic interface. A method for visualizing rotation of the γ subunit. A fluorescently labeled actin filament was attached to one protruding end of the γ subunit. The F 1 complex was then attached upside down to a coverslip. When ATP was added to the coverslip, the actin filament rotated. (After Noji et al. 1997.) Sequential images of the rotating actin filament attached to the Y subunit, as viewed in a fluorescence microscope. There is 133 ms between the images and the rotation rate is 0.5 r.p.s. (From Noji et al. 1997, courtesy of S. Noji.) http://docencia.izt.uam.mx/docencia/alva/atpaseyoshida.htm . 03-03-2024 ATP SYNTHASE CHARU KALRA

25. Paul D. Boyer Proposed passage of protons through the channel of the basal unit makes the stalk & headpiece spin like a top just as the flow of water makes a water wheel turn. The turn cycles each of 3 headpiece. catalytic sites on the Through sequential conformational changes that pickup ADP & phosphate, combine them, & release the ATP product. Nobel prize w/ walker for research into the mechanisms by which ATP synthase makes ATP. John E. Walker Used X- ray diffraction to create a 3 dimensional synthase that picture of ATP clearly verified Boyers model by showing the different head in positions as rotational ATP synthesis proceeds. Nobel prize w/ Boyer for research into the mechanisms by which ATP synthase makes ATP For their contributions to elucidation of the mechanism of ATP synthesis, Paul Boyer and John Walker shared half the Nobel prize in physiology or medicine in 1997. The other half went to Jens Skou for his pioneering work on the K + ,Na + - ATP synthase, the mammalian counterpart of the plant plasma membrane H + - ATP synthase Dr. Roshni Rajamohan 03-03-2024 ATP SYNTHASE CHARU KALRA

26. Suggested Reading: Taiz, L. and Zeiger, E. (2010) Plant Physiology. 5th Edition, Sinauer Associates. William G. Hopkins, Norman P. A. Hüner (2008) Introduction to Plant Physiology, 4th Edition, Wiley. Bhatla, Satish C, Lal Manju A. (2018) Plant Physiology, Development and Metabolism, Springer. 03-03-2024 ATP SYNTHASE CHARU KALRA