Bioenergetics is an important domain in biology. This presentation has explored ATP production and its optimum utilization in biological systems along with certain theories and experiments to give a bird's eye view of this important issue.
2. COURSE CONTENT
ATP- the Energy Currency of Cell
ATP-Synthesis - Mechanism of ATP synthesis,
Substrate level Phosphorylation,
Chemiosmotic mechanism (oxidative and photophosphorylation),
ATP synthases,
Boyer’s conformational model,
Racker’s experiment,
Jagendorf’s experiment;
Role of uncouplers.
ATP- Functions & Importance
3. ATP-THE ENERGY CURRENCY
• ATP-the high energy bond was realized by Fritz A. Lipmann(1942)for the first time and
advocated the major link in transfer of chemical energy via ATP-ADP system. Energy released
from hydrolysis of ATP( Dephosphorylation) again undergoes reversion of ATP by
phosphorylation.The entire process is governed by free energy changes.
• ▲G of ATP hydrolysis was large and negative-7300cal/mole.
• If a compound in a physical or chemical process releases free energy value of more than
5Kcal/mole i.e. ▲G is -5kcal/mole or more -ve value, then the compound is treated as energy rich
compound and if ▲G is -1to -5 kcal/mole then such compound is treated low energy compound.
The bond energy having high energy rich is called wriggle bond.
• As far ATP is concerned having two wriggle bond as depicted A-S-P-P-P
• ATP was discovered by Karl Lehmann (Germany)& C. Fiske and Y. Subharow (USA) in 1929 &
Lipmann proposed ATP as universal carrier of Chemical Energy.
5. HOW DOES ATP SYNTHESISE?
• ATP is synthesised both photosynthesis & Cellular respiration.
• During anabolism
• ATP is synthesised by photosynthetic phosphorylation in presence of photon during the day time
in the chlorophyll containing cells in general and chloroplastids( Quantasomes of Granum of
Plastids) in particular. The phosphorylation process depends upon the drain off the electrons from
the activated chlorophyll molecules excited by photon. It may be three types- cyclic, non-cyclic
and pseudo cyclic. Most of the green plants perform by non-cyclic electron transport chain for
phosphorylation while chemosynthetic bacteria prefer to cyclic pathways.
• During catabolism
• Cellular respiration in general but oxidative phosphorylation in particular along with the substrate
level phosphorylation are the two main sources of high energy rich molecules to extendd energy
for sorts of metabolic activities in cell. The substrate level phosphorylation and ATP generation
are very limited in amount and most of the ATPs are generated via oxidation phosphorylation
through ETC for electron drain off through chemiosmotic process. The detail is explored below.
7. METHODS OF CELLULAR RESPIRATION
• Cellular Respiration in living organisms take place either in absence of oxygen or in the presence of
oxygen. Respiration without oxygen is called anaerobic respiration having little efficiency mostly
found in anaerobic bacteria and the cells starved due to oxygen deficiency.
• But true cellular respiration is the complete oxidation of the respiratory substrates in presence of
ambient oxygen supply& here the maximum number of ATP-the energy currency is produced at the
complete oxidation of bio-fuels-Hydrogen.
• Aerobic respiration is comprising of the following events-
Glycolysis - consists of Preparatory phase & pay off phase takes place in Cytoplasm.
Citric acid cycle or TCA cycle of Krebs Cycle occurs in Mitochondria
Terminal respiration via Electron Transport Chain(ETC) in the inner membrane of Mitochondria i.e.
cristae.
The end products are Carbon dioxide, metabolic water & energy i.e. ATP.
9. OVERVIEW OF RESPIRATION
Life without energy is never to be imagined,
The energy for life requires for different types of biochemical reactions as the metabolism is the elexir
of life processes,
ATP is the energy currency of cell to drive the vitality,
Anaerobic reaction produce energy to a limited extent as the partial oxidation seldom produce little bit
energy,
Aerobic respiration through complete oxidation or dehydrogenation of the respiratory substrates is the
sole source of most of the biological energy sources,
Aerobic respiration takes place in two sites of cell- Cytoplasm & Mitochondria,
Cytoplasm conducts glycolytic to split up glucose molecules to pyruvic acid (3C) compound, s simple
respiratory substrates by the expenditure of 2 ATPs as preparatory phase,
The pay off phase starts by the generation of ATP initially to a lesser extent along with the production
of hydrogen sources in the different forms to produce energy in the due state,
Electron transport Chain (ETC) ultimately plays the pivotal role of energy production in the due time
by the presence of oxygen as terminal electron acceptor and to produce ATP on the basis of redox
potential.
11. CELLAND RESPIRATION
• ANAEROBIC RESPIRATION
In absence of oxygen, the aerobic respiration takes place inside the cytoplasm of the cell.
Here, glucose is converted into puruvic acid and this is being used for the production of the different
compounds depending upon the enzymes present in the cell.
The preparatory phase needs 2 ATP molecule for this conversion and little amount of energy is
produced only by the substrate level of phosphorylation.
AEROBIC RESPIRATION
The respiration takes place in presence of oxygen is aerobic respiration produces the desired amount
of energy and the sites for the respiration is as follows:
GLYCOLYSIS- Cytoplasm of the cell due to presence of enzymes,
KREB’S CYCLE- Takes place in mitochondria and major amount of cellular energy is generated
here, mitochondria number and size depends on the required work, mitochondrial network,
intermediate space with aqueous medium lies in between outer and inner membranes, inner membrane
folded to form cristae extend to mitochondrion interior, surface of the inner membrane contains small
knob-like particles, oxysomeas called coupling factors.
13. SUMMARY OF GLYCOLYSIS
Partial degradation of monomers, glucose to key intermediates, 2 mols of acetyl Co-A,
6 C containing glucose is primarily converted into 3C containing pyruvic acid by the investment of 2
ATPs as preparatory phase and 4 directs ATP molecules ( 2*2) are synthesized, called Investment
phase or Preparatory phase,
Now, 3C containing pyruvic acid undergoes decarboxylation to produce 2C containing acetyl Co-A
along with the generation of NADPH2 that acts as energy source in the due course in presence of
terminal electron acceptor, oxygen (Via ETC).
The entire conversion of 6C compound to 2C compound takes place by a number of intermediate steps
each one is catalyzed by the different specific enzymes,
The reaction is initially endothermic reactions followed by the discharge of energy in the high energy
rich bond,
This is also called EMP as far as the names of the scientists proposed it.
At the end, 2 ATPs are net produced along with 2NADH & 2 pyruvate molecules are synthesized,
Glucose + 2 NAD+ + 2 ADP + 2 Pi --> 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O). The
hydroxyl groups allow for phosphorylation. The specific form of glucose used in glycolysis is glucose
6-phosphate.
15. MITOCHONDRIA- POWER HOUSE OF CELL
Key determinants of cellular health,
Depends on cell to cell, plays role in energy distribution, metabolite biosynthesis & signaling,
Increasing the amount of cristae in the inner membrane increases the capacity for energy conversion
and free radical mediated signaling,
Electrically isolated individual cristae provide a protection mechanism to spread the limit of
dysfunction within the mitochondria,
a large, stable mitochondrial reticulum can provide a structural pathway for energy distribution and
communication across long distances yet also enable rapid spreading of localized dysfunction,
Highly dynamic mitochondrial networks allow for frequent content mixing and communication but
require constant cellular remodeling to accommodate the movement of mitochondria.
The formation of contact sites between mitochondria and several other organelles provides a
mechanism for specialized communication and direct content transfer between organelles. However,
increasing the number of contact sites between mitochondria and any given organelle reduces the
mitochondrial surface area available for contact sites with other organelles as well as for metabolite
exchange with cytosol.
17. TCA CYCLE-OVERVIEW
The tricarboxylic acid cycle (TCA) is a series of chemical reactions used in aerobic organisms to
generate energy via the oxidation of acetyl coenzyme A (CoA) derived from carbohydrates, fatty acids
and proteins.
In the eukaryotic system, the TCA cycle occurs completely in mitochondria, while the intermediates of
the TCA cycle are retained inside mitochondria due to their polarity and hydrophilicity.
Under cell stress conditions, mitochondria can become disrupted and release their contents, which act
as danger signals in the cytosol.
Of note, the TCA cycle intermediates may also leak from dysfunctioning mitochondria and regulate
cellular processes. Increasing evidence shows that the metabolites of the TCA cycle are substantially
involved in the regulation of immune responses.
The first stable product of TCA cycle, Citric acid undergoes dehydrogenation i.e. oxidation and the
hydrogen ions derived from this pathway ultimately joined wi9th the oxygen ,
This ultimately leads to the generation of proton gradient and by means of chemiosmosis process leads
to the generation of ATP by oxidative phosphorylation.
Majority of the ATPs are generated by this pathway and least are produced by the direct substrate level
phosphorylation.
18. ATP SYNTHASE
The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of
about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo.
The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial
matrix.
F0 is embedded inside the mitochondrial membrane or thylakoid membrane or cell membrane as per
occurrence while F1 is a component found towards the matrix of the mitochondria , the stroma of the
chloroplast or within the bacterial cell,
F0 is a motor driven by H+ ions travel across the membrane,
The isolated F1can not make ATP from ADP & pi but it can hydrolyze ATP to ADP & Pi----called
ATPase,
The stripped mitochondrial particles (those lacking F1 spheres but containing F0 can transfer electrons
through ETS but they longer synthesize ATP but addition of F1 spheres to the mitochondrial particles
can restore the activity,
The F1 component ( MW=360kdal)contain 9 polypeptide chain subunits of five kinds-α,β,⅋,φ,ℇ
arranged into cluster with many binding sites for ATP & ADP,
The F0 component is a hydrophilic segment of 4 polypeptide chain and it is the proton channel of
enzyme complex.
20. ATP SYNTHASE-FUNCTION
The cylindrical stalk between F0 & F1 includes many proteins including enzyme complex sensitive to
oligomycin, an antibiotic that blocks ATP synthesis by interfering the utilization of proton gradient,
The stalk is the communicating portion of the enzyme complex,
F0F1 ATPase is called ATPase because, in isolated form, it hydrolyses ATP to ADP plus Pi,
As it is important biological role in intact mitochondria to produce ATP from ADP and Pi, it is better to
called as ATP synthetase.
Paul Boyer proposed a simple catalytic mechanism to predict that F-ATPase implements a rotational
mechanism in the catalysis of ATP,
Each catalytic site of the F-ATPase would achieve and change three conformations during a complete
360º turnover and a cycle would be completed at a three different catalytic site with a rotation of 120º,
When a nucleotide binds to ATPase, it undergoes a conformational change in order to be tightly bound
to ATP,
Another conformational change brings the release of ATP,
These conformational change are accomplished by rotating the inner core of the enzyme,
The core itself is powered by the proton motive force conferred by protons crossing the mitochondrial
membrane.
22. BOYER’S CONFORMATIONAL MODEL
• The F1 unit about 80 Angstroms from the F0 subunit and both are connected to the Y subunit which
spans to the ⅋3β3 ring .energy transduction (necessary to capture the negative free energy change
associated with collapse of the proton gradient to drive the positive free energy change for ATP
synthesis) occur between the two subunits.
• Boyer in the absence of the complete structure of F1F0 ATP Synthases was able to deduce from
experimental evidence that the ⅋3β3 complex which can be viewed as three αβ dimers (with catalysis
occurring between subunits of individual dimers where ATP and ADP bind) have three different
interconvertible conformation defined as loose (L), Open (O) and tight (T) states with the names of the
strength of substrate to bind in each dimer.
• O- open state with very low affinity for substrates and has no catalytic activity,
• L-Loose state with low affinity for substrates and has no catalytic activity,
• T- tight state with high affinity for substrate and with high catalytic activity.
• The three states do not rotate with respect to the central axis , but conformationally depending on their
interaction with Y subunit which binds perpendicularly in the central junction of the ⅋3β3 ring cause
the conformation of the O,L and T states to change in situ with the orientation of the rotating Y
subunit.
23. BOYER’S CONFORMATIONAL MODEL
The conversion of the LOT conformations , their binding substrate ( ADP & Pi), the conversion of
bound ADP & Pi and the released of the product is done as par the following method:
The Collapse of the proton gradient (i.e. the proton motive force) causes the Y subunit to rotate like a
crankshaft relative to the F1 subunit, forcing the β subunit to change the conformation from the T to
the O (releasing ATP) and then to the L(binding ADP & Pi) states.
The Y subunit does not appear to undergo any significant conformational change on ATP hydrolysis as
evidenced by tritium exchange studies of amide proteins.
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 F1 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. •
25. BOYER’S CONFORMATIONAL MODEL
• 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.
• Gamma sub unit is in the form of axle, it rotates when protons flow,
• ATP synthetase is known as the smallest MOLECULAR MOTOR in the living cells,
• Conformational Coupling can explain proton translocation coupled to ATP cleavage and active
transport of metabolites coupled to membrane potential , proton gradients of ATP cleavage.
• This model can explain how the conformational model where the molecular motor play a very
significant role for the production of ATP in this regard to drive the biological pathways which are
mostly energy dependent process.
26. RACKER’S EXPERIMENT,
• Efraim Racker- identified and purified Factor-1 (F1), the first part of the ATP synthase enzyme to be
characterized.
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 Peter D. Mitchell's hypothesis that contrary to popular opinion, ATP
synthesis was not coupled to respiration through a high energy intermediate but instead by a
transmembrane proton gradient.
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.
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.
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 turn, powered
synthesis of ATP via ATP synthase.
28. JAGENDORF’S EXPERIMENT
• Proton gradient is essential for ATP synthesis and this is to be confirmed by this experiment as stated
below. Peter Mitchell in 1961 proposed CHEMIOSMOTIC THEORY.
• 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's and co-workers,
• Jagendorf's continued to carry out novel experiments that deepened our understanding of how ATP
formation is linked to the capture of light energy in a process called photophosphorylation,
• Mitchell's Chemiosmotic hypothesis is the most widely accepted theory that advocates that the proton
gradient is the only prerequisite for the synthesis of ATP in presence of ADP and iP in the availability
of ATP synthetase. This also conclusively proved that light does not require for the synthesis of ATP as
it was proposed that the light energy is required for the synthesis of ATP during the light reaction of
photosynthesis during the photosynthetic phosphorylation.
30. JAGENDORF’S EXPERIMENT
Andre T. Jagendorf's & 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.
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.
31. ROLE OF UNCOUPLERS.
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
32. ROLE OF UNCOUPLERS.
• Uncoupling proteins (UCPs) occur in the inner mitochondrial membrane and dissipate the proton
gradient across this membrane that is normally used for ATP synthesis
• 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
synthases 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 uncouplers. Other examples of
uncouplers- Carbonyl cyanide phenylhydrazone (CCP) 2,4-dinitrophenol (DNP), Carbonyl cyanide m-
chlorophenyl hydrazine (CCCP).
33. ATPYEILD FROM COMPLETE OXIDATION OF GLUCOSE
• GLYCOLYSIS ( CYTOSOL)
Phosphorylation of glucose: -1
Phosphorylation of Fructose 6-phosphate -1
Dephosphorylation of 2 moles of 1,3 DPG +2
Dephosphorylation of 2 moles of PEP +2
2 NADH formed in the oxidation of 2 moles of G-3-P
• CONVERSION OF PYRUVATE INTO ACETYL CO-A ( Inside Mitochondria)
2 NADH are formed
• CITRIC ACID CYCLE( Inside Mitochondria)
2 moles of GTP from 2 moles of Succinyl – CoA +2
6 NADH formed in the oxidation of 2 moles of isocitrate, α- ketoglutarate and malate,
2FADH2 formed in the oxidation of 2 moles of succinate
35. ATPYEILD FROM COMPLETE OXIDATION OF GLUCOSE
• OXIDATIVE PHOSPHORYLATION
2NADH formed in Glycolysis ; each yield 2 ATP ( assuming transport of NADH by malate –
oxaloacetate - aspertate shuttle) +6
2 NADH formed in oxidative decarboxylation of pyruvate; each yields 3 ATP +6
2 FADH formed in the citric acid cycle; each yields 3 ATP +4
6 NADH formed in the citric acid cycle; each yields 3 ATP +18
• ------------------------------------------------------------------------------------------------------------------------
NET YIELD PER GLUCOSE +38 ATP
36. FUNCTIONS OF ATP
Hydrolysis is the process of breaking complex macromolecules apart. During hydrolysis, water is
split, or lyses, and the resulting hydrogen atom (H+ ) and a hydroxyl group (OH– ) are added to the
larger molecule. The hydrolysis of ATP produces ADP, together with an inorganic phosphate ion (Pi),
and the release of free energy.
To carry out life processes, ATP is continuously broken down into ADP, and like a rechargeable
battery, ADP is continuously regenerated into ATP by the reattachment of a third phosphate group.
Water, which was broken down into its hydrogen atom and hydroxyl group during ATP hydrolysis, is
regenerated when a third phosphate is added to the ADP molecule, reforming ATP.
Obviously, energy must be infused into the system to regenerate ATP. Where does this energy come
from? In nearly every living thing on earth, the energy comes from the metabolism of glucose.
In this way, ATP is a direct link between the limited set of exergonic pathways of glucose catabolism
and the multitude of endergonic pathways that power living cells,
unctions of ATP The ATP is used for various cellular functions, including transportation of different
molecules across cell membranes. Other functions of ATP include supplying the energy required for
the muscle contraction, circulation of blood, locomotion and various body movements.
38. FUNCTIONS OF ATP
A significant role of ATP apart from energy production includes: synthesizing the multi-thousand types
of macromolecules that the cell requires for their survival. ATP molecule is also used as a switch to
control chemical reactions and to send messages.
• IMPORTANCE OF ATP MOLECULE IN METABOLISM
1. These ATP molecules can be recycled after every reaction.
2. ATP molecule provides energy for both the exergonic and endergonic processes.
3. ATP serves as an extracellular signaling molecule and acts as a neurotransmitter in both central and
peripheral nervous systems.
4. It is the only energy, which can be directly used for different metabolic process. Other forms of
chemical energy need to be converted into ATP before they can be used.
5. It plays an important role in the Metabolism –a life-sustaining chemical reactions including cellular
division, fermentation, photosynthesis, photophosphorylation, aerobic respiration, protein synthesis,
exocytosis, Endocytosis and motility.
39. THANKS FOR YOUR JOURNEY
• ACKNOWLEDGEMENT:
1. Google for images
2. Different web pages for content and enrichment,
3. Principles of Biochemistry- Lehninger,
4. Biochemistry- Reginald H Garrett,
5. Fundamentals of Biochemistry- Jain, Jain & Jain
6. Plant Physiology- Taiz & Zeiger
7. Plant Physiology- Mukherjee & Ghosh
8. Applied Plant Physiology- Arup Kumar Mitra
9. A text book of Botany- Hait, Bhattacharya & Ghosh
10. Plant Physiology-Devlin
• Disclaimer: This presentation has been prepared for online free study materials for academic
domain without any financial interest.
