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Phosphorylation
Submitted By: Farheen Shaikh
To: Dr. Ahmad Ali
In order to understand how the pathways for electron transport and oxidative phosphorylation
work, we need to look at the general structure of a mitochondrion.
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Summary of
decarboxylation:
2Pyruvic acid + 2 NAD+
+ 2 CoA 2 Acetyl CoA
+ 2 CO2 + 2 NADH
Decarboxylation i.e. formation of Acetyl CoA: Pyruvate produced by glycolysis enters
the mitochondrion by active transport and is converted to acetyl CoA. The remainder of the
reactions of cellular respiration occur in the mitochondrion. A carbon atom is removed from
each of the pyruvate molecules forming a two-carbon compound and CO 2. Each of the twocarbon compounds are oxidized forming NADH from NAD+. Coenzyme A is attached to each
of the two-carbon compounds producing two acetyl CoA molecules.
Citric acid (TCA) Cycle:
This is the truly aerobic part of the aerobic metabolism of glucose as this is where the oxygen
is utilized. Oxidative phosphorylation occurs on a membrane, the mitochondrial cristae, to
generate most of the ATP produced from glucose. Coenzymes from the previous reactions
pass electrons to a series of electron carrier molecules, which carry out redox reactions
resulting in the chemiosmotic generation of ATP.
NADH and FADH2 are oxidized providing electrons for redox reactions ultimately
reduce oxygen to generate ATP. The majority of the ATP is produced at this step. Electron
Transport in the mitochondria occurs in four steps at four different sites embedded in the
inner membrane, protein complexes I-IV.. Complexes I-IV has a variety of prosthetic groups
including metal ions, iron-sulphur centres, hemes, and flavins.
There are three classes of carrier molecules:
1. FMN (flavin mononucleotide): protein + flavin coenzyme
2. CoEnzyme Q: nonprotein
3. Cytochromes: protein + an iron group (most common)
1. NADH dehydrogenase (Complex I):
B. OXIDATIVE PHOSPHORYLATION:
5. F1F0-ATPase = ATP Synthase (Complex V):
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.
ATP synthase can be separated into 2 parts:
This is why the intact ATP synthase is also called the FoF1-ATPase.
When the F1-ATPase is isolated in vitro, it catalyses 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 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
The flow of protons down their concentration gradient in the intact mitochondrion
into mechanical energy the turning of the motor.
cellular pathway) and complex II (the predominantly tricarboxylic acid cycle pathway)
Depletion of tricarboxylic acid cycle intermediates plays an important role in the oxidative
phosphorylation flux control.
In respirometric assays, supplies of complex I as well as complex II are required.
Convergent electron input and reconstitution of the tricarboxylic acid are needed to achieve
maximal respiration. It is controlled also by the availability of adenosine-5-diphosphate for
the adenine nucleotide transporter in the inner mitochondrial membrane. Complex I is
suggested to be responsible for adaptive changes and physiological set up of oxidative
phosphorylation efficiency. The stoichiometric efficiency of oxidative phosphorylation is
defined by the phosphorylation, or the amount of inorganic phosphate (Pi) incorporated into
adenosine-5-triphosphate per amount of consumed oxygen.
3. Regulation of Oxidative Phosphorylation by Mitochondrial Calcium:
Stimulation of mitochondrial oxidative metabolism by Ca 2+ is now generally recognised as
important for the control of cellular ATP homeostasis. Here, we review the mechanisms
through which Ca2+ regulates mitochondrial ATP synthesis.
Calcium is believed to regulate mitochondrial oxidative phosphorylation, thereby
contributing to the maintenance of cellular energy homeostasis. Skeletal muscle, with an
energy conversion dynamic range of up to 100-fold, is an extreme case for evaluating the
cellular balance of ATP production and consumption. This study examined the role of Ca 2+in
the entire oxidative phosphorylation reaction network in isolated skeletal muscle
mitochondria and attempted to extrapolate these results back to the muscle, in vivo.
Kinetic analysis was conducted to evaluate the dose-response effect of Ca2+ on the
maximal velocity of oxidative phosphorylation [V (maxO)] and the ADP affinity. Force-flow
analysis evaluated the interplay between energetic driving forces and flux to determine the
conductance, or effective activity, of individual steps within oxidative phosphorylation.
Force-flow analysis revealed that Ca2+ activation of [V (maxO)] was distributed throughout
the oxidative phosphorylation reaction sequence. Specifically, Ca2+ increased the conductance
of Complex IV (2.3-fold), Complexes I and III (2.2-fold), ATP production/transport (2.4fold), and fuel transport/dehydrogenases (1.7-fold). These data support the notion that Ca 2+
activates the entire muscle oxidative phosphorylation cascade, while extrapolation of these
data to the exercising muscle predicts a significant role of Ca2+ in maintaining cellular energy
homeostasis.
4. Oxidative Phosphorylation Is Regulated by Cellular Energy Needs:
Oxidative phosphorylation is regulated by cellular energy demands. The intracellular [ADP]
and the mass-action ratio [ATP]/ ([ADP][Pi]) are measures of a cells energy status. The rate
of respiration (O2 consumption) in mitochondria is under tight regulation; it is generally
limited by the availability of ADP as a substrate for phosphorylation. As we saw in Figure 1813b, the respiration rate in isolated mitochondria is low in the absence of ADP and increases
strikingly with the addition of ADP; this phenomenon is part of the definition of coupling of
oxidation and phosphorylation. The intracellular concentration of ADP is one measure of the
energy status of cells.
Another, related measure is the mass-action ratio of the ATP-ADP system: [ATP]/
([ADP] [Pi]). Normally this ratio is very high, so that the ATP-ADP system is almost fully
phosphorylated. When the rate of some energy-requiring process in cells (protein synthesis,
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for example) increases, there is an increased rate of breakdown of ATP to ADP and Pi,
lowering the mass-action ratio. With more ADP available for oxidative phosphorylation, the
rate of respiration increases, causing regeneration of ATP. This continues until the mass action
ratio returns to its normal high level, at which point respiration slows again. The rate of
oxidation of cell fuels is regulated with such sensitivity and precision that the ratio [ATP]/
([ADP][Pi] fluctuates only slightly in most tissues, even during extreme variations in energy
demand. In short, ATP is formed only as fast as it is used in energy requiring cell activities.
5. An Inhibitory Protein Prevents ATP Hydrolysis during Ischemia:
In ischemic (oxygen-deprived) cells, a protein inhibitor blocks ATP hydrolysis by the ATP
Synthase operating in reverse, preventing a drastic drop in [ATP]. We have already
encountered ATP synthase as an ATP driven proton pump. As in a heart attack or stroke,
electron transfer to oxygen ceases, and so does the pumping of), catalysing the reverse of ATP
synthesis. When a cell is ischemic (deprived of oxygen), protons the proton-motive force
soon collapses. Under these conditions, the ATP synthase could operate in reverse,
hydrolysing ATP to pump protons outward and causing a disastrous drop in ATP levels. This
is prevented by a small (84 amino acids) protein inhibitor, IF1, which simultaneously binds to
two ATP synthase molecules, inhibiting their ATPase activity IF1 is inhibitory only in its
dimeric form, which is favoured at pH lower than 6.5.
In a cell starved for oxygen, the main source of ATP becomes glycolysis, and the pyruvic
or lactic acid thus formed lowers the pH in the cytosol and the mitochondrial matrix. This
favours IF1 dimerization, leading to inhibition of the ATPase activity of ATP synthase,
thereby preventing wasteful hydrolysis of ATP. When aerobic metabolism resumes,
production of pyruvic acid slows, the pH of the cytosol rises, the IF1 dimer is destabilized,
and the inhibition of ATP synthase is lifted.
6. Uncoupled Mitochondria in Brown Fat Produce Heat:
In brown fat, which is specialized for the production of metabolic heat, electron transfer is
uncoupled from ATP synthesis and the energy of fatty acid oxidation is dissipated as heat.
There is a remarkable and instructive exception to the general rule that respiration slows
when the ATP supply is adequate. Most new born mammals, including humans, have a type
of adipose tissue called brown fat in which fuel oxidation serves not to produce ATP but to
generate heat to keep the new-born warm. This specialized adipose tissue is brown because of
the presence of large numbers of mitochondria and thus large amounts of cytochromes,
whose heme groups are strong absorbers of visible light.
The mitochondria of brown fat are like those of other mammalian cells in all respects, except
that they have a unique protein in their inner membrane. Thermogenin, also called the
uncoupling protein (Table 194), Provides a path for protons to return to the matrix without
passing through the FoF1 complex As a result of this short-circuiting of protons, the energy
of oxidation is not conserved by ATP formation but is dissipated as heat, which contributes to
maintaining the body temperature of the new born. Hibernating animals also depend on
uncoupled mitochondria of brown fat to generate heat during their long dormancy
7. ATP-Producing Pathways Are Co-ordinately Regulated:
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ATP and ADP concentrations set the rate of electron transfer through the respiratory chain via
a series of interlocking controls on respiration, glycolysis, and the citric acid cycle.
The relative concentrations of ATP and ADP control not only the rates of electron transfer and
oxidative phosphorylation but also the rates of the citric acid cycle, pyruvate oxidation, and
glycolysis. Whenever ATP consumption increases, the rate of electron transfer and oxidative
phosphorylation increases. Simultaneously, the rate of pyruvate oxidation via the citric acid
cycle increases, increasing the flow of electrons into the respiratory chain. These events can
in turn evoke an increase in the rate of glycolysis, increasing the rate of pyruvate formation.
When conversion of ADP to ATP lowers the ADP concentration, acceptor control slows
electron transfer and thus oxidative phosphorylation.
Glycolysis and the citric acid cycle are also slowed, because ATP is an allosteric inhibitor of
the glycolytic enzyme phosphofructokinase-1 and of pyruvate dehydrogenase.
CONCLUSION:
Regulation of cellular bioenergetics is crucial in processes of neuroplasticity. Oxidative
phosphorylation is the most important source of adenosine-5- triphosphate; its efficacy is
determined by different mechanisms. Primary, the supply of substrates implemented by Ca2+
levels, reversible phosphorylation, allosteric inhibition of oxidative phosphorylation
subunits, fatty acids and uncoupling protein, and influences of hormones. The system of
oxidative phosphorylation does not respond to thermodynamic equilibrium, but embodies a
rate of uncoupling. Lower membrane potential (m) can result in hydrolysis of cytoplasmic
adenosine-5-triphosphate; high membrane potential (m) leads to proton leak and
increased uncoupling. Measurement of both respiration and membrane potential during action
of appropriate endogenous and exogenous substances enables the identification of the
primary sites of effectors and the distribution of control, allowing deeper quantitative
analyses. Better insight into molecular mechanisms of cellular respiration, control of
oxidative phosphorylation and its roles in neuroplasticity likely better understand function,
physiology as well as pathophysiology of various diseases.
Current Research:
Recent experimental results indicate that oxidative phosphorylation in mitochondria is not
only regulated by respiratory control, i.e., inhibition of respiration at low ATP utilization
via the electrochemical proton gradient across the inner mitochondrial membrane, but in
addition by reversible phosphorylation of respiratory chain complexes and of ATP synthase.
Thus the formation of ATP and the generation of heat by mitochondria is also controlled by
second messenger-mediated signal transduction mechanisms. The second messengers include
cAMP, calcium, and ROS leading to activation of mitochondrial protein kinases and
phosphatases. Some protein kinases (e.g., PKB = Akt, PKC) have been demonstrated to be
translocated into mitochondria after activation (phosphorylation) outside of mitochondria.
Subunit phosphorylation has been described for complexes I (NADH dehydrogenase), II
(succinate dehydrogenase), III (cytochrome c reductase), IV (cytochrome c oxidase) and V
(ATP synthase). Of particular interest is the phosphorylation of complex IV leading to an
allosteric ATP-inhibition of cytochrome c oxidase, representing a second mechanism of
respiratory control.
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BIBLIOGRAPHY:
BOOKS:
WEBSITES:
http://www.sciencedirect.com/science/article/pii/S0167488907002364
http://www.biologyreference.com/Oc-Ph/Oxidative-Phosphorylation.html
http://www.sparknotes.com/biology/cellrespiration/oxidativephosphorylation/section3.rh
ml
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/ATPsynthase.html
https://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/redox.htm
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