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UNIT II:
Intermediary Metabolism
Bioenergetics and Oxidative
Phosphorylation
Overview
• Bioenergetics describe transfer and utilization of energy
in biologic systems, it makes use of few basic ideas of
thermodynamics, particularly the concept of free energy
• Changes in free energy (ΔG) provide a measure of
energetic feasibility of a reaction & can allow prediction
of whether a reaction or process can take place
• Bioenergetics concerns only initial & final energy states
of reaction components, not mechanism or how much
time is needed for chemical change to take place
• Bioenergetics predicts if a process is possible, whereas
kinetics measure how fast the reaction occurs
II. Free Energy
- Direction & extent to which a chemical reaction proceeds
is determined by the degree to which 2 factors change
during reaction.
- These are enthalpy (ΔH, a measure of change in heat
content of reactants & products) and entropy (ΔS, a
measure of change in randomness or disorder of
reactants & products)
- Neither of these by itself is sufficient to determine
whether a chemical reaction will proceed spontaneously
in the direction written
- When combined math. enthalpy & entropy can be used
to define a 3rd quantity, free energy (G), which predicts
direction in which a reaction will spontaneously proceed
Figure 6.1
Relationship between changes
in free energy (G), enthalpy (H),
and entropy (S). T is the
absolute temperature in degrees
Kelvin (°K): °K = °C + 273.
III. Free energy change
• Change in free energy comes in 2 forms, ΔG, ΔGº. ΔG is
the more general as it predicts change in free energy &
thus direction of reaction at any specified conc of
reactants and products
• This contrasts with change in standard free energy, ΔGº
= energy change when reactants & products are at conc
of 1 mol/L [ conc of protons assumed 10-7 mol/L-i.e, pH =
7]
• Although ΔGº represents energy changes at these nonphysiologic conc’s of reactants & products, it is useful in
comparing energy changes of different reactions. Plus
ΔGº can be determined from measurement of
equilibrium constant
A. Sign of ΔG predicts the direction of a reaction
•
Change in free energy, ΔG, can be used to predict
direction of a reaction at constant temp & pressure
e.g., A ↔ B:
1. Negative ΔG: there is net loss of energy & reaction
goes spontaneously as written. Reaction is said to be
exergonic
2. Positive ΔG: net gain of energy & reaction does not go
spontaneously. Reaction is endergonic, energy must
be supplied to system to make reaction go
3. ΔG is zero: reactants are in equilibrium
Note: when a reaction is proceeding spontaneously i.e.,
free energy is being lost, reaction continues until ΔG
reaches zero & equil. is established
Figure 6.2. Change in free energy (∆G) during a reaction. A. The product has a
lower free energy (G) than the reactant. B. The product has a higher free energy
than the reactant.
B. ΔG of the forward and back reactions
- Free energy of forward reaction (A → B) is equal
in magnitude but opposite in sign to back
reaction (B → A)
C. ΔG depends on the concentration of reactants
and products
- ΔG of A → B depends on conc of reactant &
product. At const temp & pressure:
ΔG = ΔG º + RT ln [B]/[A]
R: gas constant (1.987 cal/mol.degree)
T: absolute temp (ºK)
[A] & [B]: actual conc of reactant & product
• A reaction with +ve ΔGº can proceed in forward direction
(have a –ve overall ΔG) if ratio of products/reactants
([B]/[A]) is sufficiently small
Figure 6.3. ∆G of a reaction depends on the concentration of reactant (A)
and product (B). For the conversion of glucose 6-P to fructose 6-P, ∆G is
negative when the ratio of reactant (A) to product (B) is large (top, panel A);
is positive under standard conditions (middle, panel B); and is zero at
equilibrium (bottom, panel C).
D. Standard free energy change, ΔGº
- ΔGº is called standard free energy change
because it is equal to free energy change, ΔG,
under standard conditions i.e., when reactants &
products are kept at 1 mol/L. under these
conditions ln [B]/[A] = 0, and ΔG = ΔGº + 0
1. ΔGº is predictive only under standard
conditions: because ΔGº = ΔG. However, ΔGº
can not predict direction of a reaction under
physiologic conditions, because it is composed
solely of constants (R, T, Keq) & is not altered by
changes in product or substrate conc’s
• Relationship between ΔGº and Keq:
- In A → B, equilibrium is reached when no net
chemical change takes place i.e., when A
converted to B as fast as B is to A. In this state
ratio of [B] to [A] is const. regardless of actual
conc’s of the 2 cpds
Keq = [B]eq/ [A]eq
- If the reaction A ↔ B is allowed to go to equil. at
const. temp. & pressure, then at equil. The overall
free energy change (ΔG) is zero. Therefore,
ΔG = 0 = ΔGº + RT ln [B]eq/ [A]eq, thus,
ΔGº = - RT ln Keq
ΔGº = - RT ln Keq
- The above eq. allows some simple predictions:
- If Keq = 1, then ΔGº = 0, reaction is at equil
- If Keq > 1, then ΔGº < 0, forward reaction favored
- If Keq < 1, then ΔGº > 0, back reaction favored
3. ΔGº of two consecutive reactions are additive:
ΔGº’s are additive for any sequence of
reactions, as are the ΔG’s
• E.g.,
glucose + ATP → glucose-6-P + ADP (ΔGº = -4000 cal/mol)
glucose-6-P → fructose 6-P
(ΔGº = 400 cal/mol)
________________________________________________________
glucose 6-P + ATP → fructose 6-P + ADP
(ΔGº = -3600 cal/mol)
4. ΔGs of a pathway are additive:
- This property is very important in biochemical
pathways through which substrates must pass in
a particular direction
- As long as sum of ΔGs of individual reactions is
negative, pathway can potentially proceed, even
if some of component reactions of the pathway
have a +ve ΔG. Actual rate of reactions does
depend on activity of enz’s that catalyze the
reactions
IV. ATP as an energy carrier
• Reactions/processes that have a large +ve ΔG,
e.g., moving ions against conc gradient across
CM, are made possible by coupling endergonic
movement of ions with a 2nd spontaneous
process with a large –ve ΔG, e.g., hydrolysis of
ATP
• The simplest example of energy coupling in
biologic reactions occurs when energy-requiring
& energy-yielding reactions share a common
intermediate
Figure 6.4. Mechanical model of coupling of
favorable and unfavorable processes.
A.
-
-
-
Reactions are coupled through common intermediates
Two reactions have a common intermediate when they
occur sequentially so that product of 1st is substrate for
the 2nd e.g.,
A+B→C+D
D+X→Y+Z
D is common intermediate & can serve as a carrier of
chemical energy b/w the 2 reactions
Many coupled reactions use ATP to generate a
common intermediate. These reactions may involve
ATP cleavage i.e., transfer of P group from ATP to
another molecule.
Other reactions lead to ATP synthesis by transfer of P
from an energy-rich intermediate to ADP, forming ATP
B. Energy carried by ATP
- Adenosine (adenine + ribose) + 3 P groups
- If one P removed, ADP is produced, if 2 P’s
removed  AMP
- ΔGº of hydrolysis of ATP ~ -7300 cal/mol for
each of the 2 terminal P’s. because of this large
–ve ΔGº, ATP is called a high-energy phosphate
cpd.
Figure 6.5. Adenosine triphosphate.
V. Electron Transport Chain
• Energy-rich molecules, e.g., glucose, metabolized by a
series of oxidation reactions ultimately  CO2 & H2O.
• Metabolic intermediates of these reactions donate e’s to
specific coenzymes NAD+ & FAD, to form energy-rich
reduced coenzymes, NADH & FADH2.
• Reduced coenzymes can donate a pair of e’s each to
specialized set of e-carriers, collectively called electron
transport chain (ETC)
• As e’s pass down ETC they lose much of their free
energy. Part of which can be captured and stored by
production of ATP from ADP & Pi. This process is =
oxidative phosphorylation. Remainder of free energy
is released as heat.
Figure 6.6. The metabolic
breakdown of energyyielding molecules.
A. Mitochondrion
- The ETC is present in inner mitochondrial membrane & is
the final common pathway by which electrons derived
from different fuels of the body flow to O2
Electron transport & ATP synthesis by oxphos proceed
continuously in all tissues that contain mitochondria
1. Structure of mitochondria
Components of ETC are located in IM. Although OM
contains special pores, making it freely permeable to
most ions & small molecules, IM is a specialized
structure that is impermeable to most small ions,
including H+, Na+, & K+, small molecules such as ATP,
ADP, pyruvate, and other metabolites important to
mitochondrial function
Figure 6.7
Structure of a mitochondrion
showing schematic representation
of the electron transport chain and
ATP synthesizing structures on the
inner membrane. mtDNA = mitochondrial
DNA; mtRNA =
mitochondrial RNA.
• Specialized carriers of transport systems are
required to move ions or molecules across this
memb.
• The mitoch. IM is usually rich in protein, half of
which is directly involved in e transport & oxphos
• The IM is highly convoluted. Convolutions called
“cristae”, greatly increase surface area of memb.
2. ATP synthase complexes
- These complexes of proteins are referred to inner
membrane particles and are attached to inner
surface of the mitoch. IM. They appear as
spheres that protrude into mitoch. matrix
3. Matrix of the mitochondrion
- gel-like solution in the interior of mitoch. Is 50%
protein. These molecules include E’s
responsible for oxidation of pyruvate, aa’s, fatty
acids (by β-oxidation), & those of TCA cycle.
- The synthesis of urea & heme occur partially in
matrix
- In addition, matrix contains NAD+ & FAD & ADP
& Pi
- Matrix also contains mitochondrial DNA & RNA
(mtDNA, mtRNA) & mitoch. ribosomes
B. Organization of the chain
- Mitoch. IM can be disrupted into 5 separate enz
complexes: I,II,III,IV, & V.
- Complexes I-IV each contains part of ETC,
whereas complex V catalyzes ATP synthesis.
- Each complex accepts or donates e’s to
relatively mobile electron carriers, such as
coenz. Q & cytochrome C
- Each carrier in ETC can receive e’s from an edonor, & can subsequently donate e’s to next
carrier in the chain
- e’s ultimately combine with O2 and H+’s  H2O.
This requirement for O2 makes the e-transport
process the respiratory chain, which accounts
for greatest portion of body’s use of O2.
Figure 6.8. Electron transport chain. [Note: Complex V is not shown.]
C. Reactions of the ETC
- With exception of coenz. Q, all members of
chain are proteins. These may act as enz’s as
is the case with dehydrogenases, they may
contain iron as part of an iron-sulfur center,
they may be coordinated with a porphyrin ring
as in cytochromes, or they may contain
copper, as does the cytochrome a + a3
complex
1. Formation of NADH:
- NAD+ is reduced to NADH by dehydrogenases
that remove 2 hydrogen atoms from their
substrate. Both e’s but only one proton (i.e., a
hydride ion, :H-) are transferred to NAD+,
forming NADH plus a free proton, H+.
2. NADH dehydrogenase:
- Free proton plus hydride ion carried by NADH
are next transferred to NADH dehydrogenase,
an enz complex (complex I) embedded in
mitoch. IM
- This complex has a tightly bound molecule of
flavin mononucleotide (FMN, a coenz structurally
related to FAD) that accepts the 2 H atoms (2e &
2 H+), becoming FMNH2.
- NADH dehydrogenase also contains several iron
atoms paired with sulfur atoms to make ironsulfur centers. These are necessary for transfer
of H atoms to next member of the chain,
ubiquinone (a.k.a coenz. Q)
Figure 6.9. Iron-sulfur center of NADH dehydrogenase.
3. Coenzyme Q:
- Co.Q is a quinone derivative with a long
isoprenoid tail. It is a.k.a ubiquinone because it
is ubiquitous in biologic systems
- Co.Q can accept H-atoms both from FMNH2,
produced by NADH dehydrogenase, & from
FADH2 (complex II), which is produced by
succinate dehydrogenase and acyl CoA
dehydrogenase.
4. Cytochromes
• Remaining members of ETC are cytochromes
• Each contain a heme group made of porphyrin
ring containing an atom of iron
• Unlike heme groups of Hb, the cytoch. Iron atom
is reversibly converted from its ferric (Fe3+) to
ferrous (Fe2+) form as a normal part of its
function as a reversible carrier of e’s.
• e’s are passed along chain from Co.Q to cytoch
b & c (complex III), & a + a3 (complex IV)
5. Cytochrom a + a3:
- This cytoch complex is the only e-carrier in which
heme iron has a free ligand that can react
directly with molecular O2.
- At this site, transported e’s, molecular O2, & free
protons are brought together to produce H2O
- Cytoch a+a3 (a.k.a cytochrome oxidase)
contains bound copper atoms that are required
for this complex reaction to occur
6. Site-specific inhibitors:
- Site-specific inhibitors of electron transport have
been identified & illustrated in Fig.10
- These cpds prevent passage of e’s by binding to
a component of the chain, blocking the redox
reaction
- Therefore, all e-carriers before the block are fully
reduced & those located after the block are
oxidized
- As electron transport & oxphos are tightly
coupled, site-specific inhibition of ETC also
inhibits ATP synthesis
Figure 6.10
Site-specific inhibitors of electron transport shown using a
mechanical model for the coupling of oxidation-reduction
reactions. [Note: Figure illustrates normal direction of electron
flow.]
C. Release of free energy during electron transport
- Free energy is released as e’s are transferred
along the ETC from e-donor (reducing agent or
reductant) to an e-acceptor (oxidizing agent or
oxidant)
- The e’s can be transferred in different forms e.g.,
as hydride ions (:H-) to NAD+, as H-atoms (.H)
to FMN, Co.Q, & FAD, or as e’s (.e-) to
cytochromes
1.
-
-
-
Redox pairs
Oxidation (loss of e’s) of one cpd is always
accompanied by reduction (gain of e’s) of a 2nd
substance. E.g., Fig 6.11
Such redox reactions can be written as sum of two
half-reactions: an isolated oxidation reaction & a
separate reduction reaction (Fig. 6.11)
NAD+ & NADH form a redox pair, as do FAD and
FADH2. Redox pairs differ in their tendency to lose e’s.
this tendency is a characteristic for a particular redox
pair, & can be quantitatively specified by a constant, E◦
(standard reduction potential), with units in volts
Figure 6.11. Oxidation of NADH by FMN, separated into
two component redox pairs.
2. Standard reduction potential (E◦):
- Standard reduction potentials of various redox
pairs can be listed to range from the most
negative E◦ to the most positive
- The more negative E◦, the greater the tendency
of the reductant member of the pair to lose e’s
- The more +ve the E◦, the greater the tendency
of the oxidant member of that pair to accept e’s
- Therefore, e’s flow from the pair with more –ve
E◦ to that with more +ve E◦
- E◦ values for some members of ETC, Fig.6.12
Figure 6.12
Standard reduction
potentials of some
reactions.
3. ΔGº is related to Δ E◦:
- The change in free energy is related directly to
the magnitude of the change in E◦ :
ΔGº = -nF ΔE◦
n = # of e’s transferred (1 for cytoch., 2 for NADH, FADH2, Co.Q)
F = Faraday constant (23,062 cal/volt.mol)
ΔE◦ = E◦ of the e-accepting pair minus E◦ of e-donating pair
ΔGº = change in the standard free energy
4. ΔGº of ATP:
- The standard free energy of hydrolysis of
terminal P group of ATP is -7300 cal/mol
- The transport of a pair of e’s from NADH to O2
via ETC produces 52,580 cal i.e., more than
sufficient energy is made available to produce 3
ATP from 3 ADP & 3 Pi (3 x 7300 = 21,900).
Remaining calories are released as heat
- Note: transport of a pair of e’s from FADH2 or
FMNH2 to O2 via ETC produces more than
sufficient energy to produce 2 ATP from 2 ADP &
2Pi
VI. Oxidative phosphorylation
- Transfer of e’s down ETC is energetically
favored as NADH is a strong e-donor & O2 is
an avid e-acceptor. But, flow of e’s from NADH
to O2 does not directly result in ATP synthesis
A. Chemiosmotic hypothesis (a.k.a Mitchell
hypothesis)
explains how free energy generated by
transport of e’s by ETC is used to produce ATP
from ADP + Pi
1. Proton pump:
- e-transport is coupled to phosphorylation of
ADP by transport of H+ across mitoch. IM from
the matrix to the intermembrane space
- This creates across IM an electrical gradient
(with more +ve outside memb. than on the
inside) & a pH gradient (outside of the memb is
at lower pH than inside).
- The energy generated by this H+ gradient is
sufficient to drive ATP synthesis.
- Thus, H+ gradient serves as the common
intermediate that couples oxidation to
phosphorylation
2. ATP synthase:
- The enzyme complex ATP synthase (complex V)
synthesizes ATP, using energy of H+ gradient
generated by ETC
- Note: the complex is also called ATPase,
because the isolated enz. also catalyzes the
hydrolysis of ATP  ADP + Pi
- The chemiosmotic hypothesis proposes that
after H+’s have been transferred to cytosolic
side of mitoch. IM, they re-enter mitoch. matrix
by passing through a channel in the ATP
synthase complex, resulting in synthesis of ATP
from ADP & Pi and, at the same time dissipating
the pH & electrical gradient
Figure 6.13. Electron transport chain shown coupled to the transport of protons.
[Note: Complex II is not shown.]
a. Oligomycin:
- This drug binds to the stalk of ATP synthase,
closing H+ channel, and preventing re-entry of
H+’s into mitoch. matrix
- Because pH & electrical gradients can not be
dissipated in presence of this drug, e-transport
stops because of difficulty of pumping any
more H+’s against the steep gradients
- e-transport & phosphorylation are, therefore,
again shown to be tightly coupled processes,
inhibition of phosphorylation inhibits oxidation
b. Uncoupling proteins (UCP):
- UCPs occur in mitoch. IM of mammals, including
humans
- These proteins create a “proton leak” i.e., they
allow H+’s to re-enter mitoch. matrix without
energy being captured as ATP. Note: energy is
released in form of heat
- UCP1, a.k.a thermogenin, is responsible for
activation of fatty acid oxidation and heat
production in the brown adipocytes of mammals
- Brown fat, unlike the more abundant white fat,
wastes almost 90% of its respiratory energy for
thermogenesis in response to cold, at birth, and
during arousal in hibernating animals
- However, humans have little brown fat
(except in newborn), & UCP1 does not
appear to play a major role in energy
balance.
- Other uncoupling proteins (UCP2, UCP3)
have been found in humans, but their
significance remains controversial
Figure 6.14. Transport of H+ across mitochondrial
membrane by 2,4-dinitrophenol.
c. Synthetic uncouplers:
- e-transport & phosphorylation can be uncoupled by cpds
that increase permeability of mitoch. IM to H+,s
- The classic e.g. is 2,4-dinitrophenol, a lipophilic H+
carrier that readily diffuses through mitoch memb.
- This uncoupler causes e-transport to proceed at a rapid
rate without establishing a H+ gradient, much as do
UCPs.
- Energy produced by transport of e’s is released as heat
rather than being used to synthesize ATP.
- In high doses, the drug aspirin (as well as other
salicylates) uncouples oxphos. This explains the fever
that accompanies toxic overdoses of these drugs
B. Membrane transport systems
Mitoch. IM is impermeable to most charged or
hydrophilic substances. However, it contains numerous
transport proteins that permit passage of specific
molecules from cytosol (more correctly, intermembrane
space) to mitoch matrix.
1. ATP-ADP transport:
Mitoch. IM requires special carriers to transport ADP &
Pi from cytosol into mitoch, where ATP can be
resynthesized
An adenine nucleotide carrier transports one molecule
of ADP from cytosol into mitoch, while exporting one
ATP from matrix back to cytosol.
This carrier is strongly inhibited by the plant toxin
atractyloside, resulting in depletion of
intramitochondrial ADP pool & cessation of ATP
production
Note: a phosphate carrier is responsible for transporting
inorganic phosphate from cytosol into mitoch
2. Transport of reducing equivalents:
- Mitoch. IM lacks NADH transport protein, & NADH
produced in cytosol cannot directly penetrate into mitoch.
- However, the 2 e’s of NADH (a.k.a reducing equivalent)
are transported from cytosol into mitoch using shuttle
mechanisms
- In glycerophosphate shuttle, 2 e’s are transferred from
NADH to flavoprotein dehydrogenase within mitoch IM.
This enz then donates its e’s to ETC in a manner similar
to that of succinate dehydrogenase. The
glycerophosphate shuttle, therefore, results in synthesis
of 2 ATPs for each cytosolic NADH oxidized
- This contrasts with malate-apartate shuttle, which
produces NADH (rather than FADH2) in mitoch matrix,
therefore  3 ATPs for each cytosolic NADH oxidized
Figure 6.15. Shuttle pathways for the transport of electrons across the inner
mitochondrial membrane. A. Glycerophosphate shuttle. B. Malate/aspartate shuttle.
C. Inherited defects in oxidative phosphorylation
- Thirteen of ~ 100 polyps required for oxphos are
coded for by mtDNA, whereas remaining mitoch
proteins are synthesized in cytosol & transported
into mitoch.
- Defects in oxphos are more likely a result of
alterations in mtDNA, which has a mutation rate
about 10x greater than that of nuclear DNA
- Tissues with greatest ATP requirement (e.g.,
CNS, skeletal & heart muscle, kidney, & liver)
are most affected by defects in oxphos.
- Mutations in mtDNA are responsible for
several diseases, including some cases of
mitoch. myopathies, & Leber’s hereditary
optic neuropathy, a disease in which
bilateral loss of central vision occurs as a
result of neuroretinal degeneration,
including damage to the optic nerve.
- mtDNA is maternally inherited because
mitoch from sperm cell do not enter the
fertilized egg.
Figure 6.16. Muscle fibers from a patient with a mitochondrial myopathy show
abnormal mitochondrial proliferation when stained for succinic dehydrogenase.
Summary
• Change in free energy ΔG predicts direction in which reaction will
spontaneously proceed
• If ΔG is –ve, reaction goes spont. If ΔG is +ve, reaction does not go
spont. If ΔG = 0, reactants are in equil.
• Change in free energy of forward & back reactions are equal but
opposite in signs.
• ΔGº are additive in any sequence of consecutive reactions. So,
reactions or processes that have a large +ve ΔG are made possible
by coupling with hydrolysis of ATP, which has a large –ve ΔGº
• The reduced coenz’s NADH & FADH2 each donate a pair of e’s to a
specialized set of e carriers, consisting of FMN, Co.Q, & a series of
cytoch’s collectively called ETC
• This pathway is present in mitoch. IM, & is the final common
pathway by which e’s derived from different fuels of the body flow to
O2
• The terminal cytoch a+a3, is the only cytoch capable of binding O2
• Electron transport is coupled to transport of protons (H+) across
mitoch IM, from matrix to intermembrane space.
• This process creates an electrical gradient & a pH gradient across
mitoch IM
• After H+’s have been transferred to cytosolic part of mitoch IM, they
can re-enter mitoch matrix by passing through a channel in ATP
synthase complex, resulting in synthesis of ATP from ADP & Pi, & at
same time dissipating the pH & electrical gradient
• e-transport & phosphorylation are thus said to be tightly coupled.
These processes can be uncoupled by uncoupling proteins found in
mitoch IM & by synthetic cpds e.g., 2,4-dinitrophenol & aspirin, all of
which increase permeability of mitoch IM to H+’s. Energy produced
by e-transport is released as heat rather than being used for ATP
synthesis
• Mutations in mtDNA are responsible for some cases of mitoch
diseases e.g., Leber’s hereditary optic neuropathy
Summary of key concepts for oxidative phosphorylation. [Note: Electron flow and ATP
synthesis are are envisioned as sets of interlocking gears to emphase the idea of