• The mitochondrial respiratory chain consists of a series
of sequentially acting electron carriers, most of which
are integral proteins with prosthetic groups capable of
accepting and donating either one or two electrons.
• Three types of electron transfers occur in oxidative
1. direct transfer of electrons, as in the reduction of
Fe3+ to Fe2+;
2. transfer as a hydrogen atom (H+ + e- ); and
3. transfer as a hydride ion (:H-), which bears two
• The term reducing equivalent is used to designate a
single electron equivalent transferred in an oxidation-
benzoquinone with a
long isoprenoid side
UBIQUINONE (Q or
Coenzyme Q) can accept
one electron to become
the semiquinone radical
('QH) or two electrons to
form UBIQUINOL (QH2)
• Iron associated with inorganic sulfur atoms of with
sulfur atoms of Cys residues in the protein, or both
• lron-sulfur centers :The Fe-S centers of iron-sulfur
proteins may be as simple as:
(a) with a single Fe ion surrounded by the S atoms of
four Cys residues.
Other centers include both inorganic and Cys S atoms as
in (b) 2Fe-2S or (c) 4Fe-4S centers
• View of the complex shows how cytochrome c1 and the
Rieske iron-sulfur protein project from the p surface and
can interact with cytochrome C in the intermembrane
• The process by which
electrons travel from QH2 to
Cytochrome C is known as Q-
• The Q cycle, shown in two
1. The path of electrons
through Complex II is shown
by blue arrows In the first
stage (left) Q, on the N side
is reduced to the
semiquinone radical which
in the second stage (right) is
converted to QH2.
2. Meanwhile on the P side of
the membrane two molecules
of QH2 are oxidized to Q,
releasing two protons per Q
molecule (four protons in all)
into the intermembrane space.
Each QH2 donates one electron
( via the Rieske Fe-S center) to
cytochrome cl, and one
electron( via cytochrome b ) to
a molecule of Q near the N
side, reducing it in two steps to
This reduction also uses two
protons per Q, which are taken
up from the matrix.
Path of electron through complex IV
• The 3 proteins critical to electron flow are subunits I, II
• Electron transfer through complex IV begins with
cytochrome C. 2 molecules of reduced cytochrome C
each donate an electron to the binuclear center CuA.
• From here electrons pass through heme a to the Fe-Cu
center (cytochrome a3 and CuB)
• Oxygen now binds to heme a3 and is reduced to its
peroxy derivative by 2 electrons from the Fe-Cu center.
• Delivery of 2 more electrons from cytochrome c
converts the O2-
2 to 2 molecules of water, with
consumption of 4 substrate protons from the matrix.
• At the same time, 4 protons are pumped from the
matrix by an as yet unknown mechanism
Shuttle systems indirectly convey cytosolic
NADH into Mitochondria for Oxidation
• The NADH dehydrogenase of the inner mitochondrial
membrane of animal cells can accept electrons only from
NADH in the matrix
• Malate-aspartate shuttle is the special shuttle system that
carry reducing equivalents from cytosolic NADH into
mitochondria by an indirect route
• Functions mainly in liver, kidney, and heart mitochondria
• The reducing equivalents of cytosolic NADH are first
transferred to cytosolic oxaloacetate to yield malate,
catalyzed by cytosolic malate dehydrogenase
• The malate thus formed passes through the inner
membrane via the malate--ketoglutarate transporter
• Within the matrix the reducing equivalents are passed
to NAD- by the action of matrix malate dehydrogenase
forming NADH; this NADH can pass electrons directly to
the respiratory chain
• About 2.5 molecules of ATP are generated as this pair of
electrons passes to 02.
• Cytosolic oxaloacetate must be regenerated by
transamination reactions and the activity of membrane
transporters to start another cycle of the shuttle.
• Skeletal muscle and brain use a different NADH shuttle,
the Glycerol 3 phosphate shuttle
• This alternative means of moving reducing
equivalents from the cytosol to the mitochondrial
matrix operates in skeletal muscle and the brain.
• In the cytosol, dihydroxyacetonephosphate accepts
two reducing equivalent from NADH in a reaction
catalyzed by cytosolic glycerol 3 –phosphate
• An isozyme of glycerol3 –phosphate
dehydrogenase bound to the outer face of the
inner membrane then transfers two reducing
equivalents from glycerol3 –phosphate in the
intermembrane space to ubiquinone.
• Note that this shuttle does not involve membrane
• Substrate shuttles for the transportof electrons across the
inner mitochondrial membrane. A. Glycerophosphate
shuttle. B. Malate-aspartate shuttle. DHAP =
dihydroxyacetone phosphate; NAD(H) = nicotinamide
adenine dinucleotide; FAD(H2) = flavin adenine
dinucleotide; CoQ = coenzyme Q. 21
• An uncoupling protein (UCP) is a mitochondrial inner
membrane protein that is a regulated proton channel
• An uncoupling protein is thus capable of dissipating the
proton gradient generated by NADH-powered pumping
of protons from the mitochondrial matrix to the
mitochondrial intermembrane space.
• The energy lost in dissipating the proton gradient via
UCPs is not used to do biochemical work. Instead, heat
• This is what links UCP to thermogenesis.
• UCPs are positioned in the same membrane as the ATP
synthase, which is also a proton channel.
• The two proteins thus work in parallel with one
generating heat and the other generating ATP from ADP
and inorganic phosphate, the last step in oxidative
• Mitochondria respiration is coupled to ATP synthesis
(ADP phosphorylation) but is regulated by UCPs.
• There are five types of homologs known in mammals:
• UCP1, also known as thermogenin
• SLC25A27, also known as "UCP4"
• SLC25A14, also known as "UCP5"
• Uncoupling proteins play a role in normal physiology, as in
cold exposure or hibernation, because the energy is used
to generate heat instead of producing ATP.
• Some plants species use the heat generated by uncoupling
proteins for special purposes.
• Skunk cabbage, for example, keeps the temperature of its
spikes as much as 20° higher than the environment,
spreading odor and attracting insects that fertilize the
• However, other substances, such as 2,4-dinitrophenol and
carbonyl cyanide m-chlorophenyl hydrazone, also serve the
same uncoupling function, and are considered poisonous
• Salicylic acid is also an uncoupling agent and will decrease
production of ATP and increase body temperature if taken
• Uncoupling proteins are increased by thyroid hormone,
norepinephrine, epinephrine, and leptin.
Inhibitors of Electron Transport:
• These are the inhibitors that arrest respiration by
combining with members of the respiratory chain,
rather than with the enzymes that may be involved
in coupling respiration with ATP synthesis.
• They appear to act at 3 loci that may be identical to
the energy transfer sites I, II and III. The given
below are the inhibitors of Electron transport
• It inhibits the transfer of electrons from iron-sulfur
centers in complex I to ubiquinone.
• This interferes with NADH during the creation of
usable cellular energy (ATP)
• Complex I is unable to pass off its electron to CoQ,
creating a back-up of electrons within the
• Cellular oxygen is reduced to the radical, creating
reactive oxygen species, which can damage DNA
and other components of the mitochondria
• It is the non-toxic inhibitors of Electron transport
• This is non-toxic to mammals because poorly
absorbed. Shows toxic effect in fishes.
• It is an Antibiotic.
• It is produced by species of streptomyces.
• The action is similar to Rotenone.
Barbiturates (Amytal, Seconal):
• It blocks NADH dehydrogenase and Coenzyme.Q
• These are antibiotic, produced by Streptomyces. One of the
inhibitor in ETC.
• It inhibits around site II and block electron flow between
cytochromes b and c1, which prevents ATP synthesis coupled
to the generation of a proton gradient at site II.
• About 0.07 micromole of antimycin A per gram of
mitochondrial protein is effective.
• It is identical in action to the antimycins.
• The cyanide ion (CN–) combines tightly with cytochrome oxidase,
leading to inhibition of ETC
• Azide blocks the electron flow between the cytochrome oxidase
complex and oxygen.
• Azide reacts with the ferric form (Fe3
+) of this carrier.
• H2S is toxic, with disagreeing odour gives warning.
• It inhibits Cytochrome Oxidase.
• It blocks between cytochrome oxidase and Oxygen.
• It inhibits Fe2
Inhibitors of Oxidative Phosphorylation:
• Is a polypeptide antibiotic are obtained from various
species of “Streptomyces”.
• The antibiotic is potent inhibitor to ATP synthase complex.
• binds to the Fo domain of ATP synthase, closing the proton
channel and preventing reentry of protons into the matrix,
there by preventing phosphorylation of ADP to ATP.
• Because the pH and electrical gradients cannot be
dissipated in the presence of this drug, electron transport
stops because of the difficulty of pumping any more
protons against the steep gradients .
• This dependency of cellular respiration on the ability to
phosphorylate ADP to ATP is known as respiratory control
and is the consequence of the tight coupling of these
• This antibiotic also inhibits both ETC and oxidative
• It backs oxidative phosphorylation by compelling with
ATP & ADP for a site on the ADP-ATP antiport of the
mitochondrial membranes. One of the inhibitors list
which blocks the oxidative phosphorylation.
• It is a toxin formed by bacteria (Pseudomonas) in a
coconut preparation from Java.
• It also blocks the ADP-ATP antiport.
Uncouplers of Oxidative
• Uncouplers can be defined as A substance that
uncouples phosphorylation of ADP from electron
• Uncoupling agents are compounds which dissociate the
synthesis of ATP from the transport of electrons
through the cytochrome system.
• This means that the electron transport continues to
function, leading to oxygen consumption but
phosphorylation of ADP is inhibited.
• A classic uncoupler of oxidative phosphorylation.
• was used as a weight-loss agent in the 1930s
• The substance carries protons across the inner mitochondria
• In the presence of these uncouplers, electron transport from
NADH to O2 proceeds normally, but ATP is not formed by the
• Body temperature is elevated as a result of hyper metabolism.
• When phosphorylation is uncoupled from electron flow, a
decrease in the proton gradient across the inner mitochondrial
membrane and, therefore , impaired ATP synthesis is expected.
• In an attempt to compensate for this defect in energy capture ,
metabolism and electron flow to oxygen is increased.
• This hyper metabolism will be accompanied by elevated body
temperature be cause the energy in fuels is largely wasted,
appearing as heat. 33
Dicoumarol (Vitamin.K analogue):
• Used as anticoagulant.
• Transport of Ca+2 ion into mitochondria can cause uncoupling.
• Mitochondrial transport of Ca+2 is energetically coupled to
• It is coupled with uptake of pi
• When calcium is transported into mitochondria, electron
transport can proceed but energy is required to pump the4 Ca+2
into the mitochondria. Hence, no energy is stored as ATP.
CCCP (Chloro carbonyl cyanide phenyl hydrazone):
• Most active uncoupler
• These lipid soluble substances can carry protons across the inner
• This is the example to Ionophore of oxidative
• Produced by a type of streptomyces
• It is a repeating macrocyclic molecule made up of four kinds
of residues (L-lactate, L-Valine, D-hydroxyisovalarate and D-
Valine) taken 3 times.
• Transports K+ from the cytosol into matrix and H+ from
matrix to cytosol, thereby decreasing the proton gradient.
• Excessive thyroxin hormone
• EFA deficiency
• Long chain FA in brown adipose tissue
• Unconjugated hyperbilirubinaemia
Uncoupling Proteins and the Molecular
Mechanisms of Thyroid Thermogenesis
• TH is synthesized in the thyroid gland and controlled by
thyroid peroxidase activity that regulates the iodination,
coupling, and ultimately proteolysis of tyrosine residues on
thyroglobulin to release the THs, T4 and T3, into the
• The lesser active T4 is released from the thyroid gland at
higher concentrations than the more active T3 and is locally
converted to T3 in target tissues by the actions of tissue-
• Two genes, THRA and THRB, are responsible for the
expression of distinct thyroid hormone receptors (TRs), each
of which are alternatively spliced to produce multiple
isoforms, TRα1, TRα2, TRβ1, and TRβ2, respectively.
• With the exception of TRα2, which does not bind T3
and functions to repress T3 actions, TR isoforms
mediate distinct functions (both stimulatory and
repressive) in response to and in the absence of T3.
• Integral to their functions as transcriptional regulators,
TRs bind other nuclear hormone receptors,
coactivators, and corepressors, the details of which
have been reviewed elsewhere .
• In addition to its transcriptional regulation, recent work
has also revealed that TH may regulate cell signaling
• However, it is not yet established whether and how TH
may influence body temperature apart from its role as
a ligand for thyroid hormone receptor-dependent gene
NADH:ubiquinone oxidoreductase (Complex l ). Complex I catalyzes the transfer of a hydride ion from NADH to FMN, from which two electrons pass through a series of Fe-S centers to the ironsulfur protein N -2 in the matrix arm of the complex. Electron transfer from N-2 to ubiquinone on the membrane arm forms QH2, which diffuses in to the lipid bilayer. This electron transfer also drives the expulsion from the matrix of four protons per pair of electrons The detailed mechanism that couples electron and proton transferin Complex I is not yet known but probably involves a Q cycle similar to that in Complex ll l in which QH2 participates twice per electron pair (see Fig.1 9-12). Proton flux produces an electrochemical potential across the inner mitochondria membrane N side negative, side positive) which conserves some of the energy released by the electron-transfer actions This electrochemical potential drives ATP synthesis.
Path of electrons from NADH,succinate fatty acyl-CoA, and glycerol3 –phosphate to ubiquinone. Electrons from NADH pass through a flavoprotein to a series of iron-sulfur proteins ( in Complex l ) and then to Q. Electrons from succinate pass through a flavoprotein and several Fe-S centers ( in Complex ll) on the way to Q. Clycerol3 – phosphate donates electrons to a flavoprotein (glycerol3 –phosphate dehydrogenase) the outer face of the inner mitochondrial membrane, from which they pass to Q. Acyl-CoA dehydrogenase(the first enzyme of B oxidation) transfers electrons to electron-transferring flavoprotein (ETF) from which they pass to Q via ETF:ubiquinone oxidoreductase
The complex has two distinct binding sites for ubiquinone Qn and Qp, which correspond do the sites of inhibition by two drugs that block oxidative phosphorylation. –Myxothiazol,which prevents electron flow fromQH2, to the Rieske iron-sulfur protein binds at Qp, near the 2Fe-2S center and heme b1 on the p side The dimeric structure is essential to the function of ComplexIII. The interface between monomers forms two caverns each containing a Qp site from one monomer and a Qn, site from the other. The ubiquinone intermediates move within these sheltered caverns.
The larger green structure includes the other 10 proteins in the complex
NADH in the cytosol passes 2 reducing equivalents to oxaloacetate, producing malate
Malate crosses the inner membrane via the malate alpha ketoglutarate transporter
In the matrix, malate passes 2 reducing equivalents to NAD+, and the resulting NADH is oxidized by the respiratory chain; the oxaloacetate formed from malate cannot pass directly into the cytosol
Oxaloacetate is first transminated to aspartate
Aspartate can leave via the glutamate-aspartate transporter
Oxaloacetate is regenerated in the cytosol, completing the cycle
This uncoupler cause electron transport to proceed at a rapid rate without establishing a proton gradient, much a s do the UCPs . Again, energy is released as heat rather than being used to synthesize ATP. [Note: In high doses, aspirin and other salicylates uncouple oxidative phosphorylation. This explains the fever that accompanies toxic overdoses of these drugs .]
responsible for heat production in the brown adipocytes of mammals. In brown fat, unlike the more abundant white fat, almost 90% of its respiratory energy is used for thermogenesis in response to cold in the neonate and during arousal in hibernating animals. However, humans appear to have few concentrated deposits of brown fat (except in the newborn), and UCP1 does not appear to play a major role in energy balance.
ATP are because the proton motive force across the inner mitochondrial membrane is dissipated
The electron transport cha in will s till be inhibited by cyanide .
Tissue specific mechanisms of TH-mediated thermogenesis. In response to cold exposure, NorEpinephrine released from SNS (sympathetic nervous system) nerve terminals binds β3-adrenergic receptors (β-AR) in BAT and WAT, increasing local and systemic FFA release along with an induction/activation of UCP1/3 and other potential thermogenic genes in BAT and skeletal muscle, respectively. Simultaneously, SNS stimulation activates D2 deiodinases in BAT and skeletal muscle that increase T3 levels, leading to the transactivation via TRs of thermogenic genes that ultimately govern the thermogenic capacities of BAT and skeletal muscle including PGC1α and mGPD. In BAT, UCP1 is activated by FFA release to transport protons from the mitochondrial intermembrane space (IMS) to the matrix, dissipating the proton gradient to produce heat. Similarly, we propose that skeletal muscle NST ( nonshivering thermogenesis) is activated in part by the uptake of FFA released from SNS-stimulated WAT lipolysis and UCP3 activation. MCT8, monocarboxylate transporter 8.