Oxidative Phosphorylation V

Inhibitors and Uncouplers

Inhibitors of electron transport chain
❖ Inhibitors have been very useful in determining complete
sequence of electron transport chain and understanding the
mechanism of oxidative phosphorylation.
Inhibitors of Complexes I, II, and III Block Electron
Transport
❖ Rotenone is a common insecticide that strongly inhibits the
NADH–UQ reductase. Rotenone is obtained from the roots of
several species of plants.
❖ Amytal, and other barbiturates, Piericidin A (an antibiotics)
and the widely prescribed painkiller Demerol inhibit Complex I
by inhibiting reduction of coenzyme Q and the oxidation of the
Fe-S clusters of NADH–UQ reductase.
❖ Antimycin A, inhibit complex III by blocking electron flow from
heme bH to Q, binds at QN, close to heme bH on the N (matrix)
side of the membrane.
Inhibitors of Complex I of Electron Transport Chain

❖ Myxothiazol inhibit complex III by preventing electron flow

from QH2 to the Rieske iron-sulfur protein, binds at QP, near
the 2Fe-2S center and heme bL on the P side.
❖ Cyanide, Azide, and Carbon Monoxide Inhibit Complex IV
✓ Complex IV (Cytochrome c oxidase), is specifically inhibited by
cyanide, azide, and carbon monoxide.
✓ Cyanide and azide bind tightly to the ferric form of cytochrome a3.
The inhibitory actions of cyanide and azide at this site are very potent
✓ Carbon monoxide binds only to the ferrous form. The principal
toxicity of carbon monoxide arises from its affinity for the iron of
hemoglobin. This is an important distinction between the poisonous
effects of cyanide and carbon monoxide.
✓ Because animals (including humans) carry many, many hemoglobin
molecules, they must inhale a large quantity of carbon monoxide to
die from it.
✓ These same organisms, however, possess comparatively few
molecules of cytochrome a3.
✓ Consequently, a limited exposure to cyanide can be lethal. The
sudden action of cyanide attests to the organism’s constant and
immediate need for the energy supplied by electron transport
Inhibitors of ATP synthase
❖ Inhibitors of ATP synthase include oligomycin, aurovertin,
venturicidin and dicyclohexylcarbodiimide (DCCD).
❖ Oligomycin is a polyketide antibiotic that acts directly on ATP
synthase by binding to the OSCP subunit of Fo. Oligomycin
also blocks the movement of protons through Fo.
❖ Aurovertin prevents ATP synthesis by binding to F1 of ATP
synthase.
❖ Venturicidin is highly toxic agent inhibit ATP synthase by
interacting with the Fo of the enzyme and blocking proton
passage through the FoF1 complex.
❖ Dicyclohexylcarbodiimide (DCCD) a toxic agent that block
the proton flow through Fo. a
Inhibitors of ATP synthase
❖ Oligomycin or Venturicidin are toxic antibiotics that bind to the
Fo component of ATP synthase in mitochondria
Uncouplers
❖ Agents that disrupt the tight coupling between electron transport
and the ATP synthase are called Uncouplers.
❖ Uncouplers affect ATP synthesis in a way that does not involve
direct binding to any of the proteins of the electron-transport
chain or the F1Fo–ATPase.
❖ Uncouplers act by dissipating the proton gradient across the inner
mitochondrial membrane created by the electron-transport
system.
❖ Examples are 2,4-dinitrophenol (DNP), dicumarol, and
carbonyl cyanide-p-trifluoro-methoxyphenyl hydrazone (also
known as fluorocarbonyl cyanide phenylhydrazone, or FCCP).
❖ Uncouplers are small and hydrophobic and can bind protons
reversibly.
Uncouplers
❖ They bind proton on cytosolic side and carry them to matrix side
thereby destroying the proton gradient that couples electron transport
and the ATP synthase.
❖ In mitochondria treated with uncouplers, electron transport continues
and protons are driven out through the inner membrane. But they leak
back in so rapidly via the uncouplers that ATP synthesis does not occur
and the energy released in electron transport is dissipated as heat.
❖ Valinomycin, is an antibiotic belongs to the ionophores group of
uncouplers. Ionophores bind to the ions and move them across
the membrane disturbing the electrochemical gradient.
❖ Valinomycin binds to K+ ions and allow it to pass easily through
membranes according to their electrochemical gradient.
❖ Valinomycin uncouples electron transfer from oxidative
phosphorylation by dissipating the electrical contribution (move
positive charges to the matrix) the electrochemical gradient
across the mitochondrial membrane.
Natural Uncouplers facilitate organisms to generate
heat
❖ Animals such as cold-adapted/ hibernating / newborn animals produce
large amounts of heat by uncoupling oxidative phosphorylation.
❖ These organisms possessed brown adipose tissue (brown due to large
number of mitochondria).
❖ The inner membrane of brown adipose tissue mitochondria contains
large number of an endogenous protein called thermogenin (literally,
“heatmaker”) or uncoupling protein 1 (UCP1).
❖ UCP1 creates a passive proton channel through which protons flow
from the cytosol to the matrix.
❖ Mice that lack UCP1 cannot maintain their body temperature in cold
conditions, whereas normal animals produce larger amounts of UCP1
when they are cold-adapted.
❖ Two other mitochondrial proteins, designated UCP2 and UCP3, have
similar sequence as that of UCP1 however their functions as
uncouples are clear.
Natural Uncouplers facilitate organisms to generate heat
❖ Certain plants also use the heat of uncoupled proton transport for a
special purpose.
❖ Some plants, (family Araceae) such as philodendrons, arum
lilies, and skunk cabbages contain floral spikes that are
maintained 20-40°C above ambient temperature.
❖ The warmth of the spikes serves to vaporize odiferous molecules,
which attract insects that fertilize the flowers.
❖ These plants have an alternative respiratory pathway in addition to
normal electron transport chain as that in animals. .
❖ A cyanide-resistant QH2 oxidase transfers electrons from the QH2
pool directly to oxygen, bypassing the two proton-translocating
steps of Complexes III and IV directly leading to dissipating the
energy as heat.
❖ Plant mitochondria also have an alternative NADH
dehydrogenase, resistant to the rotenone that transfers electrons
from NADH in the matrix to Q, bypassing Complex I.
❖ Plant mitochondria also have another NADH dehydrogenase, on
the external face of the inner membrane, that transfers electrons
from NADPH or NADH in the intermembrane space to
ubiquinone, again bypassing Complex I.
❖ Therefore, when electrons enter the alternative respiratory pathway
through the rotenone-insensitive NADH dehydrogenase/ external
NADH dehydrogenase /succinate dehydrogenase and pass to O2
via the cyanide-resistant alternative oxidase, energy is not
conserved as ATP but is released as heat.
❖ A skunk cabbage can use the heat to melt snow, produce a foul
stench, or attract beetles or flies
❖ Red tomatoes have very small mitochondrial membrane proton
gradients compared with green tomatoes—evidence that
uncouplers are more active in red tomatoes
References
❖Nelson DL and Cox MM, Lehninger Principles of
Biochemistry, 5th Edition 2008, W.H.
Freeman and Company, New-York​​
❖Berg, Tymoczko and Stryer, Biochemistry 5th Edit
ion W.H. Freeman and Company, New-York​​
❖Garrett and Grisham - Biochemistry (4E) – 2010,
Brooks/Cole, Cengage Learning