Inhibitors of Respiratory Chain

• Rotenone: It blocks transfer of electrons from

NADH to ubiquinone. It is a plant product
that is extremely toxic and is used by the
American Indians as a fish poison.
• Barbiturates: Besides rotenone, some
barbiturates (e.g. amytal) inhibit electron flow
through Complex I.
• Antibiotics: An antibiotic, British antilewisite
(BAL) also acts at Complex I; and another
antibiotic, antimycin A and dimercapol,
inhibits Complex III.
• Cyanide, carbon monoxide azide, and
hydrogen sulphide: These inhibitors act at
complex IV. They bind to the iron
protoporphyrin in cytochrome aa3.
• Malonate is a competitive inhibitor of
Complex II.
• Atractyloside inhibits oxidative
phosphorylation by inhibiting the transporter
of ADP into and ATP out of the
mitochondrion
 Inhibitors of Respiratory Chain
• The antibiotic oligomycin completely
blocks oxidation and phosphorylation by
blocking the flow of protons through ATP
synthase.
• Uncouplers dissociate oxidation in the
respiratory chain from phosphorylation.
These compounds are toxic in vivo,
causing respiration to become
uncontrolled, since the rate is no longer
limited by the concentration of ADP or Pi.
• The uncoupler that has been used most
frequently is 2,4-dinitrophenol, but other
compounds act in a similar manner.
• Thermogenin (or the uncoupling protein)
is a physiological uncoupler found in
brown adipose tissue that functions to
generate body heat, particularly for the
newborn and during hibernation in
animals
 Chemiosmotic Hypothesis
• The chemiosmotic theory,
proposed by Peter Mitchell
in 1961, postulates that the
two processes are coupled by
a proton gradient across the
inner mitochondrial
membrane so that the proton
motive force caused by the
electrochemical potential
difference (negative on the
matrix side) drives the
mechanism of ATP synthesis.
• As we have seen, Complexes
I, III, and IV act as proton
pumps.
 Energy Product of Respiratory Chain
• For each mol of substrate
oxidized via Complexes I, III,
and IV in the respiratory chain
(ie, via NADH), 2.5 mol of
ATP are formed per 0.5 mol of
O2 consumed; ie, the P:O ratio
= 2.5 .
• On the other hand, when 1 mol
of substrate (eg, succinate or 3-
phophoglycerate/FADH2) is
oxidized via Complexes II, III,
and IV, only 1.5 mol of ATP
are formed; that is, P:O = 1.5.
• These reactions are known as
oxidative phosphorylation at
the respiratory chain level.
 Mitochondrial MembraneTransporters

• The inner mitochondrial

membrane is freely permeable to
uncharged small molecules, such
as oxygen, water, CO2, NH3,
and to monocarboxylic acids,
such as 3-hydroxybutyric,
acetoacetic, and acetic.
• Long-chain fatty acids are
transported into mitochondria
via the carnitine system, and
there is also a special carrier for
pyruvate involving a symport
that utilizes the H+ gradient from
outside to inside the
mitochondrion.
 Mitochondrial MembraneTransporters
• The adenine nucleotide translocase
(ANT) permits inward movement of
ADP into the mitochondrial matrix, and
a simultaneous outward movement of
ATP into the intermembranous space.
• These movements are important because
ADP (and phosphate) have to enter the
mitochondrion as substrates for
oxidative phosphorylation, and ATP has
to pass from the mitochondrion to the
cytosol, where it is used for energy-
dependent processes.
• Atractyloside is toxic glycoside which
specifically inhibits the ANT system.
• Inhibition of ANT hampers the transport
of ATP, which leads to serious
consequences since ATP is required to
drive a number of cellular activities.
 Mitochondrial MembraneTransporters

• The phosphate translocase

system is functionally related to
the adenine nucleotide
translocase. It cotransports a
phosphate ion along with H+ into
the mitochondrial
matrix.Phosphate is then used for
the generation of ATP from ADP.
• Monocarboxylate carrier:
carrier transports pyruvate
produced in cytosol, mainly
through the glycolytic sequence
into mitochondria.
 Mitochondrial MembraneTransporters

• Dicarboxylate Carrier: moves

malate from site of its production
(i.e. the mitochondrial matrix)
into cytosol.
• Tricarboxylate Carrier:
transports citrate, the first
intermediate of TCA cycle,into
cytosol.
• Combined action of the
dicarboxylate and tricarboxylate
carriers play vital role in
lipogenesis.
Mitochondrial MembraneTransporters
(in Glukoneogenesis)
Mitochondrial MembraneTransporters
(in Lipogenesis)
 Ionophores
•Ionophores are lipophilic molecules that complex
specific cations and facilitate their transport
through biologic membranes for example,
valinomycin (K+).
•This is because the translocation of potassium
ions dissipates the membrane potential, which is
an essential component of the proton-motive
force.
•The classic uncouplers such as dinitrophenol are,
in fact, proton ionophores
 Shuttle Systems
• NADH cannot penetrate the
mitochondrial membrane, but it is
produced continuously in the cytosol
by 3-phosphoglyceraldehyde
dehydrogenase, an enzyme in the
glycolysis sequence
• Shuttle systems move the NADH
produced in the cytosol into the
mitochondrion.
• Specific shuttle systems,namely:
1. Malate aspartate shuttle,
2. Glycerol phosphate shuttle
• Malate aspartate shuttle: It operates
in liver and heart muscles.
• Glycerol phosphate shuttle: In
skeletal muscle and brain
Shuttle Systems
 The Creatine phosphate Shuttle
• The creatine phosphate shuttle
augments the functions of creatine
phosphate as an energy buffer by
acting as a dynamic system for
transfer of high-energy phosphate
from mitochondria in active tissues
such as heart and skeletal muscle.
• An isoenzyme of creatine kinase
(CKm) is found in the mitochondrial
intermembrane space, catalyzing the
transfer of high-energy phosphate to
creatine from ATP emerging from the
adenine nucleotide transporter.
The Citric Acid Cycle
Loo Hariyanto Raharjo, dr., Msi.
 The Citric Acid Cycle
• Tricarboxylic acid (TCA) cycle is also called Krebs
cycle or the citric acid cycle.
• It is a cyclic pathway occurring in mitochondria,
also referred to as the “central catabolic pathway”.
• Initially, the acetyl group is incorporated into
citrate, an intermediate of the TCA cycle.
• As citrate progresses through the cycle to
oxaloacetate, it is oxidized by four dehydrogenases
(isocitrate dehydrogenase, α-ketoglutarate
dehydrogenase, succinate dehydrogenase, and
malate dehydrogenase), which transfer electrons to
NAD+ or FAD. The isomerase aconitase rearranges
electrons in citrate, thereby forming isocitrate, to
facilitate an electron transfer to NAD+.
• The overall yield of energy-containing compounds
from the TCA cycle is three NADH, one FAD(2H),
and one guanosine triphosphate (GTP)
 The Citric Acid Cycle
• The high-energy phosphate bond of GTP is
generated from substrate-level
phosphorylation catalyzed by succinate
thiokinase (succinyl CoA synthetase).
• As the NADH and FAD(2H) are reoxidized
in the electron transport chain,
approximately 2.5 ATP are generated for
each NADH and 1.5 ATP for the FAD(2H).
• Consequently, the net energy yield from the
TCA cycle and oxidative phosphorylation is
about 10 high-energy phosphate bonds for
each acetyl group oxidized.
• The reactions of TCA cycle take place in
the mitochondrial matrix
 The Citric Acid Cycle
Reaction 1: Synthesis of Citrate from Acetyl CoA and
Oxaloacetate
• Citrate is produced by condensation of acetyl
CoA with oxaloacetate in an irreverssible
reaction catalyzed by the enzyme, citrate
synthase.
• Excessive citrate crosses the inner mitochondrial
membrane through specific tricarboxylate
carriers and reaches the cytosol, where it
provides acetyl CoA:

• In cytosol, acetyl CoA serves as a precursor for

fatty acid synthesis (lipogenesis). Thus, when
citrate concentration is high, implying that the
cell is adequately supplied with fuel molecules
and, therefore, further production of energy is not
required, the energy-yielding catabolic pathways
(e.g. glycolysis and TCA cycle) are inhibited.
The biosynthetic pathway (i.e. fatty acid
synthesis) is favoured at the same time.
 The Citric Acid Cycle
Reaction 2: Isomerization of Citrate
Reaction 3: Oxidative Decarboxylation of Isocitrate
• Isomerization of citrate by the enzyme
aconitase yields isocitrate. During this
reaction, a transient enzyme bound
intermediate, cis-aconitate is formed.
• The enzyme isocitrate dehydrogenase
(IDH) catalyzes removal of two hydrogen
atoms from isocitrate (i.e. oxidation) with a
concomitant release of a CO2 molecule i.e.
decarboxylation).
• α-Ketoglutarate is the reaction product.
• NAD+serves as a coenzyme in this step, and
is converted to NADH by accepting a pair
of hydrogen atoms. NADP + can also serve
as a coenzyme in this reaction.
 The Citric Acid Cycle
Reaction 4: Oxidative Decarboxylation of α-
Ketoglutarate
• The reaction is catalyzed by the enzyme α-
Ketoglutarate dehydrogenase, to produce
succinyl CoA.
• The same set of coenzymes, i.e. thiamine
pyrophosphate, lipoic acid, FAD, NAD+,
and CoASH, is used.
• Each of these performs a function
analogous to that performed in the pyruvate
dehydrogenase complex.
• The reaction releases the second CO2
molecule of the cycle and produces the
second NADH.
• The equilibrium of the reaction lies towards
the right, i.e. towards succinyl CoA
formation.
 The Citric Acid Cycle
Reaction 5: Cleavage of Succinyl CoA

• The enzyme succinyl CoA synthetase

(also called succinate thiokinase) cleaves
the high-energy thioester bond in succinyl
CoA to release large amount of free
energy, which is used to produce a GTP
molecule.
• This reaction provides an example of
substrate level phosphorylation, since the
production of a high-energy phosphate
(e.g. GTP) is coupled with enzymatic
transformation of a substrate molecule.
GTP can produce ATP by action of the
enzyme nucleoside diphosphokinase.
 The Citric Acid Cycle
Reaction 6: Oxidation of Succinate
• A pair of reducing equivalents is removed
from succinate by the enzyme succinate
dehydrogenase to form fumarate.
• FAD serves as a coenzyme in this reaction.
Unlike the other enzymes of the TCA cycle,
which are located in the mitochondrial
matrix, succinate dehydrogenase is
anchored in the inner mitochondrial
membrane.
• It catalyzes removal of a hydrogen pair
from succinate and transfers it to
FADwhich becomes FADH2.
• This in turn transfers its electrons to
ubiquinone, which becomes ubiquinol, and
is thentransferred to complex III for
oxidation.
 The Citric Acid Cycle
Reaction 7: Hydration of Fumarate
Reaction 8: Oxidation of Malate
• Addition of a water molecule to
fumarate forms malate.
• It is a reversible reaction, catalyzed by
the enzyme fumarase (fumarate
hydratase).
• Removal of a pair of reducing
equivalents from malate by the enzyme
malate dehydrogenase produces
oxaloacetate. The reaction generates the
third NADH molecule of the cycle.
• Malate to oxaloacetate conversion is the
last reaction of the cycle.
 VITAMINS PLAY KEY ROLES
IN THE CITRIC ACID CYCLE
• Four of the B vitamins are essential in the citric
acid cycle and hence energy-yielding
metabolism:
1. Riboflavin, in the form of flavin adenine
dinucleotide (FAD), a cofactor for succinate
dehydrogenase;
2. Niacin, in the form of nicotinamide adenine
dinucleotide (NAD+), the electron acceptor
for isocitrate dehydrogenase, α-ketoglutarate
dehydrogenase, and malate dehydrogenase;
3. Thiamin (vitamin B1), as thiamin
diphosphate, the coenzyme for
decarboxylation in the α-ketoglutarate
dehydrogenase reaction; and
4. Pantothenic acid, as part of coenzyme A,
the cofactor esterified to “active” carboxylic
acid residues: acetyl-CoA and succinyl-CoA.
 REGULATION OF THE TCA
CYCLE
• The rate of the TCA cycle, like that of all fuel
oxidation pathways, is principally regulated to
correspond to the rate of the electron transport
chain, which is regulated by the ATP/ADP ratio and
the rate of ATP utilization.
• Two major messengers feed information on the rate
of ATP utilization back to the TCA cycle: (a) the
phosphorylation state of ATP, as reflected in ATP
and ADP levels, and (b) the reduction state of NAD
+as reflected in the ratio of NADH/NAD+

• Within the cell, even within the mitochondrion, the

total adenine nucleotide pool (AMP, ADP, plus
ATP) and the total NAD pool (NAD+ plus NADH)
are relatively constant.
• Thus, an increased rate of ATP utilization results in
a small decrease of ATP concentration and an
increase of ADP. Likewise, increased NADH
oxidation to NAD+ by the electron transport chain
increases the rate of pathways producing NADH.
 TCA Cycle Intermediates as Biosynthetic Precursors
• After a highcarbohydrate meal, citrate efflux
and cleavage to acetyl CoA provides acetyl
units for cytosolic fatty acid synthesis.
• During fasting, gluconeogenic precursors are
converted to malate, which leaves the
mitochondria for cytosolic gluconeogenesis.
• The liver also uses TCA cycle intermediates
to synthesize carbon skeletons of amino
acids.
• Succinyl CoA may be removed from the
TCA cycle to form heme in cells of the liver
and bone marrow.
• In the brain, α-ketoglutarate is converted to
glutamate and then to γ-aminobutyric acid
(GABA), a neurotransmitter.
• In skeletal muscle, α-ketoglutarate is
converted to glutamine, which is transported
through the blood to other tissues.
 Anaplerotic Reactions
(PYRUVATE CARBOXYLASE)
• Pyruvate carboxylase is one of the major
anaplerotic enzymes in the cell. It catalyzes the
addition of CO2 to pyruvate to form oxaloacetate.
• Pyruvate carboxylase is found in many tissues,
such as liver, brain, adipocytes, and fibroblasts,
where its function is anaplerotic.
• Its concentration is high in liver and kidney
cortex, where there is a continuous removal of
oxaloacetate and malate from the TCA cycle to
enter the gluconeogenic pathway.
• Pyruvate carboxylase is activated by acetyl CoA
and inhibited by high concentrations of many acyl
CoA derivatives. As the concentration of
oxaloacetate is depleted through the efflux of TCA
cycle intermediates, the rate of the citrate synthase
reaction decreases and acetyl CoA concentration
rises.
• The acetyl CoA then activates pyruvate
carboxylase to synthesize more oxaloacetate.
 Anaplerotic Reactions
(AMINO ACID DEGRADATION)
• Alanine and serine carbons can enter
through pyruvate carboxylase.
• In all tissues with mitochondria (except
for, surprisingly, the liver), oxidation of
the two branched-chain amino acids
isoleucine and valine to succinyl CoA
forms a major anaplerotic route.
• In the liver, other compounds forming
propionyl CoA (e.g., methionine,
threonine, and odd chain length or
branched fatty acids) also enter the TCA
cycle as succinyl CoA. In most tissues,
glutamine is taken up from the blood,
converted to glutamate, and then
oxidized to α-ketoglutarate, forming
another major anaplerotic route.