Mitochondria

Mitochondria, have two membranes :

• The outer mitochondrial membrane is readily permeable to small

molecules and ions, which move freely through transmembrane channels
formed by a family of integral membrane proteins called porins.
• The inner membrane is impermeable to most small molecules and ions,
including protons ; the only species that cross this membrane do so
through specific transporters. The inner membrane bears the
components of the respiratory chain and the ATP synthase.

The mitochondrial matrix, enclosed by the inner membrane, contains

the pyruvate dehydrogenase complex and the enzymes of the citric acid
cycle, the fatty acid β-oxidation pathway, and the pathways of amino
acid oxidation—all the pathways of fuel oxidation except glycolysis,
which takes place in the cytosol.

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Oxidative Phosphorylation/Electron Transport Chain
in Mitochondria

• Oxidative phosphorylation is the process by which NADH and FADH2 are

oxidized and ATP is formed (Reduced coenzymes from the oxidation of
glucose by glycolysis, fatty acid oxidation and the citric acid cycle).

➢ The Respiratory Electron-transport Chain (ETC) is a series of enzyme

complexes embedded in the inner mitochondrial membrane, which oxidize
NADH and QH2. Oxidation energy is used to transport protons across
the inner mitochondrial membrane, creating a proton gradient
➢ Proton translocation and the development of a transmembrane proton
gradient provides the required coupling mechanism.

ATP synthase are integral protein complexes of the inner mitochondrial

membrane, is an enzyme that uses the proton gradient energy to
produce ATP by Oxidation of NADH with phosphorylation of ADP.
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 Oxidative Phosphorylation / Electron
Transport Chain
“Oxidative Phosphorylation”
➢ Making ATP by moving electrons from a higher energy
position (stored in NADH or FADH2) to a lower energy
position (the electrons of water).

➢ As electrons move from higher potential to lower,

energy is converted to a chemical/electrical gradient
(protons are pumped across a membrane).

➢ Gradient relaxes at sites (ATP synthase) where a

channel for protons drives a rotor which churns out
ATP molecules
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 Oxidative Phosphorylation / Electron Transport Chain

➢ For the oxidation of NADH, the standard biological reduction

potential is –52.6 kcal/mole. With a free energy change of –52.6
kcal/mole, it is clear that NADH oxidation has the potential for
driving the synthesis of a number of ATPs since the standard free
energy for the reaction below is +7.3kcal/mole:

ADP + Pi ——> ATP

➢ Note that: Classically, the description of ATP synthesis through
oxidation of reduced electron carriers indicated 3 moles of ATP
could be generated for every mole of NADH and 2 moles for
every mole of FADH2. However, direct chemical analysis has
shown that for every 2 electrons transferred from NADH to
oxygen, 2.5 equivalents of ATP are synthesized and 1.5 for
FADH2.

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 Diagrammatic representation of the electron transport chain (ETC)

FMN and iron-

sulfur (Fe-S)
proteins

FAD and Fe-S

proteins

cytochrome b,
cytochrome c1 Fe-S
protein and
cytochrome c

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 Electron Flow in Oxidative Phosphorylation
• Five oligomeric assemblies of proteins associated with
oxidative phosphorylation are found in the inner
mitochondrial membrane
• Complexes I-IV contain multiple cofactors, and are involved
in electron transport
• Electrons flow through complexes I-IV
• Complexes I, III and IV pump protons across the inner
mitochondrial membrane as electrons are transferred
• Complex IV reduces O2 to water
• Complex V is ATP synthase, which uses the generated proton
gradient across the membrane to make ATP
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As the two electrons pass through the proteins of complex I,
four protons (H+) are pumped into the intramembrane space
of the mitochondrion.
Similarly, four protons are pumped into the intramembrane
space as each electron pair flows through complexes III and
as four electrons are used to reduce O2 to H2O or 2
electrons for one oxygen atom, in complex IV.
The free energy released as electrons flow through complex
II is insufficient to be coupled to proton pumping. These
protons are returned to the matrix of the mitochondrion,
down their concentration gradient, by passing through ATP
synthase coupling electron flow and proton pumping to ATP
synthesis.

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 Electron Carriers
In addition to NAD (Nicotinamide nucleotides:Reduced pyridine nucleotides)
and flavoproteins, three other types of electron-carrying molecules function in
the respiratory chain:

• Ubiquinone or coenzyme Q, or simply Q, is a hydrophobic quinone a lipid-

soluble benzoquinone with a long isoprenoid side chain. Q is a lipid soluble
molecule that diffuses within the lipid bilayer, accepting electrons from
Complex I and Complex II and passing them to Complex III

• Two different types of iron-containing proteins (cytochromes and iron-sulfur

proteins).

The cytochromes of type a and b and some of type c are integral proteins of
the inner mitochondrial membrane. One striking exception is the cytochrome c
of mitochondria, a soluble protein that associates through electrostatic
interactions with the outer surface of the inner membrane.

Associated with the outer face of the inner mitochondrial membrane.

Transports electrons from Complex III to Complex IV
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 Overview of oxidative phosphorylation

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 Complex IV. Cytochrome c oxidase
• Uses four-electrons from the soluble electron carrier
cytochrome c to reduce oxygen (O2) to water (H2O)
• Uses Iron atoms (hemes of cytochrome a) and copper atoms
• Pumps two protons (H+) across the inner mitochondrial
membrane per pair of electrons, or four H+ for each O2
reduced

O2 + 4 e- + 4H+ 2 H2O

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 Cytosolic NADH

NADH that are produced by glycolysis Pass electrons to

shuttles:

• Glycerol-phosphate shuttle:
Transfers electrons from cytosolic NADH to FAD to produce FADH2

• Malate Aspartate shuttle:

Transfers electrons from cytosolic NADH to NAD+ to produce NADH

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Glycerol phosphate- shuttle Muscles and brain

Note where it enters: Q to complex III

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Cytosolic NADH 2/17/2021
Malate- Aspartate shuttle Live , kidney, heart

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 Mitochondria Complexes May Associate
in Respirasomes
Kinetic evidence showed that electron transfer through solid
state and thus resiprasome model.

There is growing experimental evidence that in the intact

mitochondrion, the respiratory complexes tightly associate
with each other in the inner membrane to form
respirasomes, functional combinations of two or more
electron-transfer complexes. Such as complex I and complex
III association.

Cardiolipin, the Lipid that is especially abundant in the inner

mitochondrial membrane, may be critical to the integrity of
respirasomes; its removal with detergents,or its absence in
certain yeast mutants, results in defective mitochondrial
electron transfer and a loss of affinity between the
respiratory
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complexes. 2/17/2021
 Oxidative Phosphorylation
➢ The free energy available as a consequence of
transferring 2 electrons from NADH or succinate to
molecular oxygen is –57 and –36 kcal/mol,
respectively.
➢ Oxidative phosphorylation traps this energy as the
high-energy phosphate of ATP.
➢ In order for oxidative phosphorylation to proceed,
two principal conditions must be met:
▪ First, the inner mitochondrial membrane must be
physically intact so that protons can only reenter
the mitochondrion by a process coupled to ATP
synthesis.
▪ Second, a proton gradient must be developed
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 Mechanism of ATP Synthase
• There are 3 active sites, one in each b subunit
• Passage of protons through the Fo channel causes the c-e-g
unit to rotate inside the a3b3 hexamer, opening and closing
the b-subunits, which make ATP
In damaged
mitochondria,
permeable to
protons, the ATP
synthase
reaction is active
in the reverse
direction acting
as a very
efficient ATP
hydrolase or
ATPase
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Transport of ATP, ADP and Pi across the
inner mitochondrial membrane
Adenine nucleotide translocase: unidirectional •
exchange of ATP for ADP (antiport)
Symport of Pi and H+ is electroneutral •

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 Adenine nucleotide and phosphate translocases

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 The P:O Ratio

molecules of ADP phosphorylated

P:O
-- ratio = ---------------------------------------
atoms of oxygen reduced
• Translocation of 3H+ required by ATP synthase
for each ATP produced
• 1 H+ needed for transport of Pi, ADP and ATP
• Net: 4 H+ transported for each ATP
synthesized
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 Calculation of the P:O ratio

Complex I III IV
#H+ translocated/2e- 4 4 2
Since 4 H+ are required for each ATP
synthesized:
For NADH: 10 H+ translocated / O (2e-)
P/O = (10 H+/ 4 H+) = 2.5 ATP/O
For succinate (FADH2) substrate = 6 H+/ O
(2e-)

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P/O = (6 H+/ 4 H+) = 1.5 ATP/O 2/17/2021
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 Sites of superoxide formation in the
respiratory chain Various respiratory complexes leak
electrons to oxygen producing
primarily superoxide anion.

This species may reduce

cytochrome c, or may be converted
to hydrogen peroxide (H2O2 and
oxygen (in both the matrix and the
intermembrane space).

Increased steady state

concentrations of O •−2may reduce
transition metals (which in turn
react with H2O 2producing
hydroxyl radicals (OH•)) or may
react with nitric oxide to form
peroxynitrite. Both OH• and
peroxynitrite are strong oxidants
which indiscriminately react with
nucleic acids lipids and proteins.
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 Inhibitors of Oxidative Phosphorylation

Name Function Site of Action

Rotenone e– transport inhibitor Complex I

Amytal e– transport inhibitor Complex I

Antimycin A e– transport inhibitor Complex III

Cyanide e– transport inhibitor Complex IV

Carbon Monoxide e– transport inhibitor Complex IV

Azide e– transport inhibitor Complex IV

2,4,-dinitrophenol Uncoupling agent transmembrane H+ carrier

Pentachlorophenol Uncoupling agent transmembrane H+ carrier

OSCP fraction of ATP
Oligomycin Inhibits ATP synthase
synthase

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 Coupling of electron transfer and ATP
synthesis in mitochondria.

In experiments to demonstrate coupling,

mitochondria are
suspended in a buffered medium and an
O2 electrode monitors O2
consumption. At intervals, samples are
removed and assayed for the
presence of ATP.

(a) Addition of ADP and Pi alone results

in little or no
increase in either respiration (O2
consumption; black) or ATP synthesis
(red). When succinate is added,
respiration begins immediately and
ATP is synthesized. Addition of cyanide
(CN), which blocks electron
transfer between cytochrome oxidase
(Complex IV) and O2, inhibits both
respiration and ATP synthesis

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 Regulation of Oxidative Phosphorylation
• The rate of respiration (O consumption)
2

in mitochondria is tightly regulated; it is

generally limited by the substrate for
phosphorylation and cellular energy
demand .
• Important substrates: NADH, O2, ADP

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 Electron Transport Chain
Four Complexes
Complex I : catalyze electron transfer from
NADH to ubiquinone
Complex II catalyze electron transfer from
succinate to ubiquinone
Complex III carries electrons from reduced
ubiquinone (QH2) to cytochrome c.
Complex IV completes the sequence by
transferring electrons from cytochrome c to
oxygen.
– Electrons ultimately reduce oxygen to water
• 2 H+ + 2 e- + ½ O2 --→ H2O
 Proton-motive force

The inner mitochondrial

membrane
separates two compartments of
different [H], resulting in
differences in chemical
concentration and charge
distribution across the
membrane. The net effect is the
proton-motive force,
Which can be calculated as shown
here.

The energy of the proton gradient is known as the chemiosmotic potential =

proton motive force PMF

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 Mitochondrial Diseases
Basis of the Disease (invisible)
• Mitochondrial diseases result from failures of the mitochondria, present in
every cell of the body except red blood cells.
• Difficult diagnosis.

• Mitochondria are responsible for creating more than 90% of the energy needed
by the body to sustain life and support growth.

• When they fail, less and less energy is generated within the cell. Cell injury and
even cell death follow. If this process is repeated throughout the body, whole
systems begin to fail, and the life of the person in whom this is happening is
severely compromised.

• The disease primarily affects children, but adult onset is becoming more and
more common.

• Diseases of the mitochondria appear to cause the most damage to cells of the
brain, heart, liver, skeletal muscles, kidney and the endocrine and respiratory
systems.

• Depending on which cells are affected, symptoms may include:

loss of motor control, muscle weakness and pain, gastro-intestinal disorders and
swallowing difficulties, poor growth, cardiac disease, liver disease, diabetes,
respiratory complications, seizures, visual/hearing problems, lactic acidosis,
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 • Mitochondrial Diseases
like toy trying to function on weak
battery that …run out

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 Complex I Deficiency or NADH-Coenzyme Q
Reductase Deficiency
Complex I, the first step in ETC, is the most common site
for mitochondrial abnormalities, representing as much as
one third of the respiratory chain deficiencies. Often
presenting at birth or in early childhood, Complex I
deficiency is usually a progressive neuro-degenerative
disorder and is responsible for a variety of clinical
symptoms, particularly in organs and tissues that require
high energy levels, such as brain, heart, liver, and skeletal
muscles.
A number of specific mitochondrial disorders have been
associated with Complex I deficiency including:
Leber’s hereditary optic neuropathy (LHON) and
Mitochondrial encephalomyopathy, lactic acidosis,
and stroke-like episodes (MELAS).

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 Mitochondrial cocktail
• Combination of vitamins
• Antioxidants
• Cofactors
• Supplements

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