Electron Transport and Oxidative

Phosphorylation

Refer to: Lehninger Principles of Biochemistry (Chapter 19)

Dr Fawaz Awad; 2018

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 ubiquinol (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.
The Electron-transport Chain (ETC) :three defining
components
 Oxidative Phosphorylation /
Electron Transport Chain
➢ NADH oxidation: the standard biological reduction potential is –
52.6 kcal/mole (ΔG = –52.6 kcal/mole)
➢ 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 (+30.5 kJ/mol):

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.
 Electrons Are Funneled to Universal
Electron Acceptors
• Oxidative phosphorylation begins with the entry of electrons into the chain of
electron carriers called the respiratory chain.

• Most of these electrons arise from the action of dehydrogenases that

collect electrons from catabolic pathways and funnel them into universal
electron acceptors—nicotinamide nucleotides (NAD or NADP) or flavin
nucleotides (FMN or FAD).

• Nicotinamide nucleotide–linked dehydrogenases catalyze reversible

reactions of the following general types:
Electron carriers
 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.

• 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 soluble cytochrome c that associates through electrostatic
interactions with the outer surface of the inner membrane.
Mobile electron carriers

1. Ubiquinone (Q)
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.
2. Cytochrome c
Associated with the outer face of the inner
mitochondrial membrane. Transports electrons
from Complex III to Complex IV.
 Ubiquinone (Q, or coenzyme Q)

Like flavoprotein carriers, Ubiquinone can

act at the junction between a two-electron
donor and a one-electron acceptor

Ubiquinone can accept one electron to

become the semiquinone radical (•QH)

Or can accept two electrons to form

ubiquinol (QH2)
Electron Carriers Function in
Multienzyme Complexes
 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
Cofactors in electron
transport
 Complex I. NADH-ubiquinone oxidoreductase
• Transfers two electrons from NADH as a hydride ion (H:-) to
flavin mononucleotide (FMN), to iron-sulfur complexes, to
ubiquinone (Q), making QH2
• About 4 protons (H+) are translocated across the inner
mitochondrial membrane per 2 electrons transferred
 Complex II. Succinate-ubiquinone oxidoreductase
• Also known as succinate dehydrogenase complex.
• Transfers electrons from succinate to flavin adenine dinucleotide (FAD) as a
hydride ion (H:-), to an iron-sulfur complex, to ubiquinone (Q), making QH2.
• Complex II does not pump protons.

Succinate + FAD → fumarate + FADH2 No proton pump

Complex II. Succinate-ubiquinone oxidoreductase

➢ Complex II contains FAD and 7–8 Fe-S proteins in 3

clusters and cytochrome b560.
➢ Complex II is commonly referred to as succinate
dehydrogenase (SDH).
➢ In addition to transferring electrons from the
FADH2 generated by SDH, complex II also accepts
electrons from the FADH2 generated during fatty acid
oxidation via the fatty acyl-CoA dehydrogenases and
from mitochondrial glycerol-3-phosphate dehydrogenase
(GPD2) of the glycerol phosphate shuttle.
Complex III. Ubiquinol-cytochrome c oxidoreductase
• Transfers electrons from QH2 to cytochrome c, mediated by iron-
sulfur and other cytochromes
• Electron transfer from
QH2 is accompanied
by the translocation
of 4 H+ across the
inner mitochondrial
membrane
 Complex IV. Cytochrome oxidase
• As happens with the complex I and complex III, complex IV
also pumps protons from the matrix to the intermembrane
space.
• However, in this case for each 2 electrons that pass through
the complex, only 2 H+ are pumped to the intermembrane
space.
 Diagrammatic representation of the electron transport chain (ETC):
• As the two electrons pass
FMN and iron- through the proteins of complex
sulfur (Fe-S) I, four protons (H+) are pumped
proteins
into the intramembrane space of
the mitochondrion.

• Similarly, four protons are

pumped into the intramembrane
FAD and Fe-S space as each electron pair
proteins
flows through complexes III and
as four electrons are used to
cytochrome b, reduce O2 to H2O or 2 electrons
cytochrome c1 Fe-S
for one oxygen atom, in
protein and
cytochrome c 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.
 Mitochondria Complexes May Associate in
Respirasomes
• Kinetic evidence showed that electron transfer is through
solid state and thus resiprasome model.

• The respiratory complexes tightly associate with each other in

the inner membrane to form respirasomes.
• Respirasomes: functional combinations of two or more
electron-transfer complexes. E.g., 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 absence results in defective mitochondrial
electron transfer and a loss of affinity between the respiratory
complexes.
 Complex V: ATP Synthase
F0F1 ATP Synthase uses the proton gradient energy for the synthesis of ATP
composed of 3 fragments
1. F1 contains the catalytic subunits. which protrudes from the inside of the inner
membrane into the matrix
2. F0 has a proton channel which localized in membrane.
3. Oligomycin sensitivity-conferring protein (OSCP), which connects F0 to F1.

• Estimated passage of 4 protons

(H+) per ATP synthesized

➢The complex binds ADP and inorganic phosphate at its catalytic site inside the mitochondrion,
and requires a proton gradient for activity in the forward direction.
➢ 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.
 The chemiosmotic model
• How is a concentration gradient of protons transformed into ATP?
• What is the chemical mechanism that couples proton flux with phosphorylation?

The chemiosmotic model is the paradigm for this mechanism: the electrochemical
energy inherent (hidden) in the difference in proton concentration and the
separation of charge across the inner mitochondrial membrane drives the synthesis
of ATP as protons flow passively back into the matrix through a proton pore in ATP
synthase.
 Oxidative Phosphorylation
➢ Oxidative phosphorylation traps the free energy as the
high-energy phosphate of ATP.

➢ In order for oxidative phosphorylation (ATP synthesis) to

proceed, two principal conditions must be met:
1. The inner mitochondrial membrane must be physically intact so
that protons can only reenter the mitochondrion by a process
coupled to ATP synthesis.

2. A proton gradient must be developed across inner mitochondrial

membrane.

➢ The energy of the proton gradient is known as the

chemiosmotic potential, or proton motive force (PMF).
 Coupling of electron transfer and ATP synthesis in
mitochondria
• In experiments to demonstrate coupling,
mitochondria are suspended in a
buffered medium.

• Addition of ADP and Pi alone → no

increase in either respiration (O2
consumption; black) or ATP synthesis
(red).

• Addition of succinate → 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.
• More surprising is the finding that the converse is also true: inhibition of ATP
synthesis blocks electron transfer in intact mitochondria
Oxidative Phosphorylation / Electron Transport
Chain
➢ Transmembrane differences in proton concentration are the reservoir
for the energy extracted from biological oxidation reactions.

➢ The ETC is made up of a chain of electron carriers with increasing

redox potential.

➢ 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
 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
Adenine nucleotide and phosphate translocases
 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
 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-)
P/O = (6 H+/ 4 H+) = 1.5 ATP/O
Electrons shuttles
 Shuttle Systems Indirectly Convey Cytosolic NADH into
Mitochondria for Oxidation

• When the inner membrane is not permeable to NADH, how can the
NADH generated by glycolysis in the cytosol be reoxidized to NAD+
by O2 via the respiratory chain?

• Special shuttle systems carry reducing equivalents from cytosolic

NADH into mitochondria by an indirect route

NADH that are produced by glycolysis Pass electrons to shuttles:

1. Glycerol-phosphate shuttle: Transfers electrons from cytosolic NADH

to FAD to produce FADH2
2. Malate Aspartate shuttle: Transfers electrons from cytosolic NADH to
NAD+ to produce NADH
 Glycerol phosphate- shuttle Muscles and brain
Note where it enters: Q to complex III

Cytosolic NADH

This shuttle delivers the reducing equivalents from NADH to ubiquinone and thus into
Complex III, not Complex I, providing only enough energy to synthesize 1.5 ATP
molecules per pair of electrons.
Malate- Aspartate shuttle Liver, kidney, heart

About 2.5 molecules of ATP are generated as this pair of electrons passes to O2.
Reactive Oxygen Species (ROS)
Sites of superoxide formation • Various respiratory complexes leak
electrons to oxygen producing
in the respiratory chain 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•−2 may reduce
transition metals (which in turn react
with H2O2 producing 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 that can
leads to their damage.
ROS formation in mitochondria and mitochondrial defenses

• When the rate of electron entry into the respiratory chain and the rate of
electron transfer through the chain are mismatched, superoxide radical
(•O-2) production increases at Complexes I and III as the partially
reduced ubiquinone radical (•Q2) donates an electron to O2.

• Superoxide acts on aconitase, a 4Fe-4S protein, to release Fe2+.

• In the presence of Fe2+, the Fenton reaction leads to formation of the

highly reactive hydroxyl free radical (•OH).

• In actively respiring mitochondria, 0,1%-4% of the O2 used in

respiration forms oxygen radicals more than enough to have lethal
effects unless the free radical is quickly removed.
 ROS formation in mitochondria and mitochondrial
defenses
Reduced glutathione (GSH) serves to keep protein
sulfhydryl groups in their reduced state, preventing
some of the deleterious effects of oxidative stress
The reactions shown in blue defend the
cell against the damaging effects of
superoxide.
GSH donates electrons for the reduction
of H2O2 and of the oxidized Cys
residues (—S—S—) of enzymes and
other proteins, and GSH is regenerated
from the oxidized form (GSSG) by
reduction with NADPH.
ROS formation in mitochondria and mitochondrial
defenses

Generated by nicotinamide Nucleotide

Transhydrogenase in the Mitochondrion or
Pentose Shunt
 Overview of mitochondrial ROS production

The formation of ROS is favored when

two conditions are met:

1. Mitochondria are not making ATP (for

lack of ADP or O2) and therefore
have a large proton-motive force and
a high ratio of QH2/Q

2. High NADH/NAD ratio in the matrix.

➢ Factors that slow the flow of electrons through the respiratory chain increase
the formation of superoxide, perhaps by prolonging the lifetime of •O2-
generated in the Q cycle
Regulation of oxidative
phosphorlation
 Regulation of Oxidative Phosphorylation

• The rate of respiration (O consumption) in

2

mitochondria is tightly regulated; it is

generally limited by the substrate for
phosphorylation and cellular energy demand.

• Important substrates: NADH, O2, ADP

Oxidative Phosphorylation ls Regulated
by Cellular Energy Needs

1. Dependence of the rate of O2 consumption on the availability of

the Pi acceptor ADP, the acceptor control of respiration, can be
remarkable.

2. In some animal tissues, the acceptor control ratio, the ratio of the
maximal rate of ADP-induced O2 consumption to the basal rate in
the absence of ADP, is at least 10. The intracellular concentration
of ADP is one measure of the energy status of cells.

3. In eukaryotes intramitochondrial ratio of the ATP-ADP system,

ATP/([ADP].[P]) is a secondary control mechanism (mass-action
ratio).

4. Another, related measure is the High ratio inhibits oxidative

phosphorylation as ATP binds to a subunit of Complex IV