Respiratory Electron Transport Chain and

Oxidative Phosphorylation
Oxidation of cofactors (NADH and FADH2)
releases energy:
NADH + H+ + 1/2O2 > NAD+ + H2O
G' = -220 kJ/mol
FADH2 + 1/2O2 > FAD + H2O
G' = -182 kJ/mol

(Note that the standard redox potential of flavin

actually depends upon its environment, as it is
tightly bound by the enzyme.)

Respiratory Electron Transport Chain

Consider that:
C6H12O6 + 6O2 > 6CO2 + 6H2O
G' = -2870 kJ/mol
Thus, the reduced cofactors from oxidation
of 1 mole of glucose store
(10 x 220 kJ) + (2 x 182 kJ) = 2564 kJ
of energy, which accounts for about 90% of
the total realizable energy (under standard
conditions).

Respiratory Electron Transport Chain

The job of the mitochondrial electron transport chain is to:
1. reoxidize these cofactors (NADH + H+ and FADH2)
2. convert this energy into a usable form that can do work
(ie. phosphorylating ADP)

These functions are achieved somewhat separately.

The reoxidation occurs in several steps.
The reduced cofactors do not donate electrons directly to O2.
The electron transport chain is composed of several electron
transfer intermediates, allowing the cell to break down the
oxidation into several parts.
Some of these steps are coupled to the synthesis of ATP.

Respiratory Electron Transport Chain

This coupling process is indirect and involves the creation of
a proton electrochemical gradient across the mitochondrial
inner membrane.
That is, protons are pumped across the membrane (in this
case, out of the matrix) concomitantly with certain electron
transfer steps.
This imbalance of protons and charge across the membrane
is discharged by ATP synthase as it phosphorylates ADP.

Introducing the players in ETC

In general, amino acid residues serve as poor redox
cofactors.
Nature has developed several cofactors adapted to
serve as electron carriers.
Some are organic molecules, some are inorganic, and
some could be termed "hybrid".
Most are bound tightly by the enzymes that catalyze the
electron transfer reactions; some serve as mobile electron
carriers.
We will spend some time getting to know them.

1. Quinones
Quinones are like flavins in that they can accept 1 or 2
electrons at a time, and they accept 1 or 2 protons as
well.
Note that after accepting 1 electron, the resulting
semiquinone is a radical (possesses 1 unpaired
electron). It may be a radical anion or may be
protonated.
After accepting a second e- and H+, it becomes the quinol
form (aka dihydroquinone).
Unlike flavins, quinol is stable and can diffuse through the
membrane as a carrier of both electrons and protons.
This ability makes it ideal for coupling electron flow to
proton pumping across the membrane.

2. Cytochromomes
Cytochromes are proteins that contain heme as a
tightly bound cofactor.
Heme is a tetrapyrrole coordinating a single atom of
Fe(II/III).
The fifth (axial) ligand is usually supplied by the
protein and the sixth (axial) ligand is often supplied
by the substrate.
In the reduced form (Fe2+), cytochromes have 3
characteristic visible absorption bands.
The lowest energy () is at 550-600 nm and disappears upon
oxidation, which can be used to monitor the redox state

Cytochromomes
There are 3 major types of heme:
Iron protoporphyrin IX: the simplest type of heme (first product of the
biosynthetic pathway)
Found in b-type cytochromes.
Heme A: one of the methyl groups is oxidized to a formyl group, and one
of the vinyl groups has been attached to an isoprenoid tail.
Found in a-type cytochromes.
Heme C: Both vinyl groups has been attached to cysteinyl sulfurs of the
cytochrome.
Found in c-type cytochromes.

a-type and b-type cytochromes are integral membrane

proteins, while c-type cytochromes can be either.

3. Iron-sulfur clusters
2 main types:
Fe2S2: 2 atoms of Fe linked by 2 sulfide bridges, each Fe
is also coordinated by 2 Cys sulfurs

Fe4S4: 4 atoms of Fe linked by 4 sulfides each forms

the corner of a distorted cube. Each Fe is also
coordinated by 1 Cys.
(Also possible to have single Fe coordinated by 4 Cys residues.)

Iron-sulfur clusters
Fe-S clusters are used as single electron carriers.
They are thought to represent one of the most ancient
prosthetic groups, as they can form spontaneously on a
renatured FeS protein in the presence of Fe(II) and sulfide
ions.
They are exclusively prosthetic groups, as they cannot
exist independently of the protein into which they are
built.
The redox potential of Fe-S clusters depends crucially
upon the environment provided by the protein. They can
vary enormously (-650 mV to +450 mV).

Electron transfer reactions

The driving force is expressed as a difference in the
standard redox potentials of the components (E'). This
is equivalent to the change in free energy.
G' = -nF E'
n = # of e- transferred
F = 96.4 kJ mol-1 V-1
E' = EA' - ED
Electron flow is favorable from donors of low potential
(NADH; E' = -320 mV) to acceptors of high potential (O2;
E' = +820 mV)

Electron transfer reactions

the overall pathway of electron transfer through the
electron transport chain:
NADH UQ cyt b cyt c1 cyt c cyt (a/a3) O2
This same order has been confirmed by experiments
that block specific electron transport steps.

Electron transport chain

Composed of 4 integral membrane complexes:
Complex I: NADH dehydrogenase
Complex II: succinate dehydrogenase
Complex III: cytochrome bc1
Complex IV: cytochrome oxidase

Electron transport chain

Basically, their jobs are to transfer electrons:
from

to

NADH
Dehydrogenase

NADH

ubiquinone

Succinate
Dehydrogenase

succinate
(via FAD)

ubiquinone

Cytochrome bc1

ubiquinone

cytochrome c

Cytochrome
oxidase

cytochrome c O2

Electron transport chain

The first 2 enzymes collect electrons as ubiquinol.
Because NADH is more strongly reducing, NADH
dehydrogenase can couple the reaction to proton pumping;
succinate dehydrogenase cannot.

This explains the difference (2.5 vs. 1.5 ATP).

The electrons are then transferred via cytochrome c (a 1ecarrier) to oxygen.

Proton pumping also occurs during the reactions catalyzed

by the last 2 enzymes enough for 1.5 ATP per 2etransferred.

Coupling electron transport to ATP synthesis

Peter Mitchel first formulated the chemiosmotic hypothesis: explains the coupling
of electron flow through mitochondrial electron transport chain to the synthesis of
ATP by formation of an electrochemical potential gradient for protons across the
inner mitochondrial membrane.
This hypothesis explained a number of observations:
1. Protons are extruded from the matrix, acidifying the intermembrane space,
when electron transport is active.

2. Electron transport creates a transmembrane electric field. For mitochondria, this

potential () -150 mV, with the inside negative. This can be measured by
examining how a lipophilic ion partitions itself across the membrane.
3. Respiration and phosphorylation are normally tightly coupled if one ceases,
then the other does also.
In the absence of ADP + Pi, electron transport rapidly ceases, because the
electrochemical gradient becomes too strong and the respiratory electron transport
chain can no longer pump protons against it.
The same happens if inhibitors of ATP synthase are added.

Energetics
If we assume that across the inner mitochondrial membrane there exists
a pH difference of 0.75 pH units
a 150 mV electric field
Then we can calculate the free energy difference of a proton moving from one side
of the membrane to the other:
G = Gin - Gout = RT ln ([H+]in/[H+]out) + F
= -2.3 RT pH + F
= (-2.3)(8.315 J K-1 mol-1)(298 K)(0.75) + (96.5 kJ mol-1 V-1)(-0.15V)
= -4.3 kJ/mole - 14.5 kJ/mole = -18.8 kJ/mole
(Note that it is dominated by the electric field term.)
The decrease in free energy when 3 or 4 protons fall down the electrochemical
potential would be:
3 H+ > 56 kJ/mole
4 H+ > 75 kJ/mole
This is more than sufficient to drive this reaction:
ADP + Pi > ATP (G' +52 kJ/mole)