Reginald H.

Garrett
Charles M. Grisham
www.cengage.com/chemistry/garrett

Chapter 20
Electron Transport

Reginald Garrett & Charles Grisham • University of Virginia

 Study Guide ETC and Oxidative Phosphorylation
Key concepts:
 Electron Transport: Electrons carried by reduced coenzymes (NADH and [FADH 2])are
passed through a chain of proteins and coenzymes to drive the generation of a proton
gradient across the inner mitochondrial membrane of eukaryotic mitochondria cytoplasmic
membrane of Bacteria
 Oxidative Phosphorylation: The proton gradient drives the synthesis of ATP
 Know that enzymes of the TCA cycle are located in the mitochondrial matrix and proteins
and coenzymes of the electron transport chain are in the inner mitochondrial membrane
 Know the concepts of standard reduction potential (ε 0), ε0’, and reduction potential ε
 Understand how reduction potential determines how the electrons will flow from the donor
to the acceptor couple in redox reactions
 Understand that good oxidizing agents have positive reduction potentials and good reducing
agents have negative reduction potentials.
 Know how reduction potentials are used to calculate free energy changes for oxidation-
reduction reactions.
 Know the proteins involved in the Mitochondrial Electron Transport Chain (Complexes I-
IV)
 Know how electron flow in the Mitochondrial Electron Transport Chain
 Know the roles of the mobile lipid-soluble electron carrier in the membrane (ubiquinone)
and water-soluble protein electron carrier in the inter-membrane space (cytochrome c)
 Understand how the reduction potentials for the components of the mitochondrial electron-
transport chain determine the flow of electron transport.
Study Guide ETC and Oxidative Phosphorylation

 Know the structure, cofactors and electron flow in Complex I, II, III and IV
 Know the reactions catalyzed by Complex I, II, III and IV
 Know in detail the Q-cycle for complex III
 Understand how electrons from cytochrome c are used in a four-electron reduction of O 2 to 2
H2O by cytochrome c oxidase
 Understand how electron transport is coupled to H + transport in electron transport chain to
generate a H+ gradient across the mitochondrial membrane.
 Know how the proton gradient drive the synthesis of ATP in ATP Synthase (F 1F0ATPase)
 Know the structure of ATP Synthase
 Understand the mechanism of rotational catalysis in ATP Synthase
 Understand how proton flow through F 0 drives rotation of the motor and synthesis of ATP in F 1
 Know that one complete rotation of the rotor is coupled to synthesis of 3 molecules of ATP
 Know the role of ATP-ADP translocase in mitochondrial membrane
 Know how to calculate the P/O ratio for mitochondrial electron transport and oxidative
phosphorylation
 Know the how to calculate the net yield of ATP resulting from glucose oxidation from glucose
oxidation (34 ATP per glucose if malate-Asp shuttle is used)
 Understand how uncouplers disrupt the coupling of electron transport and ATP synthase
How do cells oxidize NADH and [FADH2] and convert their reducing
potential into the chemical energy of ATP?
Electron Transport: Electrons carried by reduced coenzymes (NADH and [FADH 2])are passed
through a chain of proteins and coenzymes to drive the generation of a proton gradient across the
inner mitochondrial membrane of eukaryotic mitochondria cytoplasmic membrane of Bacteria

Oxidative Phosphorylation: The proton gradient drives the synthesis of ATP

The systems conserve the energy of electron transfer as chemical energy in the form of ATP
Enzymes of the TCA cycle are located in
the matrix, except succinate dehydrogenase,
which is in the inner membrane

Proteins and coenzymes of the electron

transport chain are in the inner membrane

The mitochondrion has its own genome and

ribosomes, but most mitochondrial proteins
are synthesized in the cytosol and imported
into the mitochondrion
Reduction potentials (Chapter 3.9)
The standard reduction potential (ε0) is a quantitative measure
the tendency of chemical species to be oxidized or reduced

Reduced donor Oxidized

acceptor
n electrons
Oxidized Reduced
donor acceptor

Δ Go =-nFΔεo
The standard reduction potential is directly correlated to free
energy change for the redox reaction

F = Faraday constant

 ε0= difference in reduction potentials between donor and

 ΔG0 =-n F Δεo
D ε0= ε0(acceptor couple) - ε0( donor couple)
D ε0= ε0((reduction) - ε0(oxidation)

If  ε0 is positive, electrons will flow from the donor to the

acceptor couple

The redox couple with the more negative reduction potential

will donate electrons to the couple with the more positive
reduction potential

Example:
CH3CH2OH → CH3CHO + H2

CH3CH2OH → CH3CHO + 2 H+ + 2 e- (half-cell reaction)

oxidation

+ -
 Standard reduction potential is
measured in a reaction half-cell.
The sample half-cell contains 1M
solutions of a redox couple
(reduced and oxidized forms of the
species)

The reference half- cell contains 1M

H+ in equilibrium with H2 gas at 1
atm pressure. The reference half-cell
is assigned an arbitrary standard
reduction potential of 0.0 V.

The standard reduction potentials of other redox couples are defined relative to
H+/H2 and can be positive or negative
If electrons flow from the sample cell
to the reference cell, then the
reduction potential is negative:
• oxidation is occurring in the sample
cell (ethanol oxidized to
acetaldehyde)
• reduction is occurring in the
reference cell (protons are reduced
to H2)
If electrons flow from the reference
cell to the samples cells the reduction
potential is positive:
• Reduction is occurring in the
sample cell (fumarate to succinate)
• oxidation is occurring in the
reference cell (H2 oxidized to H+)
Reduction potentials and redox reactions (Chapter 3.9)

Reduced Oxidized
donor acceptor H+
n
Ethanol electrons
Oxidized Reduced
n=2
donor acceptor H2
Acetaldehy
de
The standard reduction potential for the ethanol/acetaldehyde couple
is -0.197 V.
This means that ethanol is oxidized more readily than hydrogen (so it
is a better reducing agent, it releases its electrons more easily), and
allows us to predict the direction of the redox reaction

εo= 0 – (-0.197) = So electrons flow from ethanol to H+

+0.197.
Reduction potentials and redox reactions (Chapter 3.9)

Reduced Oxidized
donor acceptor
n
H2 electrons Fumarate
Oxidized Reduced
n=2
donor acceptor
2 H+ Succinate

The standard reduction potential for the succinate/fumarate couple is

0.031 V.
This means that hydrogen is oxidized more readily than succinate (so
it is a better reducing agent, it releases its electrons more easily),
and allows us to predict the direction of the redox reaction

εo= 0.031 – 0= + 0.031 So electrons flow from H2 to

fumarate
 εo’
The standard reduction potential εo is measured under standard conditions,
where reactants are at 1 M concentrations, and H2 is at 1 atmosphere.

This means that the pH in the reference cell is 0, since [H+] = 1 M

The usual convention in biochemistry is to measure reduction

potentials at pH 7, this is designated εo’

H+/H2 couple is -0.42 rather than zero (Table 20.1): pH=7, so [H+] = 10-7 M
Positive reduction potentials:
the compound on the
left of the reaction is a good
oxidizing agent and thus can
easily get reduced

Negative reduction potentials:

the compound on the
left is a poor oxidizing agent
and the compound on the right
of the reaction is a good
reducing agent and can easily
get oxidized
Reduction Potentials and Free Energy Changes

How Are Reduction Potentials Used to Calculate Free Energy Changes

for Oxidation-Reduction Reactions?

High ℰo' indicates a strong tendency to be reduced

Crucial equation: Go' = −nℱΔℰo'

 ℰo' = ℰo'(acceptor) - ℰo'(donor)

Electrons are donated by the half reaction with the more negative
reduction potential and are accepted by the reaction with the more
D ℰo ' positive,  Go' negative
positive reduction potential:

If a given reaction is written so the reverse is true, then the  ℰo' will be
a negative number and  Go' will be positive
 An Example

The half-reaction is conventionally written in the direction of reduction, and

the standard reduction potential is for the reduction half-reaction

The reaction with the more negative reduction potential donates electrons to
the acceptor with the less negative (more positive) potential (isocitrate is a
better reducing agent than NADH, it donates its electrons more easily).

NAD+ + isocitrate NADH + - +

H+
+ ketoglutarate CO2
NAD+ + 2H+ + 2e- NADH H+
o’ = -0.32
+ V
+ 2H+ +
-ketoglutarate + 2e- CO2 isocitrate  o’
= -0.38 V

Under standard conditions, isocitrate donates electrons to NAD+

 An Example
ε0’ = ε0’ (acceptor) - ε0’ ’ (donor)

Here, acceptor is NAD+, donor is isocitrate

ε0’ = -0.32 - (-0.38) =+

0.06V
G0’= -nF ε0’ F electric charge on one mole of electrons,
= 96.5 kJ/V.mol
G0’= (-2)(96.5 kJ/V.mol)(0.06V)
= -11.6 kJ/mol

The reaction is exergonic under standard conditions

 The reduction potential ε

The reduction potential ε depends on the concentration of reactants and to the

standard reduction potential :

ε = ε0’ + (RT/n F) ln ([ox]/[red])

R: Gas Constant
F : Faraday Constant

Basis for understanding, understand how and why

electrons flow in a particular direction in biological
systems
 In the electron transport chain, oxygen is used
(indirectly) to reoxidize NADH to NAD+:
NADH H+
+ 0.5 O2 → NAD+
+ H2 O +
The half reactions and reduction potentials are:
NAD+ + 2H+ + 2e- → NADH H+
o’ = -0.32 V
+
0.5 + 2H+ + 2e- → o’ = +0.816
O2 H 2O V
So electrons will flow from NADH to oxygen

The standard free energy change ΔGo’ for this reaction is -219 kJ/mol

The energy released is conserved as chemical energy in the form of ATP

 Mitochondrial Electron Transport
Chain

In mitochondria, the electron transport chain comprises

• four large protein complexes in the inner membrane,
• a mobile lipid-soluble electron carrier in the membrane (ubiquinone)
• a water-soluble protein electron carrier in the inter-membrane space (cytochrome c)
Complex II is succinate dehydrogenase from the TCA cycle
Electrons generally fall in energy through the chain - from complexes I and II to complex
Mitochondrial Electron Transport Chain

Electrons from NADH are passed to complex IV via cytochrome

c.
Electrons are used to reduce oxygen to water. As they
catalyze redox reactions, complexes I, III and IV also pump
protons across the mitochondrial membrane, such that the
Electron Transport Chain
Electron Transport Chain

Reduction potentials for

the components of the
mitochondrial electron-
transport chain.
Values are consensus for
animal mitochondria.
Black bars represent ℰo’;
red bars, ℰ.
 Mitochondrial Electron Transport Chain

Overview of the complexes and pathways in the mitochondrial electron-

transport chain.
Coenzyme Q is a Mobile Electron
Carrier

• It is hydrophobic as it contains a long isoprenoid tail.

• It is localized to the membrane or can be associated to enzymes as a cofactor.

• Reduced coenzyme Q (UQH2, ubiquinol) can be converted to oxidized coenzyme

Q (Q, ubiquinone) releasing 2 protons and 2 electrons

• Two one electron reactions: coenzyme Q can receive electrons from Fe-S
clusters
 Fe-S clusters in complex I

NADH dehydrogenase contains [2Fe-2S] and [4Fe-4S] clusters.

Typically, clusters participate in one electron redox reactions
Complex I Complex I carries out electron transfer from
NADH to Coenzyme Q

Complex I is also called NADH

dehydrogenase, or NADH-coenzyme Q
reductase

Contains ~43 polypeptides, 1 flavin

mononucleotide (FMN), and 9 [Fe-S]
clusters

Two electrons released from NADH are first

used to reduce FMN

Function: oxidize NADH back to NAD+, accept

high energy electrons that will form a pump
from matrix to intermembrane space

NADH + [FMN] +
H+
NADH + [FMN] + [FMNH2] +
H+ NAD+
 Complex I

Nine [Fe-S] clusters are present in the

globular portion of the enzyme
protruding the matrix

The only Q is the substrate; it is reduced

and the released into the membrane lipid
bilayer
NADH + [FMN] +
H+

NADH + [FMN] + [FMNH2] +

Complex I

Electrons are transferred form FMNH to Q

through a chain of [Fe-S] clusters, each
having a more positive reduction
potential than the last, so that electron
transfer is favorable or ‘downhill’
Reduction of Q (ubiquinone) to QH2 can
occur in two one electron reactions, thus
coenzyme Q can receive electrons from
Fe-S clusters (1 electron transfers)
The exact path of electrons through the
enzyme is yet to be established.
Quick Quiz
How many electrons can ubiquinone carry?

a) 0
b) 1
c) 2 …it’s complicated…
d) 3
e) 1 or 2
 Ubiquinone

Reduction of Q (UQ) with one electron generates the anion of the

semiquinone intermediate.
The subsequent one-electron reduction of the semiquinone intermediate
generate QH2 (UQH2)

Takes two protons (2H+) from the matrix to transform into one reduced
ubiquinol form (QH2)
The standard reduction
potential for the
FMN/FMNH2
couple is more positive
than for the
NAD+/NADH couple.
This means that under
standard conditions,
electrons
will flow from NADH to
FMN.

The true reduction

potential of the flavin is
influenced by the protein
environment, and in
complex I, the difference
between the two
potentials is much
Complex I

Protons required for Q

reduction are taken up from
the matrix.
Later, when UQH2 is reoxidized,
these protons are released in
the intermembrane space,
thus contributing to the
formation of the proton
gradient by a ‘redox loop’
mechanism
Complex I

The energy released by

electron transfer from NADH
to Q drives conformational
changes that pump 4 protons
from matrix to
intermembrane space.
These protons do not
participate in reaction
chemistry.
4 protons pumped for each
This diagram is incorrect:

There are no [Fe-S] clusters

in the membrane
associated portion of the
enzyme

This diagram envisages a

molecule of UQ bound to
the enzyme as a cofactor,
the general view is that
Fig. 20.5, p.
there is no cofactor
650 UQ in
the enzyme
It is a model of the transmembrane domain

Figure 20-5b p650

 Membrane domain: seven
subunits, 54 trans-membrane
helices

8 subunits in hydrophilic
domain
Mammalian Complex I structure
Structures of the core subunits of mammalian complex
I.

KR Vinothkumar et al. Nature 515, 80-84 (2014) doi:10.1038/nature13686

Mammalian Complex I structure
Architecture of mammalian complex I showing the
densities of the supernumerary subunits enclosing the core
domain.

KR Vinothkumar et al. Nature 515, 80-84 (2014) doi:10.1038/nature13686

Complex I : proton pumping

R Baradaran et al. Nature 494, 443-448

(2013)

A long alpha helix from the NuoL subunit acts as a piston to drive
conformational changes that couple electron transfer to proton
 Complex II: Succinate-CoQ Reductase
• Aka succinate dehydrogenase (from TCA cycle) , or flavoprotein 2 (FP 2) - FAD covalently bound (from the TCA CYCLE)
• Complex II is not a pump, and since it extracts electrons from FADH2, this is why FADH2 contributes less to ATP
synthesis than NADH.
• 4 subunits, including 2 Fe-S proteins
• Three types of Fe-S cluster:
4Fe-4S, 3Fe-4S, 2Fe-2S
• Net reaction: succinate + UQ → fumarate + UQH 2

Complex II oxidizes Succinate to

fumarate, which will then oxidize
FADH2 to FAD. Releases 2 Electrons are
then passed to Fe-S centers and then to
ubiquinone (Q) to form another QH2
Complex II: structure and electron pathway

The arrangement
of redox centers.
Electron flow is
from bottom to
top.

The
structure of
Complex II
from pig
heart.
Complex II

Succinate dehydrogenase is a b type cytochrome, it

contains a non-covalently bound iron protoporphyrin IX
(heme).
The iron can be in 2+ or 3+ oxidation states, heme
participates in 1 electron redox reactions
Heme is not on the direct electron transfer route from Fe-S
cluster to UQ
There is no net translocation of protons across the
membrane.
Succinate makes a smaller contribution to ATP synthesis
than NADH
Detour: Fatty-Acyl-CoA Dehydrogenases Also Supply
Electrons to UQ

The fatty acyl-CoA dehydrogenases are three soluble matrix

enzymes involved in fatty acid oxidation.
The fatty acyl-CoA dehydrogenase reaction, emphasizing
that the reaction involves reduction of enzyme-bound FAD
(indicated by brackets).
Complex III: Electron Transport from CoQ to Cytochrome c
• Another name for Complex III: UQ-Cytochrome c Reductase
• CoQ (UQ) passes electrons to cyt c (and pumps H+) in a unique redox cycle known as the Q
cycle
• The principal transmembrane protein in complex III is the b cytochrome - with hemes bL and bH
• Cytochromes, like Fe in Fe-S clusters, are one- electron transfer agents
• UQH2 is a lipid-soluble electron carrier
• Cytochrome c is a water-soluble electron carrier
Complex III: the Q cycle
Goal: move electrons from QH2 to cytochrome C

• Starts when UQH2 binds to the Qp site

• This UQH2 is oxidized in two one electron steps
• The first electron is transferred to an Fe-S cluster (in the Rieske subunit), This
electron is then transferred to the iron in the heme group of cytochrome c1.
(which will reduce iron from Fe3+ to Fe2+)
• Cyt C can then diffuse to complex IV
• Cytochrome C can only bind one electron so the second electron goes on a
different path
Complex III: the Q cycle

• A second UQH2 is oxidized in the Qp site  another electron enters

the Rieske center and attaches onto Cyt C.
• The second one will now attach to UQ- (product formed from the
first half) and one electron reduction UQ- to UQH2 occurs (and
also combines with 2 H+)
• UQH2 is then formed, which can be reused to repeat the entire
cycle again
Q cycle: two molecules of UQH2 are oxidized, and one UQ is
reduced, 2 molecules of cytochrome c are reduced, 2 protons are
consumed in the matrix and 4 released in the intermembrane
 Complex III: the Q cycle overview and products

Q cycle: two molecules of UQH2 are oxidized, and

one UQ is reduced, 2 molecules of cytochrome c are
reduced, 2 protons are consumed in the matrix and 4
released in the intermembrane space
Basically:
- Two QH2 are oxidized into Q, releasing 4H+
- One Q is reduced into QH2 (recycling step)
- Two cytochrome c molecules are reduced
Complex III: structure
Electrons from NADH are passed to complex IV via cytochrome
c.
Electrons are used to reduce oxygen to water. As they
catalyze redox reactions, complexes I, III and IV also pump
protons across the mitochondrial membrane, such that the
Cytochrome c is a mobile electron carrier

The structure of
mitochondrial cytochrome c.
The heme is shown at the
center of the structure. It is
covalently linked to the
protein via two sulfur atoms.
A third sulfur from a
methionine residue
coordinates the iron.
Complex IV: cytochrome c oxidase

• Cytochrome c Oxidase
• Electrons from cytochrome c are used in a four-electron reduction
of O2 to produce 2H2O
• Oxygen is thus the terminal acceptor of electrons in the electron
transport pathway
• Cytochrome c oxidase has two important groups: 2 hemes (a and
a3) and 2 copper sites
• Complex IV also transports 4 H+ across the inner mitochondrial
membrane – helps to also generate a proton gradient
• Four H+ participate in O2 reduction and four H+ are transported in
each catalytic cycle
Overall: 4cytc (Fe2+)+ 4H+ + O2 → 4cytc (Fe3+) + 2 H2O
Complex IV oxidizes cytochrome c (thus cytochrome c
oxidase)
O2 is the terminal electron acceptor and Complex IV is
Complex IV: structure

bovine
cytochrome
c oxidase
Electron Transport Chain: overview
A simple electron transport chain in E. coli

NADH NAD+ H+
+
+ 2e- NADH
UQH
dehydrogena
UQ + 2H+ +
2e- 2
se

UQH2 UQ + 2H+ + 2e-

quinol
0.5 + 2H+ + 2e- H2O
O2 oxidas
e
A simple electron transport chain in E. coli

4H+
2H+
NADH
2H quinol
+
dehydrogena
se oxidas
e

UQH
2

2e
2e U -
-
Q

NAD 2H H2
H +
O
4H 2H
0.5O2 +
NAD+ + H+ + + H+
Proton pumping accompanies electron
transfer
Study Guide ETC and Oxidative Phosphorylation

Key concepts:
 Electron Transport: Electrons carried by reduced coenzymes (NADH and [FADH 2])are
passed through a chain of proteins and coenzymes to drive the generation of a proton
gradient across the inner mitochondrial membrane of eukaryotic mitochondria cytoplasmic
membrane of Bacteria
 Oxidative Phosphorylation: The proton gradient drives the synthesis of ATP
 Know that enzymes of the TCA cycle are located in the mitochondrial matrix and proteins
and coenzymes of the electron transport chain are in the inner mitochondrial membrane
 Know the concepts of standard reduction potential (ε 0), ε0’, and reduction potential ε
 Understand how reduction potential determines how the electrons will flow from the donor
to the acceptor couple in redox reactions
 Understand that good oxidizing agents have positive reduction potentials and good reducing
agents have negative reduction potentials.
 Know how reduction potentials are used to calculate free energy changes for oxidation-
reduction reactions.
 Know the proteins involved in the Mitochondrial Electron Transport Chain (Complexes I-
IV)
 Know how electron flow in the Mitochondrial Electron Transport Chain
 Know the roles of the mobile lipid-soluble electron carrier in the membrane (ubiquinone)
and water-soluble protein electron carrier in the inter-membrane space (cytochrome c)
 Understand how the reduction potentials for the components of the mitochondrial electron-
transport chain determine the flow of electron transport.
Study Guide ETC and Oxidative Phosphorylation

 Know the structure, cofactors and electron flow in Complex I, II, III and IV
 Know the reactions catalyzed by Complex I, II, III and IV
 Know in detail the Q-cycle for complex III
 Understand how electrons from cytochrome c are used in a four-electron reduction of O 2 to 2
H2O by cytochrome c oxidase
 Understand how electron transport is coupled to H + transport in electron transport chain to
generate a H+ gradient across the mitochondrial membrane.
 Know how the proton gradient drive the synthesis of ATP in ATP Synthase (F 1F0ATPase)
 Know the structure of ATP Synthase
 Understand the mechanism of rotational catalysis in ATP Synthase
 Understand how proton flow through F0 drives rotation of the motor and synthesis of ATP in F 1
 Know that one complete rotation of the rotor is coupled to synthesis of 3 molecules of ATP
 Know the role of ATP-ADP translocase in mitochondrial membrane
 Know how to calculate the P/O ratio for mitochondrial electron transport and oxidative
phosphorylation
 Know the how to calculate the net yield of ATP resulting from glucose oxidation from glucose
oxidation (34 ATP per glucose if malate-Asp shuttle is used)
 Understand how uncouplers disrupt the coupling of electron transport and ATP synthase