Chapter 20
Electron Transport and Oxidative
Phosphorylation
Biochemistry
by
Reginald Garrett and Charles Grisham
Essential Question
 How do cells oxidize NADH and [FADH2]
 How do cells convert their reducing potential
into the chemical energy of ATP?
• Electron Transport:
– Electrons carried by reduced coenzymes,
NADH or FADH2, are passed through a
chain of proteins and coenzymes, finally
reaching O2, the terminal electron acceptor
– and to drive the generation of a proton
gradient across the inner mitochondrial
membrane
• Oxidative Phosphorylation:
– The proton gradient runs downhill to drive
the synthesis of ATP
20.1 - Where in the Cell Do Electron Transport
and Oxidative Phosphorylation Occur?
• The processes of electron transport and oxidative
phosphorylation are membrane associated
– in bacteria, is carried out at the plasma membrane
– In eukaryotic cells, happens in or at the inner
mitochondrial membrane
• 500-2500 mitochondria per cell; no mitochondria
in human erythrocytes
• The mitochondria is about 0.5 ± 0.3 micron in
diameter and from 0.5 to several micron long
Figure 20.1 (a) A drawing of a mitochondrion. (b) Tomography of a
rat liver mitochondrion. The colors represent individual cristae.
Mitochondrial functions are localized in specific
compartments
1. Outer membrane
–
–
–
–
Fatty acid elongation
Fatty acid desaturation
Phospholipid synthesis
Monoamine oxidase
2. Inner membrane
–
–
–
–
Electron transport
Oxidative
phosphorylation
Transport system
Fatty acid transport
3. Intermembrane space
–
–
Creatine kinase
Adenylate kinase
4. Martix
–
–
–
–
–
–
–
–
Pyruvate dehydrogenase
complex
TCA cycle
Glutathione
dehydrogenase
Fatty acid oxidation
Urea cycle
DNA replication
Transcription
Translation
Intermembrane space
-- creatine kinase
-- adenylate kinase
Outer membrane
-- fatty acid elongation
-- fatty acid desaturation
-- phospholipid synthesis
-- monoamine oxidase
Inner membrane (cristae)
-- electron transport
-- oxidative phosphorylation
-- transport system
-- fatty acid transport
Matrix
-- pyruvate dehydrogenase
complex
-- citric acid cycle
-- glutathione dehydrogenase
-- fatty acid oxidation
-- urea cycle
-- replication
-- transcription
-- translation
20.2 – How Is the Electron Transport Chain
Organized?
Reduction potential:
• The tendency of an electron donor to reduce its
conjugate acceptor
• The standard reduction potential, ℰo' (25℃, 1
M), is the tendency of a reductant to be reduced
or oxidized
• The standard reduction potentials are determined
by measuring the voltages generated in reaction
half-cells
※
※
※
※
※
The hydrogen electrode (pH 0) is set at 0 volts.
Negative value of ℰo' tends to donate
electrons to H electrode (oxidation)
Positive value of ℰo' tends to accept electrons
from H electrode
High ℰo' indicates a strong tendency to be reduced
• Crucial equation: Go' = - n ℱ ℰo'
 ℰo' = ℰo' (acceptor) - ℰo' (donor)
F: Faraday’s constant (96.5 kJ/mol‧V)
• Electrons are donated by the half reaction with
the more negative reduction potential and are
accepted by the reaction with the more positive
reduction potential: ℰo ' positive, Go' negative
ℰ = ℰo' + (RT/ n ℱ ) ln
[ox]
[red]
20.2 – How Is the Electron Transport Chain
Organized?
NADH (reductant) + H+ + O2 (oxidant) → NAD+ + H2O
Half-reaction:
NAD+ + 2 H+ + 2 e- → NADH + H+
ℰo' = -0.32 V
½ O2 + 2 H+ + 2 e- → H2O
ℰo' = +0.816 V
ℰo' = 0.816 – (-0.32) = 1.136 V
Go'= -219 kJ/mol
Electros move from more negative to more positive
reduction potential in the electron-transport chain
Figure 20.2
Eo΄ and E values for the
components of the
mitochondrial electrontransport chain. Values
indicated are consensus
values for animal
mitochondria. Black bars
represent Eo΄; red bars, E.
The Electron Transport Chain
The electron-transport chain involves several
different molecular species:
1.
2.
3.
4.
5.
Flavoproteins: FAD and FMN
A lipid soluble coenzyme Q (UQ, CoQ)
A water soluble protein (cytochrome c)
A number of iron-sulfur proteins: Fe2+ and Fe3+
Protein-bound copper: Cu+ and Cu2+
All these intermediates except for cytochrome c
are membrane associated
The Electron Transport Chain
The electron-transport chain can be considered to
be composed of four parts:
1.
2.
3.
4.
•
NADH-coenzyme Q reductase
succinate-coenzyme Q reductase
coenzyme Q-cytochrome c reductase
cytochrome c oxidase
Electrons generally fall in energy through the
chain - from complexes I and II to complex IV
Figure 20.3 An overview of the complexes and pathways in the
mitochondrial electron-transport chain.
Complex I Oxidizes NADH and Reduces
Coenzyme Q
NADH-CoQ Reductase or NADH dehydrogenase
• Electron transfer from NADH to CoQ
• More than 45 protein subunits - mass of 980 kD
• Path:
NADH  FMN  Fe-S  CoQ
• Four H+ transported out per 2 eIron-sulfur
proteins
Isoprene unit
10 (Mammal)
6 (Bacteria)
Figure 20.4 (a) The three oxidation states of
coenzyme Q. (b) A space-filling model of
coenzyme Q.
coenzyme Q is a mobile electron carrier
Complex II Oxidizes Succinate and
Reduces Coenzyme Q
•
•
•
•
•
Succinate-CoQ Reductase
Also called succinate dehydrogenase or
flavoprotein 2 (FAD covalently bound)
four subunits, including 2 Fe-S proteins
Three types of Fe-S cluster: 4Fe-4S, 3Fe-4S,
2Fe-2S
Path: Succinate  FADH2  2Fe2+  CoQH2
Net reaction:
succinate + CoQ  fumarate + CoQH2
ℰo'= 0.029V
Figure 20.6 A probable scheme for
electron flow in Complex II.
flavoprotein 3 (fig 20.3)
Figure 20.7 The fatty acyl-CoA dehydrogenase reaction, emphasizing that
the reaction involves reduction of enzyme-bound FAD (indicated by brackets).
Complex III Mediates Electron Transport from
Coenzyme Q to Cytochrome c
CoQ-Cytochrome c Reductase
• CoQ passes electrons to cyt c (and pumps H+) in a
unique redox cycle known as the Q cycle (Fig 20.11)
• The principal transmembrane protein in complex III is
the b cytochrome - with hemes bL(566) and bH(562)
• Cytochromes, like Fe in Fe-S clusters, are one- electron
transfer agents
• The Q cycle drives 4 H+ transport
• CoQH2 is a lipid-soluble electron carrier
• cyt c is a water-soluble mobile electron carrier
Figure 20.8 Typical visible
absorption spectra of cytochromes.
Figure 20.9 The structures of iron
protoporphyrin IX, heme c, and heme a.
Heme c
Figure 20.12 The structure of
mitochondrial cytochrome c
Complex IV Transfers Electrons from
Cytochrome c to Reduce Oxygen on the
Matrix Side
Cytochrome c Oxidase
• Electrons from cyt c are used in a four-electron
reduction of O2 to produce 2 H2O
4 cyt c (Fe2+) + 4 H+ + O2 → 4 cyt c (Fe3+) + 2 H2O
• Oxygen is the terminal acceptor of electrons in the
electron transport pathway
• Cytochrome c oxidase utilizes 2 hemes (a and a3) and 2
copper sites (CuA and CuB)
• Complex IV also transports H+ across the inner
membrane
Figure 20.15 The electron transfer
pathway for cytochrome oxidase.
Cytochrome c binds on the cytosolic side,
transferring electrons through the copper
and heme centers to reduce O2 on the
matrix side of the membrane.
Figure 20.16 Structures of the redox
centers of bovine cytochrome c oxidase.
(a) The CuA site
(b) the heme a site
(c) the binuclear CuB/heme a3 site
The Complexes of Electron Transport May
Function as Supercomplexes
• For many years, the complexes of the electron
transport chain were thought to exist and function
independently in the mitochondrial inner membrane
• However, growing experimental evidence supports
the existence of multimeric supercomplexes of the
four electron transport complexes
• Also known as respirasomes (Fig 20.18a) , these
complexes may represent functional states
• Association of two or more of the individual
complexes may be advantageous for the organism
Figure 20.18 (a) An electron microscopy image of a supercomplex
formed from Complex I, Complex III, and Complex IV (left), and a
model of the complex (right).
Figure 20.18 (b) A model for the electron-transport pathway in
the mitochondrial inner membrane. UQ/UQH2 and cyt c are
mobile carriers and transfer electrons between the complexes.
The proton transport driven by Complex I, III, and IV
Electron Transfer Energy Stored in a Proton
Gradient: The Mitchell Hypothesis
• Peter Mitchell proposed a novel idea—a proton
gradient across the inner membrane could be used to
drive ATP synthesis
• The proton gradient is created by the proteins of the
electron-transport pathway (Figure 20.19)
• This mechanism stores the energy of electron
transport in an electrochemical potential
• As proton are driven out of the matrix, the pH rises
and the matrix become negatively charged with
respect to the cytosol
Figure 20.19 The proton and electrochemical gradients
existing across the inner mitochondrial membrane.
Electron transport-driven proton pumping thus creates a pH gradient
and an electric gradient across the inner membrane, both of which
tend to attract proton back into the matrix from the cytoplasm.
20.3 – What Are the Thermodynamic
Implications of Chemiosmotic Coupling?
• How much energy can be stored in an
electrochemical gradient?
H+in  H +out
G = RT ln [c2]/[c1] + Z F y
G = RT ln [H+out]/[H+in] + Z F y
G = -2.303 RT pH + Z F y
G = 2.3 RT + F (0.18V)
G =5.9 kJ +17.4 kJ = 23.3 kJ
pH = 1
Z = +1
F = 96.485 kJ/V
y = 0.18 V
T= 37 ℃
Z : the charge on a proton
y : the potential difference across the membrane
20.4 – How Does a Proton Gradient Drive
the Synthesis of ATP?
Proton diffusion through the ATP synthase drives
ATP synthesis
• The electrochemical gradient is an energetically
favorable process, drives the synthesis of ATP
• ATP synthase also called F1F0-ATPase
– Is an enzyme, a proton pump, and a rotating
molecular motor
– Consists of two complexes: F1 and F0
1.F1 catalyzes ATP synthesis
2.F0 An integral membrane protein attached to F1
F0 includes three hydrophobic subunits denoted a,
b and c; ab2c10-15
• F0 forms the transmembrane
pore or channel through
which protons move to
drive ATP synthesis
• The a and b subunits
comprise part of the stator
and a ring of c subunits
forms a rotor of the motor
• Protons flow through the ac complex and cause the cring to rotate in the
membrane
* Bovine mitochrodria: 10; Yeast mitochondria: 10; spinach chloroplasts: 14
F1 consists of five polypeptides: a, b, g, d, and e;
a3b3gde
•The a- and b-subunits are arranged in an alternating
pattern in the hexamer which are similar but not
identical
•The noncatalytic a-sites have similar structure, but the
catalytic b-sites have three quite different
conformations
• The g is anchored to the c-subunit
rotor, then the c rotor-g complex can
rotate together relative to the (ab)
complex
• The three b subunits of F1 exist in
three different conformations
represent the three steps of ATP
synthesis
• Each site steps through the three
conformations or states to make ATP
• Three ATP are synthesized per turn
Open (O) conformation with no affinity for ligands
Loose (L) conformation with low affinity for ligands
Tight (T) conformation with high affinity for ligands.
Proton Flow Through F0 Drives Rotation of the
Motor and Synthesis of ATP
Figure 20.24 (c) A view
looking down into the
plane of the membrane.
Transported protons flow
from the inlet halfchannel to Asp61
residues on the c-ring,
around the ring, and then
into the outlet halfchannel.
Upon illumination, bacteriorhodopsin
pumped protons into these vesicles,
and the resulting proton gradient was
sufficient to drive ATP synthesis by
the ATP synthase.
Figure 20.25 The reconstituted
vesicles containing ATP synthase
and bacteriorhodopsin used by
Stoeckenius and Racker to
confirm the Mitchell
chemiosmotic hypothesis.
Inhibitors of Oxidative Phosphorylation
Reveal Insights About the Mechanism
• Rotenone inhibits Complex I - and helps
natives of the Amazon rain forest catch fish
• Cyanide (CN-), azide (N3-) and CO inhibit
Complex IV, binding tightly to the ferric
form (Fe3+) of a3
• Oligomycin is an ATP synthase inhibitors
Figure 20.27 The sites of action of several inhibitors of electron
transport and oxidative phosphorylation.
Uncouplers Disrupt the Coupling of Electron
Transport and ATP Synthase
• Uncouplers disrupt the tight coupling between
electron transport and ATP synthase
– Uncouplers are hydrophobic molecules with a
dissociable proton
– They shuttle back and forth across the inner
membrane, carrying protons to dissipate the proton
gradient
– The energy released in electron transport is
dissipated as heat
• Hibernating Animals Generate Heat by
Uncoupling Oxidative Phosphorylation
Figure 20.28 Structures of
several uncouplers, molecules
that dissipate the proton
gradient across the inner
mitochondrial membrane and
thereby destroy the tight
coupling between electron
transport and the ATP synthase
reaction.
ATP-ADP Translocase Mediates the
Movement of ATP and ADP Across the
Mitochondrial Membrane
ATP must be transported out of the mitochondria
• ATP out, ADP in - through a "translocase"
• ATP movement out is favored because the
cytosol is "+" relative to the "-" matrix
– ATP out and ADP in is net movement of a negative
charge out - equivalent to a H+ going in
– So every ATP transported out costs one H+
• One ATP synthesis costs about 3 H+. Thus,
making and exporting 1 ATP = 4H+
Figure 20.29 (a) The
bovine ATP-ADP
translocase. (b) Outward
transport of ATP (via the
ATP-ADP translocase is
favored by the membrane
electrochemical potential.
20.5 - What Is the P/O Ratio for
Mitochondrial Oxidative Phosphorylation?
P/O ratio is the number of molecules of ATP
formed per two electrons through the chain
• The number of H+ pass through the ATP
synthase to synthesize an ATP, depending on the
number of the c-subunit in the F0
• The number of c-subunit in ATP synthase ranges
from 8-15, depending on the organism
• The ratios of H+/ATP is about 2.7(8/3) to 5(15/3)
• Adding one H+ for ATP-ADP translocase raises
to 3.7 and 6
P/O ratio is the number of molecules of ATP
formed per two electrons through the chain
• The e- transport chain yields 10 H+ pumped out
per electron pair from NADH to oxygen
– 10/3.7 = 2.7 for electrons entering as NADH
– Earlier estimates the P/O ratio for NADH at 3
• For electrons entering as succinate (FADH2),
about 6 H+ pumped per electron pair to oxygen
– 6/3.7 = 1.6 for electrons entering as succinate
– Earlier estimates the P/O ratio for FADH2 at 2
20.6 – How Are the Electrons of Cytosolic
NADH Fed into Electron Transport?
• Most NADH used in electron transport is
produced in mitochondrial matrix
• Cytosolic NADH produced in glycolysis doesn't
cross the inner mitochondrial membrane
• "Shuttle systems" effect electron movement
without actually carrying NADH
1. Glycerophosphate shuttle stores electrons in glycerol3-P, which transfers electrons to FAD
2. Malate-aspartate shuttle uses malate to carry electrons
across the membrane
Figure 20.30 The glycerophosphate shuttle couples cytosolic
oxidation of NADH with mitochondrial reduction of [FAD].
Figure 20.31 The malate-aspartate shuttle.
The Net Yield of ATP from Glucose
Oxidation Depends on the Shuttle Used
• 32 ATP per glucose if glycerol-3-P shuttle used
• 34.2 ATP per glucose if malate-Asp shuttle used
• In bacteria, no extra H+ used to export ATP to
cytosol. Thus, bacteria have the potential to
produce approximately 38 ATP per glucoose
20.7 – How Do Mitochondria Mediate
Apoptosis?
Mitochondria play a significant role in the
programmed death of cells (apoptosis)
– Apoptosis is a mechanism through which certain
cells are eliminated from higher organisms
– It is central to the development and homeostasis of
multicellular organisms
– Apoptosis is suppressed through
compartmentation of the involved activators and
enzymes
– Mitochondria do this in part, by partitioning some
of the apoptotic activator molecules, e.g.,
cytochrome c
• Cytochrome c resides in the intermembrane space and
bound tightly to a lipid chain of cardiolipin
(diphosphatidylglycerol; p240) in the membrane
• Oxidation of bound cardiolipins releases cytochrome c
from the inner membrane
• Apoptotic signals, including Ca2+, ROS, a family of
caspases, and certain protein kinase, can induce the
opening of pores in the mitochondria
• Opening of pores in the outer membrane releases
cytochrome c from the mitochondria
(chapter 31;
p1100)
1. Trigger mitochrondrial
outer membrane
permeabilization (MOMP)
2. Realeasing cytochrome c
3. Binding of cytochrome c to
Apaf-1 (apoptotic proteaseactivating factor 1) in the
cytosol leads to assembly
of apoptosomes, thus
triggering the events of
apoptosis
Figure 20.33 (a) Apaf-1 is a multidomain protein,
consisting of an N-terminal CARD, a nucleotidebinding and oligomerization domain (NOD), and
several WD40 domains.
(b) Binding of cytochrome c to the WD40 domains
and ATP hydrolysis unlocks Apaf-1 to form the semiopen conformation. Nucleotide exchange leads to
oligomerization and apoptosome formation.
(c) A model of the apoptosome, a wheel-like structure
with molecules of cytochrome c bound to the WD40
domains, which extend outward like spokes.