Reading: Harper's Biochemistry pp. 130-148 Lehninger Principles of Biochemistry 3rd Ed. pp. 659-690.

1. Electron Transport and OxidativeElectron Transport and Oxidative
PhosphorylationPhosphorylation
Reading:
Harper’s Biochemistry pp. 130-148
Lehninger Principles of Biochemistry
3rd Ed. pp. 659-690

2. OBJECTIVESOBJECTIVES
To learn how NADH and FADH are re-oxidized by
molecular oxygen using the electron transport chain
in mitochondria, and how this generates a proton
gradient across the inner mitochondrial membrane.

3. Electron Transport and OxidativeElectron Transport and Oxidative
PhosphorylationPhosphorylation
OVERVIEW
- Mitrochondria contain the series of catalysts known as the electron-
transfer chain, or the respiratory chain, that collect and transport
reducing equivalents and direct them to their final reaction with
oxygen to form water.
- The free energy made available by this “downhill” (exergonic)
electron flow is coupled to the “uphill” transport of protons across a
proton-impermeable membrane (inner mitrochondrial membrane).
The free energy is captured as a transmembrane electrochemical
potential.
- The transmembrane flow of protons down their concentration
gradient through specific protein channels provides the free energy
for synthesis of ATP. This is performed by a membrane ATP
synthase that couples proton flow to phosphorylation of ADP.

4. Role of the respiratory chain of mitrochondria is the
conversion of food energy to ATP. Oxidation of the major
foodstuffs leads to the generation of reducing equivalents
(2H) that are collected by the respiratory chain for oxidation
and coupled generation of ATP.

5. The term “reducing equivalent” is used to designate a
single electron equivalent transferred in an oxidation-
reduction reaction. Three types of electron transfers
occur in oxidative phosphorylation:
1. Direct transfer of electrons, as in the reduction of Fe3+
to Fe2+
2. Transfer as a hydrogen atom (H+
+ e-
)
3. Transfer as a hydride ion (:H -
), which bears two
electrons.
Most of the electrons that enter the respiratory chain arise
from the action of dehydrogenases that collect electrons
from catabolic pathways and funnel them into universal
electron acceptors - NAD+
(or NADP+
) or flavin nucleotides
(FMN or FAD).

7. Summary of flow of electrons and protonsSummary of flow of electrons and protons
through the four complexes of thethrough the four complexes of the
respiratory chainrespiratory chain
Complexes Ι and ΙΙ
transfer electrons from
two different electron
donors (NADH and
succinate) to ubiquinone.
Complex ΙΙΙ carries
electrons from
ubiquinone to
cytochrome c, and
complex IV transfers
electrons from
cytochrome c to O2
Q
QH2
e
Mitochondrial Respiratory Chain
Intermembrane Space
e
Cyt c1
α
βεβ δ
α α
b b
γ
β
ccc a cc
F0
F1Complex I
Complex IV
F1F0-ATPase
Complex III
Cytochrome
oxidase
Cytochrome bc1
a a3
Cu B
O2 H2O
MgIII II
I
Cyt c
Cyt c
H+
H+
H+
H+
H+
H+
Cu A
e
Fe-S
NADH NAD+ + H+
FMNH2 FMN
Fe-S
e
e
e
e
e
bL
bH
Fe-S
Matrix
Qo
Qi
Q QH2
H+
H+
ADP + Pi ATP

8. Path of electrons from NADH,
succinate, fatty acyl-CoA, and glycerol
3-phosphate to ubiquinone. Electrons
from NADH pass through a
flavoprotein to a series of iron-sulfur
proteins (in Complex Ι ) and then to Q.
Electrons from succinate pass
through a flavoprotein and several Fe-
S centers (in Complex ΙΙ ) on the way
to Q. Glycerol 3-phosphate donates
electrons to a flavoprotein (glycerol 3-
phosphate dehydrogenase) on the
outer face of the inner mitochondrial
membrane, from which they pass to
Q. Acyl-CoA dehydrogenase (the first
enzyme of β oxidation) transfers
electrons to electron-transferring
flavoprotein (ETF), from which they
pass via ETF:ubiquinone
oxidoreductase to Q

10. Prosthetic groups involved in electron-Prosthetic groups involved in electron-
transfer in respiratory chaintransfer in respiratory chain
FMN - flavin mononucleotide
FAD - flavin adenine dinucleotide
Heme - iron containing prosthetic group found in cytochromes (and
other proteins)
Fe-S - iron-sulfur proteins. Iron is not associated with heme but is
found with inorganic sulfur ions and/or with sulfur of Cys residues
in protein.
Electrons move:
from NADH, succinate, or other primary electron donor
to flavoproteins
to ubiquinone
to iron-sulfur proteins
to cytochomes
to oxygen

12. Comparison of biological
oxidations with combustion.
Highly schematic illustration
showing how most of the
energy that would be released
as heat if hydrogen were
burned (a) is instead
harnessed and stored in a
form useful to the cell by
means of the electron-
transport chain in the
mitrochondrial inner
membrane (b). The rest of the
oxidation energy is released as
heat by the mitochondrion. In
reality, the protons and
electrons shown are removed
from hydrogen atoms that are
covalently linked to NADH or
FADH2 molecules.

13. Complex I: NADH to ubiquinone (coenzyme Q)Complex I: NADH to ubiquinone (coenzyme Q)
Complex Ι (NADH:
ubiquinone oxidoreductase)
catalyzes two simultaneous
and obligatory coupled
processes:
1. The exergonic transfer to
ubiquinone of a hydride ion
from NADH and a proton from
the matrix:
NADH + H+
+ Q→ NAD +
+QH2
2. The endergonic transfer of
4 protons from the matrix to
the intermembrane space

14. ComplexComplex ΙΙ is a proton pumpis a proton pump
Complex Ι can be considered a proton pump, driven by the energy
of electron transfer. The reaction it catalyzes is vectorial; it
moves protons from the matrix, which becomes more negatively
charged with the departure of protons, to the intermembrane
space, which becomes more positively charged.
The overall reaction can be written:
NADH + SHN
+
+ Q→NAD +
+ QH2 + 4HP
+
where HN = negative side of membrane (matrix)
HP = positive side of membrane (intermembrane space)
Inhibitors of Complex Ι include:
- amytol (a barbiturate)
- retenone (a plant product insecticide)
- piericidin A (an antibiotic)

15. ComplexComplex ΙΙΙΙ: Succinate to ubiquinone: Succinate to ubiquinone
Complex ΙΙ is succinate dehydrogenase, the only membrane-
bound enzyme in the citric acid cycle. It is a flavoprotein and in
the conversion of succinate to fumarate, electrons pass from
succinate, through succinate dehydrogenase via FAD, and then
through several Fe-S centers to ubiquinone.
Electrons from other sources pass into the respiratory chain at
the level of ubiquinone, but not through Complex ΙΙ; electrons
are passed from acyl-CoA dehydrogenase (the first enzyme of
β-oxidation) via flavoproteins and Fe-S centers to Q, and
electrons pass via FAD from glycol 3-phosphate
dehydrogenase to Q from the outer face of the inner
mitochondrial membrane.
The overall effect to this point is to contribute to the pool of
reduced Q (QH2).

16. ComplexComplex ΙΙΙΙΙΙ: Ubiquinone to cytochrome c: Ubiquinone to cytochrome c
Complex ΙΙΙ- cytochrome bc1 complex- couples transfer of
electrons from ubiquinol (QH2) to cytochrome c with vectorial
transport of protons from the matrix to the intermembrane space.
The complex is a dimer of identical monomers, each with 11
different subunits

17. The passage of electrons and protonsThe passage of electrons and protons
through Complexthrough Complex ΙΙΙΙΙΙ is complex andis complex and
referred to as thereferred to as the Q cycleQ cycle
The net equation is:
QH2 + 2cytc1(oxidized) + 2HN
+
Q + 2cytc1(reduced) + 4HP
+
Cytochrome c is a soluble
protein of the intermembrane
space. After its single heme
accepts an electron from
Complex ΙΙΙ, cytochrome c
moves to Complex IV to donate
the electron to a binuclear
copper center in that enzyme.

18. Complex IV: cytochrome c to OComplex IV: cytochrome c to O22
In the final step of the
respiratory chain, Complex IV,
also called cytochrome oxidase,
carries electrons from
cytochrome c to molecular
oxygen, reducing it to H2O.
Electron transport is again
coupled to the pumping of
protons into the intermembrane
space.
Overall reaction:
4cytc(reduced) + 8HN
+
+ O2
4cytc(oxidized) + 4HP
+
+ 2H2O

19. Energy ConsiderationsEnergy Considerations
The energy of electron transfer is efficiently conserved in a proton
gradient:
NADH + H+
+  O2→NAD+
+ H2O
E´°(standard reduction potential) for the redox pair -
NAD+
/ NADH = -0.32 V
O2/ H2O = +0.816 V
∆E´° for rxn = 0.816 -(-0.32) = 1.14 V
relationship between free energy and reduction potential
∆G´° = -nF∆E´°
where n = no. of electrons transferred
F = Faraday constant = 96,480 J/V·mol
∆G´° = -2(96.5 KJ/V·mol)(1.14)
= -220 KJ/mol (of NADH)
Highly exergonic; much of this energy released is used to pump protons
out of the matrix.

20. The inner mitochondrial membrane separates two
compartments of different [H+
], resulting in differences in
chemical concentration (∆pH) and charge distribution (∆Ψ)
across the membrane. The net effect generates a proton-
motive force.
For each pair of electrons transferred to O2, four protons
are pumped out by Complex Ι, four by Complex ΙΙΙ, and
two by Complex IV.

21. The mitrochondria
electron transport chain.
The electron transferring
oxidation-reduction
groups of each complex
are boxed. The direction
of electron flow is
indicated by arrows. The
sites of action of
inhibitors are indicated
by dashed lines. The
oxidation-reduction
potentials of certain
electron carriers are
indicated in parentheses.
Enzymes are italicized.