1) Oxidative phosphorylation uses electron transport chain complexes in the mitochondrial inner membrane to generate ATP from ADP and inorganic phosphate. As electrons are passed through Complexes I-IV, protons are pumped from the matrix to the intermembrane space, building an electrochemical gradient. 2) Protons flow back through ATP synthase, driving the phosphorylation of ADP to ATP in the matrix. The electron carriers, including ubiquinone and cytochrome c, shuttle electrons and protons between the complexes. 3) Oxygen is the final electron acceptor, being reduced to water along with protons in Complex IV. This chemiosmotic mechanism couples electron transport to ATP synthesis via the proton gradient across the inner mitochondrial membrane

1. 1
Oxidative phosphorylation and
photophosphorylation
keystone concepts:
• Oxidative phosphorylation is the enzymatic synthesis of ATP coupled to
the transfer of electrons (e-) to oxygen
• The mitochondrial respiratory chain is an ordered array of electron
carriers arranged in complexes
• Complex I is a transmembrane protein complex of the inner
mitochondrial membrane that accepts e- from NADH
• Complex II (succinate dehydrogenase) transfers e- from succinate to FAD
and then to the Fe-S centers and then to ubiquinone (Coenzyme Q)
• Complex III transfers e- from ubiquinone (QH2) to cytochrome c
• Complex IV receives e- from cytochrome c and passes them to the final
e- acceptor (O2)
• The chemiosmotic model explains how the proton gradient generated by
e- flow drives ATP synthesis

2. Impermeable to
ions and most
other compounds
In inner
membrane
mitochondrion
the mitochondrion contained the enzymes responsible for
electron transport and oxidative phosphorylation

3. 3
anatomy of a
mitochondrion
Where is ATP synthesized?
Where are the citric acid cycle and
β-oxidation pathways?
Which membranes are permeable?

4. Complexes of the respiratory chain

5. 5
Where do the electrons come from?
Where do they go?
• All the dehydrogenase reactions in the citric acid cycle, b-
oxidation and amino acid oxidation and glycolysis
– Hydride ion transfers + hydrogen atom transfers
• Electron carriers in addition to NAD and flavoproteins:
1. Ubiquinone (aka. Coenzyme Q) - a fat soluble mobile protein
2. Cytochromes – iron containing e- transfer proteins (in heme)
3. Iron sulfur proteins – (not in heme) but where iron is directly
associated with inorganic sulfur or the sulfur on cysteine
residues

6. NAD+, flavins and Q carry electrons and H+
Cytochromes and non-heme iron proteins carry only
electrons
NAD+, FAD undergoes only a 2 e- reaction;
cytochromes undergo only 1e- reactions
FMN, Q undergoes 1e- and 2 e- reaction
Electron carriers

7. Ubiquinone
• Coenzyme Q (CoQ, or Q) is lipid-soluble. It
dissolves in the hydrocarbon core of a
membrane.
• the only electron carrier not bound to a
protein.
• it can accept/donate 1 or 2 e-. Q can mediate
e- transfer between 2 e- and 1 e- carriers

8. 8
ubiquinone (Q, or coenzyme Q)

9. Cytochromes are electron carriers containing
hemes . Hemes in 3 classes of cytochrome (a, b, c)
differ in substituents on the porphyrin ring.
Some cytochromes(b,c1,a,a3) are part of large
integral membrane protein complexes (such as
complex III).
Cytochrome c is a small, water-soluble protein.
Cytochromes

10. 10
cytochromes

11. The heme iron can undergo 1 e- transition between
ferric and ferrous states: Fe3+ + e-  Fe2+
Copper ions besides two heme A groups (a and a3)
act as electron carriers in CuB, accepting electrons
from heme a
Cu2++e-  Cu+
Heme is a prosthetic group of cytochromes.
Heme contains an iron atom in a porphyrin ring system.

12. Cytochromes
NAD+
FMN
FeS
ubiquinoneFAD FeS
Cyt b
FeS Cyt c1 Cyt c Cyt a Cyt a3
1/2 O2
ubiquinone
proteins that accept
electrons from QH2 or
FeS
Ultimately transfers the
electrons to oxygen

13. Iron-sulfur centers (Fe-S) are prosthetic groups containing 1-4 iron
atoms
Iron-sulfur centers transfer only one electron, even if they contain
two or more iron atoms.
E.g., a 4-Fe center might cycle between redox states:
3Fe+++ + Fe++ + 1 e-  2Fe+++ + 2Fe++
Iron-sulfur Centers

14. 14
iron sulfur proteins

15. 15
OVERVIEW

16. Electron Transport chain
• The electron transport chain in the inner
mitochondrial membrane can be isolated in
four proteins complexes(I, II, III, IV).
• A lipid soluble coenzyme (Q) and a water
soluble protein (cyt c) shuttle between protein
complexes
• Electrons transfer through the chain - from
complexes I to complex IV

17. NAD+
FMN
FeS
ubiquinoneFAD FeS
Cyt b
FeS Cyt c1 Cyt c Cyt a Cyt a3
1/2 O2
ubiquinone
I
II
III IV
Mitochondrial Complexes
NADH Dehydrogenase
Succinate
dehydrogenase
CoQ-cyt c Reductase
Cytochrome Oxidase

18. 18
How are electron
transport complexes
studied?

19. Support for this order of events
1. Energetically favorable. electrons pass from lower
to higher standard reduction potentials
2. Spectra: the absorption spectrum for the reduced
carrier differs from that of its oxidized form.
carriers closer to oxygen are more oxidized.
3. Specific inhibitors. Those before the blocked step
should be reduced and those after be oxidized.
1. Assay of individual complexes: NADH can reduce complex I
but not the other complexes.

20. H+ Transport
 Complex I, III, IV drive H+ transport from
matrix to the cytosol when e- flow through,
which creates proton gradient
 Creates an electrochemical potential across
the inner membrane

21. 1.Electrons are transported along the inner
mitochondrial membrane, through a series of
electron carriers
2.Protons (indicated by + charge) are translocated
across the membrane, from the matrix to the
intermembrane space
3.Oxygen is the terminal electron acceptor,
combining with electrons and H+ ions to produce
water
4. As NADH delivers more H+ and electrons into
the ETS, the proton gradient increases, with H+
building up outside the inner mitochondrial
membrane

22. Complex I: NADH dehydrogenase
• NADH binds complex I & passes 2 electrons to
a flavin momonucleotide (FMN) prosthetic
group.
• The FMN is reduced to FMNH2. Each electron
is transferred with a proton.
• The electrons are then passed to iron-sulphur
proteins (FeS) in complex I (this is non-heme
iron). The electron is accepted by Fe3+ which is
reduced to Fe2+

24. Complex I
• Two electrons from the reduced FeS proteins
are then passed to CoQ along with 2 protons.
• The CoQ is thus reduced to CoQH2
(ubiquinol) while the FeS proteins are
oxidized back to Fe3+ state.

25. CoQ is small and lipid soluble so it is mobile in the mitochondrial
membrane. It diffuses easily and shuttles the electrons to
complex III

26. 26
complex I: NADH to ubiquinone

27. Complex II: Succinate dehydrogenase
• Complex II actually contains the enzyme
succinate dehydrogenase which catalyses the
reduction of succinate to fumarate (reaction of
the citric acid cycle).
• FAD oxidizes succinate to fumarate (FAD
becoming reduced to FADH2 as it picks up 2
electrons and 2 protons).
• FADH2 is oxidized back to FAD by passing the
electrons on to FeS proteins in complex II. The
electrons are then passed to CoQ and are passed
on to complex III

28. 28
complex II: FADH2 to ubiquinone

29. Complex III: cytochrome reductase
• Complex III contains cytochrome b, cytochrome
c1 and FeS proteins.
• Like FeS proteins, cytochromes contain bound Fe
atoms in heme.
• The iron atoms alternate between +3 and +2
oxidation states as they pass on the electrons.
• CoQH2 passes 2 electrons to cyt b causing the
Fe3+ to be reduced to Fe2+.
• The electrons are passed to the FeS protein and
then to cyt c1.

31. 31
Complex III: QH2 to cytochrome c
-not a direct
proton path
across membrane

32. Cytochrome C
• Cyt c is another small mobile protein.
• It accepts electrons from complex III (Fe3+ is
reduced to Fe2+) and shuttles them to the last
electron transport protein in the chain
(complex IV).

34. Complex IV: cytochrome oxidase
• Complex IV contains cytochrome a and
cytochrome a3 (both use Fe and Cu atoms to
handle the electrons).
• Four cytochrome c molecules pass on 4
electrons to complex IV.
• These are eventually transferred with 4 H+ to
O2 to form 2 water molecules.

35. 35
complex IV: cytochrome oxidase

36. Cytochrome C oxidase combines electrons with oxygen and 4H+
to form 2 water
Oxygen = final electron acceptor

37. Paths of H+ and e- transfer in
cytochrome c oxidase
Blue- chemical reaction of O2
reduction to water
coupled to
Red- translocation of four
protons
e- flow from red cyt c in inner
mem space  CuA center 
heme a3-CuB center reduce O2
H+ from matrix:
-shuttled to heme a3-CuB site
and consumed in production of
H2O
Or
-transloacted across mem
Intermembrane
space
Matrix

38. 38
summary of e- flow from complex I-IV
• Transfer of e- from NADH, energy is conserved in the
proton gradient (called the proton motive force)
• Energy is used to pump protons across the membrane,
which can then be used for work (ATP synthesis)

39. The proton pumps are Complexes I,
III and IV.
Protons return thru ATP synthase

40. Chemiosmotic model
• Electron transport linked to ATP synthesis
• Protons “trapped” in intermembrane space form
electrochemical gradient
• Protons flow down gradient through ATP synthase complex
– phosphorylates ADP and Pi to form ATP

41. -- The protons have a thermodynamic
tendency to return to the matrix =
Proton-motive force
The proton move back into the matrix
through the
FoF1ATP
synthase
driving
ATP synthesis.

42. ATP Synthase
• aka F1F2- ATPase
• Couples the flow of E- across the inner
mitochondrial membrane to synthesis of
ATP (reverse reaction also possible)
• 18 subunits (mammals)  “molecular
machine”

43. 43
mitochondrial ATP synthase complex: FoF1
ATP synthase complex
• Couples the flow of
e- across the inner
mitochondrial
membrane to
synthesize of ATP
(reverse reaction
also possible)
• 18 subunits
(mammals)

44. H+
catalytic
head
rod
rotor
H+
H+
H+
H+ H+
H+H+
H+
FoF1 ATP synthase
ATP
ADP P+
• Enzyme channel in
mitochondrial membrane
– permeable to H+
– H+ flow down
concentration gradient
• flow like water over
water wheel
• flowing H+ cause
change in shape of
ATP synthase enzyme
• powers binding of
Pi to ADP
ADP + Pi  ATP

45. FoF1 ATP synthase
-- ATP synthesized on matrix side.
-- electron transport complexes
and FoF1 ATP synthase arranged
on the inner membrane of the
mitochondrion facing in and lining
the membranes.

46. The return of protons “downhill”
through Fo rotates Fo
relative to F1,
driving ATP
synthesis.
-Note: Subunit 
rotates
through F1.
-Catalytic sites
are located in
the α/β interfaces

47. 47
Where do the substrates come from?
Where do the products go?

48. Mitochondrial ATP transport
• Charge difference between ATP4-
and ADP3- provides driving force
for translocation
– ATP moves from more negative
matrix to more positive
intermembrane space
– ADP moves in opposite
direction
– Reduces charge gradient across
inner membrane by 1

49. Respiratory Control
-- Most mitochondria are said to be
tightly coupled.
That is there is no electron flow
without phosphorylation and no
phosphorylation without
electron flow.
-- Substrate ADP, Pi and O2
are all necessary for
oxidative phosphorylation.

50. For example, in the absence of ADP
or O2 electron flow stops, reduced
substrate is not consumed and no
ATP is made = acceptor control.
Under certain conditions, coupling
can be lost.

51. -- Brown adipose
(fat) cells
contain natural
uncouplers to
warm animals -
cold adaptation
and hibernation.

52. 52
thermogenesis
Thermogenin or uncoupling proteins
provide a path for protons to return to the
matrix without passing through ATP
synthase

53. NADH shuttles
• NADH produced in cytosol during glycolysis
• Mitochondrial membranes impermeable to
NADH
– Reducing equivalents shuttled into
mitochondria to ETC
• Two shuttles operate:
– Glycerol phosphate shuttle
– Malate-aspartate shuttle

54. Glycerol phosphate shuttle
• Operates to minor extent
in variety of tissues, but
very important in Skeletal
muscle and brain
• Transfers reducing
equivalents held by
cytosolic NADH to FAD in
ETC

55. Malate-aspartate shuttle
• Dominant shuttle in liver, kidney and heart
• Transfers reducing equivalents held by cytosolic NADH to NAD in ETC