1. OXIDATIVE PHOSPHORYLATION
Is the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH2 to O2 by a series of electron carriers

2. Cellular respiration
Carbon fuels are oxidized in the citric acid cycle to yield electrons with high transfer potential.
This electron-motive force is converted into a proton-motive force.
The proton-motive force is converted into phosphoryl transfer potential.

3. Essence of Oxidative Phosphorylation
Oxidation and ATP synthesis are coupled by transmembrane proton fluxes.

4. Mitochondria: TWO COMPARTMENTS
The intermembrane space between the outer and the inner membranes
Oxidative phosphorylation
The matrix which is bounded by the inner membrane:
most of the reactions of the citric acid cycle and fatty acid oxidation

5. Components of the mitochondrial electron-transport chain
Large multi subunit integral membrane protein complexes or coupling sites: couple electron transfer with H+ gradient generation
Complex I: NADH-Q oxidoreductase (MW =880 kd) Coupling Site 1
Complex II: succinate-Q reductase complex (MW =140 kd)
Complex III: Q-cytochrome c oxido-reductase (MW = 250 kd). Coupling Site 2
Complex IV: cytochrome c oxidase (MW = 160 kd) Coupling Site 3.

6. Small Mobile Electron Carriers:
Ubiquinone/Ubiquinol (Q/QH2): small hydrophobic electron carriers which shuttle electrons between the large complexes and back and forth across the lipid bilayer.
Cytochrome c: is a small water soluble protein which is a mobile electron carrier and carries electrons between cytochrome bc1 complex and cytochrome c oxidase.

7. Components of the mitochondrial electron-transport chain

8. Electron flow within these transmembrane complexes leads to the transport of protons across the inner mitochondrial membrane.
Electrons are carried from NADH-Q oxidoreductase to Q-cytochrome c oxidoreductase by the reduced form of coenzyme Q (Q).
Q also carries electrons from FADH2, generated in succinate dehydrogenase or (succinate-Q reductase) in the citric acid cycle, to Q-cytochrome c oxidoreductase
Cytochrome c, a small, soluble protein, shuttles electrons from Q-cytochrome c oxidoreductase to cytochrome c oxidase (complex IV), which catalyzes the reduction of O2.
Succinate-Q reductase (Complex II), in contrast with the other complexes, does not pump protons.

9. Coenzyme Q (Q)
Q is a hydrophobic quinone that diffuses rapidly within the inner mitochondrial membrane.
Q is a quinone derivative with a long isoprenoid tail.
The number of five-carbon isoprene units in coenzyme Q depends on the species.
The most common form in mammals contains 10 isoprene units (coenzyme Q10).

10. Oxidation States of Quinones
The reduction of ubiquinone (Q) to ubiquinol (QH2) proceeds through a semiquinone anion intermediate (Q.-).
In the fully oxidized state (Q), coenzyme Q has two keto groups.
The addition of one electron and one proton results in the semiquinone form (QH·).
The semiquinone form is relatively easily deprotonated to form a semiquinone radical anion (Q·-).
The addition of a second electron and proton generates ubiquinol (QH2),

11. Thus, electron-transfer reactions of quinones are coupled to proton binding and release, a property that is key to transmembrane proton transport.

12. Flavins Oxidation States of Flavins:
The reduction of flavin mononucleotide (FMN) to FMNH2 proceeds through a semiquinone intermediate.

13. Fe-S clusters
Fe-S clusters in iron-sulfur proteins (nonheme iron proteins) play a critical role in a wide range of reduction reactions in biological systems.
Iron ions in these Fe-S complexes cycle between:
Fe2+ (reduced state)
Fe3+ (oxidized state)
Unlike quinones and flavins, iron-sulfur clusters generally undergo oxidation-reduction reactions without releasing or binding protons.

14. Several types of Fe-S clusters are known:
A single iron ion bound by four cysteine residues.
2Fe-2S cluster with iron ions bridged by sulfide ions.
4Fe-4S cluster.
Each of these clusters can undergo oxidation-reduction reactions.

15. COMPLEX I NADH-Q oxidoreductase
NADH-Q oxidoreductase (also called NADH dehydrogenase):
an enormous enzyme (880 kd)
consists of at least 34 polypeptide chains.
consists of a membrane-spanning part and a long arm that extends into the matrix.
Contains FMN and Fe-S prosthetic groups.

16. NADH is oxidized in the arm, and the electrons are transferred to reduce Q in the membrane.
The reaction catalyzed by this enzyme appears to be:

17. Cytosol
The initial step is the binding of NADH and the transfer of its two high-potential electrons to the flavin mononucleotide (FMN) prosthetic group of this complex to give the reduced form, FMNH2.
NADH + H+
NAD+
FMN
FMNH2
Matrix

18. NADH-Q oxidoreductase contains:
Electrons are then transferred from FMNH2 to a series of three 4Fe4S, the second type of prosthetic group in NADH-Q oxidoreductase.
NADH-Q oxidoreductase contains:
2Fe-2S cluster
4Fe-4S cluster
Two protons move back to the matrix
Fe3+
Fe2+
2H+
NADH + H+
NAD+
FMN
FMNH2
Matrix

19. Two hydrogen ions are pumped out of the matrix of the mitochondrion.
Cytosol
Electrons in the 4Fe4S of NADH-Q oxidoreductase are shuttled to coenzyme Q.
Two hydrogen ions are pumped out of the matrix of the mitochondrion. UQ
UQH2
2H+
Fe3+
Fe2+
2H+
NADH + H+
NAD+
FMN
FMNH2
Matrix

20. 2H+
Cytosol UQ
UQH2
The pair of electrons on bound QH2 are transferred to a 4Fe-4S center and the protons are released on the cytosolic side.
These electrons are transferred to a mobile Q in the hydrophobic core of the membrane, resulting in the uptake of two additional protons from the matrix.
2H+
Fe3+
Fe2+ UQ
UQH2
2H+
Fe3+
Fe2+
2H+
NADH + H+
NAD+
FMN
FMNH2
Matrix

21. In summary:
NADH binds to a site on the vertical arm and transfers its electrons to FMN.
These electrons flow within the vertical unit to three 4Fe-4S centers.
Then they flow to a bound Q. The reduction of Q to QH2 results in the uptake of two protons from the matrix.
4 Protons are pumped this way which is still under investigation

22. COMPLEX II succinate-Q reductase complex
It is an integral membrane protein of the inner mitochondrial membrane.
Contains three different kinds of Fe-S clusters:
2Fe-2S
3Fe-3S
4Fe-4S.

23. FADH2 is generated in the citric acid cycle by the enzyme succinate dehydrogenase, with the oxidation of succinate to fumarate.

24. Two electrons are transferred from FADH2 directly to Fe-S clusters of succinate dehydrogenase.
The electrons are then passed to (Q) for entry into the electron-transport chain.
FADH2 is generated by other reactions (such as: Glycerol phosphate dehydrogenase and fatty acyl CoA dehydrogenase).
This FADH2 also transfers its electrons to (Q), to form (QH2).

25. The succinate-Q reductase complex and other enzymes that transfer electrons from FADH2 to Q, in contrast with NADH-Q oxidoreductase, do not transport protons.
Consequently, less ATP is formed from the oxidation of FADH2 than from NADH.

26. COMPLEX III Q-Cytochrome c Oxidoreductase
The second of the three proton pumps in the respiratory chain (also known as cytochrome reductase).
The function of Q-cytochrome c oxidoreductase is to catalyze the transfer of electrons from QH2 to oxidized cytochrome c (cyt c), a water-soluble protein, and concomitantly pump protons out of the mitochondrial matrix.

27. Structure of Q-Cytochrome C Oxidoreductase (Cytochrome BC1).
This enzyme is a homodimer with 11 distinct polypeptide chains.
The major prosthetic groups, three hemes and a 2Fe-2S cluster, mediate the electron-transfer reactions between quinones in the membrane and cytochrome c in the intermembrane space.

28. Structure of Q-Cytochrome C Oxidoreductase
Q-cytochrome c oxidoreductase contains:
Cytochrome b562
Cytochrome b566
Cytochrome c1
An iron sulfur protein
At least six other subunits.
A cytochrome is an electron-transferring protein that contains a heme prosthetic group.
The iron ion of a cytochrome alternates between a reduced ferrous (+2) state and an oxidized ferric (+3) state during electron transport.

29. Structure of Q-Cytochrome C Oxidoreductase
The enzyme contains three heme prosthetic groups contained within two cytochrome subunits:
Two b-type hemes within cytochrome b:
Heme bL (L for low affinity)
Heme bH (H for high affinity)
One c-type heme within cytochrome c1 (Heme c1).
Hemes in the 3 classes of cytochrome (a, b, c) differ slightly in substituents on the porphyrin ring system.
Only heme c is covalently linked to the protein via thioether bonds to cysteine residues

30. Structure of Q-Cytochrome C Oxidoreductase
The enzyme also contains an iron-sulfur protein with an 2Fe-2S center (Rieske center).
This center is unusual in that one of the iron ions is coordinated by two his residues rather than two cysteine residues.
This coordination stabilizes the center in its reduced form, raising its reduction potential.
Finally, Q-cytochrome c oxidoreductase contains two distinct binding sites for ubiquinone termed (Qo) and (Qi), with the Qi site lying closer to the inside of the matrix.

31. The Q Cycle
The mechanism for the coupling of electron transfer from Q to cytochrome c to transmembrane proton transport.
Facilitates the switch from the two-electron carrier ubiquinol to the one-electron carrier cytochrome c.

32. QH2 Qo site QH Q Q Q.- Qi site
The first electron:
flows first to the Rieske 2Fe-2S cluster
then to cytochrome c1
finally to a molecule of oxidized cytochrome c, converting it into its reduced form.
The second electron:
transferred first to cytochrome bL
then to cytochrome bH
finally to an oxidized uniquinone bound in the Qi site.
This quinone (Q) molecule is reduced to a semiquinone anion (Q·-).
Cyt c
Cyt c1
Fe-S
QH2
Qo site QH Q H+ H+
Cyt bL
Cyt bH Q
Q.-
Qi site

33. Cyt c
Fe-S
Cyt c1
Cyt bL
Cyt bH Q
Qo site
QH2
Qi site
Q.-

34. Cyt c
Cyt c1
Fe-S
QH2 Q
Qo site H+
Cyt bL
Cyt bH
QH2
Q.-
Qi site H+

35. (QH2) binds in the Qo site, and transfers its electrons, one at a time:

36. COMPLEX IV Cytochrome c Oxidase
It catalyzes the coupled oxidation of the reduced cyt c generated by Complex III, and reduction of O2 to two molecules of H2O.
The four-electron reduction of oxygen directly to water without the release of intermediates is quite thermodynamically favorable.
DG°´ = kJ mol-1
As much of this free energy as possible must be captured in the form of a proton gradient for subsequent use in ATP synthesis.

37. Cytochrome c oxidase contains:
It consists of 13 subunits, 3 of which (subunits I, II, and III) are encoded by the mitochondrial genome.
Cytochrome c oxidase contains:
Three copper ions, arranged as two copper centers, designated A and B:
CuA/CuA: contains two copper ions linked by two bridging cysteine residues. This center initially accepts electrons from reduced cytochrome c.
CuB: is coordinated by three histidine residues, one of which is modified by covalent linkage to a tyrosine residue.
Two heme A groups:
Heme a: functions to carry electrons from CuA/CuA
heme a3: passes electrons to CuB, to which it is directly adjacent.
Together, heme a3 and CuB form the active center at which O2 is reduced to H2O.

38. The major prosthetic groups include: CuA/CuA heme a heme a3-CuB.
Cytochrome C Oxidase.
The major prosthetic groups include:
CuA/CuA
heme a
heme a3-CuB.
Heme a3-CuB is the site of the reduction of oxygen to water. .

39. Electrone transfer to CuB
FIRST ELECTRONE
Cyt c
Cyt c
CuA/CuA
Electrone transfer to CuB
Heme A
Heme a3
CuB

40. Electrone transfer to Fe in Heme a3
SECOND ELECTRONE
Cyt c
Cyt c
CuA/CuA
Electrone transfer to Fe in Heme a3
Heme A
Both CuB and Fe in Heme a3 are in reduced form
Heme a3 O O
Binding of O2
CuB
Formation of peroxide bridge O O

41. THIRD ELECTRONE Cyt c Cyt c Cleavage of O-O bond O O CuB Heme A
CuA/CuA
Heme A
Heme a3 O O
CuB
Cleavage of O-O bond H+

42. Reduction of the Ferryl group
FOURTH ELECTRONE
Cyt c
Cyt c
CuA/CuA
Heme A H+
Heme a3 O O
CuB
Reduction of the Ferryl group H+

43. Cyt c Release of water O O CuB Heme A H+ H+ Heme a3 H+ H+ H+ H+
CuA/CuA
Heme A H+ H+
Heme a3 O O
CuB H+ H+
Release of water H+ H+

44. Cyt c
Cyt c
CuA/CuA
Heme A O H+
Heme a3
CuB

46. This reaction can be summarized as
The four protons in this reaction come exclusively from the matrix.
Thus, the consumption of these four protons contributes directly to the proton gradient.
Each proton contributes (21.8 kJ mol-1) to the free energy associated with the proton gradient.

47. Proton Transport by Cytochrome C Oxidase.
Four "chemical" protons are taken up from the matrix side to reduce one molecule of O2 to two molecules of H2O.
Four additional "pumped" protons are transported out of the matrix and released on the Cytosolic side in the course of the reaction.
The pumped protons double the efficiency of free-energy storage in the form of a proton gradient for this final step in the electron-transport chain.

48. How these protons are transported through the protein?
Still under study.
However, two effects contribute to the mechanism:
Charge neutrality tends to be maintained in the interior of proteins.
Thus, the addition of an electron to a site inside a protein tends to favor the binding of a proton to a nearby site.
Conformational changes take place, particularly around the heme a3-CuB center, in the course of the reaction cycle.
These changes must be used to allow protons to enter the protein exclusively from the matrix side and to exit exclusively to the cytosolic side.

49. Thus, the overall process catalyzed by cytochrome c oxidase is.
molecular oxygen is an ideal terminal electron acceptor, because its high affinity for electrons provides a large thermodynamic driving force.

50. Reactive oxygen species or ROS.
The reduction of O2 is safe because:
Cytochrome c oxidase does not release partly reduced intermediates by holding O2 tightly between Fe and Cu ions.
However, partial reduction generates small amounts of hazardous compounds (ROS).
Superoxide anion
peroxide.

51. These TOXIC derivatives of molecular oxygen are scavenged by protective enzymes e.g. superoxide dismutase.
This enzyme catalyzes the conversion of two of the superoxide radicals into hydrogen peroxide and molecular oxygen.
The H2O2 formed by superoxide dismutase and by other processes is scavenged by catalase, that catalyzes the dismutation of H2O2 into H2O and O2.

52. A Proton Gradient Powers the Synthesis of ATP
So far, we have considered the flow of electrons from NADH to O2, an exergonic process.
Next, we consider how this process is coupled to the synthesis of ATP, an endergonic process?

55. ATP synthase
The synthesis of ATP is carried out by A molecular assembly in the inner mitochondrial membrane called:
ATP synthase
mitochondrial ATPase
F1F0 ATPase
Complex V

56. How is the oxidation of NADH coupled to the phosphorylation of ADP?
Chemiosmotic Hypothesis:
Complex I, Complex III and Complex IV pump protons across the inner mitochondrial membrane.
Pumping uses the energy liberated from the oxidation of NADH and FADH2
Pumping generates a membrane potential because it generates an electrochemical gradient
Negative inside, positive outside
Alkaline inside, acidic outside

57. The difference in proton conc
The difference in proton conc. (pH) and the electrical potential together represent a form of stored energy called (proton-motive force).
This proton-motive force drives the synthesis of ATP by ATP synthase.

58. How can the energy associated with a proton gradient be quantified?X+
Outer side
Inner side
Conc. C1
Charge Z
Conc. C2
+ + + -
DV = potential across the membrane (V)
DG is given by:

59. Under typical mitochondrial conditions:
pH outside is 1.4 units lower than inside. (log10(c2/c1))
The membrane potenial is 0.14V. (outside +ve)
Z=+1
DG = 5.2 kcal mol-1 (21.8 kJ mol-1).
Thus, each proton that is transported out of the matrix to the cytosolic side corresponds to 5.2 kcal mol-1 of free energy.

60. The respiratory chain and ATP synthase are biochemically separate systems, linked only by a proton-motive force
Testing the Chemiosmotic Hypothesis. ATP is synthesized when reconstituted membrane vesicles containing bacteriorhodopsin (a light-driven proton pump) and ATP synthase are illuminated.
The orientation of ATP synthase in this reconstituted membrane is the reverse of that in the mitochondrion

61. Structure of ATP Synthase
The F1 sector contains the catalytic sites of the synthase.
F0 sector acts as a proton translocator.

62. The C ring is tightly linked to the g and e subunits

63. Recall that the number of c subunits in the c ring appears to range between 10 and 15.
Each 360-degree rotation of the g subunit leads to the synthesis and release of three molecules of ATP (1/120o).
Thus, if there are 10 c subunits in the ring (as was observed in a crystal structure of yeast mitochondrial ATP synthase), each ATP generated requires the transport of 10/3 = 3.33 protons. If there are 12 c ring, then 12/3 = 4 H+
For simplicity, we will assume that 3 protons must flow into the matrix for each ATP formed, but we must keep in mind that the true value may differ.

64. Many Shuttles Allow Movement Across the Mitochondrial Membranes
NAD+ must be regenerated for glycolysis to continue.
How is cytosolic NADH reoxidized under aerobic conditions?
The inner mitochondrial membrane is impermeable to NADH and NAD+.
Thus electrons from NADH, rather than NADH itself, are carried across the mitochondrial membrane.

65. The glycerol 3-phosphate shuttle

66. FADH2 transfers its electrons to Q, which then enters the respiratory chain as QH2.
1.5 rather than 2.5 ATP are formed (FAD is the elect. Acceptor)
The use of FAD enables electrons from cytosolic NADH to be transported into mitochondria.
The price of this transport is one molecule of ATP/2e-.
This glycerol 3-phosphate shuttle is especially prominent in muscle and enables it to sustain a very high rate of oxidative phosphorylation.

67. The malate-aspartate shuttle
In heart and liver

68. The malate-aspartate shuttle
This shuttle, in contrast with the glycerol 3-phosphate shuttle, is readily reversible.
Consequently, NADH can be brought into mitochondria by the malate- aspartate shuttle only if the NADH/NAD+ ratio is higher in the cytosol than in the mitochondrial matrix.
This versatile shuttle also facilitates the exchange of key intermediates between mitochondria and the cytosol.

69. The ATP-ADP translocase
Also called adenine nucleotide translocase or ANT
The highly charged ATP and ADP do not diffuse freely across the inner mitochondrial membrane.
The in and out flows of ATP and ADP are coupled by the translocase, which acts as an antiporter.

70. The translocase, contains a single nucleotide-binding site that alternately faces the matrix and cytosolic sides of the membrane

71. ATP and ADP (both devoid of Mg2+) are bound with nearly the same affinity.
In actively respiring mitochondrion the membrane potential is positive.
The rate of binding-site eversion from the matrix to the cytosolic side is more rapid for ATP than for ADP because ATP has one more negative charge.
The membrane potential and hence the proton-motive force are decreased by the exchange of ATP for ADP, which results in a net transfer of one negative charge out of the matrix.
ATP-ADP exchange is energetically expensive; about a quarter of the energy yield from electron transfer by the respiratory chain is consumed to regenerate the membrane potential.

72. Mitochondrial Transporters for Metabolites Have a Common Tripartite Motif
Transporters (also called carriers) are transmembrane proteins that move ions and charged metabolites across the inner mitochondrial membrane.

73. The Regulation of Cellular Respiration Is Governed Primarily by the Need for ATP
The best current estimates for the number of protons pumped out of the matrix per electron pair is:
4 by NADH-Q oxidoreductase
2 by Q-cytochrome c oxidoreductase
4 by cytochrome c oxidase
Three protons are pumbed through the ATP synthase for each molecule of ATP.
One proton is consumed (neutralized) in transporting ATP from the matrix to the cytosol.
Hence, about 2.5 molecules of ATP are generated as a result of the flow of a pair of electrons from NADH to O2.

74. The Need for ATP determines the rate of Oxidative Phosphorylation
Electrons do not usually flow through the electron-transport chain to O2 unless ADP is simultaneously phosphorylated to ATP.
Oxidative phosphorylation requires a supply of NADH (or FADH2), O2, ADP, and Pi.
The most important factor in determining the rate of oxidative phosphorylation is the level of ADP.

75. The rate of oxygen consumption by mitochondria increases markedly when ADP is added and then returns to its initial value when the added ADP has been converted into ATP

77. Stages at which Oxidative Phosphorylation Can be Inhibited
Specific inhibitors of electron transport were invaluable in revealing the sequence of electron carriers in the respiratory chain.

78. rotenone and amytal block electron transfer in NADH-Q oxidoreductase
Prevents utilization of electrons from NADH but not FADH2
Antimycin A interferes with electron flow from cytochrome bH in Q-cytochrome c oxidoreductase
Prevents utilization of electrons from NADH and FADH2
cyanide (CN-), azide (N3-), and carbon monoxide (CO) block electron flow in cytochrome c oxidase

79. ATP synthase also can be inhibited
Oligomycin and dicyclohexylcarbodiimide (DCCD) prevent the influx of protons through ATP synthase.
If actively respiring mitochondria are exposed to an inhibitor of ATP synthase, the electron-transport chain ceases to operate.
Indeed, this observation clearly illustrates that electron transport and ATP synthesis are normally tightly coupled.

80. Uncoupling of mitochondria
The coupling of electron transport and phosphorylation in mitochondria can be disrupted (uncoupled) by 2,4-dinitrophenol and certain other acidic aromatic compounds.
These substances carry protons across the inner mitochondrial membrane.

81. Regulated Uncoupling Leads to the Generation of Heat
Uncoupling is a means of generating heat to maintain body temperature in hibernating animals, in some newborn animals (including human beings), and in mammals adapted to cold.
Brown adipose tissue, which is very rich in mitochondria is specialized for this process.
The inner mitochondrial membrane of these mitochondria contains a large amount of uncoupling protein (UCP).

82. UCP-1 forms a pathway for the flow of protons from the cytosol to the matrix.
In essence, UCP-1 generates heat by short-circuiting the mitochondrial proton battery. UCP-2 & UCP-3 also have been identified and may have a role in energy homeostasis.
This dissipative proton pathway is activated by free fatty acids liberated from triacylglycerols in response to hormonal signals, such as b-adrenergic agonists