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Summary of Chapter 17
1. Mitochondrion has outer and inner membranes. Inner compartment is called matrix where citric acid
cycle and oxidative phosphorylation are carried out.
2. Inner membrane is impermeable to most hydrophilic substances, except for O2, CO2 and H2O.
3. NADH produced in cytosol by glycolysis is transported by two kinds of cytoplasmic shuttle systems:
1. Glycerophosphate shuttle: Cytosolic NADH reduces indirectly a FAD in the membrane to
FADH2.
The electrons released from FADH2 are put into the electron transport chain. Through
this
shuttle, one NADH is converted to two ATPs.
2. Malate-aspartate shuttle: Cytosolic NADH is utilized to reduce oxaloacetate to malate which can
pass through the inner membrane by the malate-α-ketoglutarate antiport. The malate brought in
the mitochondrion is utilized to reduce the mitochondrial NAD+ to NADH. The oxidized
malate,
oxaloacetate, is aminated to aspartate that is transported to the cytosol by the glutamateaspartate antiport carrier and converted to oxaloacetate. Through this shuttle, NADH → 3ATP
4.
Intermembrane space space
Cyto c
e
Q
I
e
-
e
-
-
IV
III
e
NA D H
-
II
NA D
+
S ucci na te
e
-
O2
H2O
F um a ra te
Matrix
When NADH is oxidized at the complex I, 2e- flow into the electron-transport chain and travel to the
complex IV. At the complex IV, ½O2 is reduced by the 2e- to produce H2O. Half-cell reactions at the
both ends are:
NADH → NAD+ + H+ + 2e∆E°’ = 0.315 V
½O2 + 2H+ + 2e- → H2O
∆E°’ = 0.815 V
Overall:
NADH + ½O2 + H+ → NAD+ + H2O
∆E°’ = 1.130 V ∆G°’ = -218 kJ/mol
5. Electrons are carried between the complexes by CoQ (ubiquinone) and cytochrome c.
6. O2 consumption of mitochondria is measurable by an oxygen electrode.
7. Electron transport in Complex I is inhibited by rotenone and amytal. Similarly, antimycin A and
CN- inhibit the electron transport in Complex III and Complex IV, respectively.
8. The electron flow in the electron-transport chain has been extensively studied by using the above
inhibitors and substituted electron donor fuels (β-hydroxybutyrate, succinate, tetramethyl-pphenylenediamine (TMPD) and ascorbic acid). This experiment should be reviewed.
9. ATP synthesis (phosphorylation) and O2 consumption (oxidation) are closely coupled, i.e., without
ADP and Pi, O2 consumption is minimal.
10. Using the inhibitors and the electron donor fuels, the ATP production per O2 (P/O ratio) are
determined.
- 3 ATP are produced when the 2e- from oxidation of NADH travel through Complex I → Complex III
→ Complex IV (P/O ratio = 3).
- 2 ATP are produced when the 2e- from oxidation of succinate travel through Complex II → Complex
III → Complex IV (P/O ratio = 2).
- 1 ATP is produced when the 2e- travel through Complex IV (P/O ratio = 1).
11. Complexes I, III and IV are multisubunit transmembrane proteins.
- Complex I (NADH-Coenzyme Q reductase) contains Fe-S clusters and FMN (flavin
mononucleotide).
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- Complex II (Succinate-Coenzyme Q reductase) is succinate dehydrogenase complex and contains FeS clusters, FAD and cytochrome b.
- Complex III (Coenzyme Q-cytochrome c reductase) contains Fe-S cluster, cytochrome bL (b562) and
bH (b566) and one cytochrome c1.
- Complex IV (Cytochrome c oxidase) contains [2Cu-2S] cluster (CuA), cytochrome a, cytochrome a3CuB binuclear complex.
12. NADH, CoQ, FAD and FMN are two-electron carriers, while cytochromes and Fe-S clusters are oneelectron carriers.
13. There are three types of Fe-S clusters: [Fe-S], [2Fe-2S], and [4Fe-4S]. Oxidation states of irons are
between +2 and +3. For example, [4Fe-4S] cluster in ferredoxin has:
1 Fe(II) and 3 Fe(III) in the oxidized form
2 Fe(II) and 2 Fe(III) in the reduced form
14. Cytochromes are electron-transport heme proteins.
e- → [Fe(III)→Fe(II)] → e- → [Fe(III)→Fe(II)] → e- → [Fe(III)→Fe(II)] → e- There are three types of hemes: heme a and b are unbound prosthetic groups with two His
coordination, whereas heme c is covalently bound prosthetic group with one His and one Met
coordination.
- The reduced hemes have 3 characteristic absorption bands (α, β, γ) between 400 and 600 nm.
- The absorption maxim of α bands are used to nomenclature, such as cytochrome b566 --- this
cytochrome has a b-type heme and an absorption maximum of α-band at 566 nm.
15. Cytochrome c is a peripheral membrane protein and carries electron from cytochrome c1 of Complex
III to CuA of Complex IV (cytochrome c oxidase).
16. Cytochrome c oxidase receives four consecutive reduced cytochrome c2+ molecules and concomitant
four-electron reduction of one O2 molecule to 2H2O.
- Electron flow: Cytochrome c → CuA → Heme a → Heme a3-CuB binuclear complex
- O2 molecule binds between Fe of heme a3 and Cu of CuB, and is reduced to H2O.
17. The free energy (~218 kJ/mol) of electron transport is converted to the [H+] gradient between the
matrix (low [H+]) and intermembrane space (high [H+]).
- Proton pump mechanism: When electrons pass through the Complex I, III and IV , the conformations
of the proteins are changed. These conformational changes change pK values of side chains of
several amino acid residues. A series of proton association to low pK residue and proton dissociation
from high pK residue results in the proton transport from the matrix to intermembrane space.
18. Chemiosmotic hypothesis: The free energy of electron transport is converted to an electrochemical
H+ gradient across the inner mitochondrial membrane. This electrochemical potential of H+ gradient
is harnessed to synthesize ATP. This hypothesis has been supported by 4 key observations:
1. An intact inner membrane is required for oxidative phosphorylation.
2. Inner membrane is impermeable to H+, OH-, K+, and Cl-.
3. There is a measurable electrochemical gradient across the inner membrane.
4. “Uncouple” agents do not disrupt the electron transport, but inhibit the ATP synthesis.
19. When protons are pumped from chamber A to chamber B across a membrane, the free energy change
∆G can be described below. H+(A) → H+(B)
∆G = RT ln
[ H + ( B)]
+ ZF∆Ψ = −2.3RT ( pH ( B) − pH ( A)) + ZF∆Ψ
[ H + ( A)]
where Z, F and ∆Ψ are charge on the proton (+1), Faraday constant, and membrane potential,
respectively. ∆Ψ = Ψ(B) - Ψ(A)
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20. ATP is synthesized by proton-translocating ATP synthase (Complex V) composed of two major
substructures, F1 and F0.
H
+
H
e
+
H
+
H
+
-
I
III
IV
F0
V
II
NADH
H
NAD
+
+
Succi nate
H
+
H
Fumarate
O2
+
F1 F 1
ADP + Pi
H2 O
H
+
ATP
- F1 is a peripheral membrane protein composed of 9 subunits, α3β 3γδε, and catalyzes ATP synthesis
from ADP + Pi. F1 itself cannot catalyze ATP synthesis without F0, but can hydrolyze ATP.
- F0 is a transmembrane protein composed of 9 - 12 subunits. The H+ transport channels are passed
through the F0. The proton transport through F0 is inhibited by oligomycin and DCCD
(dicyclohexylcarbodiimide). The DCCD molecule reacts with the Glu residue in lipid
environment.
21. ATP synthesis is driven by conformational changes in F1, which might be driven by the rotation of
the γδε subunits.
- There are three catalytic sites between α and β subunits in (αβ)3 of F1.
- The catalytic sites take three states:
1. Tightly binding state: ATP is synthesized
2. Loosely binding state:
ADP and Pi are bound
3. Open binding state: ATP is released
22. Tight coupling between electron transport and oxidative phosphorylation in mitochondrion depends
on the impermeability of the inner mitochondrial membrane.
23. “Uncouple” agents, 2,4-dinitrophenol (DNP) and carbonylcyanide-ptrifluoromethoxyphenylhydrazone (FCCP), bear H+ and diffuse into the matrix. Thus, these
uncouple agents reduce the electrochemical potential across the inner mitochondrial membrane, and
consequently inhibit ATP synthesis.
24. Uncoupling protein (UCP) called thermogenin is a transmembrane proton channel protein in the
mitochondrial inner membrane. When H+ passes through the UCP, heat is generated instead of ATP.
25. ATP production is controlled by:
- [NADH]/NAD+] ratios, and ATP mass action ratio, [ATP]/([ADP]·[Pi]).
- ATP, ADP and Pi transport systems between matrix and cytosol.
27. The rate-determining enzymes in glycolysis (PFK) and citric acid cycle (pyruvate dehydrogenase,
citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase) are regulated by ATP,
ADP, NADH or certain metabolites, such as citrate. For example, citric acid cycle is slowed down
at isocitrate dehydrogenase and α-ketoglutarate dehydrogenase by increasing the [NADH]/[NAD+]
and/or [ATP]. Thus, the [citrate] is increased, and the excess citrates leave the mitochondria to
cytosol and inhibit the PFK activity. Consequently, the glycolysis activity is slowed down.
28. Undetectable heart attack (myocardial infarction) and stroke can be detected by measuring H-type
lactate dehydrogenase in blood and/or intracellular pH. Why ?
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