Chapter 18

Electron
transport
&
oxidative
phosphorylation

1
Figure 14-3
 e- transport & oxidative phosphorylation
H+
Glucose
2 NAD+ 2 ADP - +
6 CO2 8 NADH, 2 FADH2 6 O2
2 NADH 2 ATP - +
Acetyl-coA
e- transport - +
2 Pyr - +
2 Pyr 8 NAD+, 2 FAD 12 H2O - +
2H+ Gluc
ADP
Pi
H+
ADP ATP
Malate/Asp Mitochondria
Ox/Phos
shuttle
- or –
Glycerol-P
shuttle
H+ Pi ADP ATP

Mechanical or chemical work 2

 e- transport & ox/phos occur on
mitochondrial inner membrane
• Mitochondria is an
ancient endosymbiot
– DNA, ribosomes etc.

• Completely
domesticated
– Only make a few
proteins
– Critical functions tightly
controlled by nucleus
3
Figure 18-2 part 2
 Mitochondrial membranes
• Outer membrane has
porins.
– Freely permeable up
to 10K

• Inner membrane
permeable only to
– H20
– CO2
– O2
– Everything else must
have a transporter
4
Figure 18-4
 e- transport & oxidative phosphorylation
H+
Glucose
2 NAD+ 2 ADP - +
6 CO2 8 NADH, 2 FADH2 6O2
2 NADH 2 ATP - +
Acetyl-coA
e- transport - +
2 Pyr - +
2 Pyr 8 NAD+, 2 FAD 12 H2O - +
2H+ Gluc
ADP
Pi
H+
ADP ATP
Malate/Asp Mitochondria -4 Ox/Phos
shuttle
- or –
Glycerol-P
shuttle
H+ Pi ADP ATP
-3

Mechanical or chemical work 5

Two ways to move electrons on NADH
from cytoplasm into mitochondria
• Malate/Aspartate shuttle
– completely reversible
– cytoplasmic NADH  3 ATP

• Glycerol phosphate shuttle

– Irreversible
– cytoplasmic NADH  2 ATP

6
 Malate/Asp shuttle was shown in Ch 16
we used top part to move OXAL + 2 e- from mito  cytoplasm

• Can move 2e- from

malate into the
mitochondria

• Note: This diagram

does not explain
origin of N for
OXAC  Asp

Note: I removed the PEP transporter

since it is irrelevant for this discussion
7
Figure 16-20
 Better diagram of Malate/Asp shuttle
(Lehninger, Principles of Biochemistry, 4th ed.)

v
v v v

v v

v
v

8
 Glycerol phosphate shuttle
irreversible, feeds cyoplasmic NADH
directly into mitochondrial e- transport chain

cytoplasm mitochondria

Used in flight
muscles of insects
& human brain

Recall FADH2 has lower

energy than NADH
• FADH2 2 ATP

9
Figure 18-5
 Ca +2 transporters
Cytoplasm Mitochondria

↑ Km
Ca+2 ↑ Vmax passive

↓ Km
↓ Vmax pump
H+

• Muscle contraction  ↑ Ca+2 in cytoplasm

• Need to ↑ Ca+2 in mito to activate Pyr DH & TCA cycle

• Afterwards, low Km system pumps most Ca+2 back out

10
Lecture #15

Monday Oct. 1

11
 e- transport & oxidative phosphorylation
H+
Glucose
2 NAD+ 2 ADP - +
6 CO2 8 NADH, 2 FADH2 6O2
2 NADH 2 ATP - +
Acetyl-coA
e- transport - +
2 Pyr - +
2 Pyr 8 NAD+, 2 FAD 12 H2O - +
2H+ Gluc
ADP
Pi
H+
ADP ATP
Malate/Asp Mitochondria -4 Ox/Phos
shuttle
- or –
Glycerol-P
shuttle
H+ Pi ADP ATP
-3

Mechanical or chemical work 12

 Electron transport
NADH + H+ + ½ O2  NAD+ + H20
• Oxidation:
NADH + H+  NAD+ + 2e- + 2H+
• Reduction:
½ O2 + 2e- + 2H+  H20
• Do in a series of small steps
• Link with transport of 10 H+ out of mito.

Page 593 13
How much energy is released or
consumed in a redox reaction?

Chapter 14.3 B
Pages 463 - 467

14
 ΔGo’ = -nF ΔE
ΔGo’ = -nF (Eo’acceptor - Eo’donor)

n = # of e- transferred
F = Faraday’s constant (96.5 kJ/volt)
Eo’ = std. reduction potential
Electron affinity, measured in volts
Measured relative to H2 electrode at pH 0
H2 gas  2e- + 2H+

Due to way units are defined, must use - nFΔE

15
 Reduction  Low energy electrons

“Happy” to get e-
Reduction rxn gives off large
amount of energy

Spontaneous
electron
flow

Will only except e- if

large amount of
Reduction High energy electrons
energy is added
Table 14-4 page 466 16
 NADH + H+ + ½ O2  NAD+ + H20
• Reduction:
½ O2 + 2e- + 2H+  H20
• Oxidation:
NADH + H+  NAD+ + 2e- + 2H+

ΔGo’ = -nF (Eo’acceptor - Eo’donor)

Which is the donor and which is the acceptor?

17
 NADH + H+ + ½ O2  NAD+ + H20
• Reduction: e- acceptor (this exact reaction is in Eo’ table)
½ O2 + 2e- + 2H+  H20
• Oxidation: e- donor (Eo’ table shows reverse of this reaction)
NADH + H+  NAD+ + 2e- + 2H+
ΔGo’ = -nF (Eo’acceptor - Eo’donor)
= -2F (0.815 – (-0.315))
= - 218 kJ/mol
Subtract the one running backwards
negative
Don’t forget to include # of e- 18
How many ATP could you
potentially make?
Would you want to
be 100% efficient?

19
 Path from NADH to oxygen
involves three e- transfer complexes
(complex II is a “side door” used for FADH2 rxns)

In the process, 10 H+ are pumped out of mitochondria

20
Figure 18-8
 High energy electrons

The three
e- transport
complexes
have
progressively
↑ e- affinity

(i.e. Lower energy)

Low energy electrons 21

Figure 18-7
 Complex 1
• ΔGo’ = -nF (Eo’acceptor - Eo’donor)

• ΔGo’ = -2F (0 volts- (-0.3 volts))

• ΔGo’ = -2F (0.3 volts)

F ≈ 100 kJ/volt

• ΔGo’ = -60 kJ  enough to make an ATP

22
 Within each
complex
e- follow a
multistep path

Each step has

increasing
e- affinity

23
 Complex I
NADH - coenzyme Q oxidoreductase
Pumps 4 H+ across mito. inner membrane / 2e-

Reduced by NADH e- carriers

Oxidized by coenzyme Q FMN (≈ FAD) & FeS
24
Figure 18-8
 Iron sulfur prosthetic groups are used for
these high energy electrons
(a.k.a. nonheme iron)

Do not need to know structures

25
Page 598
 Know
structures
Q
QH∙
QH2
Do not worry
about isoprene
“R” group
26
Figure 18-10b
 Complex III
Coenzyme Q –cytochrome C oxidoreductase
Pumps 4 H+ across mito. inner membrane / 2e-

Reduced by QH2 e- carriers

Oxidized by Cyt C FeS & cytochromes
27
Figure 18-8
 Various cytochromes contain
different versions of heme

28
Box 18-1c, page 603
 Structure/function of
complex III is
understood
reasonably well

Figure 18-14
29
 Text version
of Q cycle

Allows
complex III
to transport
4H+ with
only 2 e-
30
Figure 18-15
 Inner mito
Cytoplasm membrane Mitochondrial matrix
(actually inter membrane space)

2H+ Q:H2
1e- Simpler
Q∙-
version
Q∙- of Q
2H+
Q:H2 cycle
1e-

Thus far, 2 QH2 have transported 4 H+ and 2 e-

but we are not yet finished 31
 Inner mito
Cytoplasm membrane Mitochondrial matrix
(actually inter membrane space)

2H+
1e-
Q:H2
Q∙- 2H+ Q:H2
Q∙- Q
2H+
1e- Q:H2
Net result
Q:H2
4 H+
2 e- Q
32
 Complex IV Cytochrome C oxidase
Pumps 2 H+ across mito. inner membrane / 2e-
(but always works in batches of 4 e-)

Reduced by Cyt C e- carriers

Oxidized by O2 cytochromes & Cu
33
Figure 18-8
 Some intermediates in O2  H20
are extremely reactive free radicals
• O2 + 1 e-  O2-∙ (superoxide radical)
• O2-∙ + 1 e- + 2H+  H2O2 (hydrogen peroxide)
• H2O2 + 1 e-  OH- + ∙OH (hydroxyl radical)
• H+ + ∙OH + 1e-  H20
Complex IV transfers electrons in batches of 4
Shields rest of cell from reactive intermediates
34
 We are not going to cover the
detailed mechanism of complex IV

Figure 18-19
35
 What about complex II ?
Succinate-coenzyme Q oxidoreductase
(Figure from Lehninger, Principles of Biochemistry, 4th ed.)

Complex II is a “side door” for FADH2 reactions, e.g. succinate DH

No protons are pumped out of the mitochondria by complex II
36
 Recall from Chapter 17: TCA cycle
Succinate DH

Succinate DH is a component
of e- transport complex II

37
Page 582
Lecture 2: Alcohols ≈ Alkenes

FADH2

NADH

Alcohols have lower energy

Electrons bind oxygen better than carbon
38
 High energy electrons
FADH2
has lower
energy than
NADH

e- from FADH2
can not go
through
complex 1
(and thus 4 less protons are
pumped out of the
mitochondria per 2 e-)

Low energy electrons 39

Figure from Garrett & Grisham
As we will see in chapter 20, essentially same
reaction is also used during lipid degradation

40
 Recall: Glycerol phosphate shuttle
Feeds e- from cytoplasmic NADH directly into
mitochondrial e- transport chain
cytoplasm mitochondria

Used in the flight

muscles of insects
and human brain

Recall FADH2 has lower

energy than NADH

FADH2 2 ATP

41
Figure 18-5
Note: Glycerol phosphate shuttle feeds into Q from cytoplasm
whereas succinate  fumarate occurs inside the mitochondria

NADH NAD+
cytoplasm
Glycerol-P shuttle

mitochondria

Both bypass complex I, thus 6 rather than 10 protons are pumped out / 2 e-
(thus making 2 ATP / 2e- rather than 3 ATP 2e-) 42
 Complex 1 can be
selectively inhibited by
the fish poison
rotenone & amytal

Stops e- transfer from

NADH in mitochondrial
matrix, but not from
succinate DH or
glycerol P shuttle
43
Figure 18-7
 Electron transport  proton motive force
Concentration component (Δ pH) = 0.75 units
Charge component (Δ) = 0.17 volts

≈Figure 18-20
from Lehninger, Fund. of Biochem, 4th ed. A good study summary44
 How many protons to make an ATP?
G = 2.3 RT(pH) + ZF = 21.5 kJ/mol
Recall Gphysiological for ATP synth ≈ 40-50
 about 2-3 H+ per ATP

Note: can easily estimate relative contribution of ΔpH & Δ volts

using equations we already know.
• G = - RT ln Keq
= 2.3 RT(pH) ≈ 6 (0.75) ≈ 4 kJ/mol

• G = - nFE = -(number of e- transferred) F (volts)

= - (1) (≈100) (- 0.168) ≈ 17 kJ/mol
More exact calculation on page 613  22.1 total 45
 Expectations
NADH  10H+  3 ATP + 1 H+
– Complex I  4 H+
– Complex III  4 H+
– Complex IV  2 H+

FAH2  6H+  2 ATP

– Complex I - not used
– Complex II  0
– Complex III  4 H+
– Complex IV  2 H+

Reality
NADH ≈ 2.5 ATP
FADH2 ≈ 1.5 ATP
• Transport costs
• Leakage 46
 Measuring ATP per 2 e- transferred

ADP + Pi + ½ O2 + NADH + H+  ATP + H20 + NAD+

NADH 2 e-

Measure ↓ O2 ↑ ATP
mitochondria

P/O ratio = P / 2 e- ratio ≈ 2.5

47
 Lecture 16

Wednesday, Oct. 3

48
 How can a proton gradient make ATP?
Peter Michell’s “crazy” idea (see Box 18-2, page 611)
H+
Glucose
2 NAD+ 2 ADP - +
6 CO2 8 NADH, 2 FADH2 6O2
2 NADH 2 ATP - +
Acetyl-coA
e- transport - +
2 Pyr - +
2 Pyr 8 NAD+, 2 FAD 12 H2O - +
2H+ Gluc
ADP
Pi
H+
ADP ATP
Malate/Asp Mitochondria
Ox/Phos
shuttle
- or –
Glycerol-P
shuttle
H+ Pi ADP ATP

Mechanical or chemical work 49

 ATP Synthase
Mito. proton pumping ATP synthase
matrix F1/F0 ATPase

9 H+

3 x 120o
movements

1 revolution

3 (ADP + Pi ATP)

50
Figure 18-26
ATP synthase in the EM

51
 X-ray
structure
shows
atomic
detail

52
Figure 18-23
 Rotation of the γ subunit distorts the α and β subunits
This changes binding affinity for ADP + Pi and ATP

α and β subunits
do not rotate

← γ subunit
rotates

53
Figure 18-22a
 As γ-subunit rotates, it changes the
conformation of the α and β subunits

54
 Movement of γ-subunit causes
αβ subunits to have three distinct
conformations
Kd ≈10-5 M Kd ≈10-12 M

O = nonbinding site
Figure 18-24 L = loose binding site
T = tight binding site

Each of the 3 sites goes through all

three conformations with each 360 55
degree rotation
 Does Kd 10-5  10-12 really provide
enough energy to make ATP?

Δ G = -RT lnKeq ≈ -6 log Keq

ADP + Pi  ATP
Δ Gphysiological (mitochondria) ≈ 40
Keq ≈ 106.6

Kd 10-5  10-12 = 107 increase

 Enough energy to convert ADP + Pi  ATP
56
 Movement of γ-subunit causes
αβ subunits to have three distinct
conformations
Kd ≈10-5 M Kd ≈10-12 M

O = nonbinding site
Figure 18-24 L = loose binding site
T = tight binding site

Each of the 3 sites goes through all

three conformations with each 360 57
degree rotation
 What happens next?
• γ-subunit again rotates 120 degrees powered
by movement of 3 H+

• All binding sites again change conformation.

– Nonbinding  Loose
• Will now bind ADP + Pi
– Loose  Tight
• ADP + Pi  ATP
– Tight  Nonbinding
• ATP will be ejected
58
 Movement of γ-subunit causes
αβ subunits to have three distinct
conformations
Kd ≈10-5 M Kd ≈10-12 M

O = nonbinding site
Figure 18-24 L = loose binding site
T = tight binding site

Each of the 3 sites goes through all

three conformations with each 360 59
degree rotation
 ATP Synthase
Mito. proton pumping ATP synthase
matrix F1/F0 ATPase

9 H+

3 x 120o
movements

1 revolution

3 (ADP + Pi ATP)

60
Figure 18-25
 What happens when ATP consumption is low?
H+
Glucose
2 NAD+ 2 ADP - +
6 CO2 8 NADH, 2 FADH2 6O2
2 NADH 2 ATP - +
Acetyl-coA
e- transport - +
2 Pyr - +
2 Pyr 8 NAD+, 2 FAD 12 H2O - +
2H+ Gluc
ADP
Pi
H+
ADP ATP
Malate/Asp Mitochondria
Ox/Phos
shuttle
- or –
Glycerol-P
shuttle
H+ Pi ADP ATP

Mechanical or chemical work 61

 Remember “little” Johnny?

Epinephrine + exercise Excess food + no exercise

62
 Low blood glucose  High blood glucose
 If we added a controlled “H+ leak”, could we eat
all we want, not exercise, and still lose weight?
Glucose H+
2 NAD+ 2 ADP - +
6 CO2 8 NADH, 2 FADH2 6O2
2 NADH 2 ATP - +
Acetyl-coA
e- transport - +
2 Pyr - +
2 Pyr 8 NAD+, 2 FAD 12 H2O - +
2H+ Gluc
ADP
Pi
H+
Malate/Asp
shuttle ADP ATP Ox/Phos
- or – Mitochondria
Glycerol-P
shuttle
H+ Pi ADP ATP
H+

Mechanical or chemical work 63

 Uncouplers drain H+ gradient
without making ATP
↓ H+ conc. ↑ H+ conc.

Flow of H+ into mito matrix

Would this make an effective diet pill?

64
Figure 18-29
 Dinitrophenol, a chemical currently used
Clin Toxicology 43: 281 as an insecticide, is known to uncouple
(2005) mitochondrial oxidative phosphorylation.
In the 1930s, physicians prescribed it for
Pediatric fatality weight loss, but this was discontinued
when reports of cataracts, deaths, and
following ingestion of other adverse outcomes came to light.
dinitrophenol: We describe in our report the overdose
postmortem and fatality of a teenager who purchased
identification of a the product as a weight loss dietary
“dietary supplement” supplement by mail order. Her death,
despite prompt medical treatment,
underscores the danger of dinitrophenol.
Yale School of Easy accessibility and apparent resurgent
Medicine interest in dinitrophenol as a weight loss
agent is extremely timely and troubling.
65
 Uncoupling
proteins
(UCP)

Drain H+ gradient
without making ATP
 HEAT

A carefully
regulated process

66
Box 18-4 figure 2
 Brown Fat
• New born mammals
• Bears etc.

• Fat
acetyl-coA
NADH, FADH2
H+ gradient
 drain via UCP
 HEAT
67
 Respiration uncoupling and metabolism
in the control of energy expenditure
Centre National de la Recherche Scientifique, Paris
Proc Nutri Soc 64: 47-52 (05)

• Ectopic expression of UCP was directed to white

adipose tissue or skeletal muscle of transgenic mice.

As expected, these animals developed resistance to

diet-induced obesity. In particular, expression of
UCP1 in skeletal muscle induces a marked uncoupling
of respiration and a stimulated fatty acid oxidation.

68
 Adult humans
do have
brown fat.
Activated in
response to
exposure to cold
temperatures

More in young and

lean than old and fat

Be cool, loose weight

Nature 458: 839-840, April 16, 2009
 Humans have multiple UCP
UCP1 Keeps babies (and adults) warm
• Brown fat  heat
• Knock out mice are cold sensitive

UCP2
• Broadly expressed (including brain)
• Proposed to protect neurons against free-radical induced
death

UCP3
• Expressed in muscle
• Over-expression  lean mice
70
 Oxygen addiction
• Aerobic metabolism allows production of
much more ATP/glucose

• We have become dependent on this higher

energy output
– Pumps
– Swelling, leakage and death

• Also must deal with reactive oxygen species

71
 Recall from discussion of e- transport complex IV
Some intermediates in O2  H20
are extremely reactive free radicals

• O2 + 1 e-  O2-∙ (superoxide radical)

• O2-∙ + 1 e- + 2H+  H2O2 (hydrogen peroxide)
• H2O2 + 1 e-  OH- + ∙OH (hydroxyl radical)
• H+ + ∙OH + 1e-  H20

72
 Anti-oxidant mechanisms
• Super-oxide dismutase
2 O2-• + 2H+  H2O2 + O2
Amylothophic lateral sclerosis (ALS, Lou Gehrig)
• Catalase
2 H2O2  2 H2O + O2
• Glutathione peroxidase
2 GSH + R-O-O-H  GSSG + ROH + H2O
(GSSG + NADPH  2 GSH + NADP+)
recall discussion of people with ↓G6PDH activity (Ch 15, pentose-P)

• Se, Vit E, Vit C, uric acid 73