Biochemistry I
Lecture 33
November 17,2015
Lecture 33: Electron transport, ATP synthesis (Oxidative Phosphorylation), Anaerobic
Metabolism
Glucose
Electron Transport:

The energy captured in
glycolysis, TCA cycle, and
fatty acid oxidation on NADH
and FADH2 is converted to a
proton gradient across the
inner mitochondrial
membrane.
The energy stored in this
gradient is used to produce
ATP.

Glucose
Glycolysis
ATP
Pyruvate
NADH
Pyruvate
Fatty acids
triglycerides
Fatty Acid
activation
Acyl-CoA
Electron
Transport
Acyl-CoA
CO2
FADH2
NADH
Fatty Acid
Oxidation
Citrate
Acetyl-CoA
Oxidative
Phosphorylation
O2
NADH
NADH
Citric Acid
Cycle
FADH2
Pathway
Glycolysis
TCA cycle
Fatty Acid Ox,
NAD+/NADH
Glyceraldehyde 3-phosphate
dehydrogenase
Pyruvate dehydrogenase
Isocitrate dehydrogenase
-ketoglutarate dehydrogenase
Malate dehydrogenase
hydroxyacyl-CoA dehydrogenase
H
COHN2
Within above
pathways
H
+
H
N
H
N
NADH
FAD/FADH2
Succinate dehydrogenase
Acyl-CoA dehydrogenase
O
H
COHN2
H
CO2
H
H
NADH
CO2
H
H
N
O
N
O
N
H
N
O
H
N
N
N
N
H
NAD+ (Oxidized)
Electron
Transport
H
N
O
H
H
H
COHN2
FAD (Oxidized)
NADH (Reduced)
H
COHN2
H
H
H
+
N
H
H
O
N
N
FADH2 (Reduced)
H
O
N
H
N
O
N
N
N
N
H
NADH (Reduced)



NAD+ (Oxidized)
FADH2 (Reduced)
FAD (Oxidized)
In most organisms the electrons from these compounds are deposited on oxygen, reducing it
to water. Note that the oxygen only serves as a final acceptor of electrons in this process.
In many organisms other compounds besides oxygen can serve as electron sinks, allowing
organisms to perform 'oxidative' phosphorylation in the absence of O2.
The actual synthesis of ATP is from a proton gradient across the inner membrane that is
generated during the transfer of electrons to oxygen.
The oxidation of NADH releases a lot of energy:
Oxidation of NADH
Reduction of oxygen
Tot. Reaction
NADH  NAD+ + 2 e- + 2H+
2e- + 2H+ + (1/2) O2  H2O
NADH + (1/2) O2  H2O + NAD+
1
G = -60 kJ/m.
G= - 156 kJ/m.
-200KJ/mol
Biochemistry I
Lecture 33
November 17,2015
Key Components in Electron Transfer:
1. Inorganic carriers of electrons
b) Fe in heme – e.g. cytochrome C
a) Iron-sulfur centers (e.g. succinate
dehydrogenase)
Methionine
S
S
Cys
S
Fe
Cys S
S
Fe2+
S Cys
Fe
S
H3C
N Fe2+N
N
N
H3C
H3C
Cys
R1
Fe3+
N
Fe2+
R1
Fe3+
CH3
N
H
2. Organic Carriers of electrons:
a) Coenzyme Q. Coenzyme Q is a non-polar
electron carrier that diffuses freely in the fluid
mitochondrial membrane. R group is non-polar.
 Can participate in one or two electron redox
transactions, two electron reduction shown
on the right
O
OH
OMe
CH3
OMe
CH3
OMe
R
OMe
R
O
Coenzyme Q
OH
ubiquinol
(hydroquinone)
Oxidized
Reduced
Electron Transport: Gibbs Energy & Flow
Chemical
Potential
NADH
Complex I
Succinate
Complex IIA [FADH2]
Q
Fatty acid ox.
Complex IIB [FADH2]
Complex III
Complex IIA = succinate dehydrogenase (TCA cycle)
Complex IIB = acyl-CoA dehydrogenase (fatty acid oxidation)
Cytochrome C
Glucose
Acyl-CoA
Cytosol
Pyr
Outer membrane
Inner membrane
Cytochrome C
III
I
IIB
IV
IIA
FADH2
FAD
FADH
2
Pyr AcylCoA
Succinate
NADH
acetyl-CoA
TCA
Fumarate
Succinate
Dehydrogenase
NADH
FADH2
FAD
NADH
2
Mito Matrix
Complex IV
Biochemistry I
Lecture 33
November 17,2015
Complex I: NADH-CoQ oxidoreductase
 Four protons/NADH are pumped from
the inside (matrix) to the intermembrane
space.
Complex II: Succinate-CoQ oxidoreductase
 Succinate dehydrogenase of the citric
acid cycle is part of this complex.
 Two
electrons
from
FADH2
are
transferred to CoQ via Fe-S clusters,
generating CoQH2.
 Does not pump any protons.
Complex III: CoQH2-cytochrome c
oxidoreductase
 Transfers electrons from CoQH2 to
cytochrome c one electron at a time.
 Four protons are pumped/NADH or
FADH2
Cytochrome C: Shuttles one electron from III to
IV.
Complex IV: Cytochrome c oxidase
 Accepts 4 e- , one at at a time from cytochrome c.
 Donates a total of four electrons/O2.
 Site of oxygen reduction to water.
i) Produces 2 water molecules/O2 molecule.
ii) Pumps an additional two protons/NADH or FADH2.
Energy Stored in the Proton Gradient
The energy 'stored' in a concentration gradient can be
considered to consist of two parts: GTOTAL  GCONC  GELEC
Defining the reaction direction from inter-membrane space (out)
to the matrix (in):
i) The Gibbs energy due to a concentration difference across a
sealed membrane. The Gibbs energy is:
G  G 0  RT ln
4 H+/2e
I
2 H+/2e
4 H+/2e
II
QH2
III
Q
NADH
NAD+
IV
O2
H2O
[ X IN ]
[ X IN ]
0
0
 ( IN
  OUT
)  RT ln
[ X out ]
[ X out ]
Since the standard chemical potential (0) for the species ([X]) is
the same on both the inside and the outside of the membrane:
GCONC  RT ln
[ X ] IN
[ X ]OUT
This is the amount of energy that is released when the
concentration gradient moves towards equilibrium.
ii) Movement of a charged particle through a voltage difference. The free energy associated with
moving a particle of charge Z, through a voltage difference (=ΔV), is:
GELEC = ZF
 Z = the charge on the transported ion (+1 in the case of the proton)
 F is Faraday's constant, 96,494 C/mol. C=coulomb
  is the voltage difference across the membrane, in volts. This voltage difference is often
referred to as the membrane potential:  = VIN –
[ H  ] IN
GTOTAL  RT ln
 ZF
VOUT.
[ H  ]OUT
The total Gibbs free energy is the sum of these two terms:
Example Calculation: Typical values across the inner mitochondrial membrane are:
[H+]IN/[H+]out = 0.1 (pH=6.5 outside, 7+ on the outside of the membrane:
G  (8.31)(300 ) ln( 0.1)  (1)(96,000 )(0.150 )
 5.7 k J / mol  14.4k J / mol
 20 k J / mol
3
Biochemistry I
Lecture 33
November 17,2015
ATP Synthesis:
ATP synthesis is attained by coupling the free
energy of a proton gradient to the chemical
synthesis of
ATP. The enzyme that
accomplishes this coupling is called ATPsynthase (also known as FoF1 ATPase)
3 H+ = 1 ATP synthesized
Structural Features:
1. The Fo Complex
 Membrane-spanning, multi-protein
complex.
 Responsible for coupling the movement of
three protons to 120° rotations of the γsubunit.
2. The F1 Complex
 Attached to Fo, it protrudes into the
mitochondrial matrix.
 Composed of five different subunits: 33
 The  subunit is the shaft at the center of the
3 3 disk.  rotates 120o every time 3
protons pass through the complex.
 The  subunits are asymmetric due to their
interactions with the -subunit.
1. One conformation of the  subunit has
very low affinity for both ADP and ATP.
Everything is released.
2. One conformation of the  subunit has
high affinity for ADP and Pi.
3. One conformation of the  subunit makes
ATP lower in energy than ADP+Pi.
High affinity
ADP+Pi





High affinity
ATP

Low Affinity
How the motor works:
 Every time three proton move through the
complex, the  subunit rotates 120°.
 The rotation of  subunit changes the
conformation of the β-subunits such that the
Gibbs energy of the bound ADP + Pi becomes
higher than the energy of ATP, thus ATP
forms spontaneously from the bound ADP
and Pi.
 The newly-formed ATP is released with the
transport of three additional protons.
 The actual synthesis, or formation of the bond
between ADP and PI, is catalyzed by
conformational changes of the -subunit that
occur as a consequence of the rotation.
 Since all three β subunits are functioning at
the same time, the transport of 9 protons in a
complete cycle produces 3 ATP.
High affinity
for ADP+Pi
HIgh affinity
for ATP (ATP
lower in energy
than ADP & Pi)
Low Affinity
NADH
FADH2
~10 protons pumped
~6 protons pumped
~ 3 ATP
~2 ATP
4
Biochemistry I
Lecture 33
November 17,2015
Anaerobic Metabolism and the Fate of Pyruvate.
Anaerobic Metabolism:
NAD+ is required as the electron
acceptor in glycolysis.
Which step in glycolysis does this
occur?
Glucose
NAD+
People
NADH
How is NAD+ regenerated?
Pyr
Lactate
Ala
[pyruvate
dehydrogenase]
What happens to glycolysis if NAD+
cannot be regenerated?
Yeast
Fatty Acids
Acetyl CoA
NADH
[citrate
synthase]
TCA Cycle
NAD+
ethanol
Other Fates of Pyruvate
i) Pyr can be converted to Acetyl CoA, a one way reaction in humans.
a) acetyl CoA can be oxidized by the TCA cycle.
b) acetyl CoA can be used to synthesize fatty acids (via citrate), which are then used to make
triglycerides.
ii) Pyruvate can be converted to alanine in a one-step transaminase reaction.
iii) Pyruvate can be used to make oxaloacetate, to replace the
carbons that are removed from the citric acid cycle by
anabolic processes (this reaction is the first step in
gluconeogenesis).
Cooperation between muscle and liver cells during active
exercise (Cori cycle).
a) During intense exercise muscle tissue cannot get sufficient oxygen to process the NADH
produced in metabolism.
b) Pyruvate is reduced to lactate, to regenerate NAD+ for glycolysis.
c) The lactate travels to the liver, where it is oxidized to pyruvate, used to make more glucose,
which is then returned to the muscle.
Muscle Cell
Liver Cell
Glucose
Hormone
Receptor
Glucose
Transporter
glycogen
synthase
Glycogen
Glucose
Lactate
Lactate
Transporter
Glycogen
F6P
Glycolysis
F6P
Glycolysis
PFK
Lactate
F16P
NAD+
NADH
Lactate
Transporter
Glucose
glycogen
phosphorylase
(Glu - Pyr)
Lactate
Glucose
Transporter
glycogen
synthase
Glucose
glycogen
phosphorylase
(Glu - Pyr)
Hormone
Receptor
PFK
Gluconeogenesis
(Pyr-Glucose)
bisPhosphatase
F16P
NAD+
NAD+
NADH
NADH
Pyruvate
Pyruvate
5
NAD+
NADH