Chapter 6
Biological Oxidation

Oxidation
removal of electrons

Reduction
gain of electrons

NADH and FADH2
formed in glycolysis, fatty acid oxidation, and citric acid
cycle can be used for reductive biosynthesis
Biological Oxidation



The deducing potential of
mitochondrial NADH is most
often used to supply the
energy for ATP synthesis via
oxidative phosphorylation.
Oxidation of NADH with
phosphorylation of ADP to
form ATP are processes
supported by the
mitochondrial electron
transport assembly and ATP
synthase witch are integral
protein complexes of the inner
mitochondrial membrane.
Principles of Reduction/Oxidation
(Redox) Reactions

Redox reactions
involve the transfer of
electrons from one
chemical species to
another.
Principles of Reduction/Oxidation
(Redox) Reactions


Oxidation of NADH
by the electron
transport chain
NADH + (1/2)O2 +H+  NAD+ + H2O
The reduction
potential is –52.6
kcal/mol
Principles of Reduction/Oxidation
(Redox) Reactions

ADP + Pi  ATP
is + 7.3 kcal/mole
Direct chemical analysis
has shown that for every
2 electrons transferred
from NADH to oxygen,
2.5 equivalents of ATP
are synthesized and 1.5
for FADH2

Principals of Reduction/Oxidation (Redox)
Reactions
Redox reactions involve the transfer of electrons from one
chemical species to another. The oxidized plus the
reduced form of each chemical species is referred to as an
electrochemical half cell. Two half cells having at least one
common intermediate comprise a complete, coupled,
redox reaction. Coupled electrochemical half cells have
the thermodynamic properties of other coupled chemical
reactions. If one half cell is far from electrochemical
equilibrium, its tendency to achieve equilibrium (i.e., to
gain or lose electrons) can be used to alter the equilibrium
position of a coupled half cell. An example of a coupled
redox reaction is the oxidation of NADH by the electron
transport chain:
NADH + (1/2)O2 + H+ -----> NAD+ + H2O
The thermodynamic potential of a chemical reaction is calculated
from equilibrium constants and concentrations of reactants and
products. Because it is not practical to measure electron
concentrations directly, the electron energy potential of a redox
system is determined from the electrical potential or voltage of
the individual half cells, relative to a standard half cell. When the
reactants and products of a half cell are in their standard state
and the voltage is determined relative to a standard hydrogen
half cell (whose voltage, by convention, is zero), the potential
observed is defined as the standard electrode potential, E0. If the
pH of a standard cell is in the biological range, pH 7, its potential
is defined as the standard biological electrode potential and
designated E0'. By convention, standard electrode potentials are
written as potentials for reduction reactions of half cells. The free
energy of a typical reaction is calculated directly from its E0' by
the Nernst equation as shown below, where n is the number of
electrons involved in the reaction and F is the Faraday constant
(23.06 kcal/volt/mol or 94.4 kJ/volt/mol):
DG0' = -nFDE0'
For the oxidation of NADH, the standard biological reduction
potential is -52.6 kcal/mole. With a free energy change of 52.6 kcal/mole, it is clear that NADH oxidation has the
potential for driving the synthesis of a number of ATPs
since the standard free energy for the reaction below is
+7.3kcal/mole:
ADP + Pi ------> ATP
Classically, the description of ATP synthesis through oxidation
of reduced electron carriers indicated 3 moles of ATP
could be generated for every mole of NADH and 2 moles
for every mole of FADH2. However, direct chemical
analysis has shown that for every 2 electrons transferred
from NADH to oxygen, 2.5 equivalents of ATP are
synthesized and 1.5 for FADH2.
The final piece of the puzzle
Electron transport
and
Oxidative phosphorylation
Take a deep breath and push on
Major Energy
Pathways
Glycolysis
Anaerobic
Lactate
pyruvate
Glucose
Galactose
Fructose
Mannose
Fatty Acids
Aerobic
1 FADH2
Acetyl-CoA
Amino Acids
Krebs Cycle
3 NADH
O2
Oxidative phosphorylation
H2
Electron Transport and Oxidative Phosphorylation
1. The absolute heart of aerobic metabolism
2. Three Functional Phases
Electron transfer from NADH, FADH2 to O2
Energy preserved as a proton gradient
Proton gradient energy makes ATP
We are making ATP from ADP and Pi by tapping
the oxidative energy generated in the transfer of
electrons to O2
Anatomy of Mitochondria
Mitochondria are composed of a dual membrane system:
Outer: Porous to all molecules < 10 kDa
Inner: Transporter-dependent transport

Diagrammatic representation of the flow of electrons from either NADH or
succinate to oxygen (O2) in the electron transport chain of oxidative
phosphorylation. Complex I contains FMN and 22-24 iron-sulfur (Fe-S)
proteins in 5-7 clusters. Complex II contains FAD and 7-8 Fe-S proteins in 3
clusters and cytochrome b560. Complex III contains cytochrome b,
cytochrome c1 and one Fe-S protein. Associated with complex III by
electrostatic interaction is cytochrome c, the ultimate electron acceptor in
complex III. Complex IV contains cytochrome a, cytochrome a3 and 2
copper ions. As the two electrons pass through the proteins of complex I,
four protons (H+) are pumped into the intramembrane space of the
mitochondrion. Similarly, four protons are pumped into the intramembrane
space as each electron pair flows through complexes III and as four
electrons are used to reduce O2 to H2O in complex IV. The free energy
released as electrons flow through complex II is insufficient to be coupled
to proton pumping. These protons are returned to the matrix of the
mitochondrion, down their concentration gradient, by passing through ATP
synthase coupling electron flow and proton pumping to ATP synthesis.
Complex I - NADH-Q reductase


The first step in the electron transport chain is the
oxidation of NADH to NAD+. The electrons are
transferred to flavin mononucleotide (FMN), producing
the reduced form of this compound (FMNH2):


The reduced FMNH2 is oxidized back to FMN by
transferring the electrons to an iron-sulfur cluster.
These clusters are contained in iron-sulfur proteins (or
non-heme iron proteins): they contains either one, two
or four iron molecules coordinated to the sulfhydryl
groups of four cysteine residues, with two or four
inorganic sulfide groups in the case of the two and
four iron clusters, respectively. The iron in these
clusters cycles between the +2 and +3 states.

The electrons in these clusters are then transferred to
a tightly-bound coenzyme Q (or ubiquinone (Q))
molecule, reducing it to form ubiquinol. Ubiquinone
has a long isoprenoid tail (50 carbons in mammals)
which anchors it to the mitochondrial membrane in
the case of the mobile form:

The electrons from this bound ubiquinol are transferred through two
iron-sulfur clusters to mobile ubiquinone located in the inner
mitochondrial matrix. These molecules can then shuttle around in the
membrane to pass the electrons to another protein complex. The net
result of this transfer is four protons being pumped out of the matrix and
into the intermembrane space for each molecule of NADH which is
oxidized:


Complex II - Succinate - coenzyme Q reductase
The second complex in the electron transport chain is an enzyme of the TCA cycle
which uses a tightly bound FAD to oxidize succinate to fumarate. The electrons
from this reaction are passed through an Fe-S center before being transferred to
mobile ubiquinone in the mitochondrial membrane. Similarly, electrons from the
FAD-mediated oxidation of fatty acids and glycerol 3-phosphate are passed to
mobile, membrane ubiquinone. No protons are pumped out during these
reactions because the free-energy change is too small.
The ubiquinol formed by complexes I and II
can migrate to complex III and transfer their
electrons to cytochrome c in the next step of
this process.
Complex III - Cytochrome
reductase


Complex III (cytochrome reductase, ubiquinolcytochrome c reductase) is used to transfer the
electrons from ubiquinol, oxidizing it back to
ubiquinone, and passes these electrons to cytochrome c
in a two-step process:

The first half of this reaction is the migration of ubiquinol to the Qp site
of cytochrome c reductase. Two electrons and two protons are released,
resulting in an oxidation to a semiquinone intermediate and finally to
ubiquinone, which can leave the site and enter the membrane pool. One
electron is passed to an iron-sulfur protein, through cytochrome c1 and
finally to mobile cytochrome c in the intermembrane space. The other
electron is passed through cythochromes bL and bH, reducing
ubiquinone to a semiquinone intermediate in the Qn site of the enzyme.


In the second step of this reaction, another molecule of ubiquinol enters
the Qp site and is oxidezed to ubiquinone in the same manner as in step
one. This time, however, the second electron is used to reduce the
semiquinone intermediate to ubiquinol, pulling two protons out of the
matrix and returning ubiquinol to the membrane pool. The net result for
these reactions is four protons being pumped out of the matrix for each
molecule of ubiquinol which is oxidized. The reason for the complexity of
this process is to transfer the two electrons from ubiquinol to two
molecules of the one-electron carrier, cytochrome c.
Cytochrome c contains a heme group attached to the protein by thioether
linkages:
Complex IV - cytochrome c
oxidase


Cytochrome c is reduced in complex III, and is
oxidized by complex IV, cytochrome c oxidase, in a
process which results in two more protons being
pumped out of the mitochondrial matrix:

Two molecules of the reduced form of cytochrome c pass their
electrons to a copper-heme a complex and then to a copper-heme
a3 group. This last group is responsible for the reduction of
oxygen to produce water in a multi-step reaction which uses four
electrons and four protons for each molecule of oxygen which is
reduced:

The heme of cytochrome a is slightly different than
that of cytochrome c, having a long, hydrophobic side
chain:


The electron transport chain is used to
oxidize NADH and reduce molecular
oxygen, resulting in the production of
water and regenerating NAD+. The net
reaction is:
This energy is used to create phosphoryl
potential in ATP by ATP synthase.
ATP synthase

How is ATP made?
ADP + Pi
ATP + H2O
FoF1 ATPase Complex (ATP Synthase)
1. An ATP making machine
2. Driven by a proton gradient
3. Attached to the inner mitochondria membrane
F1 = stalk and lollypop
Fo = base
How is the energy of Oxidation Preserved
for the synthesis of ATP?
ANS: Electron transfer to oxygen is accompanied
by the formation of a high energy proton gradient.
The Gradient arises by having protons pumped
from the matrix side of the mitochondria to
the inner membrane spaces
Back flow of the protons to the matrix leads
to the synthesis of ATP.
H+
3 non-equivalent sites
Matrix
F1
FO
Intermembrane space
FOF1 ATPase (ATP Synthase)
Binding-Change Model
ab
Loose Site
(ADP and Pi bind)
ADP + Pi
Open Site
(ATP is released)
ATP
F1
Tight Site
(ATP is formed and
held)
ATP
ab
ab
3-Site Model of ATP Synthesis
The flow of protons through F1 makes the sites
alternate much like a spinning propeller.
P/O Ratios
What is it?
P is phosphate taken up (incorporated into ATP)
O is the oxygen taken up (measured as atomic oxygen)
(Equated to a pair of electrons traveling to O2)
What is the significance?
Compares substrate efficacy to form ATP
Examples:
NADH
FADH2
Succinate
P/O
~3
~2
~2
Assumed to be whole
intergers based on
the “coupling site”
model of ATP
synthesis
Chemiosmotic Adjustment to P/O






10 protons are pumped for each electron pair from
NADH
6 protons are pumped for each electron pair from
FADH2
4 protons are required to make one ATP
1 of the 4 is used in transport of ADP, Pi and ATP
across mitochondrial membrane
Therefore, 10/4 or 2.5 is the P/O ratio for NADH
Therefore, 6/4 or 1.5 is the P/O ratio for FADH2
Inhibitors and Uncouplers
Table 1. Inhibitors of Respiration and Oxidative Phosphorylation
Site-Specific
Target Complex
Carbon monoxide
Cyanide
Sodium Azide
Rotenone
Antimycin A
Amytal
IV
IV
IV
I
III
I
Phosphorylation
Oligomycin
Fo
Uncouplers
2,4-Dinitrophenol (DNP)
Trifluorocarbonylcyanide
Phenylhydrazone (FCCP)
Proton gradient
Proton gradient
Any compound that
stops electron
transport will stop
respiration…this
means you stop
breathing
Electron transport can
be stopped by
inhibiting ATP
synthesis
An uncoupler breaks
the connection between
ATP synthesis and
electron transport
What is an Uncoupler?
Uncouplers break the
connection between
electron transport and
phosphorylation
Electron transport is a motor
Phosphorylation is the transmission
Uncouplers let you put the car in NEUTRAL
Table 2. Action of Inhibitors on Respiration and Phosphorylation
Agent or Condition
1. Inhibit electron transport……….
2. Inhibit phosphorylation………..
3. Increase proton gradient……….
4. Decrease proton gradient………
5. Add DNP………………………
6. Add Oligomycin……………….
7. Add Oligomycin + DNP………
O2 uptake
ATP synthesis
2,4-dinitrophenol – a proton ionophore
OH
OH
O
NO2
NO2
NO2
H+
NO2
Matrix
NO2
O
NO2
H+
NO2
NO2
Inner Membrane
Brown Adipose
Tissue
Uncoupling
a proton gradient
from FOF1 ATPase
Produces Heat!
Thermogenin
Staying Alive Energy Wise









We need 2000 Cal/day or 8,360 kJ of energy per day
Each ATP gives 30.5 kJ/mole of energy on hydrolysis
We need 246 moles of ATP
Body has less than 0.1 moles of ATP at any one time
We need to make 245.9 moles of ATP
Each mole of glucose yields 38 ATPs or 1160 kJ
We need 7.2 moles of glucose (1.3 kg or 2.86 pounds)
Each mole of stearic acid yields 147 ATPs or 4,484 kJ
We need 1.86 moles of stearic acid (0.48 kg or 1.0
pound of fat)
Control
of
Oxidative phosphorylation
What makes us breathe faster?
How does ATP synthesis in the mitochondria adjust to
the needs of the cell?
Regulation of Oxidative Phosphorylation
Since electron transport is directly coupled to proton translocation, the flow
of electrons through the electron transport system is regulated by the magnitude
of the PMF. The higher the PMF, the lower the rate of electron transport, and vice
versa. Under resting conditions, with a high cell energy charge, the demand for
new synthesis of ATP is limited and, although the PMF is high, flow of protons
back into the mitochondria through ATP synthase is minimal. When energy
demands are increased, such as during vigorous muscle activity, cytosolic ADP
rises and is exchanged with intramitochondrial ATP via the transmembrane
adenine nucleotide carrier ADP/ATP translocase. Increased intramitochondrial
concentrations of ADP cause the PMF to become discharged as protons pour
through ATP synthase, regenerating the ATP pool. Thus, while the rate of electron
transport is dependent on the PMF, the magnitude of the PMF at any moment
simply reflects the energy charge of the cell. In turn the energy charge, or more
precisely ADP concentration, normally determines the rate of electron transport
by mass action principles. The rate of electron transport is usually measured by
assaying the rate of oxygen consumption and is referred to as the cellular
respiratory rate. The respiratory rate is known as the state 4 rate when the
energy charge is high, the concentration of ADP is low, and electron transport is
limited by ADP. When ADP levels rise and inorganic phosphate is available, the
flow of protons through ATP synthase is elevated and higher rates of electron
transport are observed; the resultant respiratory rate is known as the state 3 rate.
Thus, under physiological conditions mitochondrial respiratory activity cycles
between state 3 and state 4 rates.
WHAT IS THE ATP MASS ACTION RATIO?
[ATP]
[ADP][Pi] = ATP mass action ratio
High: Energy sufficient, Signifies high ATP
Low: Energy debt, Signifies high ADP or low ATP
HIGH Mass Action Ratio:
Oxidized cytochrome C [C3+] is favored
Cytochrome oxidase is low because of low C2+
O2 uptake low
LOW Mass Action Ratio:
Reduced cytochrome C [C2+] is favored
Cytochrome oxidase stimulated because of high C2+
Oxygen uptake high
Control of Oxidative Phosphorylation
Equilibrium
½NADH + Cyt c (Fe3+) + ADP + Pi
½ NAD+ + Cyt c (Fe2+) + ATP
Keq =
[NAD+] ½
[NADH]
[c2+]
[c3+]
DGo’= 0
ATP
[ADP][Pi]
[ATP] can control its own production
Cytochrome c oxidase step is irreversible and is controlled by
reduced cytochrome c (c2+)
Because of equilibrium, concentration of c2+ depends on
[NADH]/[NAD+] and [ATP]/[ADP][Pi]
Control of Cytochrome Oxidase (Cox)
[c2+]
=
3+
[c ]
NADH
ATP mass action ratio
[NADH] ½ [ADP][Pi] Keq
[ATP]
[NAD+]
Mass Action ration
NADH
[c2+]/[c3+]
ADP
[c2+]/[c3+]
ATP
[c2+]/[c3+]
equilibrium
Stimulates Cox
equilibrium
Stimulates Cox
equilibrium
Stimulates Cox
equilibrium
Suppresses Cox
Cytochrome oxidase controls the rate of O2 uptake which
means this enzyme determines how rapidly we breathe.
Energy from Cytosolic NADH
In contrast to oxidation of mitochondrial NADH, cytosolic NADH when oxidized via the electron
transport system gives rise to 2 equivalents of ATP if it is oxidized by the glycerol phosphate shuttle
and 3 ATPs if it proceeds via the malate aspartate shuttle. The glycerol phosphate shuttle is coupled
to an inner mitochondrial membrane, FAD-linked dehydrogenase, of low energy potential like that
found in Complex II. Thus, cytosolic NADH oxidized by this pathway can generate only 2 equivalents
of ATP. The shuttle involves two different glycerol-3-phosphate dehydrogenases: one is cytosolic,
acting to produce glycerol-3-phosphate, and one is an integral protein of the inner mitochondrial
membrane that acts to oxidize the glycerol-3-phosphate produced by the cytosolic enzyme. The net
result of the process is that reducing equivalents from cytosolic NADH are transferred to the
mitochondrial electron transport system. The catalytic site of the mitochondrial glycerol phosphate
dehydrogenase is on the outer surface of the inner membrane, allowing ready access to the product
of the second, or cytosolic, glycerol-3-phosphate dehydrogenase.
In some tissues, such as that of heart and muscle, mitochondrial glycerol-3-phosphate
dehydrogenase is present in very low amounts, and the malate aspartate shuttle is the dominant
pathway for aerobic oxidation of cytosolic NADH. In contrast to the glycerol phosphate shuttle, the
malate aspartate shuttle generates 3 equivalents of ATP for every cytosolic NADH oxidized.
In action, NADH efficiently reduces oxaloacetate (OAA) to malate via cytosolic malate
dehydrogenase (MDH) . Malate is transported to the interior of the mitochondrion via the aketoglutarate/malate antiporter. Inside the mitochondrion, malate is oxidized by the MDH of the TCA
cycle, producing OAA and NADH. In this step the cytosolic, NADH-derived reducing equivalents
become available to the NADH dehydrogenase of the inner mitochondrial membrane and are
oxidized, giving rise to 3 ATPs as described earlier. The mitochondrial transaminase uses glutamate
to convert membrane-impermeable OAA to aspartate and a-ketoglutarate. This provides a pool of aketoglutarate for the aforementioned antiporter. The aspartate which is also produced is translocated
out of the mitochondrion.
Oxygen Radicals
Partially reduced oxygen species
Molecular Oxygen
..
..O
O2
O2
..
..O
::
..
O..
Octet Rule
::
..
O..
Unpaired electron
-
= O2
Superoxide Anion
What is a Free Radical ?
Any chemical species with one of more unpaired
electrons…….
Highly Reactive
Powerful Oxidant
Short half life (nanoseconds)
Can exist freely in the environment
EXAMPLES OF FREE RADICALS
H.
Hydrogen atom
O2 .
Superoxide (oxygen centered)
OH . Hydroxyl radical (most reactive)
.
NO Nitric Oxide
PRO-OXIDANTS
Fe2+ + H2O2
Ascorbic acid + Fe2+
Paraquat
Agent Orange
Ozone
(Generates Free Radicals)
Generates hydroxyl radical
Generates hydroxyl radical
Generates superoxide radical
Generates superoxide radical
Generates hydroxyl radical
WHAT ARE ANTIOXIDANTS?
ENZYMES
Superoxide dismutase
Catalase
Peroxidases
O2H2O2
R-OOH