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 reducing 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 E 0'.
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 E 0' 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):
G0' = -nFE0'
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 Glucose
Glycolysis
Galactose
Anaerobic Fructose
Mannose
Lactate pyruvate
Fatty Acids
Aerobic Acetyl-CoA Amino Acids

Krebs Cycle
1 FADH2 3 NADH

O2
Oxidative phosphorylation
H2O
Electron
Electron Transport
Transport and
and Oxidative
Oxidative Phosphorylation
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
Weare
aremaking
makingATP
ATPfrom
fromADP
ADPandandPPi iby
bytapping
tapping
the
theoxidative
oxidativeenergy
energygenerated
generatedin
inthe
thetransfer
transferofof
electrons
electronsto
toOO2
2
Anatomy of Mitochondria
Mitochondria are composed of a dual membrane system:
Outer: Porous to all molecules < 10 kDa
Inner: Transporter-dependent transport
 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, ubiquinol-
cytochrome 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.
 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
  Loose Site
(ADP and Pi bind)
ADP + Pi

Open Site F1 Tight Site

(ATP is released) ATP (ATP is formed and
held)
ATP 


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: P/O
Assumed to be whole
NADH ~3 intergers based on
FADH2 ~2 the “coupling site”
Succinate ~2 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 Any
Anycompound
compoundthatthat
Site-Specific Target Complex
stops
stopselectron
electron
transport
transportwill
willstop
stop
Carbon monoxide IV
Cyanide IV
respiration…this
respiration…this
Sodium Azide IV means
meansyou
youstop
stop
Rotenone I breathing
Antimycin A III breathing
Amytal I
Electron
Electrontransport
transportcan
can
Phosphorylation
be
bestopped
stoppedbyby
Oligomycin Fo inhibiting
inhibitingATP
ATP
Uncouplers synthesis
synthesis
2,4-Dinitrophenol (DNP) Proton gradient An
Anuncoupler
uncouplerbreaks
breaks
Trifluorocarbonylcyanide
Phenylhydrazone (FCCP) Proton gradient the
theconnection
connectionbetween
between
ATP
ATPsynthesis
synthesisand
and
electron
electrontransport
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 O2 uptake ATP synthesis

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………

2,4-dinitrophenol – a proton
ionophore OH O
O OH
NO2 NO2 NO2 NO2

H+
H+
NO2 NO2 NO2 NO2

Matrix 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
Control of
of Oxidative
Oxidative Phosphorylation
Phosphorylation
Equilibrium
Equilibrium
½NADH + Cyt c (Fe3+) + ADP + Pi
½ NAD+ + Cyt c (Fe2+) + ATP Go’= 0

[NAD+] ½ [c2+] ATP

Keq = [NADH] [c3+] [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) ATP
ATPmass
massaction
actionratio
ratio

[c2+] [NADH] ½ [ADP][Pi] Keq

= [ATP]
[c ]
3+
[NAD+]

NADH Mass Action ration equilibrium

Stimulates Cox
NADH [c2+]/[c3+] equilibrium
Stimulates Cox
ADP [c2+]/[c3+] equilibrium
Stimulates Cox
ATP [c2+]/[c3+] equilibrium
Suppresses Cox
Cytochrome
Cytochrome oxidase
oxidase controls
controls the
the rate
rate of
of O
O22 uptake
uptake which
which
means
means this
this enzyme
enzyme determines
determines how
how rapidly
rapidly we
we breathe.
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 -
ketoglutarate/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 -ketoglutarate. This provides a pool of -
ketoglutarate for the aforementioned antiporter. The aspartate which is also produced is translocated
out of the mitochondrion.
Oxygen Radicals
Partially reduced oxygen species
Molecular Oxygen
.. ..
O2 ..O :: O..
O2 Octet Rule

Unpaired
Unpaired electron
electron
.. ..
..O :: O.. = O 2-
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 (Generates Free Radicals)

Fe2+ + H2O2 Generates hydroxyl radical

Ascorbic acid + Fe2+ Generates hydroxyl radical

Paraquat Generates superoxide radical

Agent Orange Generates superoxide radical

Ozone Generates hydroxyl radical

 WHAT
WHAT ARE
ARE ANTIOXIDANTS?
ANTIOXIDANTS?

ENZYMES
Superoxide dismutase O2-
Catalase H2O2
Peroxidases R-OOH