Chapter 6 Biological Oxidation

1
Chapter 6Biological 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

2
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.

3
Principles of Reduction/Oxidation (Redox)
Reactions

• Redox reactions involve the transfer of electrons
  from one chemical species to another.

4
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

5
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

6
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 -----gt NAD H2O

7

• 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'

8

• 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 ------gt 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.

9
The final piece of the puzzle
Electron transport and Oxidative phosphorylation
Take a deep breath and push on
10
Major Energy Pathways
Galactose Fructose Mannose
Anaerobic
pyruvate
Lactate
Amino Acids
Aerobic
Oxidative phosphorylation
11
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
12
Anatomy of Mitochondria
Mitochondria are composed of a dual membrane
system
Outer Porous to all molecules lt 10 kDa
Inner Transporter-dependent transport
13
(No Transcript)
14

• 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.

15
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)

16

• 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.

17

• 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

18

• 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

19

• 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.

20
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.
21
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

22

• 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.

23

• 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

24
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

25

• 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

26

• The heme of cytochrome a is slightly different
  than that of cytochrome c, having a long,
  hydrophobic side chain

27

• 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.

28
ATP synthase

29
How is ATP made?
FoF1 ATPase Complex (ATP Synthase)
1. An ATP making machine
2. Driven by a proton gradient
3. Attached to the inner mitochondria membrane
30
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.
31
3 non-equivalent sites
H
Matrix
F1
FO
Intermembrane space
FOF1 ATPase (ATP Synthase)
Binding-Change Model
32
ab
(ADP and Pi bind)
ADP Pi
F1
Open Site
(ATP is released)
(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.
33
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 intergers based on the
coupling site model of ATP synthesis
34
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

35
Inhibitors and Uncouplers
Any compound that stops electron transport will
stop respirationthis means you stop breathing
Electron transport can be stopped by inhibiting
ATP synthesis
An uncoupler breaks the connection between ATP
synthesis and electron transport
36
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
37
(No Transcript)
38
2,4-dinitrophenol a proton ionophore
H
H
Inner Membrane
Matrix
39
Brown Adipose Tissue
Uncoupling a proton gradient from FOF1
ATPase Produces Heat!
40
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)

41
Control of Oxidative phosphorylation
What makes us breathe faster?
How does ATP synthesis in the mitochondria adjust
to the needs of the cell?
42
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.
43
WHAT IS THE ATP MASS ACTION RATIO?
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
44
Control of Oxidative Phosphorylation
Keq
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/ADPPi
45
Control of Cytochrome Oxidase (Cox)
Keq

NADH
Mass Action ration
equilibrium
Stimulates Cox
c2/c3
NADH
equilibrium
Stimulates Cox
ADP
c2/c3
equilibrium
Stimulates Cox
ATP
c2/c3
equilibrium
Suppresses Cox
Cytochrome oxidase controls the rate of O2 uptake
which means this enzyme determines how rapidly we
breathe.
46
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 a-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
a-ketoglutarate. This provides a pool of
a-ketoglutarate for the aforementioned
antiporter. The aspartate which is also produced
is translocated out of the mitochondrion.
47
Oxygen Radicals
Partially reduced oxygen species
Molecular Oxygen
.
.
.
.
O2
O O
.
.
.
.
O2
Octet Rule
Superoxide Anion
48
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
49
EXAMPLES OF FREE RADICALS
Hydrogen atom
Superoxide (oxygen centered)
Hydroxyl radical (most reactive)
Nitric Oxide
50
PRO-OXIDANTS
(Generates Free Radicals)
Generates hydroxyl radical
Fe2 H2O2
Ascorbic acid Fe2
Generates hydroxyl radical
Paraquat
Generates superoxide radical
Generates superoxide radical
Agent Orange
Generates hydroxyl radical
Ozone
51
WHAT ARE ANTIOXIDANTS?
ENZYMES
O2-
Superoxide dismutase
Catalase
H2O2
R-OOH
Peroxidases