Inhibitors and Uncouplers
Any
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
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
Text p519
Brown Adipose
Tissue
Uncoupling
a proton gradient
from FOF1 ATPase
Produces Heat!
Thermogenin
Staying Alive Energy Wise
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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?
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+]
Go’= 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.
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
VITAMINS
Vitamin E (tocopherols)
Beta Carotene (pro-vitamin A)
Vitamin C
Quiz 1
4 Questions
5 Points
1. Other than Fructose-6-PO4 and Fructose-1,6
bisPO4, name another phophate ester of fructose.
Fructose 1-PO4 or Fructose 2,6-bisPO4
2. Other than glycerate-1,3 bisPO4, name another high
energy intermediate derived from glucose in glycolysis.
PEP
3. Name a compound in the Krebs cycle, which when
oxidized to CO2 and H2O gives rise to 30 ATPs
Succinyl-CoA
4. Name 2 pentoses that are found in the pentose
phosphate pathway.
Ribose-5-PO4 Ribulose-5-PO4 Xylulose-5-PO4