Chapter 14 (part 2)
Oxidative phosphorylation
Proton Motive Force (Dp)
• PMF is the energy of the proton concentration gradient
• The chemical (DpH= pHin – pHout) potential and the
electrical potential(DY= Yin – Yout) contribute to PMF
•
•
DG = nfDY and DG = –2.303nRT DpH
DG for transporting 1 H+ from inner membrane space
to matrix = DG = nfDY –2.303nRTDpH
•
Dp = Dp = DG/nF
•
Dp = Dy –(0.059)DpH
Proton Motive Force (Dp)
• What contributes more to PMF, DY or DpH?
• In liver DY=-0.17V and DpH=0.5
•
Dp = Dy –(0.059)DpH = -0.17-(0.059)(0.5V)
•
Dp = -0.20 V
•
DY/Dp=(-0.17V/-0.20V) X 100% = 85%
• 85% of the free energy is derived form DY
Proton Motive Force (Dp)
• How much free energy generated from
one proton?
•
DG = nFDP = (1)(96.48kJ/Vmole)(-0.2V)
= -19 kJ/mole
• To make 1 ATP need 30 kJ/mole.
• Need to translocate more than one
proton to make one ATP
• ETC translocates 10 protons per NADH
ATP Synthase
• Proton diffusion through the protein drives
ATP synthesis!
• Two parts: F1 and F0
Racker & Stoeckenius
confirmed Mitchell’s
hypothesis using vesicles
containing the ATP
synthase and
bacteriorhodopsin
Binding Change Mechanism
•
•
•
•
ADP + Pi <-> ATP + H2O
In catalytic site Keq = 1
ATP formation is easy step
But once ATP is formed, it binds very
tightly to catalytic site (binding constant
= 10-12M)
• Proton induced conformation change
weakens affinity of active site for ATP
(binding constant = 10-5)
Binding Change Mechanism
•
•
•
•
Different conformation at 3 catalytic sites
Conformation changes due to proton influx
ADP + Pi bind to open-site in exchange for ATP
Proton driven conformational change (loose site)
causes substrates to bind more tightly
• ATP is formed in tight-site.
• Requires influx of three protons to get one ATP
ATPase is a Rotating Motor
• Bound a,b,g subunits
to glass slide
• Attached a
fluroescent actin
chain to g subunit.
• Hydrolysis of ATP to
ADP + Pi cause
filament to rotate
120o per ATP.
How does proton flow cause
rotation?
Active Transport of ATP, ADP
and Pi Across Mitochondrial Inner
Membrane
• ATP is synthesized in the matrix
• Need to export for use in other
cell compartments
• ADP and Pi must be imported into
the matrix from the cytosol so
more ATP can be made.
• Require the use of transporters
Transport of ATP, ADP and Pi
• Adenine nucleotide translocator = ADP/ATP antiport.
• Exchange of ATP for ADP causes a change in DY due to
net export of –1 charge
• Some of the energy generated from the proton gradient
(PMF) is used here
• Pi is imported into the matrix with a proton using a
symport.
• Because negative charge on the phosphate is canceled by
positive charge on proton no effect on DY, but effects
DpH and therefore PMF.
Transport of ATP, ADP and Pi
• NRG required to export 1 ATP and import 1
ADP and 1 Pi = NRG generated from influx of
one proton.
• Influx of three protons required by ATPase to
form 1 ATP molecule.
• Need the influx of a total of 4 protons for
each ATP made.
P/O Ratio
• The ratio of ATPs formed per oxygens reduced
• e- transport chain yields 10 H+ pumped out per
electron pair from NADH to oxygen
• 4 H+ flow back into matrix per ATP to cytosol
• 10/4 = 2.5 for electrons entering as NADH
• For electrons entering as succinate (FADH2),
about 6 H+ pumped per electron pair to oxygen
• 6/4 = 1.5 for electrons entering as succinate
Regulation of Oxidative
Phosphorylation
• ADP is required for respiration (oxygen
consumption through ETC) to occur.
• At low ADP levels oxidative phosphorylation low.
• ADP levels reflect rate of ATP consumption and
energy state of the cell.
• Intramolecular ATP/ADP ratios also impt.
• At high ATP/ADP, ATP acts as an allosteric
inhibitor for Complex IV (cytochrome oxidase)
• Inhibition is reversed by increasing ADP levels.
Uncouplers
• Uncouplers disrupt the tight coupling between
electron transport and oxidative phosphorylation
by dissipating the proton gradient
• Uncouplers are hydrophobic molecules with a
dissociable proton
• They shuttle back and forth across the
membrane, carrying protons to dissipate the
gradient
• w/o oxidative-phosphorylation energy lost as
heat
• Dinitrophenol once used as diet drug, people ran
107oF temperatures
H
NO2
O2N
OH
NO2
O2N
O
Physiological Uncoupling
• Uncoupling of ETC and Ox-phos occurs in
animals as a means to produce heat =
nonshivering thermogenesis.
• Impt. In hibernating mammals, neborn animals
and mammals adapted to cold
• Occurs in brown adipose tissues (rich in
mitochondria)
• Uncoupling protein (UCP) = channel to allow
influx of protons to matrix (dissipates proton
gradient)
Uncoupling in Plants
• Plants generate heat during fruit ripening
and to emit odors to attach pollinators.
• Plants can by pass normal ATP generating
ETC
• Alternative ETC in plants does not pump
protons, just transfers electron.
• All plant have this pathway, actual
physiological reason not known