ATP synthesis : The F1F0-ATPase
Hypotheses on the mechanism of ATP synthesis in mitochondria:
“Substrate level phosphorylation” -- coupling of ATP synthesis
to an enzymatic reaction (as in glycolysis--requires a high-energy
phosphate bond)
Stress membrane conformation -- evidence: differences in membrane
structure in mitochondria when provided substrate (pyruvate) or not
“Chemiosmotic hypothesis” (Peter Mitchell): H+ passage across
membrane powers ATP synthesis
ATP synthesis depends on a peripheral membrane protein
Inner mitochondrial
(inside-out) vesicles
capable of ATP
synthesis--note the
headpiece of the
“F0F1 ATPase”
(ATP synthase)
Removal of the
headpieces gives
vesicles that can’t
make ATP
Adding purified
headpieces restores the
ability to
make ATP
ÒChemiosmotic hypothesisÓ(Peter Mitchell): H+
passage across membrane powers ATP synthesis
AO
How does it work?
AO
AO
Clues:
AO
1. Rever sed coupling: ATP hydrolysis powers H+
flow.
AO
AO
AO
Add ATP





Membrane vesicle containing ATP synth ase
+
AOAO
(outside)
+
+
AO H+ +
Solution of acrydine orange (AO)
AO
(membrane-permeable fluo rescent dye)
AO+
Add ATP, vesicle interior becomes acidic
ATP
ADP + Pi
AO AO +, trapped inside vesicle
High concentration of AO quenches fluorescence
2. H + flow in artificial vesicle powers ATP synthesis.
Light
H+
Bacteriorhodopsin
H+ H+
H+
Add ADP
H+ H+
H+
H+
ADP + Pi
ATP
H+
3. Uncouplers — compounds that permeabilize the mitochondrial
inner membrane to H+ inhibit ATP synthesis (and allow rapid oxidation
of NADH and substrates like pyruvate)
cytosol
FCCP
intermembrane
space
FCCP
FCCP-H+
H+
H+
matrix
H+
H+
H+
H+
(FCCP: carbonylcyanide p-trifluoromethoxyphenylhydrazone)
4. Rever sible oxygen exchange
18
O from H218O incorporated into HPO42- in the
presence of the ATP synthase
Without unidirectional H+ flow, ADP + Pi
ATP +
H2O occurs reversibly, implying that the ap plication of
energy is not at the formation of the ADP~P bond(!)
Note the rotor (base) and stator (head) and rotation
F1
F0
Hypothesis: ADP + Pi
ATP + H2O occurs on head;
H+ flow turns rotor; rotor/rotation stimulates ATP release
QuickTime™ and a
decompressor
are needed to see this picture.
rotor
rotation
QuickTime™ and a
decompressor
are needed to see this picture.
rotor
rotation
QuickTime™ and a
decompressor
are needed to see this picture.
Figure 1 Observation system for the c subunit rotation in F0F1.
Y Sambongi et al. Science 1999;286:1722-1724
Movie: http://www.sciencemag.org/site/feature/data/1045705a.mov
Qu i c k T i m e ™ a n d a
T I F F (L Z W ) d e c o m p re s s o r
a re n e e d e d to s e e th i s p i c t u re .
Movie of the ATPase model -- Mechanical part of respiration.
http://multimedia.mcb.harvard.edu/anim_mitochondria.html
Model for H+-induced rotation from the textbook:
What is the P:O ratio?
ATP formed: 1/2 O2 (2 e-) taken up (NADH oxidized)
For TCA cycle, early estimates: per NADH, 3; per
succinate (FAD), 2
4
4
2
Best data: per NADH, 2.5; per succinate (FAD), 1.5
Book: 10 H+ per NADH, 4 H+ per ATP = 2.5
(Related to the Fo structure : 12 H+/Fo rotation, 3
ATP/Fo rotation = 4 H+/ATP; recall 10 H+/NADH;
(10 H+/NADH)/(4 H +/ATP) = 2.5 ATP/NADH)
Recent evidence: H+/ATP varies from 12/3 (4) in
animals to 15/3 (5) in microbes, depending on the
number of Fo subuni ts in the Fo ring in the
membrane. [See Science 330:12 (1 Oct 10) or PNAS
107:16823]
(How many ATP per FADH2?)
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
ATP from cytoplasmic NADH
There is a problem with NADH from glyc olysis: inner
mitochondrial membrane is not permeable to NADH
Glycerophosphate shuttle (animals):
Electron input at Complex II FAD: expect 1.5 ATP/NADH
Malate-aspartate shuttle (animals and plants)
Malate-OAA shutt le (plants)
NAD+
NADH
Periplasm:
malate
OAA
Matrix:
malate
OAA
NAD+
NADH
How much ATP/NADH?
Summary
Mitochondrial electron transport produces H+
gradient
+
 H gradient rotates the ATP synth ase
 ATP synth ase rotation forces ATP synth esis
(release)
 NADH reducing power from cytopl asm must be
shut tled into the mitocondrion
 ATP/glucose ratio depends on shuttle and on Fo
