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ATP synthesis : The F1F0-ATPase
Hypotheses on the mechanism of ATP synthesis in mitochondria:
“Substrate level phosphorylation” -- coupling of ATP synthesisto an enzymatic reaction (as in glycolysis--requires a high-energyphosphate bond)
Stress membrane conformation -- evidence: differences in membranestructure 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) vesiclescapable of ATPsynthesis--note theheadpiece of the “F0F1 ATPase”(ATP synthase)
Removal of theheadpieces givesvesicles that can’tmake ATP
Adding purifiedheadpieces re-stores the ability tomake ATP
“Chemiosmotic hypothesis” (Peter Mitchell): H+ passage across membrane powers ATP synthesis How does it work? Clues: 1. Reversed coupling: ATP hydrolysis powers H+ flow.
• Membrane vesicle containin g ATP synthase (outs )ide
• Solution of acryd ine orange (A )O (membrane-permea ble fluores cent dye)
• Add AT ,P vesicle interior becomes acidic • AO AO+, trapped inside vesicle • Highconcentration of AO quenches fluorescence
AOAOAO
AOAO
AO+
ATP ADP + Pi
H+
AOAO
AO+
AO+
AO+
AO+
Add ATP
ADP + Pi ATP
H+
Add ADP
H+ H+H+
H+
Light
Bacteriorhodopsin
H+
H+
H+ H+
2. H+ flow in artificial vesicle powers ATP synthesis.
3. Uncouplers — compounds that permeabilize the mitochondrial inner membrane to H+ inhibit ATP synthesis (and allow rapid oxidationof NADH and substrates like pyruvate)
FCCP
H+ H+
FCCP
H+
H+
H+
H+
FCCP-H+
(FCCP: carbonylcyanide p-trifluoromethoxyphenylhydrazone)
cytosol
intermembranespace
matrix
4. Reversible oxygen exchange 18O from H2
18O incorporated into HPO42- in the presence of the ATP synthase Without unidirec tional H+ flow, ADP + Pi ATP + H2O oc curs reve rs ibly, implying tha t the ap plicat ion of ener gy is not at the formation of the ADP~P bond( !)
Note the rotor (base) and stator (head) and rotation
Hypothesis: ADP + Pi ATP + H2O occurs on head; H+ flow turns rotor; rotor/rotation stimulates ATP release
F0
F1
rotorrotation
rotorrotation
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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
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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 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 subunits in the Fo ring in the membrane. [See Science 330:12 (1 Oct 10) or PNAS 107:16823]
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(How many ATP per FADH2?)
4 4 2
ATP from cytoplasmic NADH There is a problem with NADH from glycolysis: inner mitochondrial membrane is not permeable to NADH
Glycerophosphate shuttle (animals):
Electron input at Complex II FAD: expect 1.5 ATP/NADH
Malate-OAA shuttle (plants)
NAD+ NADH Periplasm: malate OAA Matrix: malate OAA
NAD+ NADH
Malate-aspartate shuttle (animals and plants)
How much ATP/NADH?
Summary
| Mitochondrial electron transport produces H+ gradient
| H+ gradient rotates the ATP synthase | ATP synthase rotation forces ATP synthesis
(release) | NADH reducing power from cytoplasm must be
shuttled into the mitocondrion | ATP/glucose ratio depends on shuttle and on Fo