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 containing ATP synthase (outside)

• Solution of acrydine orange (AO) (membrane-permeable fluorescent dye)

• Add ATP, vesicle interior becomes acidic • AO AO+, trapped inside vesicle • High concentration 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 H218O incorporated into HPO42- in the presence of the ATP synthase Without unidirectional H+ flow, ADP + Pi ATP + H2O occurs reversibly, implying that the application of energy 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

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

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]

(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-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