Conserved acidic residue in the c-subunit: is it water
accessible?
The conservative Glu (or Asp, in case of E. coli) residue
essential for the proton translocation through FO is
situated on the second transmembrane helix of subunit c
somewhere in the lipid bilayer. The data on E. coli enzyme
indicate that the conservative acidic residue is buried in the
hydrophobic layer, while the experimental evidence obtained on the P.
modestum sodium-translocating ATP synthase suggests that it is
water-accesible from the negatively charged side of the coupling
membrane.
The question has further implications, for each case favors slightly
different functional mechanism. If the proton-binding site is
inaccessible on most of the c-subunits, then the
functional mechanism proposed by Junge (1997) is more correct:
after the proton enters through one half-channel, it goes almost 360
degrees bound to the c-subunit, and only then is
released through the other half-channel. On the contrary, if the
proton-binding sites are water-accessible, the one-channel mechanism
proposed by Dimroth (1998) is more correct.
The very top of the Gamma subunit: fixed or free?
It was shown recently in W. Junge's group that neither truncation
nor S-S cross-linking of the gamma-subunit C-terminal region (up to 12
residues from the C-terminus) seriously hinders catalysis. So far it
was generally accepted that gamma subunit C-terminus is rotating in a
"molecular bearing" composed of the 6 beta-barrels at the "top" of the F1.
New data suggest that the gamma subunit C terminus can be
firmly fixed in the beta-barrel "crown" at the "top" of the F1.
The rotation in such a case is presumably going around covalent bonds
in the alpha-helical region of the gamma subunit below the fixed
C-terminal region.
H+/ATP ratio
How many protons should be transported through ATP synthase to
make one ATP molecule? Does this number differ from organism to
organism? Is it constant at all? Sometimes this hot point is narrowed
to the question "3 or 4?"
Stoichiometry of c-subunits
10? 11? 14? 12? Different for different organisms? Variable
amount? May be it depends on the state of the cell?
Elasticity
How can the energy of several consequently transported protons be
stored to serve as input for catalytic act? How can 10 c-subunit
oligomer be coupled with 3-site catalytic F1? A possible
explanation is an elastic transmission between proton transporting
machinery and catalytic headpiece. After each subsequent proton is
transported, the elastic tension is built up in the enzyme. When this
tension reaches a certain threshold value, ATP is released and the
enzyme relaxes. I love this hypothesis, but I have to admit that there
is no firm experimental evidence for it so far.
Hydrolysis/Synthesis: different pathways?
Many inhibitors of ATP hydrolysis do not markedly affect ATP
synthesis. Moreover, concentration of phosphate, one of the ATP
hydrolysis products, has no effect on the hydrolysis rate, which is
very strange in terms of classical enzymology. It is quite possible
that ATP synthase undergoes a major conformational change when
switching from synthesis to hydrolysis. It is also possible that the
reaction pathway can differ for synthesis and hydrolysis.
Conformational changes upon energization: are the
synthesis/hydrolysis conformations of the enzyme identical?
It was shown by electron microscopy that upon the energization of
the membrane ATP synthase changes it's conformation: the F1
headpiece shifts from the plane of the membrane by as much as 10
Angstrom (1,
2).
The mechanism and the details of the transition are still unclear.
