F1.Fo ATPase

1

• F1.Fo ATPase
• ATP synthase (or F1.Fo-ATPase) couples the back
  diffusion of H from the P-phase to the N-phase
  to the production of ATP.
• - Maintains ATP ? ADP Pi mass action ratio ten
  orders of magnitude away from equilibrium.
• Closely related ATPases (V1.Vo-ATPase) utilize
  the hydrolysis of ATP to pump protons.
• Proton gradient used to increase acidification
  levels.
• F1.Fo-ATPase can also be driven in reverse
  direction.
• Control mechanisms switch it off when DH 0.
• A highly conserved protein present in
  mitochondria, chloroplasts, and both aerobic and
  photosynthetic bacteria.

2

• Electron microscopy
• Observe knobs projecting from the matrix side of
  the membrane.
• When submitochondrial particles are washed with
  urea the knobs were lost from the membrane.
• The stripped submitochrondrial particles were
  incapable of ATPsynthesis.
• The membrane bound portion (Fo) acted as
  proton-gradient uncoupler.
• The knobs (or Fraction 1 F1-ATPase) could not
  synthesize ATP but catalyzed the conversion of
  ATP to ADP.
• The active site for ATP synthesis lies in the
  F1-ATPase domain.
• - After reconstruction, ATP synthesis was
  reproduced when incorporated with a proton pump.
• ATP synthesis is coupled to proton pumping.

3

• Subunits of F1 ATP-synthase
• F1-ATPase (soluble) contains three copies of
  subunit a and three of subunit b, and one each of
  g, d and e.
• Three active sites (one in each of the b
  subunits) were recognized by sequence comparisons
  and mutation studies.
• Three additional ATP binding sites (one in each
  of the a subunits) don't appear to be functional.
• The b subunit may (sometimes) have ATPase
  activity in the absence of the other subunits.
• g subunit acts as a stalk with e associated.
• d subunit contributes to the stator.

4

• Subunits of Fo ATP-synthase
• Fo-ATPase contains one copy of subunit a, two
  copies of subunit b, and ten (or eleven or twelve
  ....) copies of subunit c.
• Contains one glutamic acid (or aspartic residue)
  within an otherwise hydrophobic sequence
• Believed to lie in the middle of the bilayer.
• Compare with other proton pumps, eg.
  bacteriorhodopsin or cytochrome c oxidase.
• All subunits are required to create a proton
  pumping channel.

5

• Active site hydrolysis of ATP
• ATP hydrolysis is more easily studied than ATP
  synthesis.
• 18O labeled waters (oxygens in red) were used and
  showed
• At low ATP significant phosphate produced with
  two 18 0 atoms.

6

• Boyer's interpretation
• The hydrolysis of ATP to ADP at the F1-ATPase
  active site is (to some extent) reversible even
  without input energy.
• In solution the reverse reaction is
  undetectably slow.
• The rate of release of ADP from the active site
  is slow relative to the rate of resynthesis of
  ATP at the active site.
• Tightly bound ADP and Pi form ATP with little
  change in DG.
• How can you make ATP without input energy?
• You don't make free ATP but rather bound ATP.
• ATP binding energy is perturbed so as to obey
  thermodynamics (hence the 57 kJ/mol cost of ATP
  production is recovered).
• More complex experiments indicated ATP binding
  at one site enhanced the release of ADP from
  another.
• - A conformational change in the protein changes
  in the binding affinity of ATP so as to release
  ATP.

7

• The structure of F1-ATPase
• The a and b subunits are arranged symmetrically
  like an orange.
• Subunit g passes through the middle.
• Three active sites observed with three different
  substrates
• ''Open'' empty site.
• ''Loose'' site, with bound ADP.
• ''Tight'' site, with bound AMP-PNP (a
  non-hydrolysable ATP analogue).
• Suggested that the enzyme operates by rotational
  catalysis.
• - Rotation of the g subunit inside the a3-b3
  hexamer facilitates the binding of the substrate
  and the release of the product.

8

• Observation of rotation of ATP
• Each b subunit was engineered to contain a large
  his-tag, which was bound to a nickle surface.
• The g subunit was engineered to contain a
  biotinylated cysteine.
• A flourescent actin rod (about 1 mu long) was
  attached (through the biotin) to the subunit g.
• In the presence of ATP the fluorescent rod
  rotated (observed through a video
  camera/microscope).

• - Without ATP present it moved randomly.
• Hydrolysis of ATP by F1-ATPase causes g subunit
  to rotate.
• - Fo-ATPase acts as a biological windmill.
• Rotation transferred by the g subunit and
  enables ATP to be released from the active site.

9
Movie from a Japanese group
10

• Partial structure of Fo-ATPase
• Crystals structure from S. cerevisiae
  mitochondria
• An (almost) symmetric ring of 10 c subunits.
• Each subunit an a-helical hairpin (so have an
  inner an outer ring of 10 a-helices)
• The outer a-helices are slightly kinked inwards
  at the centre.

11

• Interhelical loops of six to seven subunits in
  close contact with the F1-ATPase g d central
  stalk subunits.
• - Suggests the c-ring stalk rotate together
  during catalysis.
• Subunits a b were not observed (lost during
  crystallisation).
• - No visible proton-translocation pathway.
• - No visible stator which counters the tendency
  of a3-b3 to co-rotate with g.

12

• Side-chains could not be unambiguously assigned.
  
• - Length of helices cross-linking studies used.
• - Conserved Aspartate/Glutamate would lie about
  halfway along the outer-ring a-helix.
• Stock, Leslie Walker, Science 286, 1700-1705
  (1999) (Most important references cited within
  this paper).

13

• Mechanistic implications
• C-terminal a-helix contains the conserved
  Asp/Glu essential for proton translocation.
• - Probably lies on the outside surface the a/c
  subunit interface forms the proton translocation
  pathway.
• Ten (rather than nine or twelve) c-subunits
  visible in Fo-ATPase.
• Suggests the H/ATP stoichiometry is
  non-integral.
• Non-intiger for F1 and Fo subunits suggests a
  low-friction (no deep energy minima) rotation
  mechanism.
• Rotation fueled by the proton gradient.
• - Protonation/deprotonation pathways enabling the
  c-ring to slip past the a-subunit could provide a
  rotation mechanism.

14

• ATP/ADP Carrier
• ATP resynthesis occurs in the mitochondrial
  matrix.
• ATP is exported into the cytoplasm ADP is
  imported into the matrix.
• One-to-one stoichiometry.
• Exchange is accomplished by a single protein,
  the ADP/ATP carrier.
• Called mitochondria complex VI.
• Human bovine isoforms have 90 sequence
  identity Yeast 50.
• Functional dimer.
• All ADP/ATP carriers exhibit a consensus
  sequence, RRRMMM, that is absent from other
  mitochondrial carriers.
• ADP/ATP carrier is a paradigm for mitochondrial
  carriers.

.
15

• Structure of the ADP/ATP carrier
• Six transmembrane a-helices tilted relative to
  the membrane to each other.
• Form a barrel define a cone-shaped depression
  accessible from the outside.
• Cavity has a diameter of 20 Å and a depth of 30
  Å.
• The nucleotide carriers signature (RRRMMM) is
  located at the bottom of this depression.
• - Transport substrates bind to the bottom of the
  cavity.
• - Translocation requires a transition from a
  pit to a channel conformation.

16

• Charge distribution
• Asymmetric distribution of charges within the
  cavity.
• ADP/ATP carrier signature, RRRMMM, spans the
  thinnest part of the protein in a strategic
  location for the transport.
• Attraction of ADP towards the matrix against an
  electrostatic potential is aided by the
  distribution of positive charges within the
  protein cavity.

17

• ADP ATP binding sites
• CATR (an inhibitor binds where ADP binds) was
  located deep in the cavity off the
  pseudo-threefold axis.
• Bound by many hydrogen bonds.
• Tight binding of CATR explains why its a lethal
  poison.
• The conformation of the carrier for ATP binding
  from the matrix is probably different from the
  ADP-binding conformation.
• - ATP binding site spectulated.

18

• Mechanism
• Functions as an active dimer.
• Each monomer can bind either ADP from the
  outside or ATP or from the inside.
• Transport takes place upon cooperative ADP ATP
  binding.
• Nucleotide binding favours binding of a second
  nucleotide from the opposite side.
• ATP binding can desabilize the salt bridges
  induce conformational changes.
• Prolines may act as hinges which straighten
  odd-numbered helices pull open the channel.
• The MMM motif occupies a bulky volume that may
  act as a plug.
• ADP/ATP transport in mitochondria also depends
  upon the membrane potential nucleotide
  concentrations.

19

• Unanswered questions
• What is the structure of the stator which
  prevents the free rotation
• of the F1 domain?
• What is the structure of the proton
  translocation pathway?
• How does this proton-pump act in reverse so as
  to capture a proton wind'' and drive a
  rotational motion?
• Do underlying principles from bacteriorhodopsin/c
  ytochrome c oxidase relate to proton pumping by
  Fo-ATPase?
• What is the second conformation of the ADP/ATP
  carrier?