f1fo atp synthase

8/9/2019 F1Fo ATP Synthase

1/29

F1Fo ATP Synthase

Copyright 1999-2004 by Joyce J. Diwan.

All rights reserved.

Molecular Biochemistry I


2/29

F1F

oATP Synthase of mitochondria, chloroplasts, bacteria:

When the electrochemical H+ gradient is favorable,

F1Fo couples ATP synthesis to spontaneous H+

fluxtoward the side of the membrane where F1protrudes(e.g., toward the mitochondrial matrix).

If there is no (pH or(= to drive the forward reaction,

Keq favors the reverse, ATP hydrolysis (ATPase).

ADP+Pi ATP

F1

Fo

3H+

ATPase with H+

gradient dissipated

matrix

intermembranespace

ADP + Pi ATP

F1

Fo

3 H+matrix

intermembranespace

ATP synthesis with (pH & (=

+ + +


3/29

Roles of major subunits were established in studies ofsubmitochondrial particles (SMP).

If mitochondria are treated with ultrasound, the innermembrane fragments and reseals as vesicles, with F1 onthe outside. Since F1 of intact mitochondria faces theinterior matrix space, these SMP are said to be inside out.

By EM with negativestaining, F

1appears

as "lollipops" on the

inner mitochondrial

membrane, facing thematrix.

mitochondrionultrasound

SMF1


4/29

If F1

is removed from SMP, the membrane containing Fobecomes leaky to H+.

Adding back F1 restores normal low permeability to H+.

Thus Fo includes a proton channel.

F1, the catalyticsubunit, if separatedfrom SMP catalyzesATP hydrolysis (the

spontaneous reactionin the absence of anenergy input).

mitochondrionultrasound

SMPF1


5/29

Inhibitors of F1Fo, that block H+ transport coupled

to ATP synthesis or hydrolysis, include:

oligomycin, an antibiotic

DCCD (dicyclohexylcarbodiimide), a reagentthat reacts with carboxyl groups in hydrophobicenvironments, forming a covalent adduct.

Eitheroligomycin or DCCD blocks the H+ leakin

membranes depleted of F1.

Thus oligomycin and DCCD inhibit the ATPSynthase by interacting with Fo.


6/29

Looking down at the

membrane, they are arrangedas a ring of alternating E & Fsubunits. (K to be discussedlater.)

The complete subunit composition of the ATP Synthase

was first established in E. coli, which has an operon thatencodes genes for all subunits.

F1 in E. coli consists of 5 polypeptides with stoichiometry

E3,F3,K,H,I (named in order of decreasing mol. weights).E & F subunits (513 & 460 aa in E. coli) are homologous.

E E

E

F

F FK

F1 in cross

section


7/29

There are three nucleotide-binding catalytic sites,located at EF interfaces but predominantly involvingresidues of theF subunits.

Each of the three E subunits contains a tightly boundATP, but is inactive in catalysis.

Adenine nucleotides bind to E & F subunits with Mg++.

E E

E

F

F FK

F1 in cross

section


8/29

Fo is a complex of integral membrane proteins. The stoichiometry of subunits in E. coli Fo is a, b2, c10.

Mammalian F1Fo is slightly more complex than the

bacterial enzyme.Since names were originally assigned based only onapparent MW, some subunits were given different namesin different organisms.

Bovine H subunit is homologous to E. coli I subunit. Bovine "OSCP" is homologous to E. coli H subunit.

Bovine I subunit is unique.


9/29

The binding change mechanism of energy coupling wasproposed by Paul Boyer.

He shared the Nobel prize for this model that accountsfor the existence of3 catalytic sites in F1.

For simplicity, only the catalyticF subunits are shown.

It is proposed that an irregularly shaped shaft linkedto Fo rotates relative to the ring of 3 F subunits.

The rotation is driven by flow ofH

+

through Fo.

ADP+Pi ATP

ATP

ATP ADP

+PiATP ADP

+ Pi

ATP

opentight

binding

loosebinding

repeat

Binding Change Mechanism


10/29

ADP+Pi ATP

ATP

ATP ADP

+PiATP ADP

+ Pi

ATP

opentight

binding

loosebinding

repeat


The conformation of eachF subunit changes sequentiallyas it interacts with the rotating shaft.

EachF subunit is in a different stage of the catalytic cycleat any time. E.g, the green subunit sequentially changes to:

a loose conformation in which the active site can looselybind ADP + Pi a tight conformation in which substrates are tightly

bound and ATP is formed

an open conformation that favors ATP release.


11/29

The K subunit includes a bent helical loop that constitutesa "shaft" within the ring ofE & F subunits.

Shown is bovine F1 treated with DCCD to yield crystalsin which more of the stalk is ordered, allowing structure

determination. Colors: E, F, K, H, I.

F1 ATPase PDB 1E79

side view base view

Supportingevidence:

1. The crystalstructure ofF1 was solvedby J. Walker,

who sharedthe Nobelprize.


12/29

Note the wide base of the rotary shaft, including part ofK as well as H and I subunits.

Recall that the bovine H subunit, which is at the base of

the shaft, is equivalent to I of bacterial F1.

F1 ATPase PDB 1E79

side view base view

Bovine F1(DCCD-

treated)


13/29

In crystals of F1 not treated with DCCD, less of the shaft

structure is solved, but ligand binding may be observedunder more natural conditions.

The 3F subunits are found to differ in conformation &

bound ligand.

T o vie s o F1 (K subunit red) ile 1 OW


14/29

Bound to oneF subunit is a non-hydrolyzable ATPanalog (assumed to be the tight conformation).

Bound to anotherF subunit is ADP (loose).

The thirdF subunit has an empty active site (open).

This is consistent with the binding change model, which

predicts that each F subunit, being differently affected by

the irregularly shaped rotating shaft, will be in a differentone of 3 stages of the catalytic cycle.

ADP+Pi ATP

ATP

ATP ADP

+PiATP ADP

+ Pi

ATP

opentight

binding

loosebinding

repeat



15/29

Explore with Chime the structure of bovine F1 withbound ADP and AMPPNP.

The non-hydrolyzable AMPPNP is used as a substitute

for ATP, which would hydrolyze during crystallization.

AMPPNP (ADPNP) ATP analog

N

NN

N

NH2

O

OHOH

HH

H

CH2

H

OPOPNP-O

O

O- O-

O O

O-

H


16/29

F subunits of F1 were tethered to a glass surface.

A fluorescent-labeled actin filament (yellow) was attachedto the protruding end of the K subunit (shaft).

Video recordings showed the actin filament rotating like a

propeller. The rotation was ATP-dependent.

EFF

K

Rotation ofK relative to E & F

2. Rotation of the

K shaft relative to

the ring ofE & F

subunits was

demonstrated by

Noji, Yasuda,

Yoshida &

Kinoshita.


17/29

Some observations indicate that each 120o step consistsof90o & 30o substeps, with a brief intervening pause.

Proposals have been made correlating these substepswith particular stages of the reaction cycle, such as ATPbinding and Pi release.

EFF

K

Rotation o K relative to E F

Studies usingvaried techniqueshave shown ATP-induced rotation

to occur in discrete120o steps, withintervening pauses.


18/29

Although the binding change mechanism is widelyaccepted, some details of the reaction cycle are stilldebated.

View an animation of ATP synthesis based onobserved variation in conformation of F1 subunitsattributed to rotation of the K shaft.


19/29

The c subunit of Fo has a hairpin

structure with 2 transmembraneE-helices & a short connecting loop.

The small c subunit (79 aa in E.coli) is also called proteolipid,

because of its hydrophobicity.ne E-helix includes an Asp or Glu

residue whose carboxyl reacts with

DCCD(Asp61 in E coli).

Mutation studies have shown thatthis DCCD-reactive carboxyl, in themiddle of the bilayer, is essentialforH+ transport through Fo.

Asp61

Fo subunit c

PDB 1A91


20/29

The structure of the entire F1Fo

complex has not beendetermined at atomic resolution.

At right: a low resolutionpartial structure of yeast F1

with attached Fo c subunits.View this file by Chime.

Count the number of Fo csubunits, arranged in a ring.

Look for the Asp near themiddle of one transmembranesegment of each c subunit.

Partial

structures

of F1, Fo

PDB 1Q01


21/29

Mitochondrial ATP Synthase E. coli ATP Synthase

These images depicting models of ATP Synthase subunitstructure were provided by John Walker. Some equivalentsubunits from different organisms have different names.


22/29

Mitochondrial F1Fo E. coli F1Fo

The proposed "stator" of this molecular motor consists ofthe ring of 3 E & 3 F subunits of F1, the a subunit of Fo, &several subunits that link these together (b, d, F6, & OSCP

in bovine mitochondrial F1Fo; or 2 b, & H in E. coli).

Each of the 2 Fob subunits is

predicted toinclude 1 trans-membraneE-helix & a very

polar, chargedE-helical domainthat extends outfrom the

membrane.


23/29


Although linking subunits have been identified, structuraldata is still lacking regarding the nature of the connectionbetween the a subunit & the ring ofE & F subunits.


24/29


The proposed "rotor consists of the ring of 10 c, plus

the internal F1 stalk(K, H, I in bovine mitochondria; or

K & I in E. coli).


25/29

The a subunit of Fo

(271amino acid residues inE. coli)is predicted from hydropathyplots, to include severaltransmembrane E-helices.

It has been proposed that thea-subunit forms 2 half-channels or proton wires(each a series of protonatable

groups or embedded waters),that allow passage of protonsbetween the two membranesurfaces & the bilayer interior.

a

subunit

ring of

c subunitsH

+

H+


26/29


27/29

As the ring of 10 c subunitsrotates, the c-subunit carboxyls

relay protons between the2 a-subunit half-channels.

This allows H+ gradient-drivenH+ flux across the membrane

to drive the rotation.

a

subunit

ring of

c subunitsH

+

H+


28/29

Proposed mechanism:R

otation of the ring of c subunitsmay result from concerted swiveling movements of thec-subunit helix that includes Asp61 & transmembranea-subunit helices with residues that transfer H+ to or fromAsp61, as protons are passed from or to each half-channel.

asubunit

ring ofc subunits

H+

H+

Asp61

Fo subunit c

P 1A91

Websitewith some

relevantanimations.


29/29

Recent experimental observations and datasimulations have provided the basis for more

detailed proposals regarding mechanisms by whichprotein conformational changes are linked to protontranslocation and to catalysis.

But many questions remain unanswered.

f1fo atp synthase

Documents