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Proton gradient generation by ETS

Endergonic process
Free energy change of transporting H+ from mitochondrial matrix
to intermembrane space (against membrane potential &
electrochemical gradient) is expressed as:

G = 2.3 RT [pH(in) - pH(out)] + ZF

Where Z= charge on the proton


F = Faradays constant
= membrane potential
Passage of approx. 3 protons is required to synthesize 1 equiv of ATP
plus one proton needed to transport ATP out and ADP in for total of 4.
We will see that this process is about 50% efficient = energy out/possible
(Top) A representation of the complexes of the mitochondrial respiratory chain and the proton circuit
that links the respiratory chain complexes with the ATP synthase (Bottom) Equivalent circuit

Published by AAAS D. Nicholls, Sci. Aging Knowl. Environ. 2002, pe12 (2002)
How mitochondrial function changes with cellular energy demand.
Note that Reactive Oxygen Species (ROS) is highest with low energy production!!
The amount of information you receive is proportional to how surprised you are

D. Nicholls, Sci. Aging Knowl. Environ. 2002, pe12 (2002)

Published by AAAS
Electron Micrographs of Mitochondrial Membrane

F1 Particles, sonication inverts

Lollipop structures extending


into mitochondrial matrix are
F1 ATPase particles
ATP synthase protein complex

Two functional F1 and F0 subunits:

F1 = water soluble peripheral membrane protein


composed of 5 types of subunits
9 units of 33 component
subunit contains catalytic site for ATP synthesis
subunit is required for binding F1 to F0

F0 = water insoluble transmembrane protein


contains as many as 8 different subunits (E. Coli has 3)
and a proton translocation channel
Electron microscopy of E. Coli ATP synthase (F1Fo-ATPase)
F1
subunit

Peripheral connector

stalk

F0
subunit

Dumbbell-shaped structure
F1 and F0 joined by 45 A central stalk & a less substantial
peripherally located connector
X-ray structure of F1-ATPase from bovine
heart mitochondria

3340-residue (371 kDa) protein

80 A high

30A-long
stem
F1 and subunits are 20%
90 A-long -helix of -subunit identical, but have nearly
protrudes into 15A deep dimple identical folds
centrally located at the top of the
& arranged alternately
sphere
The C subunits of F0 form a transmembrane ring that contacts the F1 stalk

E.Coli F0 subunit contains three


types of transmembrane subunits, a,
b, c
Forms an a1b2c9-12 complex

Hydrophobic c subunits
associate to form a ring
with the ab2 subunit at its
periphery
Protons move through a/c interface and cause rotation of c/
Binding change mechanism of ATP synthesis

Three phases:

(1) Translocation of protons carried out by F0

(2) Catalysis & formation of the phosphoanhydride bond of


ATP carried out by F1.

(3) Coupling of the dissipation of the proton gradient with ATP


synthesis, requiring interactions of F1 with F0.
Energy-dependent binding change mechanism for ATP synthesis

F1 has 3 chemically identical but conformationally distinct interacting protomers.


O = open conformation, catalytically inactive
L = loose binding for ligands, catalytically inactive
T = tight binding for ligands, catalytically active
Energy-dependent binding change mechanism for ATP synthesis
(Be sure to understand this)
ATP synthesis takes place in 3 steps (Several protons required/step!!):
1. Binding of ADP + Pi to L site
2. Energy-dependent conformational change converting binding site L
to T, T to O, and O to L
3. Synthesis of ATP at T site and release of ATP from O site

Enzyme returns to its initial state after 2 more passes of this reaction sequence
Energy that drives conformational change is transmitted to the catalyic 33
assembly via rotation of the assembly (= in E.coli nomenclature)
Model of the E. Coli F1F0-ATPase
Rotor = -c12 ring
complex
Stator = ab2-33
complex

Protons entering from the


outside bind to c subunit
where it interacts w/ a
subunit & exit to the inside
after the c ring has made a
nearly full rotation

The b2 complex functions


to prevent the 33
assembly from rotating
with the subunit
Dicyclohexylcarbodiimide

Lipid-soluble carboxyl reagent that inhibits proton transport


through mammalian Fo by reacting with a single Glu residue on one
side of the Fo subunits.

Implies that a carboxylic acid group is located in the lipid


environment buried in the membrane and still involved in proton
transport.
bacterial nomenclature, same
as the mitochondrial matrix
Binding changes are driven by the
rotation of the catalytic 33
assembly with respect to other
portion of the F1F0-ATPase

subunit, thought to rotate w/in the


fixed 33 assembly, acts as a
molecular cam shaft in linking the
proton gradient-driven rotational
motor to the conformational
changes in the catalytic sites of F1.

Three protons per step, 12 protons


per turn = # C subunits. Proton
coming in from intermembrane
space requires a full turn to come
out in the matrix.
4 H+/ATP and
2.5 ATP/NADH
Stoekenius/Racker
Reconstitution Exp.
Only two proteins
reconstituted in
synthetic
phospholipid vesicles.
Bacteriorhodopsin
generates proton
gradient and F1F0
makes ATP.
Best proof of Mitchell
Hypothesis that
convinced the few
remaining doubters.
Rotation of the -c ring with
respect to the ab2-33 stator
elegantly demonstrated using
His-tagged subunits &
fluorescent labels

F1F0-ATPase converts
chemical to mechanical energy
w/ nearly 100% efficiency

Observation of the rotation of


a 3.6 m-long actin filament
in the presence of 5 mM
MgATP seen through
fluorescence microscope
Uncoupling of oxidative phosphorylation
e- transport and oxidative phosphorylation (ATP synthesis)
normally tightly coupled due to impermeability of the
inner mitochondrial membrane to the diffusion of H+.
Thus only way for H+ to re-enter the mitochondrial matrix
is through the F0 subunit of ATP synthase

Uncoupling of H+ flow back to the matrix & ATP


synthesis by compounds such as 2,4 dinitrophenol (DNP),
or FCCP (carbonyl cyanide-p-
trifluoromethoxyphenylhydrasone)
Renders the mitochondrial membrane permeable to H+
Binds protons on the acidic side of the membrane &
diffuse through
Uncouplers are proton-transporting ionophores
Hormonally controlled uncoupling in Brown adipose
tissue to generate heat,
called nonshivering thermogenesis
Mechanism of heat generation in brown fat cells involves regulated
uncoupling of oxidative phosphorylation in their mitochondria

These mitochondria contain a channel protein called thermogenin (or


uncoupling protein UCP1), homodimer of 307-residue subunits
Acts as a channel to control permeability of the inner mitochondrial membrane
to protonsdumps proton gradient anf waste heat rises from 50% to 100%.
UCP2 and UCP3 may help to control weight

In cold adapted animals, thermogenin constitutes 15% of brown fat inner


mitochondrial membrane proteins (cold exposureor exercise--also increases
the NUMBER of mitochondria!)
Flow of H+ through thermogenin channel inhibited by physiological
concentration of purine dinucleotide/trinucleotide ratios: (ADP/ATP,
GDP/GTP)
But inhibition is overcome by free fatty acidsfat helps keep you warm
Heat generation in brown fat mitochondria activated by
free fatty acids
FFA counteract inhibitory effects of purine dinucleotides,
thereby stimulating H+ flux through the thermogenin
protein channel
Uncouples e- transport from oxidative phosphorylation
Concentration of free fatty acids in brown adipose tissue
controlled by the hormone norepinephrine w/ cAMP as
secondary messenger