Transcript Oxidative phosphorylation: ATP synthesis

Section 5:
Bioenergetics
3. Oxidative phosphorylation:
ATP synthesis
10/14/05
The proton-motive force (Dp)
pumping of H+ results in DpH
 since H+ is charged, effect includes net transfer of charge, i.e.,
this H+ transport is electrogenic
 combination of DpH & separation of charge (an electric membrane potential, Em) is known as the proton-motive force (Dp)
 redox-driven

Em: ~ 140 millivolts; more positive outside
DpH: ~ 1.4 pH units lower outside than matrix [H+]out ≈ 25 [H+]m
 this
Dp is a form of energy that is used to make ATP (slides 3-)
+140 mV
pHm - 1.4
H2O
Em
pHm
1
MATRIX
Alberts et al., Fig. 14-19
2e– +
½O2
Dp & ATP synthesis



this Dp equivalent to DG' of ~5.2 kcal/mol H+
since 3 H+ used to synthesize 1 ATP, available free
energy DG'  3 × 5.2  15.6 kcal/mol ATP
sufficient to produce
[ATP]/[ADP] >103
at equilibrium
actual
ratio in resting
muscle ~20

device that couples Dp
to ATP production
called ATP synthase
(composed of F0 & F1 units)
2
Alberts et al.,
Fig. 14-27
ATP synthase
 F1:
catalytic unit
subunits: 33
 subunit has
1 ATP synthesizing site
each

 subunits link
matrix
 pairs to F0
transmembrane H+
conducting channel
subunits: ab2c10
 F0:


3
H+ flow drives rotation
of rotor: c10, , & 
Fig. 18.27
rotation of  drives each  subunit in turn to release
tightly bound ATP
H+ path through membrane
c subunit subunit a
cytosolic
half-channel
c ring & a subunit structure
each
c subunit has 2 membrane-spanning
 helices
–
 midway along 1 helix: asp COOH↔COO
a subunit has 2 half-channels
asp
Fig. 18.34
matrix
halfchannel
H+ path
H+
from cytosol diffuses via half-channel
to asp on c ring subunit (c1)
this subunit can now move to
interface membrane, allowing
c ring to rotate
c ring
c9 now interfaces matrix half-channel,
subunit a
allowing H+ to diffuse into matrix
4
c1
c9
Fig. 18.36
H+ flow drives rotation of c ring
matrix
5
cannot rotate in
either direction
can rotate
clockwise
Fig. 18.35
ATP synthase
H+
 F1:
catalytic unit
subunits: 33
 subunit has
1 ATP synthesizing site
each

 subunits link
 pairs to F0
H H H H
matrix
H+
transmembrane H+
conducting channel
subunits: ab2c10
 F0:


3
H+ flow drives rotation
of rotor: c10, , & 
Fig. 18.27
rotation of  drives each  subunit in turn to release
tightly bound ATP
Binding-change mechanism of ATP synthesis





rotation of  drives release of tightly bound ATP
3 active sites cycle through 3 structural states:
O, open; L, loose-binding; T, tight-binding
at T site, ADP + Pi  ATP, but ATP can’t dissociate
 rotation causes T  O, L  T, O  L
as a result of the T  O structural change,
ATP can now dissociate from what is now an O site
O
3
1
ATP
2
ADP + Pi
6
T
L
L
120° rotation of 
(counterclockwise)
T
3
1
2
ATP
O
T
O
Fig. 18.32
Transport across inner mitochondrial membrane



Dp also drives flow of substances across inner membrane
transported by specific carrier proteins
cotransport: coupled transport of 2 substances
symport:
Alberts et al., Fig. 14-21
both move
voltage
gradient (Em)
in same
drives ADP-ATP
direction
exchange
CH3CCOO– + H+
O
HPO4= + H+
antiport:
7
each moves
in opposite
direction
ADP-ATP
exchange
pH gradient
drives
pyruvate
import
pH gradient
drives
phosphate
import
Summary of ATP synthesis & translocation of
ATP,
ADP
& Pi
8
Lehninger et al., Fig. 18-24
ATP yield
H+ pumped/e– pair
NADH-Q reductase (I)
4
cytochrome reductase (III) 2
cytochrome oxidase (IV) 4
ATP-ADP-Pi transport
ATP synthesis
H+ used
1
3
net ATP yield/e– pair:

via FADH2
(0 + 2 + 4)/(1 + 3) = 1.5

via NADH (matrix)
(4 + 2 + 4)/(1 + 3) = 2.5
energy efficiency (via NADH): (2.5 × 7.3)/53 = 34%
NADH + ½O2 + 2.5ADP + 2.5Pi  NAD+ + H2O + 2.5ATP
9
Oxidative phosphorylation: control
Vmax of e– transport chain very high;
coupling to H+ transport-ADP phosphorylation limits V
 major control factor of Vox phos: [ADP]
 Vox phos varies hyperbolically as the concentration
of 1 of its substrates (ADP) varies

Vox phos  [ADP] under
resting conditions
[ATP]/[ADP] ~20
 as [ADP] rises above
its KM, slope diminishes,
so that Vox phos
approaches a maximum
(saturation)
 ATP demand > supply
10
Vox phos

[ADP]
rest sustainable exercise
Inhibitors of oxidative phosphorylation
 inhibition of e–
transport chain
NADH-Q
reductase
–
 amytal blocks e transfer
–
 e entry & transfer via FADH2 enzymes unaffected
cytochrome oxidase
II
 CO: Fe form
–
III form
 CN : Fe
 in both cases, oxidase prevented from conversion to
other form
 inhibition of ATP synthesis
oligomycin
inhibits synthase
because phosphorylation coupled to oxidation, e– transfer
11 also inhibited
Uncoupling
 H+
gradient dissipated by
leakage of H+ across inner
membrane, bypassing synthase
 result: Vphosphorylation ,
Voxidation
 uncouplers
ionophores: small mobile carriers of ions across membranes
e.g., dinitrophenol: H+ ionophore
thermogenin: transmembrane
protein in brown adipose tissue
function: to generate heat
other
12
mitochondrial proteins:
variants may account for
differences in weight gain/loss
Lehninger et al., Fig 19-28
Dp, a widely used energy source
Electric potential
DE
Active
transport
Heat
production
PROTON GRADIENT
Dp
Flagellar
rotation
NADPH
synthesis
ATP
~P
Fig. 21-37
Next:
Section 6:
Carbohydrate metabolism