1. The Proton-Motive Force
CHAPTER 21
The Proton-Motive Force

2. Figure 21.1: Chemiosmotic hypothesis: electron transport via
the respiratory chain pump protons into intermembrane space.
These protons can be used to do work in making ATP as they
are transported back into the matrix through ATPase.
It takes 3 H+ transported into the matrix to make one ATP molecule

3. Complex V: ATP Synthase
F0F1 ATP Synthase uses the proton gradient energy for the
synthesis of ATP
Structure is composed of a “knob-and-stalk” structure
F1 (knob) contains the catalytic subunits for ATP production
F0 (stalk) has a proton channel which spans the membrane
Passage of protons through the F0 (stalk) into the mitochondrial
matrix is coupled to ATP formation
Estimated passage of
3 protons (H+) per
ATP synthesized

4. Figure 21.3 Knob-and-stalk structure of ATP synthase
A molecular machine
Intermembrane
space
H+ enter from the Intermembrane
space into subunit a.
Binding of this H+ induces a
clockwise rotation of the c ring
and the g subunit.
rotation of the g subunit also
induces conformational changes
in the a3b3 hexamer
A second H+ waiting in the lower
channel of subunit a is released into
the matrix.
Figure 14.14
The knob-and-stalk structure of ATP synthase. (a) The F1 component is on the inner face of the membrane. The F0 component, which spans the membrane, forms a proton channel at the a–c interface. The passage of protons through this channel causes the rotor (shaded) to rotate relative to the stator. The torque of these rotations is transmitted to F1 where it is used to drive ATP synthesis. (b) Molecular structure of the rotor and a portion of the stator of ATP synthase of Saccharomyces cerevisiae. The study of many molecular structures like this, based on X-ray crystallography, assisted in deduction of the complete model shown in (a). [PDB 1QO1].
Matrix

5. Figure 21.3 Knob-and-stalk structure of ATP synthase
A molecular machine
Intermembrane
space
The binding site for the ADP/ATP lies
in the clefts between adjacent a and b
subunits in the a3b3 cylinder.
The active site for ATP synthesis is
mostly part of the b subunits
One 120o rotation of the c-g subunits
requires the translocation of one proton.
Figure 14.14
The knob-and-stalk structure of ATP synthase. (a) The F1 component is on the inner face of the membrane. The F0 component, which spans the membrane, forms a proton channel at the a–c interface. The passage of protons through this channel causes the rotor (shaded) to rotate relative to the stator. The torque of these rotations is transmitted to F1 where it is used to drive ATP synthesis. (b) Molecular structure of the rotor and a portion of the stator of ATP synthase of Saccharomyces cerevisiae. The study of many molecular structures like this, based on X-ray crystallography, assisted in deduction of the complete model shown in (a). [PDB 1QO1].
Matrix

6. Figure 21.7: A closer look at subunits c and a.
H+ enters from intermembrane space
Figure 21.9
The pH is
lower on
the intermem.
side vs. matrix
Protonation of the Asp residue makes it more hydrophobic. This induces a change in the c unit were the Asp unit
Interacts more with the membrane. As this change occurs a rotation of one subunit c occurs. When a protonated Asp moves into the Matrix half channel
it is released due to the lower pH the result is that the negative Asp now interacts more with the subunit a half channel
H+ exit into the matrix space

7. Figure 21.8: Proton movement across the membrane

8. Figure 21.8: Proton movement across the membrane
Negative charged
Asp

9. Figure 21.8: Proton movement across the membrane
Neutral Asp
Each proton that passes results in a 120 degree rotation of c subunit.

10. The binding Change Mechanism
The mechanism of ATP synthesis from ADP and Pi
The binding Change Mechanism
Open: new ATP is released
and ADP and Pi bind
2. Loose: bound ADP and Pi
cannot be released
3. Tight: condensation of ADP
and Pi is favored to form ATP.
The ATP formed is very tightly
bound.
Figure 14.15
Binding change mechanism of ATP synthase. The different conformations of the three catalytic sites are indicated by different shapes. ADP and Pi bind to the yellow site in the open conformation. As the shaft rotates in the counter-clockwise direction (viewed from the cytoplasmic/matrix end of the F1 component), the yellow site is converted to a loose conformation where ADP and Pi are more firmly bound. Following the next step of the rotation, the yellow site is converted to a tight conformation and ATP is synthesized. Meanwhile, the site that had bound ATP tightly has become an open site, and a loose site containing other molecules of ADP and Pi has become a tight site. ATP is released from the open site, and ATP is synthesized in the tight site.
Figure 21.4

11. Figure 21.5 The binding Change Mechanism
The mechanism of ATP synthesis from ADP and Pi
Figure 21.5 The binding Change Mechanism
Follow the yellow region of one
b subunit.

12. Figure 21.16 Transport of ATP, ADP and Pi across the
Inner mitochondrial membrane
- Once ATP is made, it must be sent out to the cytosol and more ADP
and Pi must be transported into the matrix.
Adenine nucleotide translocase: unidirectional exchange of ATP
for ADP (antiport)
Start
here
Figure 14.17
Transport of ATP, ADP, and Pi across the inner mitochondrial membrane. The adenine nucleotide translocase carries out unidirectional exchange of ATP for ADP (antiport). The symport of Pi and H+ is electroneutral.

13. Figure 21.16 Transport of ATP, ADP and Pi across the
Inner mitochondrial membrane
- Antiport of Pi and OH- occurs resulting in an electrical neutral
translocation. (or symport with a H+)
Figure 14.17
Transport of ATP, ADP, and Pi across the inner mitochondrial membrane. The adenine nucleotide translocase carries out unidirectional exchange of ATP for ADP (antiport). The symport of Pi and H+ is electroneutral.

14. Figure 21.16: The Energy change regulates the use of fuels
1 H+ translocated
3 H+ per ATP
synth.
10 H+ per O reduced

15. Regulation of Oxidative phosphorylation
Overall rate of oxidative phosphorylation depends upon
substrate availability and cellular energy demand
Important substrates: NADH, FADH2, ADP
Resting state:
ADP levels low
NADH and FADH2 are
not oxidized via elec. transport
CAC slows down
Oxidative Phosph. Slows down
Active state:
ADP levels rise
NADH and FADH2 begin being
oxidized via elec. transport
CAC is more active
Oxidative Phosph. increases
Electrons do not flow to O2 unless ATP is in demand

16. P:O Ratio = molecules of ADP phosphorylated atoms of oxygen reduced
The relationship between oxygen consumption (respiration)
and ATP synthesis (phosphorylation)
The P:O Ratio
P:O Ratio = molecules of ADP phosphorylated atoms of oxygen reduced
Translocation of 3 H+ are required by ATP synthase for each
ATP produced
1 H+ is needed for transport of Pi, ADP and ATP
NET: 4 H+ transported for each ATP synthesized
and transported

17. Calculation of the P:O ratio
Complex I III IV
#H+ translocated/2e
Recall that two species supplied 2 e- each for proton translocation:
NADH and succinate (FADH2)
For NADH: 10 H+ translocated/O (2 e-)
P/O = (10 H+/4 H+) = 2.5 ATP/O
For succinate: (FADH2  QH2) substrate = 6 H+/O (2 e-)
P/O = (6 H+/4 H+) = 1.5 ATP/O

18. Glycerol 3-phosphate shuttle
One method electrons from cytoplasmic NADH produce ATP
Figure 21.11 The glycerol 3-phosphate shuttle. Electrons from NADH can enter the mitochondrial electron-transport chain by reducing dihydroxyacetone phosphate to glycerol 3-phosphate. Electron transfer to an FAD prosthetic group in a membrane-bound glycerol 3-phosphate dehydrogenase reoxidizes glycerol 3-phosphate. Subsequent electron transfer to Q to form QH2 allows these electrons to enter the electron-transport chain.

19. Malate – Aspartate shuttle
Another method electrons from cytoplasmic NADH produce ATP
Figure 21.12 The malate-aspartate shuttle.

21. Assignment
Read Chapter 21
Topics not covered: