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At the end of the electron transport chain, oxygen
receives the energy-spent electrons, resulting in
the production of water.
½ O2 + 2 e- + 2 H+ → H2O
Oxygen is the final electron acceptor.
Almost immediately oxidized into H2O
How is this coupling accomplished?
was originally thought that ATP generation was somehow
directly done at Complexes I, III and IV.
We now know that the coupling is indirect in that a proton
gradient is generated across the inner mitochondrial membrane
which drives ATP synthesis.
Mitochondrial respiratory chain:
Complex I:
- Transfers e- from NADH to quinone pool & pumps H+.
Complex II:
- Transfers e- from succinate to quinone pool & H+ released.
Complex III:
- Transfers e- from quinol to cytochrome c & pumps H+.
Complex IV:
- Accepts e- from cytochrome c, reduces O2 to H2O & pumps H+.
Complex V:
- Harvests H+ gradient & regenerates ATP.
Spontaneous
electron flow
through each of
complexes I, III,
& IV is coupled to
H+ ejection from
the matrix.
Matrix
H+ + NADH NAD+ + 2H+
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
++
4H+
4H+
cyt c
2H+ 4
Intermembrane Space
A total of 10 -12 H+ are ejected from the mitochondrial matrix per 2
e- transferred from NADH to oxygen via the respiratory chain.
Matrix
H+ + NADH NAD+ + 2H+
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
++
4H+
4H+
cyt c
2H+ 4
Intermembrane Space
Complex I (NADH Dehydrogenase) transports 4H+ out
of the mitochondrial matrix per 2e- transferred from
NADH to CoQ.
Matrix
H+ + NADH NAD+ + 2H+
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
++
4H
+
+
4H
cyt c
2H+
4
Intermembrane Space
Complex III (bc1 complex):
H+ transport in complex III involves coenzyme Q (CoQ).
A very strong electrochemical gradient is
established with few H+ in the matrix and many in
the intermembrane space.
The cristae also contain an ATP synthase complex
through which hydrogen ions flow down their
gradient from the intermembrane space into the
matrix.
The flow of three H+ through an ATP synthase
complex causes a conformational change, which
causes the ATP synthase to synthesize ATP from
ADP + P.
High [H+] +
+ + +
+ +
High [H+] + +
Inner
Membrane
Matrix
++ +
H+ +
- H+ _
H+
+
H+ + _
H
H
H+ Low [H+]
H+
+
+
+
H
+
+ + +
H+ H
Cytoplasm
Outer
Membrane
Intermembrane
Space
+ + ++ + +
Cristae
Generation of a pH gradient ([H+]) and charge difference
(negative in the matrix) across the inner membrane constitute
the protonmotive force that can be used to drive ATP
synthesis and transport processes.
Mitochondria produce ATP by chemiosmosis, so
called because ATP production is tied to an
electrochemical gradient, namely an H+
gradient.
Once formed, ATP molecules are transported out
of the mitochondrial matrix.
Chemiosmotic Theory --Peter Mitchell
A proton gradient is
generated with energy
from electron transport
by proton pumping by
Complexes I,III, IV
from the matrix to
intermembrane space
of the mitochondrion.
The protons have a thermodynamic tendency to return to the
matrix = Proton-motive force
The protons diffuse back into the matrix through the
FoF1ATP synthase complex. The free energy release drives
ATP synthesis.
The proton pumps are Complexes I, III and IV.
Protons return thru ATP synthase
The Domains
Hydrophobic F0 domain sits in the membrane - performs proton
translocation
Hydrophillic F1 portion protrudes from membrane - performs ATP
synthesis/hydrolysis
3 alternating alpha
and beta subunits
http://nobelprize.org/chemistry/laure
ates/1997/illpres/boyer-walker.html
http://www.bioc.aecom.yu.edu/labs/girvlab/ATPase/ATPsynthase.mov
ATP synthase: a rotating molecular motor.
a, b, , , and  subunits constitute the stator of the motor, and the c, , and 
subunits form the rotor. Flow of protons through the structure turns the rotor and
drives the cycle of conformational changes in  and  that synthesize ATP.
Animations
http://www.stolaf.edu/people/giannini/flashanimat/metabolism/atps
yn1.swf
Protons cross membrane through the ATP synthase enzyme
http://www.stolaf.edu/people/giannini/flashanimat/metabolism/atps
yn2.swf
Rotary motion of ATP synthase powers the synthesis of ATP
The “stalk” rotates in 120°increments
causes the units in the F1 domain to
contract and expand
The structural changes facilitate the
binding of ADP and Pi to make ATP
Each subunit goes through 3 stages
Open State – releases any ATP
Loose State – ADP and Pi molecules
enter the subunit
Tight State – the subunit contracts to
bind molecules and make ATP
ATP synthesis at F1 results from repetitive conformational
changes as  rotates
 rotates 1/3 turnenergy for ATP release
Interesting Facts
Contains 22722 atoms
23211 bonds connected
as 2987 amino acid groups
120 helix units and 94 sheet units
Generates over 100 kg of ATP daily (in humans)
One of the oldest enzymes-appeared earlier then
photosynthetic or respiratory enzymes
Smallest rotary machine known
Uncoupling. The compound 2,4 dinitrophenol (DNP) allows H+
through the membrane and uncouples.
Blocking. The antibiotic oligomycin blocks the flow of H+
through the Fo, directly inhibiting ox-phos.
Regulation of Respiration -> Primarily by Need for ATP
ATPase inhibited by:
Oligomycin and
Dicyclohexylcarbodiimi
de (DCCD)
Chemiosmosis
Production of ATP in Electron Transport
H+ (Protons) generated from NADH
Electrochemical Gradient Formed between membranes
Electrical Force (+) & pH Force (Acid) thus gradient formed
ATPase enzyme that channels H+ from High to Low concentration
3 ATP/NADH
2 ATP/NADH
Overall reaction using NADH funnel
NADH + H++ 3ADP + 3Pi+ ½O2 
NAD+ + 4H2O + 3ATP
For the flavoprotein-CoQ funnel,
Measure only about 2 ATP produced for each FADH2
Quantify P/O ratio
Definition: # Pi taken up in phosphorylating ADP per atom
oxygen (½O2)
per 2e-.
NADH
FADH2
3
2
What about energy and ATP stoichiometry? -- measured
-- 220 kJ/mole from NADH oxidation
-- ATP produced: ADP + Pi  ATP
G°= +30.5 kJ/mole
-- measure and find about 3 ATP produced for each NADH,
which enters. (a little less)
[3×(30.5)/220]×100 = 41% efficiency
Shuttling ATP, ADP and Pi
ATP is required in the cytosol
ADP  IN
OUT  ATP
(Exchange)
So for every ATP transported to cytosol,
an ADP must be transported into matrix
Mitochondrial transporters
3 similar 100-residue units (A,B,C)
6 membrane-spanning segments
Adding Up the ATP from Cellular
Respiration
Cytosol
Mitochondrion
Glycolysis
Glucose
2
Pyruvic
acid
2
AcetylCoA
Krebs
Cycle
Electron
Transport
Maximum
per
glucose:
by direct
synthesis
by
direct
synthesis
by
ATP
synthase
Figure 6.14
Food
Polysaccharides
Sugars
Glycerol
Fats
Fatty acids
Proteins
Amino acids
Amino groups
Glycolysis
AcetylCoA
Krebs
Cycle
Electron
Transport
Figure 6.13