BIO 402/502 Advanced Cell &
Developmental Biology I
Section I: Dr. Berezney
Lectures 6 & 7
Electron Transport,
Oxidative Phosphorylation
& the ATP Synthase
ATP Synthesis
ATP synthase
• Chemical energy produced in the
cell is stored as ATP
Electron transport chain
• ATP synthesis occurs at the inner
mitochondrial membrane of
ATP synthase
eukaryotes and the cell membrane
in prokaryotes.
• In plants, ATP is synthesized along
the thylakoid membranes
• Photosynthesis is a process by
which plants convert light energy
into ATP for glucose production .
Electron transport chain
Electron transport
chain
ATP synthase
ATP synthase
Proton Gradient Across the
Membrane: “Chemiosmosis”
• It is the universal mechanism of ATP production which
involves the production of a proton motive force (pmf)
based on a proton gradient across the membrane.
• Energy to establish this electrochemical proton gradient
is provided by the energy released as electrons move to
lower energy levels down the electron transport chain
and the coupling of this free energy to the movement of
protons across the IMM against the proton gradient
[from matrix to IMS]
• ATP is synthesized by the ATP synthase FoF1 complex :
protons move with the proton gradient through FoF1 to
generate ATP [from IMS to matrix]
Glycolysis
•
•
ATP Generation
Conversion of glucose to pyruvate
Net synthesis of 2 ATP by substrate level
phosphorylation
Krebs Cycle
•
•
Converts pyruvate to acetyl CoA & carbon dioxide
10 molecules of coenzymes NADH and 2 of FADH2
are produced. Results in synthesis of 30 ATP and 4
ATP molecules, respectively in the respiratory chain.
Electron Transport (Respiratory) Chain
•
•
The reduced coenzymes enter into the respiratory
chain of the inner mitochondrial membrane
Electron transport along the chain generates a proton
electrochemical gradient and this is used to produce
ATP
Electron Transport
Chain
• Stepwise movement of
electrons along the inner
mitochondrial membrane
respiratory chain proceeds
from a lower to a higher (+)
redox potential (E0).
• Redox changes in mV are
converted to free energy
changes by the formula
G0 = -nF E0;
F=23.063 (Kcal/Vmol
I
II
III
H+ in [matrix]
H+ out [IMS]
****[ΔEo= Eo(red) - Eo(ox)]
IV
• -G corresponds to
release of free energy and
hence more negative values
represent higher energy
levels of the transporting
electrons
Electron Transport Chain contd…
--------------------------------------------------------------------------------------------
The Four Electron Transport Complexes in the Inner
Mitochondrial Membrane Respiratory Chain
--------------------------------------------------------------------------------------------(a) Complex I
NADH-CoQ reductase ……… G = -16.6 kcal/mol (ATP)
(b) Complex II Succinate CoQ reductase …. G = -1.6 kcal/mol (no
ATP)
(c) Complex III CoQ- Cytochrome c reductase, G = -10.1 kcal/mol (ATP)
(d) Complex IV Cytochrome c oxidase …………G = -19.8 kcal/mol (ATP)
The Four Complexes of the Respiratory Chain
Glucose 10 NADH and 2 FADH2; NADH 3 ATP; FADH2  2 ATP;Total ATP = 34
Complex III
11 subunits
~240 kDa
Complex I
45 subunits
~1000 kDa
Complex II
4 subunits
~125 kDa
Complex IV
13 subunits
~ 200 kDa
Calculation of ∆G for electrochemical gradient
and the pmf
Free energy released during electron transport in the RC is stored as a proton
generated electrochemical gradient across the membrane which is composed
of two components:
(a) An electric potential (Ge = zF  Em )
(b) Proton chemical gradient ( Gc= (2.3) RT log [H+] I = - (2.3) RT  pH )
[H+]o
(c) If  pH = 1, and ΔEm= - 160 mV:  Gt =  Ge +  Gc = zF  Em - (2.3) RT  pH = - 3.7+
- 1.4 = - 5.1 kcal/mol for 1 proton (mol) to move down the electrochemical gradient
Proton motive force (pmf) is the proton electrochemical gradient across
the membrane expressed in volts or mV [Remember: ΔGo= zFΔEo].
(a) Proton motive force =  - 2.3 RT  pH =  - 59  pH ;  is the membrane
electrical potential.
F
(b)
Assume respiring mitochondria have a  of -160 mV and  pH is 1. Thus
pmf or Δp = -160 mV - (59) (1) = - 160 – (59) = - 219 mV.
(c)
ΔG = zF ΔE = 23.06 ( - 0.219 V) = - 5.1 kcal/mol
Proton Motive Q Cycle
CoQ binds 2 protons
on matrix side
One electron is
transported via FeS
protein and cyt c1 to
cyt c
The other electron
goes through cyt b’s
to CoQ . This CoQ is
fully reduced by
repeating this step.
# protons transported by 100 CoQ molecules is:
200+100+50+25+12.5 + … n = 400
Cytochrome C Oxidase (complex IV) Transport
Structure of the Cytochrome C Oxidase
Monomer
• The heme groups are
shown in blue and red
and copper sites in green
• The catalytic core
consists of I yellow, II
blue, III pink
• The entire complex
consists of 13 subunits
Structure of Beef Heart Cytochrome Oxidase
3 dimensional structure of beef heart cytochrome
oxidase at 2.8 angstrom resolution
The protein is a dimer of two 13 monomers
ATP Synthase: An Electrical
Mechanochemical Molecular
Complex
On the the Inner Mitochondrial Membrane (IMM),
the F0F1 Complex or ATP Synthase Uses the
Proton Gradient Generated by Electron Transport
of the Respiratory Chain to Synthesize ATP
Electron transport chain
Evidence that electron transport in mitochondria is
coupled to proton translocation
Change in pH indicates that for each electron pair transported from
NADH to oxygen, 10 protons are transported out of the matrix.
Correlating with the return of pH to normal is the synthesis of ATP by
the mitochondria. Proton transport is abolished by addition of mild
detergent that makes the IMM permeable.
Demonstrating Function for Mitochondria F1 Particles
This “reconstitution
experiment” demonstrates
that F1 are required for
ATP synthesis and not for
electron transport in the
mitochondrial vesicles
F1
Liposome Experiment
Demonstrating Coupling of Electron
Transport by Cytochrome Oxidase
to Proton Translocation and FoF1 as
the ATP synthase
• The measured pH indicates that two
protons are translocated per reduced
oxygen atom.
• No ATP synthesis in presence of ADP
and Pi .
Not Shown:
• If FoF1 complexes are inserted into one
of the RC complexes , then ATP synthesis
can be measured in correlation with
electron transport This is evidence that
FoF1 is the ATP synthase
The Famous Jagendorf Experiment
ATP Synthesis
in Thylakoid
Membranes
An artificially imposed
pH gradient across the
chloroplast thylakoid
membrane can drive
ATP synthesis in the
absence of electron
transport !!!!!
3 D Model of ATP Synthase:
An Electrical MechanoChemical Molecular Complex
• The Fo portion is composed of
integral transmembranous proteins
a, b and 9-14 copies of c which
forms a ring-like structure in the
plane of the membrane.
• The F1 head piece is composed of a
hexagonal array of alternating 
and  subunits, a central  protein
with a helical coil that associates
with  and  proteins and extends
into the c protein ring in the Fo.
Atomic Force Microscopy of C-subunit Ring
Structures Isolated from Chloroplast ATP Synthase
and Inserted Into Liposomes
Synthesis of ATP: Rotary Catalysis
• ATP is synthesized by coupling the energy liberated during
proton translocation through the FoF1 to a motive force that rotates
the C ring structure and the attached  subunit.
• -subunits contain the catalytic sites of ATP synthesis. 120 degree
units of rotation of the  protein around the stationary /
hexagonal array results in altered associations of the  protein
with the  protein forming the L, T and O states for the 3 β-subunits.
ATP is produced in the T state where the ∆G = ~ 0.
• Each rotation of 360 degrees of the γ subunit results in 3 ATP, one
for each β-subunit.
∆G = ~ 0
The model shows the rotation as arbitrarily clockwise.
Direct Visualization of Rotary Catalysis
Add ATP
F1 complex
F0F1 complex
From these results it was determined that the gamma protein rotates
like the camshaft in a rotary motor at a maximum rate of ~8,000 rpm!
Magnetic bead experiments shows rotation in the opposite direction
(clockwise) for ATP production!
A revolving magnetic field to rotate clockwise a magnetic bead
attached to the gamma subunit of a single F1 complex
results in ATP synthesis.
Revolving electromagnetic field
F1 complex
•Clockwise rotation led to the synthesis of 3 ATP’s for every 360o turn.
•When the magnetic field was switched off, the gamma subunit revolved
in the reverse direction, driven by the recently synthesized ATP.
Speculative Model for
Coupling of Proton
Transport to the Rotation
of the c Ring of Fo
• Proton binding to Asp61 of csubunit [on IMS side] induces a
conformational change in the c
subunit that causes the ring to
move by 30-40 degrees. Each
subsequent c picks up a proton.
• Bound protons are carried in full
circle rotating 30-40 degrees at a
time and are then released into the
matrix compartment and the c
subunit is free to bind another
proton. For one rotation of c-ring:
12 protons (30°); 9 protons (40°)
• Site directed mutagenesis of
Asp61 prevents proton tranlocation
across F0.