Download Lecture 16 (Parker) - Department of Chemistry ::: CALTECH

You can check-out any time you like,
But you can never leave! ”(as pyruvate)
Mitochondria (in green) is an Energy Capsule
How does the Mitochondria Produce ATP?
Oxidative Phosphorylation couples electron transport with ATP
synthesis via a proton gradient
CAC
electrons
Highly
permeable
Selectively
permeable
All Mitochondria have Genomes
Oxidative phosphorylation depends upon electron transfer
A strong reducing agent (NADH) is poised to donate electrons and
has a negative reduction potential
A strong oxidizing reagent (O2) is ready to accept electrons and
has a positive reduction potential
Apparatus to measure of redox potential:
Electrons travel through the agar bridge with a
voltmeter measuring the potential
Reducing
agent
oxidant
The respiratory chain consists of four complexes:
Three proton pumps and a physical link to the citric acid cycle:
Three large protein complexes called: NADH-Q oxioreductase
(complex I), Q-cytochrome c oxioreductase (complex III) and
cytochrome c oxidase (complex IV).
Electron flow within these trans-membrane complexes leads to the
transport of protons across the inner mitochondrial membrane.
A fourth large protein complex called succinate-Q reductase
(complex II) contains the succinate dehydrogenase that generates
FADH2 in the citric acid cycle and does not pump protons.
Components of the mitochondrial electrontransport chain
CAC
Coenzyme Q
Components of the mitochondrial electrontransport chain
CAC
Coenzyme Q
Coenzyme Q (ubiquinone) is hydrophobic and diffuses rapidly
within the inner mitochondrial membrane
Electrons are carried from NADH-Q oxidoreductase (complex I) to
Q-cytochrome c oxidoreductase (complex III) by the reduced form
of Q; QH2
NADH-Q oxidoreductase (complex I) is a huge (>900kD) enzyme
consisting of 46 polypeptide chains. This proton pump is
composed of both mitochondrial and nuclear gene products.
Electrons from the FADH2 generated by the citric acid cycle are
transferred to Q by Succinate-Q reductase (complex II does not
pump protons) then to Q-cytochrome c oxidoreductase (complex
III)
The electrons of NADH enter the chain at NADH-Q oxidoreductase
(complex I):
The reduction of Q
results in the addition
of two protons
forming QH2
One H+
After binding on the matrix side NADH transfers electrons to the
FMN (flavine monoucleotide) prosthetic group of the enzyme
complex
Electrons are then transferred to a series of iron-sulfur clusters
The flow of two electrons from NADH to Q leads to the pumping of
four hydrogen ions out of the matrix to the inter-membrane space:
In complex III electrons from QH2 are transferred to oxidized
cytochrome c and a total of 4 protons are pumped out of the
mitochondrial matrix
Cytochrome c is a water soluble protein and is located in the intermembrane space. Cytochrome c can accept only one electron. All
cytochromes are electron-transferring proteins that contain a heme
as the prosthetic group.
Complex III also contains two cytochromes termed b and c1.
Cytochrome b has two heme groups and cytochrome c1 has one.
In addition complex III has a Rieske iron-sulfur center located near
the cytoplasmic face of the enzyme (inter-membrane space).
Complex III consists of Q-cytochrome c oxioreductase
Release
electrons to
cytochrome c
Lower
affinity
for
electrons
High
affinity for
electrons
Intermembrane
space
11 subunits
250 kD
The Q cycle
QH2 enters complex III and ultimately transfers one of its electrons
to cytochrome c with two protons being pumped into the intermembrane space.
The other electron is transferred to another Q residing in a second
binding site.
The second half of the cycle an additional QH2 binds and transfers
one electron to cytochrome c and the other to the reduced Q.. Two
additional protons are pumped out of the matrix with two additional
protons taken up from the matrix completing the reduction of Q. to
QH2.
QH2 can then enter the Q pool of the membrane, a net pumping of
two protons per QH2 into the inter-membrane space results.
The Q cycle of complex III
Complex IV cytochrome c oxidase catalyzes the reduction of
molecular oxygen to water
The last of the three proton pumping complexes catalyzed the
transfer of electrons of the reduced form of cytochrome c to
molecular oxygen.
The requirement of molecular oxygen for this reaction is the reason
we must breath oxygen.
Cytochrome c oxidase is well understood at the structural level: it
consists of 13 subunits, three of which are encoded by the
mitochondrial genome.
Complex IV cytochrome c oxidase
Inter-membrane
area
CuA/CuA first
accepts electrons
from cytochrome c
A total of four cytochrome c electrons are needed to reduce
O2 to water
Reduced Fe and Cu
The Peroxide Bridge
Complex IV
pumps one
proton for each
cytochrome c
oxidized
The
‘chemical’
protons are
used in the
reaction with
O2
Electron Transport Chain
(3)
(1)
High ADP/
low ATP
Pyruvate/ADP/
pyruvate
dehydrogenase
A problem in using molecular oxygen is the potential
generation of a reactive oxygen species
Superoxide dismutase
H2O2 is
converted to
H2O and O2 by
catalase
Metabolizing molecular oxygen is risky
Proteins are ‘wire-like’ in the
transfer of electrons
Cytochrome c is highly conserved in evolution
21 of 104 residues have
been invariant for more
than 5000,000 years
Cytochrome c
Evolutionary
Tree
A Proton Gradient Powers the Synthesis of ATP
Mitchell’s Chemiosmotic Hypothesis
Electron transport and ATP synthesis are coupled by a proton
gradient across the inner mitochondrial membrane.
In this model the transfer of electrons through the respiratory
chain leads to the pumping of protons from the matrix to the
cytoplasmic side of the inner mitochondrial membrane.
ATP synthase is powered by the energy rich unequal distribution
of protons called the proton-motive force.
The proton-motive force can be considered as two components: a
chemical gradient represented as a pH gradient and a charge
gradient also generated by the unequal distribution of H+ across
the inner mitochondrial membrane.
Reconstituting a proton pump with ATP Synthase
Light-driven
proton pump
ATP synthase will
synthesize ATP in
the presence of light
and ADP + Pi
The ATP
Synthase
Machine
Inter-membrane
space
Inner mitochondrial
membrane
Matrix
ATP Synthase is composed of proton-conducting unit and a
catalytic unit
The F0 component is embedded in the inner mitochondrial
membrane and is proton-conducting.
The F1 component protrudes into the mitochondrial matrix and
contains the catalytic activity.
The F1 complex of ATP Synthase
The F1 complex contains the catalytic activity of the synthase, F1 subunits in the
absence of F0 have ATPase activity.
The F1 complex consists of five types of polypeptide chains: a3, b3, g, d and e.
The a and b subunits are arranged in a hexameric ring, both bind nucleotides but
only the b subunit is catalytic.
The g subunit includes a helical coiled coil that extends into the center of the a3b3
hexamer.
The g subunit breaks the symmetry of the a3b3 hexamer, each of the b subunits is
distinct by virtue of its interactions with g. Distinguishing the three b subunits is
crucial for the mechanism of ATP synthesis. Each of the three b-g interactions is
different, resulting in a different function for b depending on the surface of g that it
is interacting with.
The F0 complex of ATP synthase
F0 is a hydrophobic segment that spans the inner mitochondrial
membrane and contains the proton channel of the complex.
This channel consists of a ring of 10-14 c subunits that are imbedded
in the membrane.
A single a subunit binds to the outside of the stalk and spans the
length of the membrane.
F0 and F1 are connected by the central ge stalk and an exterior
column of one a subunit, 2b subunits and the d subunit.
c
The ATP
Synthase
Machine
g
Inter-membrane
space
Inner mitochondrial
membrane
Matrix
a
b
Proton flow through the ATP synthase leads to the release of tightly
bound ATP
This reaction
happens in the
absence of proton
flow
Isotopic labeling experiments have shown that there are equal
amounts of ATP and ADP in the active site in the absence of a
proton gradient:
The role of proton
flow is to release
ATP
The flow of protons drives the release of ATP from a b subunit
ATP synthase has two functional parts one moving and one
stationary.
The moving parts consists of the c ring and the ge stalk.
The stationary components are the rest of the molecule.
The movement of the ge stalk results in different surface
presentation of the asymmetrical g subunit to the three b subunits.
The three steps in ATP synthesis are performed sequentially by b
depending upon the surface of g presented to the subunit.
c
The ATP
Synthase
Machine
g
Inter-membrane
space
Inner mitochondrial
membrane
c ring g and e
are in motion
rotating in a
clock-wise
direction
Matrix
a
b
The three steps of ATP synthesis are:
1)  ADP and Pi binding
2)  ATP synthesis
3)  ATP release
g induces three different conformations of b:
The L or loose conformation which binds ADP and Pi
The T or tight conformation which tightly binds ATP forcing the
reaction of ADP+Pi to ATP to occur
The final conformation is O or open which will release nucleotides
The pathway
is L-T-O
Single molecule experiments have shown that isolated F1
components a b g tethered to a glass surface will rotate in the
presence of ATP
Components of the proton-conducting of ATP synthase
H+ enters the cytoplasmic channel in the a subunit allowing
rotation to go in a clockwise direction
Once in the a channel the H+ moves into the adjacent c
subunit in association with the aspartic acid residue in the
middle of the c subunit
The proton can then move into the matrix channel of the a
subunit and exit into the matrix after a complete rotation
Each 360o turn of the c
ring results in the
passage of ten H+ with
the synthesis of three
ATPs.
Overview of Oxidative Phosphorylation
Cytoplasmic NADH can also enter the respiratory chain but
only indirectly by the glycerol 3-phosphate shuttle:
Needed for
glycolysis
Complex II
The entry of ADP into the mitochondria is coupled to the exit of
ATP by ATP-ADP translocase
Structure of Mitochondrial Transporters
Three tandem
repeats of a
100-amino acid
module
Each repeat
has two transmembrane
segments
Multiple Mitochondrial Transporters Exist
Activated by ADP
pyruvate
dehydrogenase
generates Acetyl
CoA