Transcript Principles of BIOCHEMISTRY

Mitochondrial Electron
Transport
• The cheetah, whose
capacity for
aerobic metabolism
makes it one of the
fastest animals
Fatty Acids
Acetyl Co A
Pyruvate
Glucose
Citric acid
cycle supplies
NADH and
FADH2 to the
electron
transport
chain
Amino Acids
Reduced coenzymes NADH and FADH2 are
formed in matrix from:
(1) Oxidative decarboxilation of pyruvate to
acetyl CoA
(2) Aerobic oxidation of acetyl CoA by the
citric acid cycle
(3) Oxidation of fatty acids and amino acids
The NADH and FADH2 are energy-rich
molecules because each contains a pair of
electrons having a high transfer potential.
The reduced and oxidized forms of NAD
The reduced and oxidized forms of FAD
Electrons of NADH or FADH2 are used to
reduce molecular oxygen to water.
A large amount of free energy is liberated.
The electrons from NADH and FADH2 are not
transported directly to O2 but are transferred
through series of electron carriers that undergo
reversible reduction and oxidation.
The flow of electrons through carriers leads to
the pumping of protons out of the mitochondrial
matrix.
The resulting
distribution of
protons
generates a pH
gradient and a
transmembrane
electrical
potential that
creates a
protonmotive
force.
ATP is synthesized when protons flow back to the
mitochondrial matrix through an enzyme complex
ATP synthase.
The oxidation of fuels and the phosphorylation of
ADP are coupled by a proton gradient across the
inner mitochondrial membrane.
Oxidative
phosphorylation is
the process in which
ATP is formed as a
result of the
transfer of electrons
from NADH or
FADH2 to O2 by a
series of electron
carriers.
OXIDATIVE PHOSPHORYLATION IN
EUKARYOTES TAKES PLACE IN MITOCHONDRIA
Two membranes:
outer membrane
inner membrane (folded into
cristae)
Two compartments:
(1) the intermembrane space
(2) the matrix
The outer membrane
is permeable to small
molecules and ions
because it contains
pore-forming protein
(porin).
The inner membrane
is impermeable to ions
and polar molecules.
Contains transporters
(translocases).
Location of mitochondrial complexes
• Inner mitochondrial membrane:
Electron transport chain
ATP synthase
• Mitochondrial matrix:
Pyruvate dehydrogenase complex
Citric acid cycle
Fatty acid oxidation
THE ELECTRON TRANSPORT CHAIN
Series of enzyme complexes (electron carriers)
embedded in the inner mitochondrial membrane,
which oxidize NADH2 and FADH2 and transport
electrons to oxygen is called respiratory
electron-transport chain (ETC).
The sequence of electron carriers in ETC
NADH
FMN
Fe-S
succinate FAD Fe-S
Co-Q
Fe-S
cyt b
cyt c1
cyt c
cyt a
cyt a3
O2
High-Energy Electrons: Redox Potentials
and Free-Energy Changes
In oxidative phosphorylation, the electron
transfer potential of NADH or FADH2 is
converted into the phosphoryl transfer
potential of ATP.
Phosphoryl transfer potential is G°' (energy
released during the hydrolysis of activated phosphate compound). G°' for ATP = -7.3 kcal mol-1
Electron transfer potential is expressed as E'o,
the (also called redox potential, reduction
potential, or oxidation-reduction potential).
E'o (reduction potential) is a measure of how easily a
compound can be reduced (how easily it can accept
electron).
All compounds are compared to reduction potential of
hydrogen wich is 0.0 V.
The larger the value of E'o of a carrier in ETC the better
it functions as an electron acceptor (oxidizing factor).
Electrons flow through the ETC components spontaneously
in the direction of increasing reduction potentials.
E'o of NADH = -0.32 volts (strong reducing agent)
E'o of O2 = +0.82 volts (strong oxidizing agent)
NADH
FMN
Fe-S
succinate FAD Fe-S
Co-Q
Fe-S
cyt b
cyt c1
cyt c
cyt a
cyt a3
O2
Important characteristic of ETC is the amount of
energy released upon electron transfer from one
carrier to another.
This energy can be calculated using the formula:
Go’=-nFE’o
n – number of electrons transferred from one carrier
to another;
F – the Faraday constant (23.06 kcal/volt mol);
E’o – the difference in reduction potential between
two carriers.
When two electrons pass from NADH to O2 :
Go’=-2*96,5*(+0,82-(-0,32)) = -52.6 kcal/mol
THE RESPIRATORY CHAIN
CONSISTS OF FOUR
COMPLEXES
Components of electrontransport chain are arranged
in the inner membrane of
mitochondria in packages
called respiratory
assemblies (complexes).
FMN
II
III
IV
III
I
NADH
I
Fe-S
II
succinate FAD Fe-S
Co-Q
Fe-S
cyt b
IV
cyt c1
cyt c
cyt a
cyt a3
O2
The energy is released not in a single step of electron
transfer but in incremental amount at each complex.
26.8
Energy released at three specific steps in the chain is
collected in form of transmembrane proton gradient and
used to drive the synthesis of ATP.
Complexes I-IV
• Mobile coenzymes: ubiquinone
(Q) and cytochrome c serve as
links between ETC complexes
• Complex IV reduces O2 to water
Complex I (NADH-ubiquinone oxidoreductase)
Transfers electrons from NADH to Co Q (ubiquinone)
Consist of:
- enzyme NADH dehydrogenase (FMN - prosthetic group)
- iron-sulfur clusters.
NADH reduces FMN to FMNH2.
Electrons from FMNH2 pass to a Fe-S clusters.
Fe-S proteins convey electrons to ubiquinone.
QH2 is formed.
The flow of two electrons from NADH to coenzym Q leads
to the pumping of four hydrogen ions out of the matrix.
NADH
FMN
matrix
Fe-S
Iron-sulfur clusters
contains two or four iron
ions and two or four
inorganic sulfides.
Clusters are coordinated
by four cysteine residues.
Iron ions in Fe-S
complexes cycle between
Fe2+ or Fe3+ states.
NADH-Q oxidoreductase - an
enormous enzyme consisting of
34 polypeptide chains.
L-shaped (horizontal arm lying
in the membrane and a vertical
arm that projects into the
matrix).
Complex II (succinate-ubiquinon oxidoreductase)
Transfers electrons from succinate to Co Q.
Form 1 consist of:
- enzyme succinate dehydrogenase (FAD – prosthetic group)
- iron-sulfur clusters.
Succinate reduces FAD to FADH2.
Then electrons pass to Fe-S proteins which reduce Q to QH2
Form 2 and 3 contains enzymes acyl-CoA dehydrogenase
(oxidation of fatty acids) and glycerol phosphate dehydrogenase
(oxidation of glycerol) which direct the transfer of electrons
from acyl CoA to Fe-S proteins.
Complex II does not contribute to proton gradient.
All electrons must pass through the ubiquinone (Q)ubiquinole (QH2) pair.
Ubiquinone Q:
- lipid soluble molecule,
- smallest and most hydrophobic of all the carriers
- diffuses within the lipid bilayer
- accepts electrons from I and II complexes and
passes them to complex III.
Complex III (ubiquinol-cytochrome c oxidoreductase)
Transfers electrons from ubiquinol to cytochrome c.
Consist of: cytochrome b, Fe-S clusters and cytochrome c1.
Cytochromes – electron transferring proteins containing a
heme prosthetic group (Fe2+  Fe3+).
Oxidation of one QH2 is accompanied by the translocation
of 4 H+ across the inner mitochondrial membrane. Two H+
are from the matrix, two from QH2
Q-cytochrome c
oxidoreductase is a dimer.
Each monomer contains
11 subunits.
Q-cytochrome c
oxidoreductase contains
three hemes: two b-type
hemes within cytochrome b,
and one c-type heme within
cytochrome c1.
Enzyme also contains an
iron-sulfur protein with an
2Fe-2S center.
Q cycle
two molecules of QH2 are oxidized to form two
molecules of Q,
one molecule of Q is reduced to QH2,
two molecules of cytochrome c are reduced,
four protons are released on the cytoplasmic side,
two protons are removed from the mitochondrial
matrix
Complex IV (cytochrome c oxidase)
Transfers electrons from cytochrome c to O2.
Composed of: cytochromes a and a3.
Catalyzes a four-electron reduction of molecular oxygen (O2) to
water (H2O):
O2 + 4e- + 4H+  2H2O
Translocates 2H+ into the intermembrane space
Cytochrome c
oxidase
consists of 13
subunits and
contains two
hemes (two
iron atom) and
three copper
ions, arranged
as two copper
centers.
The Catalytic Cycle of Cytochrome c Oxidise
The four protons used for the
production of two molecules of
water come from the matrix.
The consumption of these four
protons contributes to the
proton gradient.
Cytochrome c oxidase pumps
four additional protons from
the matrix to the cytoplasmic
side of the membrane in the
course of each reaction cycle
(mechanism under study).
Totally eight protons are
removed from the matrix in
one reaction cycle (4 electrons)
Cellular Defense Against Reactive
Oxygen Species
If oxygen accepts four electrons - two molecules of H2O are
produced
single electron - superoxide anion (O2.-)
two electrons – peroxide (O22-).
O2.-, O22- and, particularly, their reaction products are harmful to
cell components - reactive oxygen species or ROS.
DEFENSE
superoxide dismutase (manganese-containing version in
mitochondria and a copper-zinc-dependent in cytosol)
O2.- + O2.- + 2H+ = H2O2 + O2
catalase
H2O2 + H2O2 = O2 + 2 H2O
antioxidant vitamins: vitamins E and C reduced glutathione
This complex mechanical art fo
can be viewed as a metaphor for t
molecular apparatus underly
electron transport and ATP synthe
by oxidative phosphorylatio
OXIDATIVE PHOSPHORYLATION
A PROTON GRADIENT POWERS
THE SYNTHESIS OF ATP
The transport of electrons from NADH or FADH2
to O2 via the electron-transport chain is exergonic
process:
NADH + ½O2 + H+  H2O + NAD+
FADH2 + ½O2  H2O + FAD+
Go’ = -52.6 kcal/mol for NADH
-36.3 kcal/mol for FADH2
How this process is coupled to the synthesis of ATP
(endergonic process)?
ADP + Pi  ATP + H2O
Go’=+7.3 kcal/mol
The Chemiosmotic Theory
• Proposed by Peter Mitchell in the
1960’s (Nobel Prize, 1978)
• Chemiosmotic theory: electron
transport and ATP synthesis
are coupled by a proton
gradient across the inner
mitochondrial membrane
Mitchell’s postulates for chemiosmotic theory
1. Intact inner mitochondrial membrane is required
2. Electron transport through the ETC generates a proton
gradient
3. ATP synthase catalyzes the phosphorylation of ADP in a
reaction driven by movement of H+ across the inner
membrane into the matrix
Overview of oxidative phosphorylation
+
+
+
-
-
-
+
+
+
-
As electrons flow through complexes of ETC, protons are
translocated from matrix into the intermembrane space.
The free energy stored in the proton concentration gradient is
tapped as protons reenter the matrix via ATP synthase.
As result ATP is formed from ADP and Pi.
An artificial system demonstrating the basic
principle of the chemiosmotic hypothesis
Synthetic vesicles
contains
bacteriorhodopsin and
mitochondrial ATP
synthase.
Bacteriorhodopsin protein that pumps
protons when illuminated.
When the vesicle is
exposed to light, ATP is
formed.
ATP Synthase
Two units, Fo and F1 (“knob-andstalk”; “ball on a stick”)
F1 contains the catalytic subunits
where ADP and Pi are brought
together for combination.
F0 spans the membrane and serves as
a proton channel.
Energy released by collapse of proton
gradient is transmitted to the ATP
synthesis.
• F1 contains 5 types of
polypeptide chains a3b3gde
• Fo - a1b2c10-14
(c subunits form
cylindrical, membranebound base)
• Fo and F1 are
connected by a ge stalk
and by exterior column
(a1b2 and d)
• The proton channel –
between c ring and a
subunit.
• there are 3 active sites,
one in each b subunit
• c-e-g unit forms a “rotor”
• a-b-d-a3b3 unit is the
“stator”
• passage of protons
through the Fo channel
causes the rotor to spin
• rotation of the g subunit
inside the a3b3 hexamer
causes domain
movements in the bsubunits, opening and
closing the active sites
Each b subunit
contains the catalytic
site.
At any given time,
each site is in
different
conformation: open
(O), loose (L) or
tight (T).
O conformation binds
ADP and Pi
The affinity for ATP
of T conformation is
so high that it
converts ADP and Pi
into ATP.
Binding-Change Mechanism of ATP Synthase
1. ADP and Pi bind to an open site
2. Passage of protons causes each of three sites to change
conformation.
3. The open conformation (containing the newly bound ADP and Pi)
becomes a loose site. The loose site filled with ADP and Pi
becomes a tight site. The ATP containing tight site becomes an
open site.
4. ATP released from open site, ADP and Pi form ATP in the tight
site
Experimental observation of ATP synthase
rotation
• Fluorescent protein
arm (actin) attached
to g subunits
• a3b3 subunits bound
to a glass plate
• Arm seen rotating
when ATP added
(observed by
microscopy)
OXIDATIVE PHOSPHORYLATION
Oxidative
phosphorylation
creates ATP which
is the universal
currency of free
energy in biological
systems
MOVEMENT ACROSS THE
MITOCHONDRIAL MEMBRANES
Electrons from Cytosolic NADH Enter
Mitochondria by Shuttles
NADH is
generated in the
cytosol in
glycolysis.
The inner mitochondrial membrane is impermeable to
NADH and NAD+.
Electrons from NADH, but not NADH itself, are carried
across the mitochondrial membrane.
Two shuttles move electrons: glycerol 3-phosphate
shuttle and malate-aspartate shuttle
Glycerol 3-phosphate shuttle
Active in
skeletal
muscles and
brain.
Electrons
enter the
electrontransport
chain via
complex II.
Therefore
only 1.5
molecules of
ATP are
produced.
Malate-aspartate shuttle
Active in heart and liver. 2.5 molecules of ATP are produced.
Active Transport of ATP, ADP and Pi Across the
Inner Mitochondrial Membrane
• ATP must be transported to the cytosol, and ADP and Pi must
enter the matrix
• ADP/ATP carrier, adenine nucleotide translocase, exchanges
mitochondrial ATP4- for cytosolic ADP3• The exchange causes a net loss of -1 in the matrix (draws some
energy from the H+ gradient)
• Phosphate (H2PO4-) is transported into matrix in symport with
H+. Phosphate carrier draws on pH.
• Both transporters consume proton-motive force
Mechanism of ATP and ADP Transport
ATP-ADP translocase is abundant in the inner mitochondrial
membrane (about 14% of the protein)
The entry of ADP into the matrix is coupled to the exit of ATP.
Mitochondrial Transporters
ATP-ADP translocase – antiport of ATP and ADP
Phosphate carrier – antiport of H2PO4- and OH- (symport of H2PO4- and H+)
Dicarboxylate carrier – antiport of malate, succinate, or fumarate and H2PO4Tricarboxylate carrier – antiport of citrate and H+ and malate
Pyruvate carrier – antiport of pyruvate and OH- (symport of pyruvate and H+)
REGULATION OF OXIDATIVE
PHOSPHORYLATION
Coupling of Electron Transport with ATP Synthesis
Electron transport is tightly coupled to phosphorylation.
ATP can not be synthesized by oxidative phosphorylation
unless there is energy from electron transport.
Electrons do not flow through the electron-transport chain
to O2 unless ADP is phosphorylated to ATP.
Important substrates: NADH, O2, ADP
Intramitochondrial ratio ATP/ADP is a control mechanism
High ratio inhibits oxidative phosphorylation as ATP
allosterically binds to a subunit of Complex IV
Respiratory control
The most important factor in determining the rate of
oxidative phosphorylation is the level of ADP.
The regulation of the rate of oxidative
phosphorylation by the ADP level is called respiratory
control
Uncoupling of Electron Transport with ATP Synthesis
Uncoupling of oxidative phosphorylation generates heat to maintain
body temperature in hibernating animals, in newborns, and in mammals
adapted to cold.
Brown adipose tissues is specialized for thermogenesis.
Inner mitochondrial membrane contains uncoupling protein (UCP), or
thermogenin.
UCP forms a pathway for the flow of protons from the cytosol to the
matrix.
Uncouplers
• Uncouplers are lipid-soluble aromatic weak acids
• Uncouplers deplete proton gradient by transporting
protons across the membrane
2,4-Dinitrophenol: an uncoupler
• Because the negative charge is delocalized over the ring,
both the acid and base forms of DNP are hydrophobic
enough to dissolve in the membrane.
Specific inhibitors of electron
transport chain and ATP-synthase
Specific inhibitors of electron
transport are invaluable in revealing
the sequence of electron carriers.
Rotenone and amytal block electron
transfer in Complex I.
Antimycin A interferes with electron
flow thhrough Complex III.
Cyanide, azide, and carbon monoxide
block electron flow in Complex IV.
ATP synthase is inhibited by
oligomycin which prevent the influx of
protons through ATP synthase.
ATP Yield
Ten protons are pumped out of the matrix during
the two electrons flowing from NADH to O2
(Complex I, III and IV).
3
Six protons are pumped out of the matrix during
the two electrons flowing from FADH2 to O2
4
(Complex 4III and
IV).
2
Translocation of 3H+ required by ATP synthase for each ATP
produced
1 H+ needed for transport of Pi.
Net: 4 H+ transported for each ATP synthesized
For NADH: 10 H+/ 4H+) = 2.5 ATP
For FADH2: 6 H+/ 4 H+ = 1.5 ATP