Download Complex III

Functions
Complex I
•Transport of electrons from NADH to
ubiquinone
•Electron source: NADH
•Co-factor: Flavin mononucleotide
• Transport: via eight redox groups,
iron–sulphur clusters
Complex I
Functions Cont.
Electron acceptor: Ubiquinone
Ubiquinone function: Transfers of
electrons to next complex in the
chain (Complex III)
Simultaneous shunting of protons
across inner mitochondria membrane
to intermembrane space
Stoichiometry: 4H+/2e-
In the first step of electron transfer, the hydride ion is transferred to FMN,
forming is then oxidized in two steps via a semiquinone intermediate.
The two electrons are transferred one at a time to the next oxidizing agent, an
iron–sulfur cluste
These Fe–S clusters provide a channel for electrons, directing them to the
membrane-bound portion of the complex where ubiquinone (Q) accepts
electrons one at a time passing through a semiquinone anion intermediate
before reaching its fully reduced state, ubiquinol
Q and are lipid soluble cofactors. They remain within the lipid bilayer and can
diffuse freely in two dimensions.
One of the reasons for the complicated electron transport chain within
complex I is to carry electrons from an aqueous environment to a hydrophobic
environment within the membrane.
In the first step of electron transfer, the hydride ion is
transferred to FMN, forming is then oxidized in two steps
via a semiquinone intermediate. The two electrons are
transferred one at a time to the next oxidizing agent, an
iron–sulfur cluster.
These Fe–S clusters
provide a channel for electrons, directing them to the membrane-bound portion
of
the complex where ubiquinone (Q) accepts electrons one at a time passing
through
a semiquinone anion intermediate before reaching its fully reduced state,
ubiquinol
In complex I, there are 4 protons translocated
across the membrane for every pair of
electrons that pass from NADH to QH 2. .
These do not include the protons required for
ubiquinone reduction.
The proton pump is probably an antiporter
located in the membrane-bound module. The
mechanism of proton translocation is not clear
Complex II
Mitochondrial Electron Transport
The entry point for electrons from
FADH2 of flavoproteins is
Ubiquinol (QH2).
Succinate-Q reductase complex
Complex II
•Also called succinate dehydrogenase:
–
A component of the TCA cycle in mitoch.
in both eukaryotic cells and prokaryotic
organisms
Complex II
•Complex II has 4 or 5 polypeptides, all
encoded by the nuclear genome
•In mitochondria has 4 subunits. 2 integral
membrane proteins: the large cyto. b,
cybL or C, subunit and the small cybS or
D subunit and the iron-sulfur protein
•5 mitochondrial complexes, I to V,
complex II the only one with no
subunits encoded by the mitochondrial
genome.
Complex II
This protein provides two centers for
oxidation/reduction reactions
•FAD
FADH2
•Fe3+
Fe2+
Complex II
•Function: Mitochondrial respiratory chain
• Composition: 4 Subunits; All nuclear
encoded
•Flavoprotein: FAD (SDHA; Fp)
•Functions: Catalytic site; Covalently
bound FAD cofactor
•Iron-Sulfur protein: SDHB (Ip)
•Function: Electron transfer between
FAD and membrane-bound
quinone
•Structure: Contains three different
iron-sulphur clusters
•Cytochrome b subunits: SDHC ;
SDHD
Integral membrane proteins:
Bind Fp & Ip to matrix
Location of Complex II
Matrix side of mitochondrial inner
membrane
Binding to membrane is dependent
on 2 small (15.5 and 13.5 kDa)
proteins SDHC & SDHD
Complex II contains three identical multisubunit
enzymes that associate to form a trimeric
structure that is firmly embedded in the
membrane
The overall shape resembles a mushroom with
its head projecting into the interior of the
membrane compartment
Most of them have a bound heme b molecule and this subunit is often
called cytochrome b. All of the membrane subunits have a Q binding site
positioned near the interior surface of the membrane at the point where the
head subunits are in contact with the membrane subunits.
The sequence of reactions for the transfer of two electrons from succinate to Q
begins with the reduction of FAD by a hydride ion. This is followed by two one
electron transfers from the reduced flavin to the series of three iron–sulfur
clusters
In those species with a cytochrome b anchor, the heme group is not
part of the electron transfer pathway.
Very little free energy is released in the reactions
catalyzed by complex II. This means that the
complex cannot directly contribute to the
proton concentration gradient across the
membrane. Instead, it supplies electrons from
the oxidation of succinate midway along the
electron transport sequence.
• Q can accept electrons from complex I or II
and donate them to complex III and thence to
the rest of the electron-transport chain.
• Reactions in several other pathways also
donate electrons to Q. one of them, the
reaction catalyzed by the glycerol 3-phosphate
dehydrogenase complex
Complex III
COMPLEX III (CYTOCHROME REDUCTASE)
Location of Complex III: Inner
mitochondrial membrane
•Composition
•Nuclear subunits
•Number: 10
•Components
Cytochrome c1 (CYC1)
Ubiquinone-binding protein (UQPC)
Ubiquinol-cytochrome c reductase
core protein II (UQCRC2)
Rieske FeS protein
•Mitochondrial subunits
•Number: 1
•Component: Cytochrome b
•Complex III Functions
•Transfers electrons from ubiquinol to
cytochrome c
•Coupled with transfer of electrons across
inner mitochondrial membrane
•Contains 3 redox centers
•Cytochrome b
•Cytochrome c
•Rieske FeS protein
Associations
•Supercomplex of Complexes I, III, IV
• Association of Complexes III & IV
may be stabilized by cardiolipin
Complex III:
PDB
1BE3
membrane
Half of the homodimeric
structure is shown.
Approximate location of
the membrane bilayer is
indicated.
Not shown are 2 CoQ
binding sites, one near
heme bH & the other near
heme bL.
The b hemes are
positioned to provide a
Fe-S
pathway for electrons
across the membrane.
Complex III
(bc1 Complex)
heme bH
heme bL
heme c1
The Rieske iron-sulfur
center (Fe-S) has a
flexible link to the rest
of the complex.
PDB
1BE3
Complex III
(bc1 Complex)
It changes position
during e- transfer.
membrane
Fe-S extracts an e- from
CoQ, & then moves
closer to heme c1, to
which it transfers the e-.
(Fe-S protein in green.) Fe-S
heme bH
heme bL
heme c1
Complex III is an obligate
homo-dimer.
Fe-S in one half of the
dimer interacts with
bound CoQ & heme c1 in
the other half of the
dimer.
Arrows point at:
Fe-S in the half of
complex colored
white/grey
heme c1 in the half of
complex with proteins
colored blue or green.
PDB-1BGY
Complex III
homo-dimer
Fe-S
heme c1
Matrix
H+ + NADH NAD+ + 2H+
2 e
Q
I
2H+ + ½ O2 H2O
––
III
IV
++
4H
+
+
4H
cyt c
2H+
Intermembrane Space
Complex III (bc1 complex):
H+ transport in complex III involves
coenzyme Q (CoQ).
O
O
CH3O
CH 3
CH3O
CH 3
CH3O
(CH 2 CH
O
C
CH 3
e
CH 2)nH
CH 3
CH3O
(CH 2 CH
O
coenzyme Q
C
CH 2)nH
coenzyme Q •
e + 2 H+
OH
CH3O
CH 3
CH 3
CH3O
(CH 2 CH
OH
C
CH 2)nH
coenzyme QH2
The “Q cycle” depends on:
 mobility of CoQ in the lipid bilayer
 existence of binding sites for CoQ within the
complex that stabilize the semiquinone radical,
Q·.
matrix
Q
Q Cycle:
2 H+
Q.
QH2
QH2
cyt bH
Complex III
cyt bL
Q
e

e

Q·  Fe-S
2 H+
intermembrane space
cyt c1
cyt c
As depicted above, electrons enter complex III via
coenzyme QH2, which binds at a site on the positive
side of the inner mitochondrial membrane, adjacent
to the intermembrane space.
matrix
Q
2 H+
Q.
QH2
QH2
QH2 gives up
cyt bH
one e that is
Complex III
transferred via

e
hemes bL & bH to
cyt bL

a bound Q on
e

Q
Q·  Fe-S
cyt c1
the other side of
+
2
H
the membrane. intermembrane space
cyt c
Loss of one e- to the b hemes, and release of 2 H+ to the
intermembrane space, generates a Q·- radical in the site
adjacent to the intermembrane space.
Q·- becomes Q as it gives up a second e- to the Rieske
iron-sulfur center (Fe-S).
matrix
2 H+
Fe-S is
reoxidized by
Q
Q.
QH2
QH2
electron
cyt bH
transfer to
Complex III
cytochrome
c1, which
e
cyt bL

passes the
e
 
Q
Q·
Fe-S
cyt c1
electron out of
the complex to
2 H+
cyt c
cytochrome c. intermembrane space
Some evidence suggests instead a concerted reaction in
which e- transfer from QH2 to Fe-S & cytochrome bL is
essentially simultaneous.
But there is agreement about the overall reaction cycle.
It takes 2
matrix
2 H+
cycles for

.
Q
Q
QH2
QH2
CoQ bound
at a site
cyt bH
near the
Complex III
matrix to
e
be reduced
cyt bL

e
to QH2, as
 
Q
Q·
Fe-S
cyt c1
2e- are
2 H+
transferred
cyt c
intermembrane space
from the b
hemes , and 2H+ are extracted from the matrix
compartment.
In 2 cycles, 2 QH2 enter the pathway & one is regenerated.
matrix
Overall
reaction
catalyzed by
complex III,
including net
inputs &
outputs of the
Q cycle :
Q
2 H+
Q.
QH2
QH2
cyt bH
Complex III
cyt bL
Q
e

e

Q·  Fe-S
2 H+
intermembrane space
cyt c1
cyt c
QH2 + 2H+(matrix) + 2 cyt c (Fe3+) 
Q + 4H+(outside) + 2 cyt c (Fe2+)
Per 2e- transferred through the complex to cyt c, 4H+ are
released to the intermembrane space.
Complex III contains two copies of the enzyme and is firmly
anchored to the membrane by a large number of that span the
lipid bilayer .
The functional enzyme consists of three main subunits:
cytochrome c1 cytochrome b, and the Rieske iron–sulfur
protein (ISP). Other subunits are present on the inside
surface but they do not play a direct role in the
ubiquinol:cytochrome c oxidoreductase reaction.
The reaction begins when QH2 binds to the
Q o site in the cytochrome b subunit. QH2 is
oxidized to the semiquinone and a single
electron is passed to the adjacent Fe–S
complex in the ISP subunit. From there, the
electron transfers to the heme group in
cytochrome c1 This transfer is facilitated by
movement of the head group of ISP
Soluble cytochrome c is reduced by transfer of an electron
from the membrane-bound cytochrome subunit of
complex III.
In this reaction, the terminal electron acceptor is cytochrome
c. This molecule serves as a mobile electron carrier
transferring electrons to complex IV, the next component
of the chain.
The oxidation of QH2 at the Q0 site is a two-step process with a
single electron transferred at each step.
The path of electrons from the second step, oxidation
of the semiquinone intermediate, follows a different route than
the first electron. In this case, the electron is passed
sequentially to two different b-type hemes within the
membrane portion of the complex. The first heme group bL
has a lower reduction potential and the second heme bH has a
higher reduction potential
The bH heme is part of the Qi site where a molecule of Q is reduced to
QH2 in a two-step reaction that involves a semiquinone intermediate.
A single electron is transported from bL(at the site) to bH(at the site) to Q
to produce the semiquinone. Then,
a second electron is transferred to reduce the semiquinone to QH2
The second electron is derived from the oxidation of a second molecule of
QH2 at the site Q0 . This second oxidation of QH2 also results in the
reduction of a second molecule of cytochrome c
since the two electrons from the second follow separate paths. The net
result is that the oxidation of two molecules of QH2 at the Q0site
produces two molecules of reduced cytochrome c and regenerates a
molecule of QH2 at the Qi site.
Four protons are produced during the oxidation of two
molecules of QH2 at the Q o site. These protons are
released to the exterior of the membrane
compartment
and they contribute to the proton gradient that is
formed during membrane associated electron
transport.
Complex IV
COMPLEX IV, CYTOCHROME c
OXIDASE SUBUNIT I; MTCO1
Alternative titles; symbols
CYTOCHROME c OXIDASE I; COI
• 1 of 3 mitochondrial DNA (mtDNA)
encoded subunits (MTCO1, MTCO2,
MTCO3) of respiratory Complex IV
•located within the mitochondrial inner
membrane and is the third and final
enzyme of the ETC of MOP
COMPLEX IV
•It collects electrons from reduced cyto. C
and transfers them to O2 to give water
and The energy released is used to
transport protons across the
mitochondrial inner membrane
•Complex IV is composed of 13
polypeptides. Subunits I, II, and III
(MTCO1, MTCO2, MTCO3) are encoded
by mtDNA while subunits IV, Va, Vb,
VIa, VIb, VIc, VIIa, VIIb, VIIc, and VIII
are nuclear encoded
Complex IV Composition & Related Proteins
Nuclear subunits
 Number: 10
Presumed to play a structural &
regulatory role in COX
Mitochondrial subunits
 Number: 3
Largest COX subunits
 Form catalytic core of COX
 Contain the 3 copper atoms & 2 heme
A molecules
Serve as prosthetic groups in
holoenzyme
 Directly involved in electron transfer
This complex catalyzes the oxidation of the
reduced cytochrome c molecules produced by
complex III. The reaction includes a
four-electron reduction of molecular oxygen to
water and the translocation of four protons
across the membrane.
The total mass of mammalian complex IV is
greater than 400 kDa. Additional subunits in
the eukaryotic complexes play a role in
assembling complex IV and in stabilizing the
structure.
The core structure of cytochrome c oxidase is formed from the
three conserved subunits—I, II, and III.
These polypeptides are encoded by mitochondrial genes in all
eukaryotes. Subunit I is almost entirely embedded in the
membrane. The bulk of this polypeptide consists of 12
transmembrane
There are three redox centers buried within subunit I—two of
them are a-type hemes (heme-a and a3 ), and the third is a
copper atom The copper atom is in close
to the iron atom of forming a binuclear center where the
reduction of molecular oxygen takes place.
Subunit II has two transmembrane helices that anchor it
to the membrane.
Most of the polypeptide chain forms a domain located
on the exterior surface of the membrane. This domain
contains a copper redox center 1CuA2
The external domain of subunit II is the site where
cytochrome c binds to cytochrome c oxidase.
Subunit III has seven transmembrane helices and
is completely embedded in the membrane.
There are no redox centers in subunit III and it
can be artificially removed without loss of
catalytic activity.
Its role in vivo is to stabilize subunits I and II and
help protect the redox centers from
inappropriate oxidation–reduction reactions.
Electrons are transferred one at a time from the
site to the heme a prosthetic group in subunit I.
From there they are transferred to the heme
binuclear center. The two heme groups (a and )
have identical structures but differ in their
standard reduction potentials
One oxygen atom is bound to the iron atom of the group
and the other is bound to the copper atom.
Subsequent protonation and electron transfer results
in the release of a water molecule from the copper
site followed by release of a second water molecule
from the iron ligand.
The overall reaction requires the uptake of four protons
from the inside surface of the membrane
Cytochrome c Oxidase
ATP Synthase
ATP Synthase
Proton diffusion
through the protein
drives ATP
synthesis!
Two parts: F1 and F0
Racker & Stoeckenius
confirmed Mitchell’s
hypothesis using
vesicles containing
the ATP synthase
and
bacteriorhodopsin
ATP synthase,
embedded in
cristae of the
inner
mitochondrial
membrane,
includes:
ADP + Pi
ATP
F1
+
3H
matrix
Fo
intermembrane
space
 F1 catalytic subunit, made of 5
polypeptides with stoichiometry a3b3gde.
 Fo complex of integral membrane
proteins that mediates proton transport.
F1Fo couples
ATP synthesis to
H+ transport
into the
mitochondrial
matrix.
ADP + Pi
ATP
F1
3 H+
matrix
Fo
intermembrane
space
Transport of least 3 H+ per ATP is required, as
estimated from comparison of:
DG for ATP synthesis under cellular
conditions (free energy required)
 DG for transfer of each H+ into the matrix,
given the electrochemical H+ gradient
(energy available per H+).
In spite of their name, F-type ATPases
are responsible for synthesizing, not
hydrolyzing, ATP. They are membranebound
and have a characteristic knob-and-stalk
structure
Rotation of the γ subunit inside α β the hexamer
alters the conformation of the subunits,
opening and closing the active sites. The a, b,
and γ subunits form an arm that also attaches
the component to the oligomer. This unit is
termed the “stator.” Passage of protons through
the channel at the interface between the a and c
subunits causes the rotor assembly to spin in
one direction relative to the stator. The entire
structure is often called a molecular motor.
Rotation of the subunit within the component
takes place in a stepwise, jerky manner where
each step is 120° of rotation. As the c-ring
rotates it twists the shaft until enough tension
builds up to cause it to snap into the next
position within the hexamer. If the c-ring has
10 subunits then a complete rotation requires
the translocation of 10 protons and results in
the production of 3 ATP molecules.
Enzyme that
actually makes
the ATP
The mechanism of ATP synthesis from ADP
In 1979 Paul Boyer proposed the binding change
mechanism based on observations suggesting
that the substrate and product binding
properties of the active site could change as
protons moved across the
membrane.
The oligomer of ATP synthase contains three
catalytic sites. At any given time, each site
can be in one of three different
conformations.
The three conformations are:
(1) open: newly synthesized ATP can be released
and can bind,
(2) loose: bound cannot be released, and
(3) tight:
1. One molecule of ADP and one molecule of bind to an
open site.
2. Rotation of the shaft causes each of the three catalytic
sites to change conformation.
The open conformation (containing the newly bound
ADP and Pi) becomes a loose site. The loose site,
already filled with ADP and becomes a tight site. The
ATP-bearing tight site becomes an open site.
3. ATP is released from the open site, and ADP and
condense to form ATP in the tight site.
ADP + Pi ATP
Matrix
H+ + NADH NAD+ + 2H+
2 e
Q
I
2H+ + ½ O2 H2O
––
III
IV
Fo
++
4H+
F1
4H+
cyt c
2H+
3H+
Intermembrane Space
The Chemiosmotic Theory of oxidative
phosphorylation, for which Peter Mitchell
received the Nobel prize, states that
coupling of ATP synthesis to respiration
is indirect, via a H+ electrochemical
gradient.
ADP + Pi ATP
Matrix
H+ + NADH NAD+ + 2H+
2 e
Q
I
2H+ + ½ O2 H2O
––
III
IV
Fo
++
4H+
F1
4H+
cyt c
2H+
3H+
Intermembrane Space
Chemiosmotic theory - F1Fo ATP synthase:
Non-spontaneous ATP synthesis is coupled to
spontaneous H+ transport into the matrix.
The pH & electrical gradients created by respiration are
the driving force for H+ uptake.
H+ return to the matrix via Fo "uses up" pH & electrical
gradients.
Matrix
H+ + NADH NAD+ + 2H+
2 e
Q
I
Simplified
depicting:
2H+ + ½ O2 H2O
––
III
IV
++
4H
+
+
4H
cyt c
2H+
Intermembrane Space
Ejection of a total of 20H+ from the matrix per 4etransferred from 2 NADH to O2 (10H+ per ½O2).
Not shown is OH- that would accumulate in the matrix as
protons, generated by dissociation of water
(H2O
 H+ + OH-), are pumped out.
Also not depicted is the effect of buffering.
Transport of ATP, ADP, & Pi

ATP produced in the mitochondrial matrix must exit to the
cytosol to be used by transport pumps, kinases, etc.

ADP & Pi arising from ATP hydrolysis in the cytosol must
reenter the matrix to be converted again to ATP.

Two carrier proteins in the inner mitochondrial membrane
are required.

The outer membrane is considered not a permeability
barrier. Large outer membrane VDAC channels are assumed
to allow passage of adenine nucleotides and Pi.
MITOCHONDRIAL TRANSPORT
Electroneutral Transport
•Inner membrane is highly impermeable, it contains
many transport proteins to control the movement of
substances into and out of the matrix.
Most of the transport systems require the exchange
of molecules, and most of these exchanges occur
with molecules having the same charge; this is
termed electroneutral transport.
Electroneutral Transport cont.
-For instance
pyruvate (-1 charge), which enters the matrix
for further metabolism via pyruvat dehydrogenase
or pyruvate carboxylase, exchanges with a
negatively charged hydroxyl ion (OH ).
-
Phosphate (PO4 ), which enters the matrix for
synthesis of ATP) also can exchange with OH .
-
Citrate, a tricarboxylic acid, has a negative 3 (3 )
charge at physiological pH, but exchanges with
malate, a dicarboxylic acid (2 ). To maintain
electroneutrality of this exchange, a proton
accompanies the citrate thus neutralizing one of
+
its negative charges (citrate3 + H exchanges
for malate2 ).
Electrogenic Transport
•There are some transport systems in which the
exchange of molecules involves unequal charges.
•When there is the net movement of charge
across the membrane, this is termed
electrogenic transport.
•In mitochondria, net negative charges move out
of the matrix while net positive charges must
move into the matrix by following the charge
gradient
•Recall that the pumping of protons creates a
gradient of negative inside to positive outside. So
that the more negatively charged matrix will
favor the net outward movement of negative
charge.
•The most important electrogenic transporter is
the adenine nucleotide transporter
•ATP produced in the mitochondrial matrix by
oxidative phosphorylation is needed in the
cytoplasm for energy-requiring processes such as
Muscle contraction
Lipogenesis
Cholesterol synthesis
 Gluconeogenesis
•Hence, it is obligatory that ATP only be
transported into the cytoplasm while ADP moves
into the mitochondria matrix where it can be
phosphorylated to ATP
Electrogenic transport system in mitochondria.
The adenine nucleotide exchange ensures that
ATP4- produced in the mitochondrial matrix is
transported to the cytoplasm where it is needed.
Adenine nucleotide transporter
•ADP bears a negative three charge whereas ATP
has a negative four charge at physiological pH
•Thus, the exchange of ATP4- moving out with
ADP3- moving in creates a net charge of negative
one moving into the intermembrane space.
•The charge gradient facilitates this exchange of
ADP for ATP. The reverse exchange cannot occur
in functional mitochondria because it would
require the more negative ATP molecule to move
towards the more negative environment of the
matrix, a process that cannot occur
Phosphate Can Be Obtained by H+ Symport or OH– Antiport
Active Transport of ATP, ADP,
and Pi Across the Mitochondrial Membrane
Because the inner mitochondrial membrane is impermeable to
charged substances, a transporter is required to allow ADP to
enter and ATP to leave mitochondria.
• This transporter is called the adenine nucleotide translocase.
• Some of the free energy of the proton concentration
gradient is expended to drive this transport process.
The phosphate carrier does not draw on the
electrical component of the protonmotive
force, but does draw on the concentration
difference (pH).
The combined energy cost of transporting ATP
out of the matrix and ADP and into it is
approximately equivalent to the influx of
one proton.
ADP + Pi ATP
ATP4
matrix
lower [H+]
__
Adenine
nucleotide
++
translocase
3 H+
ATP4 ADP3 H2PO4 H+
energy
requiring
reactions
ADP + Pi
higher [H+]
cytosol
(ADP/ATP carrier) is an antiporter that catalyzes exchange
of ADP for ATP across the inner mitochondrial membrane.
At cell pH, ATP has 4 () charges, ADP 3 () charges.
ADP3/ATP4 exchange is driven by, and uses up,
membrane potential (one charge per ATP).
The P/O Ratio
The P/O ratio
is the ratio of molecules phosphorylated to atoms of oxygen
reduced.
Four protons are translocated by complex I, four by complex III,
and two by complex IV. Thus, for each pair of electrons that
pass through these complexes from NADH to a total of ten
protons are moved across the membrane.
Matrix
H+ + NADH NAD+ + 2H+
2 e
Q
I
2H+ + ½ O2 H2O
––
III
IV
++
4H
+
Intermembrane Space
+
4H
cyt c
+
2H
The P/O ratio for succinate is
only since electrons contributed by succinate
oxidation do not pass
through complex I.
The ratio of protons translocated intermembrane
space per pair of electorns transferred by
coupled electron- transport complexes is 4:1
for complex I, 4:1 for complex III, and 2:1 for
complex IV.These measurment can be used to
calculate the ratio of molecules of ADP
phosphorylated to atoms of oxygen reduced,
called P:O ratio.
•
•
•
1.
2.
When mitochondriaNADH is the substrate for the
respiratory electron chain, 10 protnsare exported to
the cytosol, and the P:O ratiois 10÷4=2.5
The P:O ratio for succinateis 6÷4=1.5
These P:O values should be consider maximum
values- the effeciency of energy conservation is not
100% because:
protons can slowly leak back into moitochondria the
adenine nucleotide translocase and other transport
processes consume the energy of the proton
concentration gradient.
Contributes its electrons at a lower energy level
Uncouplers and Inhibitors
Much of our knowledge of mitochondrial function results from the study
of toxic compounds. Specific inhibitors were used to distinguish the
electron transport system from the phosphorylation system and helped
to define the sequence of redox carriers along the respiratory chain. If
the chain is blocked then all the intermediates on the substrate side of
the block become more reduced, while all those on the oxygen side
become more oxidised. It is easy to see what has happened because the
oxidised and reduced carriers often differ in their spectral properties. If
a variety of different inhibitors are available then many of the
respiratory carriers can be placed in the correct order.
There are six distinct types of poison which may affect mitochondrial function:
1) Respiratory chain inhibitors (e.g. cyanide, antimycin, rotenone & TTFA)
block respiration in the presence of either ADP or uncouplers.
2) Phosphorylation inhibitors (e.g. oligomycin) abolish the burst of oxygen
consumption after adding ADP, but have no effect on uncoupler-stimulated
respiration.
3) Uncoupling agents (e.g. dinitrophenol, CCCP, FCCP) abolish the
obligatory linkage between the respiratory chain and the phosphorylation
system which is observed with intact mitochondria.
4) Transport inhibitors (e.g. atractyloside, bongkrekic acid, NEM) either
prevent the export of ATP, or the import of raw materials across the the
mitochondrial inner membrane.
5) Ionophores (e.g. valinomycin, nigericin) make the inner membrane
permeable to compounds which are ordinarily unable to cross.
6) Krebs cycle inhibitors (e.g. arsenite, aminooxyacetate) which block one or
more of the TCA cycle enzymes, or an ancillary reation.
Inhibitors of ETC:
1- Antimycin A:
Antimycin A binds to the Qi site of Complex III (the enzyme cytochrome c
oxidoreductase), in the cytochrome b subunit.
The inhibition of Complex III by Antimycin A result in the formation of large quantities of
the toxic free radical, Superoxide.
Antimycin blocks the flow of electrons from semiquinone to ubiquinone in the Q-cycle
of complex III in oxidative phosphorylation. By doing so it inhibits the electron transport
pathway thus preventing the consumption of oxygen (which occurs at Complex IV) and
disrupting the proton gradient across the inner membrane. It is the disruption of the
proton gradient that prevents the production of ATP as protons are
unable to flow through the ATP synthase complex.
2- Rotenone:
works by interfering with the electron transport chain in mitochondria.
Specifically, it inhibits the transfer of electrons from Fe-S centers in
Complex I to ubiquinone. This prevents NADH from being converted into
usable cellular energy (ATP).
3- 2,4-Dinitropheno
Ionophores that disrupt the proton gradient by carrying protons
across the membrane. This uncouples proton pumping from ATP
synthesis.[3] Ionophores that disrupt the proton gradient by carrying
protons across the membrane. This uncouples proton pumping
from ATP synthesis.[3]
4- Malonate:
is a powerful inhibitor of cellular respiration, because it binds to the
active site of the succinate dehydrogenase in the citric acid cycle but does
not react, since it does not have the -CH2-CH2- group (as in succinate)
which is required for dehydrogenation.
It is the only enzyme that participates in both the citric acid cycle and the
mitochondrial electron transport chain (in this role it is often called
Complex II).
5- oligimycin:
inhibits ATP synthase by blocking its proton channel, which is
necessary for oxidative phosphorylation of ADP to ATP (energy
production).
6- Cyanide, Carbon monoxide:
Inhibit the electron transport chain by binding more strongly than oxygen to
the Fe–Cu center in cytochrome c oxidase, preventing the reduction of
oxygen.
7- C-Ceramide:
Ceramide is a lipid second messenger that mediates the effects of tumor
necrosis factor and other agents on cell growth and differentiation.
Ceramide is believed to act via activation of protein phosphatase, prolinedirected protein kinase, or protein kinase C
An investigation of the site of ceramide action revealed that the activity
of respiratory chain complex III is reduced by C2-ceramide with halfmaximum effect at 5-7 µM. In contrast, N-acetylsphinganine (C2dihydroceramide)
10th home work
Name a poison that can keep ADP from exchanging
with ATP.
• What happens to the energy change in the cellwhen
this poison is working
• Does electron flow speed up , stay the same or
what?
11th home work
The C subunits of the Fo component of Fo F1 ATP synthase
form an ion channel across the inner mitochondrial
membrane. When certain glutamate aspartate residues
of a C subunit react eith dicyclohexylcarbodiimide
(DCCD), the subunit is unable to participitate in proton
transport.
(a) What is the effect of DCCD on electron transport and
respiration in suspentions of intact mitochondria?
(b) What happens when dinitrophenol is subsequently
added to DCCD treated mitochondria?
Electron Shuttle System
ELECTRON SHUTTLE SYSTEMS
•In our discussion of glycolysis, you learned that
under aerobic conditions, NADH produced during
glycolysis must be oxidized by the mitochondria.
•In this way, the cell regenerates NAD+ for the
glyceraldehyde-3-phosphate dehydrogenase
reaction and produces additional energy by
feeding electrons to the respiratory chain.
•Since there is no transporter to move NADH
directly into the matrix, oxidation of cytoplasmic
NADH by mitochondria must occur indirectly
either via the malate-aspartate or the αglycerol phosphate electron shuttle.
The malate – aspartate shuttle
The malate-aspartate shuttle
(1)Reduces oxaloacetate (OAA) to malate. The αketoglutarate (KG) transporter
(2)Exchanges malate for KG. Mitochondrial malate
dehydrogenase
(3)Generates intramitochondrial NADH by oxidation of
malate to oxaloacetate. Mitochondrial aspartate
aminotransferase
(4) catalyzes the transfer of an amino group from
glutamate (glu) to oxaloacetate to produce KG and
aspartate (asp). KG is transported out on its
translocase (2) and aspartate is transported out on
the unidirectional aspartate translocase
(5). Cytoplasmic aspartate aminotransferase
(6) regenerates oxaloacetate for reaction (1) by
transferring the amino group from aspartate to KG
producing glutamate, which is transported into the
matrix in exchange for aspartate (5).
Inner
Membrane
Intermembrane
Space
Matrix Side
ATP4-
ADP3-
Glutamate1- + H+
Adenine nucleotide
translocase
Aspartate1- Aspartate translocase
Electrogenic Translocases
Electrogenic transport systems of mitochondria.
The malate-aspartate shuttle Cont.
•Electrons from cytoplasmic NADH are used to
reduce oxaloacetate to malate, which is then
transported into the matrix.
•Thus malate carries the electrons from NADH
into the mitochondria.
•In the matrix, NADH is produced by the
oxidation of malate to oxaloacetate in the citric
acid cycle via malate dehydrogenase.
•This shuttle is irreversible because the transport
of aspartate out of the matrix in exchange with
glutamate moving into the matrix is electrogenic
just as is the exchange of ATP with ADP
The malate-aspartate shuttle Cont.
•While these amino acids normally carry the
same charge, the exchange is electrogenic
because a proton neutralizes to zero the negative
charge on the glutamate.
•Thus aspartate1- exchanges for glutamate0 so
that there is a net outward movement of a
negative charge.
•It is important that this transporter operate in
only one direction (unidirectional) to ensure that
electrons from mitochondrial NADH do not move
to the cytoplasm in these tissues.
Glycerol Phosphate Shuttle
Glycerol Phosphate Shuttle
•Thus, electrons are fed directly to coenzyme Q.
Since complex I is bypassed, this shuttle
produces one fewer ATP from glycolytic NADH
than occurs with the malate-aspartate shuttle.
•Also unlike the other shuttle, the electron
carrier, glycerol 3-phosphate, never permeates
the inner membrane but interacts instead with
the transmembrane glycerol 3-phosphate
dehydrogenase.
Glycerol Phosphate Shuttle
•The glycerol phosphate (GP) shuttle occurs
almost exclusively in the liver.
•Electrons from cytoplasmic NADH reduce DHAP
to (GP), which in turn carries the electrons to the
respiratory chain.
•Electrons reach the respiratory chain via glycerol
phosphate dehydrogenase that is bound to the
inner membrane.
•This enzyme contains a FAD prosthetic group, as
we showed for succinic dehydrogenase of the
citric acid cycle.
Glycerol Phosphate Shuttle
Cytoplasmic glycerol 3-phosphate dehydrogenase (1) oxidizes NADH.
Glycerol 3-phosphate dehydrogenase in the inner mitochondrial
membrane (2) reduces bound FAD to FADH2.
Superoxide Anions
the superoxide radical hydroxyl radical
and hydrogen peroxide All of these species are highly toxic to
cells. They are produced by flavoproteins, quinones, and iron–
sulfur proteins. Almost
all of the electron transport reactions produce small amounts of
these reactive
species, especially If superoxide radical is not rapidly removed
by superoxide
dismutase it will cause the breakdown of proteins and nucleic
acids.
The rapidity of this process is typical of electron
transfer reactions. In this case, a copper ion is
the only electron transfer agent bound to the
enzyme. The copper ion is reduced by
superoxide anion and it then reduces another
molecule of
The hydrogen peroxide formed can be converted
to and by the action of catalase
Some bacteria species are obligate anaerobes.
They die in the presence of oxygen
because they cannot deplete reactive oxygen
species that arise as a by-product of oxidation–
reduction reactions. These species do not have
superoxide dismutase.
All aerobic species have enzymes that scavenge
reactive oxygen molecules.
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