1. 1. carbon fuels are oxidized in the citric acid cycle to yield electrons with high transfer...
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1. Carbon fuels are oxidized in the citric acid cycle to yield electrons with high transfer potential.
2. This electron-motive force is converted into a proton-motive force.
3. The proton-motive force is converted into phosphoryl transfer potential.
Oxidation and ATP synthesis are coupled by transmembrane proton fluxes.
1. The intermembrane space between the outer and the inner membranes Oxidative phosphorylation
2. The matrix which is bounded by the inner membrane: most of the reactions of the citric acid
cycle and fatty acid oxidation
1. Large multi subunit integral membrane protein complexes or coupling sites: couple electron transfer with H+ gradient generation› Complex I: NADH-Q oxidoreductase (MW
=880 kd) Coupling Site 1› Complex II: succinate-Q reductase complex
(MW =140 kd)› Complex III: Q-cytochrome c oxido-
reductase (MW = 250 kd). Coupling Site 2› Complex IV: cytochrome c oxidase (MW =
160 kd) Coupling Site 3.
2. Small Mobile Electron Carriers:
› Ubiquinone/Ubiquinol (Q/QH2): small hydrophobic electron carriers which shuttle electrons between the large complexes and back and forth across the lipid bilayer.
› Cytochrome c: is a small water soluble protein which is a mobile electron carrier and carries electrons between cytochrome bc1 complex and cytochrome c oxidase.
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Electron flow within these transmembrane complexes leads to the transport of protons across the inner mitochondrial membrane.
Electrons are carried from NADH-Q oxidoreductase to Q-cytochrome c oxidoreductase by the reduced form of coenzyme Q (Q).
Q also carries electrons from FADH2, generated in succinate dehydrogenase or (succinate-Q reductase) in the citric acid cycle, to Q-cytochrome c oxidoreductase
Cytochrome c, a small, soluble protein, shuttles electrons from Q-cytochrome c oxidoreductase to cytochrome c oxidase (complex IV), which catalyzes the reduction of O2.
Succinate-Q reductase (Complex II), in contrast with the other complexes, does not pump protons.
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Q is a hydrophobic quinone that diffuses rapidly within the inner mitochondrial membrane.
Q is a quinone derivative with a long isoprenoid tail.› The number of five-carbon isoprene units in
coenzyme Q depends on the species.› The most common form in mammals contains 10
isoprene units (coenzyme Q10).
The reduction of ubiquinone (Q) to ubiquinol (QH2) proceeds through a semiquinone anion intermediate (Q.-).
In the fully oxidized In the fully oxidized state (Q), coenzyme Q state (Q), coenzyme Q has two keto groups.has two keto groups.
The addition of one The addition of one electron and one electron and one proton results in the proton results in the semiquinone form semiquinone form (QH·).(QH·).
The semiquinone form is The semiquinone form is relatively easily relatively easily
deprotonated to form a deprotonated to form a semiquinone radical semiquinone radical
anion (Q·-).anion (Q·-).
The addition of a The addition of a second electron and second electron and proton generates proton generates ubiquinol (QH2),ubiquinol (QH2),
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Thus, electron-transfer reactions of quinones are coupled to proton binding and release, a property that is key to transmembrane proton transport.
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Oxidation States of Flavins:› The reduction of flavin mononucleotide (FMN)
to FMNH2 proceeds through a semiquinone intermediate.
Fe-S clusters in iron-sulfur proteins (nonheme iron proteins) play a critical role in a wide range of reduction reactions in biological systems.
Iron ions in these Fe-S complexes cycle between:› Fe2+ (reduced state)› Fe3+ (oxidized state)
Unlike quinones and flavins, iron-sulfur clusters generally undergo oxidation-reduction reactions without releasing or binding protons.
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Several types of Fe-S clusters are known:
A. A single iron ion bound by four cysteine residues.B. 2Fe-2S cluster with iron ions bridged by sulfide ions.C. 4Fe-4S cluster.
Each of these clusters can undergo oxidation-reduction reactions.
NADH-Q oxidoreductase (also called NADH dehydrogenase):› an enormous enzyme
(880 kd)› consists of at least 34
polypeptide chains.› consists of a membrane-
spanning part and a long arm that extends into the matrix.
› Contains FMN and Fe-S prosthetic groups.
NADH is oxidized in the arm, and the electrons are transferred to reduce Q in the membrane.
The reaction catalyzed by this enzyme appears to be:
1. The initial step is the binding of NADH and the transfer of its two high-potential electrons to the flavin mononucleotide (FMN) prosthetic group of this complex to give the reduced form, FMNH2.
NADH + H+NAD+
FMNFMNH2
Matrix
Cytosol
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2. Electrons are then transferred from FMNH2 to a series of three 4Fe4S, the second type of prosthetic group in NADH-Q oxidoreductase.
NADH-Q oxidoreductase contains:› 2Fe-2S cluster› 4Fe-4S cluster
Two protons move back to the matrix NADH + H+NAD+
FMNFMNH2
Fe3+ Fe2+
2H2H++2H2H++Matrix18
3. Electrons in the 4Fe4S of NADH-Q oxidoreductase are shuttled to coenzyme Q.
Two hydrogen ions are pumped out of the matrix of the mitochondrion.
NADH + H+NAD+
FMNFMNH2
Fe3+ Fe2+
UQUQH2
2H2H++2H2H++ 2H2H++2H2H++Matrix
Cytosol
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4. The pair of electrons on bound QH2 are transferred to a 4Fe-4S center and the protons are released on the cytosolic side.
5. These electrons are transferred to a mobile Q in the hydrophobic core of the membrane, resulting in the uptake of two additional protons from the matrix. NADH + H+NAD+
FMNFMNH2
Fe3+ Fe2+
UQUQH2
Fe3+ Fe2+
UQUQH2
2H2H++2H2H++ 2H2H++2H2H++ 2H2H++2H2H++
2H2H++2H2H++
Matrix
Cytosol
1. NADH binds to a site on the vertical arm and transfers its electrons to FMN.
2. These electrons flow within the vertical unit to three 4Fe-4S centers.
3. Then they flow to a bound Q. The reduction of Q to QH2 results in the uptake of two protons from the matrix.
4 Protons are pumped this way which is still under investigation
It is an integral membrane protein of the inner mitochondrial membrane.
Contains three different kinds of Fe-S clusters:1. 2Fe-2S 2. 3Fe-3S3. 4Fe-4S.
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FADH2 is generated in the citric acid cycle by the enzyme succinate dehydrogenase, with the oxidation of succinate to fumarate.
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Two electrons are transferred from FADH2 directly to Fe-S clusters of succinate dehydrogenase.
The electrons are then passed to (Q) for entry into the electron-transport chain.
FADH2 is generated by other reactions (such as: Glycerol phosphate dehydrogenase and fatty acyl CoA dehydrogenase).
This FADH2 also transfers its electrons to (Q), to form (QH2).
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The succinate-Q reductase complex and other enzymes that transfer electrons from FADH2 to Q, in contrast with NADH-Q oxidoreductase, do not transport protons.
Consequently, less ATP is formed from the oxidation of FADH2 than from NADH.
The second of the three proton pumps in the respiratory chain (also known as cytochrome reductase).
The function of Q-cytochrome c oxidoreductase is to catalyze the transfer of electrons from QH2 to oxidized cytochrome c (cyt c), a water-soluble protein, and concomitantly pump protons out of the mitochondrial matrix.
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This enzyme is a homodimer with 11 distinct polypeptide chains.The major prosthetic groups, three hemes and a 2Fe-2S cluster, mediate the electron-transfer reactions between quinones in the membrane and cytochrome c in the intermembrane space.
Q-cytochrome c oxidoreductase contains:› Cytochrome b562› Cytochrome b566› Cytochrome c1› An iron sulfur protein› At least six other subunits.
A cytochrome is an electron-transferring protein that contains a heme prosthetic group.
The iron ion of a cytochrome alternates between a reduced ferrous (+2) state and an oxidized ferric (+3) state during electron transport.
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The enzyme contains three heme prosthetic groups contained within two cytochrome subunits:› Two b-type hemes within cytochrome b:
Heme bL (L for low affinity)
Heme bH (H for high affinity)
› One c-type heme within cytochrome c1 (Heme c1).
Hemes in the 3 classes of cytochrome (a, b, c) differ slightly in substituents on the porphyrin ring system.
Only heme c is covalently linked to the protein via thioether bonds to cysteine residues
The enzyme also contains an iron-sulfur protein with an 2Fe-2S center (Rieske center).
This center is unusual in that one of the iron ions is coordinated by two his residues rather than two cysteine residues.› This coordination stabilizes the center in
its reduced form, raising its reduction potential.
Finally, Q-cytochrome c oxidoreductase contains two distinct binding sites for ubiquinone termed (Qo) and (Qi), with the Qi site lying closer to the inside of the matrix.
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The mechanism for the coupling of electron transfer from Q to cytochrome c to transmembrane proton transport.
Facilitates the switch from the two-electron carrier ubiquinol to the one-electron carrier cytochrome c.
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H+ H+Qo site
Cyt c
Fe-S
Cyt c1
Cyt bL
Cyt bH
Qi siteQ
The first electron:1. flows first to the Rieske 2Fe-2S
cluster2. then to cytochrome c13. finally to a molecule of oxidized
cytochrome c, converting it into its reduced form.
The second electron:1. transferred first to cytochrome bL
2. then to cytochrome bH
3. finally to an oxidized uniquinone bound in the Qi site.
This quinone (Q) molecule is reduced to a semiquinone anion (Q·-).
QH2 QHQ
Q.-
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Fe-S
Cyt c1
Cyt bL
Cyt bH
Cyt c
Qo siteQH2
Qi siteQ.-
Q
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H+ H+
H+ H+
Cyt c
Fe-S Cyt c1
Cyt bL
Qo site
Cyt bH
QH2
Qi siteQ.-QH2
Q
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It catalyzes the coupled oxidation of the reduced cyt c generated by Complex III, and reduction of O2 to two molecules of H2O.
The four-electron reduction of oxygen directly to water without the release of intermediates is quite thermodynamically favorable.
G°´ = -231.8 kJ mol-1 As much of this free energy as possible must be
captured in the form of a proton gradient for subsequent use in ATP synthesis.
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It consists of 13 subunits, 3 of which (subunits I, II, and III) are encoded by the mitochondrial genome.
Cytochrome c oxidase contains:› Three copper ions, arranged as two copper centers,
designated A and B:1. CuA/CuA: contains two copper ions linked by two bridging
cysteine residues. This center initially accepts electrons from reduced cytochrome c.
2. CuB: is coordinated by three histidine residues, one of which is modified by covalent linkage to a tyrosine residue.
› Two heme A groups: 1. Heme a: functions to carry electrons from CuA/CuA
2. heme a3: passes electrons to CuB, to which it is directly adjacent.
Together, heme a3 and CuB form the active center at which O2 is reduced to H2O.
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Cytochrome C Oxidase.
The major prosthetic groups include:
1. CuA/CuA
2. heme a
3. heme a3-CuB.
Heme a3-CuB is the site of the reduction of oxygen to water.
.
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Cyt cCyt c
CuA/CuA
Heme A
Hem
e a
3 CuB
Electrone transfer Electrone transfer to Cuto CuBB
FIRST ELECTRONE
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Cyt cCyt c
CuA/CuA
Heme A
Hem
e a
3 CuB
O O
O O
Electrone transfer Electrone transfer to to FeFe in in Heme aHeme a33
Both Both CuBCuB and and FeFe in in Heme aHeme a33 are in are in reduced formreduced form
Binding of Binding of O2O2
Formation of Formation of peroxide bridgeperoxide bridge
SECOND ELECTRONE
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Cyt cCyt c
CuA/CuA
Heme A
Hem
e a
3 CuB
O O
H+
THIRD ELECTRONE
Cleavage of O-O Cleavage of O-O bondbond
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Cyt cCyt c
CuA/CuA
Heme A
Hem
e a
3 CuB
O O
H+
H+
FOURTH ELECTRONE
Reduction of the Reduction of the Ferryl groupFerryl group
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Cyt c
CuA/CuA
Heme A
Hem
e a
3 CuB
O O
H+
H+
H+ H+
H+
H+
Release of waterRelease of water
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Cyt cCyt c
CuA/CuA
Heme A
Hem
e a
3 CuB
O O
H+
H+
H+
H+
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The four protons in this reaction come exclusively from the matrix.
Thus, the consumption of these four protons contributes directly to the proton gradient.
Each proton contributes (21.8 kJ mol-1) to the free energy associated with the proton gradient.
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Four "chemical" protons are taken up from the matrix side to reduce one molecule of O2 to two molecules of H2O.
Four additional "pumped" protons are transported out of the matrix and released on the Cytosolic side in the course of the reaction.
The pumped protons double the efficiency of free-energy storage in the form of a proton gradient for this final step in the electron-transport chain.
Still under study. However, two effects contribute to the
mechanism:1. Charge neutrality tends to be maintained
in the interior of proteins.› Thus, the addition of an electron to a site
inside a protein tends to favor the binding of a proton to a nearby site.
2. Conformational changes take place, particularly around the heme a3-CuB center, in the course of the reaction cycle.› These changes must be used to allow protons
to enter the protein exclusively from the matrix side and to exit exclusively to the cytosolic side.
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Thus, the overall process catalyzed by cytochrome c oxidase is.
molecular oxygen is an ideal terminal electron acceptor, because its high affinity for electrons provides a large thermodynamic driving force.
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The reduction of O2 is safe because: Cytochrome c oxidase does not release
partly reduced intermediates by holding O2 tightly between Fe and Cu ions.
However, partial reduction generates small amounts of hazardous compounds (ROS).› Superoxide anion› peroxide.
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These TOXIC derivatives of molecular oxygen are scavenged by protective enzymes e.g. superoxide dismutase.
This enzyme catalyzes the conversion of two of the superoxide radicals into hydrogen peroxide and molecular oxygen.
The H2O2 formed by superoxide dismutase
and by other processes is scavenged by catalase, that catalyzes the dismutation of H2O2 into H2O and O2.
So far, we have considered the flow of electrons from NADH to O2, an exergonic process.
Next, we consider how this process is coupled to the synthesis of ATP, an endergonic process?
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The synthesis of ATP is carried out by A molecular assembly in the inner mitochondrial membrane called:› ATP synthase› mitochondrial ATPase
› F1F0 ATPase
› Complex V
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Chemiosmotic Hypothesis: Complex I, Complex III and Complex IV
pump protons across the inner mitochondrial membrane.› Pumping uses the energy liberated from the
oxidation of NADH and FADH2› Pumping generates a membrane potential
because it generates an electrochemical gradient Negative inside, positive outside Alkaline inside, acidic outside
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The difference in proton conc. (pH) and the electrical potential together represent a form of stored energy called (proton-motive force).
This proton-motive force drives the synthesis of ATP by ATP synthase.
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G is given by:
X+ X+
Outer side Inner side
Conc. C1
Charge Z
Conc. C2
+
+
+
-
-
-
V = potential across the membrane (V)
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Under typical mitochondrial conditions:› pH outside is 1.4 units lower than inside. (log10(c2/c1))
› The membrane potenial is 0.14V. (outside +ve)› Z=+1
G = 5.2 kcal mol-1 (21.8 kJ mol-1). Thus, each proton that is transported out of the
matrix to the cytosolic side corresponds to 5.2 kcal mol-1 of free energy.
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Testing the Chemiosmotic Hypothesis. ATP is synthesized when reconstituted membrane vesicles containing bacteriorhodopsin (a light-driven proton pump) and ATP synthase are illuminated.
The orientation of ATP synthase in this reconstituted membrane is the reverse of that in the mitochondrion
The F1 sector contains the catalytic sites of the synthase.
F0 sector acts as a proton translocator.
Consists of five types of polypeptide chains (3, 3, , , and ).
The and subunits, make up the bulk of the F1, and are arranged
alternately in a hexameric ring. Both bind nucleotides but only the
subunits participate directly in catalysis.
The subunit includes a long -helical coil that extends into the center of the 33 hexamer.
breaks the symmetry of the 33 hexamer:› each of the subunits is
distinct by virtue of its interaction with a different face of .
Distinguishing the three subunits is crucial for the mechanism of ATP synthesis.
Consists of a ring comprising from 10 to 15 c subunits that are embedded in the membrane.
A single a subunit binds to the outside of this ring.
The proton translocation depends on a subunit and the c ring.
The F0 and F1 sectors are connected in two ways:› by the internal stalk.› by peripheral stalk.
The peripheral stalk consists of b2 + subunit.
1. A moving unit, or rotor, consisting of the c ring and the stalk.
2. A stationary unit, or stator, composed of the remainder of the molecule.
ATP synthesis mechanism:
The rate of incorporation of O18 into Pi showed that about equal amounts of bound ATP and ADP are in equilibrium at the catalytic site, even in the absence of a proton gradient
Changes in the properties of the three subunits allow:
1. ADP and Pi binding2. ATP synthesis3. ATP release
The concepts of this initial proposal is refined by more recent crystallographic data:
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Interactions with the subunit make the three subunits inequivalent:
1. One subunit can be in the T, or tight, conformation.› Binds ATP with great avidity.› Its affinity for ATP is so high that it will convert bound ADP and
Pi into ATP with an equilibrium constant near 1, › The conformation of this subunit is sufficiently constrained
that it cannot release ATP.2. A second subunit will be in the L, or loose, conformation.
› This conformation binds ADP and Pi.› It, too, is sufficiently constrained that it cannot release bound
nucleotides.3. The final subunit will be in the O, or open, conformation.
› This form can exist with a bound nucleotide in a structure that is similar to those of the T and L forms,
› But it can also convert to form a more open conformation and release a bound nucleotide.
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Suppose subunit is rotated by 120 degree counterclockwise.1. The T conformation will convert into O conformation:
ATP will be released
2. The L conformation will convert into T conformation: The bound ADP+Pi will be converted into ATP.
3. The O conformation will convert into L conformation: It will trap the bound ADP+Pi. So that it will not escape.
A simple experimental system consisting of cloned subunits only
The were engineered to contain N-terminal polyhistidine tags, The tags allowed the 33 assembly to be immobilized on a glass
surface that had been coated with nickel ions. The subunit was linked to a fluorescently labeled actin filament The addition of ATP caused the to rotate in a counterclockwise
direction, which could be seen directly under fluorescent microscope.
When Asp 61 of C subunit is in contact with the hydrophobic part of the membrane, the residue must be in the neutral aspartic acid form, rather than in the charged, aspartate form.
Protons can pass into either of these channels, but they cannot move completely across the membrane.
Suppose that the Asp 61 residues of the two c subunits that are in contact with a half-channel have given up their protons so that they are in the charged aspartate form.
The c ring cannot rotate in either direction, because such a rotation would move a charged aspartate residue into the hydrophobic part of the membrane.
A proton can move through either half-channel to protonate one of the aspartate residues.
It is much more likely to pass through the channel that is connected to the cytosolic side of the membrane.
The entry of protons into the cytosolic half-channel is further facilitated by the membrane potential of +0.14 V (positive on the cytoplasmic side)
If the aspartate residue is protonated to its neutral form, the c ring can now rotate, but only in a clockwise direction.
› The newly protonated aspartic acid residue becomes in contact with the membrane.
› The charged aspartate residue moves from contact with the matrix half-channel to the cytosolic half-channel
› A different protonated aspartic acid residue moves from contact with the membrane to the matrix half-channel.
The proton can then dissociate from aspartic acid and move through the half-channel into the proton-poor matrix to restore the initial state.
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The difference in proton concentration and potential on the two sides of the membrane leads to different probabilities of protonation through the two half-channels, which yields directional rotational motion.
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Recall that the number of c subunits in the c ring appears to range between 10 and 15.
Each 360-degree rotation of the subunit leads to the synthesis and release of three molecules of ATP (1/120o).
Thus, if there are 10 c subunits in the ring (as was observed in a crystal structure of yeast mitochondrial ATP synthase), each ATP generated requires the transport of 10/3 = 3.33 protons. If there are 12 c ring, then 12/3 = 4 H+
For simplicity, we will assume that 3 protons must flow into the matrix for each ATP formed, but we must keep in mind that the true value may differ.
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NAD+ must be regenerated for glycolysis to continue.
How is cytosolic NADH reoxidized under aerobic conditions?
The inner mitochondrial membrane is impermeable to NADH and NAD+.
Thus electrons from NADH, rather than NADH itself, are carried across the mitochondrial membrane.
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FADH2 transfers its electrons to Q, which then enters the respiratory chain as QH2.
1.5 rather than 2.5 ATP are formed (FAD is the elect. Acceptor)
The use of FAD enables electrons from cytosolic NADH to be transported into mitochondria.
The price of this transport is one molecule of ATP/2e-.
This glycerol 3-phosphate shuttle is especially prominent in muscle and enables it to sustain a very high rate of oxidative phosphorylation.
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In heart and liver
This shuttle, in contrast with the glycerol 3-phosphate shuttle, is readily reversible.
Consequently, NADH can be brought into mitochondria by the malate- aspartate shuttle only if the NADH/NAD+ ratio is higher in the cytosol than in the mitochondrial matrix.
This versatile shuttle also facilitates the exchange of key intermediates between mitochondria and the cytosol.
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Also called adenine nucleotide translocase or ANT
The highly charged ATP and ADP do not diffuse freely across the inner mitochondrial membrane.
The in and out flows of ATP and ADP are coupled by the translocase, which acts as an antiporter.
The translocase, contains a single nucleotide-binding site that alternately faces the matrix and cytosolic sides of the membrane
ATP and ADP (both devoid of Mg2+) are bound with nearly the same affinity.
In actively respiring mitochondrion the membrane potential is positive.
The rate of binding-site eversion from the matrix to the cytosolic side is more rapid for ATP than for ADP because ATP has one more negative charge.
The membrane potential and hence the proton-motive force are decreased by the exchange of ATP for ADP, which results in a net transfer of one negative charge out of the matrix.
ATP-ADP exchange is energetically expensive; about a quarter of the energy yield from electron transfer by the respiratory chain is consumed to regenerate the membrane potential.
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Transporters (also called carriers) are transmembrane proteins that move ions and charged metabolites across the inner mitochondrial membrane.
Many mitochondrial transporters consist of three similar 100-residue units. These proteins contain six putative membrane-spanning segments
The best current estimates for the number of protons pumped out of the matrix per electron pair is:› 4 by NADH-Q oxidoreductase› 2 by Q-cytochrome c oxidoreductase› 4 by cytochrome c oxidase
Three protons are pumbed through the ATP synthase for each molecule of ATP.
One proton is consumed (neutralized) in transporting ATP from the matrix to the cytosol.
Hence, about 2.5 molecules of ATP are generated as a result of the flow of a pair of electrons from NADH to O2.
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Electrons do not usually flow through the electron-transport chain to O2 unless ADP is simultaneously phosphorylated to ATP.
Oxidative phosphorylation requires a supply of NADH (or FADH2), O2, ADP, and Pi.
The most important factor in determining the rate of oxidative phosphorylation is the level of ADP.
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The rate of oxygen consumption by mitochondria increases markedly when ADP is added and then returns to its initial value when the added ADP has been converted into ATP
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Specific inhibitors of electron transport were invaluable in revealing the sequence of electron carriers in the respiratory chain.
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rotenone and amytal block electron transfer in NADH-Q oxidoreductase
Antimycin A interferes with electron flow from cytochrome bH in Q-cytochrome c oxidoreductase
cyanide (CN-), azide (N3-), and carbon monoxide (CO) block electron flow in cytochrome c oxidase
Prevents utilization of electrons from NADH but not FADH2
Prevents utilization of electrons from NADH and FADH2
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Oligomycin and dicyclohexylcarbodiimide (DCCD) prevent the influx of protons through ATP synthase.
If actively respiring mitochondria are exposed to an inhibitor of ATP synthase, the electron-transport chain ceases to operate.
Indeed, this observation clearly illustrates that electron transport and ATP synthesis are normally tightly coupled.
The coupling of electron transport and phosphorylation in mitochondria can be disrupted (uncoupled) by 2,4-dinitrophenol and certain other acidic aromatic compounds.
These substances carry protons across the inner mitochondrial membrane.
Uncoupling is a means of generating heat to maintain body temperature in hibernating animals, in some newborn animals (including human beings), and in mammals adapted to cold.
Brown adipose tissue, which is very rich in mitochondria is specialized for this process.
The inner mitochondrial membrane of these mitochondria contains a large amount of uncoupling protein (UCP).
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UCP-1 forms a pathway for the flow of protons from the cytosol to the matrix.
In essence, UCP-1 generates heat by short-circuiting the mitochondrial proton battery. UCP-2 & UCP-3 also have been identified and may have a role in energy homeostasis.
This dissipative proton pathway is activated by free fatty acids liberated from triacylglycerols in response to hormonal signals, such as -adrenergic agonists