Download Biochemistry 6/e

Oxidative
phosphorylation
Structure of mitochondria
outer mt. mb.:
permeable for molecules
Mwt < 5000 g/mol,
‘cause contains porin
protein = VDAC =
voltage-dependent
anion channel
inner mt. mb.:
is the most impermeable mb.
among all mb-s,
have biggest protein content,
only those ions, molecules
can cross that have transporter
(except O2, CO, CO2, NO)
green = mitoch.
Function of mitochondria: the energy factory
A sedentery male of 70 kg uses 83 kg ATP/day, but posses only about 250 g ATP.
It is possible only if each ATP molecule cycles 300 times /day,
meaning ATP is synthetized from ADP + P, then degraded to them.
Mitochondria tend to form network
mitoch. without ADP
during resting respiration
mitoch. with ADP
during active respiration
Mitochondrial DNA contain different amount of genes in
different organisms, more and more genes are transfered to
nuclear DNA in eucaryotes
It is the cause
of lous-born
typhus.
Suggested:
all existing
mitoch. are
derived from
ancestor of
R.p. as a result
of one
endosymbiontic
event.
(circles = DNA)
prowazekii
Standard redoxelectrode and standard H2-electrode :
H2-electrode is anode and Fe2+/Fe3+ electrode is catode
ε⁰ =
= ε⁰
Direction of reactions in our cells
The sequence of components of electron transport
chain is determined by their reduction potential
NAD + H+ + 2e- → NADH
E°’ = - 0.32 V
½ O2 + 2 H+ + 2 e- → H2O E°’ = + 0.82 V
½ O2 + NADH + H+ → H2O + NAD
ΔG°’ = - nF Δ E°’
ΔG°’ = - 2×96500×0.82 – [(-2)×96500×(-0.32)]
ΔG°’ = - 220 kJ/mol
This amount of energy is liberated when
1 atom of oxygen is reduced to water
by electrone-transport chain and
NADH donates the 2 electrones
Synonyms of the names of electron transport chain complexes
= complex I = NADH dehydrogenase (trivial name)
systemic name
(is a wrong name) correct sytemic name is:
succinate: coenzyme Q oxidoreductase
= complex II = succinate dehydrogenase (trivial n.)
systemic name
trivial name
= complex III = cytochrome c reductase (trivial n.)
= complex IV = cytochrome C : O2 oxidoreductase
(systemic name)
O2
Composition of NADH-CoQ oxidoreductase,
= complex I
α-ketoglut.
isocitrate
and other citirc acid cycle
dehydrogenases,
PDHC,
deh. in FA β-oxidation
glutamate deh. etc.
can produce NADH
NADH + Q + 5 H+matrix → NAD+ + QH2 + 4H+cytoplasm
FMN and iron-sulfur proteins, the nonheme iron proteins
FMN can take up H-atoms
in stepwise manner and can
take up and donate electron
and proton separatly
Fe2+ ↔ Fe3+ + e-
Ubiquinone, the ubiquitous quinone =
coenzyme Q = Q10
Long isoprene tail makes it lipophilic, it is found in the membrane of aerobic bacteria,
plants, animals etc. as well.
Succinate dehydrogenase, the complex II
is the enzyme of citrate cycle
comlex III = cytochrome c reductase and Q cycle
Fe2+ ↔ Fe3+ + e-
Q-cycle makes both electron of ubiquinone
to be usful for one electron acceptor of
cytochrome c.
QH2 + 2 Cyt.cox + 5 H+matrix →
Q + 2 Cyt.cred + 4H+cytoplasm
Cytochrome b has the same heme as in hemoglobin,
cytochrome c1 heme is different, it is attached to Cys.
Comlex III = cytochrome c reductase and Q cycle
Oxidation of this UQH2 occurs in two steps. First, an electron from UQH2 is
transferred to the Rieske protein and then to cytochrome c1. This releases
two H+ to the cytosol and leaves UQ ×- , a semiquinone anion form of UQ,
at the Qp site. The second electron is then transferred to the bL heme,
converting UQ×- to UQ. The Rieske protein and cytochrome c1 are similar
in structure; each has a globular domain and is anchored to the inner
membrane by a hydrophobic segment. However, the hydrophobic segment
is N-terminal in the Rieske protein and C-terminal in cytochrome c1.
The electron on the bL heme facing the cytosolic side of the membrane
is now passed to the bH heme on the matrix side of the membrane. This
electron transfer occurs against a membrane potential of 0.15 V and is
driven by the loss of redox potential as the electron moves from bL (Eo' = 0.100V) to bH(Eo' = +0.050V). The electron is then passed from bH to a
molecule of UQ at a second quinone-binding site, Qn, converting this UQ to
UQ×-. The resulting UQ×- remains firmly bound to the Qn site. This
completes the first half of the Q cycle (Figure 21.12a).
The second half of the cycle (Figure 21.12b) is similar to the first half,
with a second molecule of UQH2 oxidized at the Qp site, one electron being
passed to cytochrome c1 and the other transferred to heme bH and then to
heme bH. In this latter half of the Q cycle, however, the bH electron is
transferred to the semiquinone anion, UQ×- at the Qn site. With the addition
of two H+ from the mitochondrial matrix, this produces a molecule of UQH2,
which is released from the Qn site and returns to the coenzyme Q pool,
completing the Q cycle.
The Q Cycle Is an Unbalanced Proton Pump
Why has nature chosen this rather convoluted path for electrons in
Complex III? First of all, Complex III takes up two protons on the matrix side
of the inner membrane and releases four protons on the cytoplasmic side
for each pair of electrons that passes through the Q cycle. The apparent
imbalance of two protons in for four protons out is offset by proton
translocations in Complex IV, the cytochrome oxidase complex. The other
significant feature of this mechanism is that it offers a convenient way for a
two-electron carrier, UQH2, to interact with the bL and bH hemes, the
Rieske protein Fe-S cluster, and cytochrome c1, all of which are oneelectron carriers.
Cytochrome oxidase = complex IV
4 Cyt.cred + 8 H+matrix + O2 → 4Cyt.cox + 4H+cytoplasm+ 2H2O
4
2
The conservative structure of cytochrome c is
used to draw the evolutionary tree
cytochrome c2
photosynthetic
bacterium
cytochrome c550
Chemiosmotic theory: proton gradient powers
ATP synthesis
Hypothesis by Peter Mitchell
in 1961
Proof by an artificial system
intermembrane
space
inner
membrane
matrix
ATP synthase complex
It is a small motor:
the rotor continously stirs clockwise:
c ring (10-14 c proteins) strictly
bound to γε subunit
the stator does not move:
a + b + δ + 3α + 3β
ATP synthase catalyzes ATP formation
Enzyme-bound ATP forms
readily in the absence of
proton-motive force, but ATP is
hydrolysed after.
Conclusion: proton movement
through ATP synthase is
necessary for ATP detachment,
not for ATP synthesis
Paul Boyer: The conformation of β-subunit denpends
on which side of γ-subunit is turned to it
L = loose conformation
entrapes ADP + P
T = tight conformation
can form ATP and
strictly binds it
O = open conf.
ATP can leave
H+
How proton movement stirs the c-ring?
H+
When aspartate takes up proton it
becomes uncharged and prefers the
hydrophobic membrane to interact,
therefore the C-ring is stired,
the next Asp can take up another H+
then after a whole cycle the H+ leaves
the C-ring to the halfchannel of a-subunit
Cytoplasmic NADH produced in glycolysis can
enter to mitochondria by shuttles
Especially prominent in
skeletal muscle to provide
sufficient energy for
contraction
Cytoplasmic NADH produced in glycolysis can
enter to mitochondria by shuttles
glyceraldehyde-3P dehydrogenase in glycolysis
malate dehydrogenase
ASAT
α-ketoglutarate transporter
Glu + H+ /Asp antiporter
MDH
ASAT
NADH dehydrogenase = complex I
Important in heart
and liver
ADP-ATP translocase/antiporter
ANT= adenine nucleotide transporter
ATP used as energy currency in cytoplasm
atractyloside
bongkrekic acid
15% of mitochondrial
inner membrane
proteins is this protein
ATP produced in matrix
side by ATP synthase
~ ¼ of H+ -gradient
energy is spent for
ATP/ADP exchange
Type and function of substrate anion carriers,
ion transporters
energy requiring processes
lipid synthesis
gluconeogenesis
And other
amino acid,
nucleotide,
coenzyme,
fatty acid transporters
~ 40 types all together
PDHC
ATP synthesis
H2PO4- imb. →HPO42-matrix + H+
Transports protons into mito.
And not OH- is exchanged.
Biochimica et Biophysica Acta (BBA) – Bioenergetics Volume 1777, Issues 7–8, July–August 2008, Pages 564–578
Or 5, if malate-Asp
shuttle works
Or 32
What Is the P/O
Ratio for Mitochondrial Electron Transport and
Oxidative Phosphorylation?
The P/O ratio is the number of molecules of ATP formed in oxidative
phosphorylation (No of P built into ATP) per two electrons flowing through a defined segment of
the electron transport chain (the number of oxygen atom that is reduced to water by
respiratory chain: 4 e- + 4 H+ + O2 = 2 H2O).
If we accept the value of 10 H+ transported out of the matrix (4 + 2 + 4 H+ by 1. 3. 4.
complex) per 2 e- passed from NADH to O2 through the electron transport chain, and
also agree (as above) that 4 H+ are transported into the matrix per ATP synthesized
and translocated (1/4 of proton gradient is for adenine nucleotide exchange), then the
mitochondrial P/O ratio is 10/4 = 2.5, for the case of electrons entering the electron
transport chain as NADH. This is somewhat lower than earlier estimates, which placed
the P/O ratio at 3 for mitochondrial oxidation of NADH.
For the portion of the chain from succinate/glycerol-3P/fatty acyl-CoA dehydr. to O2,
the H+/2 e- ratio is 6 (the 1st complex does not work), and the P/O ratio if FADH2 is
oxidized would be 6/4 = 1.5; earlier estimates placed this number at 2. The
consensus of experimental measurements of P/O ratios for these two cases has been
closer to the more modern values of 2.5 and 1.5.
Many biochemists have been reluctant to accept the notion of nonintegral P/O ratios. At some point, as we
learn more about these complex coupled processes, it may be necessary to reassess the numbers.
Calculation of the energy of electrochemical proton gradient
ΔG = RT ln c2/c1 + zFΔE
ΔG = 8.3×10-3 kJ/molK× 310 K ×2.3 ×1.4 + 1 × 96500 kJ/molV × 0.14 V
ΔG = 21.8 kJ/mol
energy is necessary to transport out 1 H+ from matrix to intermembrane space
(at body temperature, when pH difference is the usual 1.4 and membrane potential
is 0.14 V)
Hypothetically speaking, how much energy does a eukaryotic cell extract from
the glucose molecule? Taking a value of 50 kJ/mol for the hydrolysis of ATP
under cellular conditions, the production of 32 ATP per glucose oxidized yields
1600 kJ/mol of glucose . The cellular oxidation (combustion) of glucose yields
ΔG = -2937 kJ/mol. We can calculate an efficiency for the pathways of complete
oxidation of glucose by glycolysis, PDHC, the TCA cycle, electron transport, and
oxidative phosphorylation of
Regulation of oxidative phosphorylation
Electron-transport chain and
ATP synthesis are tightly coupled,
the ETC is a near equilibrium
process: the supply of reducing
equivalents/substrates,
of ADP and P determine
the speed of electon transport
and ATP synthesis as well.
Uncopuling proteins generate heat, uncoupler
molecules are poisons
cold →CNS →
adrenalin
proton gradient is
dissipated without
ATP synthesis, only
heat is generated by:
UCP-1 = thermogenin
in brown adipocyte
mitochondrial inner mb.
UCP-2: many tissues
UCP-3: muscle,
brown fat
Uncoupling molecule:
can transport protons back to
mitochondria, ETC is accelerated,
but no ATP synthesis, just heat
Inhibitors of respiratory chain, ATP synthase and transporters
atractyloside
bongkrekic acid
oligomycin
DCCD
The inhibitory actions of cyanide and azide at this site are
very potent, whereas the principal toxicity of carbon
monoxide arises from its affinity for the iron of hemoglobin.
Herein lies an important distinction between the poisonous
effects of cyanide and carbon monoxide.
Because animals (including humans) carry many, many
hemoglobin molecules, they must inhale a large quantity of
carbon monoxide to die from it. These same organisms,
however, possess comparatively few molecules of
cytochrome a3. Consequently, a limited exposure to cyanide
can be lethal. The sudden action of cyanide attests to the
organism's constant and immediate need for the energy
supplied by electron transport.
Oligomycin and DCCD Are ATP Synthase Inhibitors
Inhibitors of ATP synthase include dicyclohexylcarbodiimide
(DCCD) and oligomycin (Figure 21.29). DCCD bonds
covalently to carboxyl groups in hydrophobic domains of
proteins in general, and to a glutamic acid residue of the c
subunit of Fo×, the proteolipid forming the proton channel of
the ATP synthase, in particular. If the c subunit is labeled with
DCCD, proton flow through Fo× is blocked and ATP synthase
activity is inhibited. Likewise, oligomycin acts directly on the
ATP synthase. By binding to a subunit of Fo×, oligomycin
also blocks the movement of protons through Fo×.
Rotenone is a common insecticide that strongly inhibits the
NADH-UQ reductase. Rotenone is obtained from the roots of
several species of plants. Tribes in certain parts of the world have
made a practice of beating the roots of trees along riverbanks to
release rotenone into the water, where it paralyzes fish and
makes them easy prey. Ptericidin, Amytal, and other barbiturates,
mercurial agents, and the widely prescribed painkiller Demerol
also exert inhibitory actions on this enzyme complex. All these
substances appear to inhibit reduction of coenzyme Q and the
oxidation of the Fe-S clusters of NADH-UQ reductase.
2-Thenoyltrifluoroacetone and carboxin and its derivatives
specifically block Complex II, the succinate-UQ reductase.
Antimycin, an antibiotic produced by Streptomyces griseus,
inhibits the UQ-cytochrome c reductase by blocking electron
transfer between bH and coenzyme Q in the Qn site. Myxothiazol
inhibits the same complex by acting at the Qp site.
Cyanide, Azide, and Carbon Monoxide Inhibit Complex IV
Complex IV, the cytochrome c oxidase, is specifically inhibited by
cyanide (CN-), azide (N3-), and carbon monoxide (CO). Cyanide
and azide bind tightly to the ferric form of cytochrome a3,
whereas carbon monoxide binds only to the ferrous form.
SUPPLEMENT, not obligatory material
SUPPLEMENT, not obligatory material
Mitochondria, ER, peroxisome,
cytoplasm generates
reactive oxigen species (ROS)
Mitochondrial ROS production and defense.
SUPPLEMENT,
not obligatory material
Figure 2. Superoxide (O2.-) generated by
a respiratory chain is mostly released to a
matrix at complex I and IMS at complex III
(indicated by stars). O2.- can naturally
dismute to hydrogen peroxide (H2O2) or is
enzymatically dismuted by matrix MnSOD
(1) or Cu/ZnSOD (2) in a IMS or cytosol.
H2O2 is detoxified in a matrix by catalase
(3), a thioredoxin/thioredoxin peroxidase
system (4), or a glutathione/glutathione
peroxidase system (5). Alternately, H2O2
can react with metal ions to generate a
highly reactive hydroxyl radical (.OH) via
Fenton chemistry (6). O2.- is not
membrane permeable but can pass
through ion channels (solid lines),
whereas H2O2 can pass freely through
membranes (dashed lines). IMM, inner
mitochondrial
membrane;
IMS,
intermembrane space; OMM, outer
mitochondrial membrane; O2.-, superoxide;
H2O2, hydrogen peroxide; MnSOD,
manganese
superoxide
dismutase;
Cu/ZnSOD,
copper/zinc
superoxide
dismutase; CAT, catalase; THD, NADH
transhydrogenase;
TR,
thioredoxin
reductase; TPx, thioredoxin peroxidase;
TRxred, reduced thioredoxin; TRxox,
oxidized thioredoxin; GSH, glutathione;
GSSG, glutathione disulfide; IMAC, inner
membrane ion channel; VDAC, voltage
dependant
anion
channel;
∆Ψm,
membrane potential
[Frontiers in Bioscience 14, 1182-1196, January 1, 2009]
SUPPLEMENT,
not obligatory material
[Frontiers in Bioscience 14, 1182-1196, January 1, 2009]
Figure 1. Mitochondrial Ca2+ dynamics and stimulation of the TCA cycle and oxidative phosphorylation. Metabolite and ion transport are
represented by thin arrows passing through membrane carriers, metabolic pathways by thick arrows, respiratory chain electron transport by
broken arrows, and TCA cycle enzymes by shaded ovals. Mitochondrial Ca2+ influx and efflux mechanisms are displayed in yellow. Enzymes
and complexes stimulated by Ca2+ are displayed in green, and the respiratory chain is displayed in blue. The mitochondrial outer membrane has
been omitted for clarity. MCU, mitochondrial calcium uniporter; mRrR, mitochondrial ryanodine receptor; RAM, rapid mode; PTP, permeability
transition pore; ANT, adenine nucleotide transporter; PDH, pyruvate dehydrogenase; CS, citrate synthase; ACON, aconitase; ICDH, isocitrate
dehydrogenase; a-KGDH,a-ketogluterate dehydrogenase; SDH, succinate dehydrogenase; FUM, fumarase; MDH, malate dehydrogenase; DYm,
membrane potential