Download F 1

13. ATP synthase was first identified
by dissociation and reconstitution
studies
• Abundant knoblike protruding structures were
observed on the matrix side of the inner
mitochondrial membrane by EM (Racker in 1960).
• The inside-out submitochondrial particles with the
“knobs” are capable of both electron transferring
and ATP synthesis.
• When the protruding F1 part was removed by
agitation, electron transferring could still occur, but
neither proton gradient nor ATP are produced.
• ATP synthesis reappeared when F1 was reconstituted
back (the solubilized F1 alone can catalyze ATP
hydrolysis, thus was originally named as F1ATPase).
• F1 was the first essential factor identified for
oxidative phosphorylation.
14. Isotope exchange experiments
revealed that the G`0 for ATP
synthesis on purified F1 is close to zero!
• When solubilized F1 (act as a ATPase) was
incubated with ATP in the presence of 18O-labeled
H2O, three or four 18O atoms were incorporated into
the Pi, indicating that ATP formation/hydrolysis are
readily reversible and multiple rounds of ATP
formation and hydrolysis occurred on the enzyme!
• Measurement of Kd values: ATP has a much higher
affinity than ADP to the enzyme (10-12 M vs 10–5 M).
• The proton gradient was thus proposed to drive the
release of ATP from the enzyme surface.
The 18O exchange
experiment:
Readily reversible
reactions
Keq = 2.4
G`0 = 0
The G`0 for ATP
synthesis on purified
F1 is close to zero!
(Paul Boyer)
Release of ATP from ATP synthase was proposed to be the
major energy barrier for ATP synthesis
15. ATP synthase comprises a proton
channel (Fo) and a ATPase (F1)
• The F1 part consists of nine subunits of five types:
a3b3gde.
• The knoblike F1 portion is a hexamer of alternating
a and b subunits (arranged like the segments of an
orange), which sits atop the single rod-shaped g
subunit.
• The Fo portion consists three types of subunits:
ab2c10-12.
• The c subunits, each forming two transmembrane
helices, form a donut-shaped ring in the plane of the
membrane.
• The leg-and-foot-shaped ge subunits stands firmly
on the ring of c subunits.
• The two b subunits of Fo seem to connect to the ab
hexamer via the d subunit of F1.
• The proton channel is believed to lie between the a
subunit and the ring of c subunits.
• X-ray crystallography revealed that the three b
subunits of F1 assumes three different conformations,
with bound ADP, ATP analog, or empty respectively
(John Walker, 1994, Nature, 370:621-628)!
a
b
The ten c subunits of Fo
(The yeast FoF1 structure)
The g subunit of F1
The ATP synthase
comprises a proton
channel (Fo) and a
ATPase (F1)
ADP
Rod-shaped g subunit.
App(NH)p
Empty
Each b sununit of ATP synthase can
assume three different conformations!
16. The binding-change model was
proposed to explain the action
mechanism of ATP synthase
• The model was proposed by Paul Boyer in 1973
(PNAS, 70:2837-2839), based on kinetic and binding
studies (before the 3-D structure of bovine F1 or
yeast FoF1 was determined).
• Downhill proton movement through Fo will drive the
rotation of the c-subunit ring and the asymmetrical
g subunits, which will cause each of the three b
subunits to interconvert between the three
conformations, as a result, each of them take turns to
take up ADP + Pi, synthesize ATP, and release ATP.
• Rotations of the g subunit and the c subunits of the
F1 unit in three discrete steps of 120o (powered by
ATP hydrolysis catalyzed by the b subunits) have
been directly observed using fluorescence
microscopy by Dr. Kazuhiko Kinosita in 1997
(Nature, 386:299-302) .
• The estimation of H+ consumption for each ATP
formed is 4 (among which one is consumed for Pi
transport), thus about 2.5 ATP/NADH, 1.5
ATP/FADH2.
• The chemiosmotic coupling allows nonintegral
stoichiometries of O2 consumption (or NADH and
FADH2 oxidation) and ATP synthesis.
b-ATP
g
b-ADP
b-empty
g
The binding-change
model proposed by
Paul Boyer
g
Rotation of the g
subunit and the
ring of c subunits
in the FoF1 complex
was observed by
in vitro studies
using fluorescence
microscopy
Rotation of the g subunit and the ring of c subunits
in the FoF1 complex was directly observed by in vitro
studies using fluorescence microscopy
17. The energy stored in the proton
gradient can be used to do other work
• The ADP, Pi, and pyruvate are believed to be
transported into, and ATP out the mitochondrial
matrix by using the proton gradient.
• The rotary motion of the bacterial flagella is
energized directly by the proton gradient across the
plasma membrane.
• The thermogenin on the inner mitochondrial
membrane of brown fat tissue cells uses the protongradient to produce heat to maintain body
temperature for hibernating animals, newborn
animals and mammals adapted to cold
(thermogenesis).
The protonmotive force
is used for
active transport
through the
inner
membrane of
the mitochondria
The rotary motion of the bacterial flagella
is energized directly by the proton
gradient across the cytoplasmic
Membrane.
Heat is generated in
Brown fat through the
action of thermogenin,
an uncoupling protein.
18. Electrons in NADH generated in
cytosol is shuttled into mitochondria
to enter the respiratory chain
• This is usually fulfilled by the malate-aspartate
shuttle system in liver, kidney and heart, using the
malate-a-ketoglutarate and the glutamate-aspartate
transporters.
• Electrons of NADH in the cytosols of skeletal
muscle and brain are often shuttled into the matrix
by using the glycerol 3-phosphate shuttle system,
which delivers the electrons to complex III, thus
releasing less amount of energy for proton gradient
generation.
• Electrons of NADH generated in plant cytosol enter
the respiratory chain directly with no need of
shuttling due to the presence of an externally
oriented NADH dehydrogenase.
19. The pathways leading to ATP
sysnthesis is coordinately regulated
• The rate of the respiration is generally controlled by
the availability of ADP and since ADP acts as the
acceptor of Pi, this way of regulation is thus called
“acceptor control”.
• ATP, NADH; ADP, AMP, Pi, NAD+ regulates rate of
fuel oxidation at further upstream steps.
• The first intermediate of citric acid cycle acts to
inhibit the glycolysis pathway at PFK-1!
• The ratio [ATP]/([ADP][Pi]) fluctuates only slightly
in most tissues due to a coordinated regulation of all
the pathways leading to ATP production.
The interlocking regulation of glycolysis,
pyruvate oxidation, the citric acid cycle,
and oxidative phosphorylation by the
relative levels of ATP/NADH, ADP, AMP,
Pi, and NAD+.
20. Photosynthetic organisms generate
ATPs (and NADPH) via
photophosphorylation.
• The molecular mechanism of photophosphorylation
is remarkably similar to that of oxidative
phosphorylation: also mediated via a acrossmembrane proton gradient generated using energy
released from stepwise electron flow through a
series of similar electron carriers (cytochromes,
quinones, and Fe-S proteins), located on the
thylakoid (类囊体) membranes of chloroplasts or
plasma membrane of the photosynthetic bacteria.
• The electron donor in photophosphorylation, H2O, is
a poor electron donor (Eo`= +0.82 V) and needs to
be first charged by using light energy to provide
electrons of high potential energy.
• Protons are pumped by a protein complex similar to
complex III (cytochrome c reductase, cytochrome
bc1 complex) of mitochondria.
• The excess energy-rich ATP and NADPH generated
by photophosphorylation (or the light reactions) is
further stored in stable energy-rich carbohydrates
through the carbon-assimilation (the second phase of
photosynthesis ) reactions occurring in the stroma
of chloroplasts.
• The carbohydrates are then used by heterotrophic
organisms as energy and carbon sources.
Stage III
Stage II
Thylakoid
Stage I
Stroma
Stage I
Solar energy is the ultimate source of all
biological energy
Thylakoid
Stroma
Stage IV
21. It took a long time for humans to
understand the chemical process of
photosynthesis
•
•
•
•
O2 is produced by plants (1780).
Light is needed for plants to produce O2 (1786).
CO2 is taken up by plants (1790s).
H2O is taken up during CO2 fixation because the
sum of weights of organic matter and O2 is much
more than the weight of CO2 consumed, water is the
only other substance present (1790s).
• Plants convert solar energy into chemical free
energy (1842).
• Experiment with leaf extract containing
chloroplasts revealed that absorbed light energy
causes electrons to flow from H2O to an artificial
electron acceptor, e.g., dichlorophenolindophenol,
or Hill reagent (NADP+ was found to be the
acceptor in chloroplasts later); CO2 is not required
for this process; therefore O2 could not be
produced from CO2 (1930s, Hill);
• Radio isotope tracer experiments revealed that CO2
is added to ribulose-1,5-bisphosphate in a cyclic
pathway before it is used for glucose synthesis
(1950s, Calvin).
22. The major light absorbing
pigments on thylakoid membrane is
chlorophylls (叶绿素）
• Chlorophylls (a and b) were found to resemble the
heme group of hemoglobin, being polycyclic planar
polytenes, except that the central Fe2+ is replaced by
a Mg 2+; a 21-carbon alcohol called phytol (叶绿醇)
is attached to a carboxyl group on the
protoporphyrin ring; there exists an extra nonpyrrole ring.
• The light absorbing pigments in algae (海藻) and
photosynthetic bacteria, bacteriochlorophylls, are
very similar to that of higher plants.
• Carotenoids (类胡萝卜素), absorbing light at
wavelengths distinct from chlorophylls, act as
accessory pigments on thylakoid membranes.
• Chlorophyll is always associated with specific
proteins to form light-harvesting complexes (LHCs).
• The absorption spectra of chlorophyll a and b
overlap with the action spectrum of photosynthesis
in chloroplasts.
• Cyanobacteria (蓝藻细菌) and red algae use openchain tetrapyrroles, called phycobilins (藻胆色素),
to absorb light at wavelengths between 520-630 nm.
• All these photopigments show strong absorption in
the visible range of light.
The light absorbing
pigments in higher
plants, algae, and
photosynthetic
bacteria are all
heme-like molecules.
Carotenoids （shown here are b-carotene
And lutein), act as accessory pigments on
thylakoid membranes
Phycobilins
Chlorophyll a
A light-harvesting
complexe (LHCII)
Lutein (叶黄素）
Chlorophyll b
alga
O2 attracting bacteria
The action spectrum
of photosynthesis in alga
overlaps with the
absorption spectra
of chlorophyll a and b.
Light absorbing pigments in cyanobacteria
and red algae, phycobilins, are open-chain
tetraparrole polytenes
23. Photons absorbed by many
chlorophylls funnel into one
reaction center via exciton transfer
• A saturating light flash was found to lead to the
production of only one O2 per 2500 chlorophyll
molecules (using chlorella cells, 1932).
• The photosynthetic unit (or photosystem) concept
was thus proposed: photons absorbed by many
antenna pigments funnel via exciton (激发子)
transfer to one reaction center (in picoseconds) here
light energy is transduced to chemical energy
through charge separation, after which electron flow
begins and proton gradient will then be produced.
Possible way of pigment
arrangement in a
photosystem
Special pair
of chlorophyll a
Charge separation at the reaction center
may be caused by the absorption of one
photon from one chlorophyll molecule
24. Two types of photochemical
reaction centers revealed in bacteria
• The phototransduction machinery of photosynthetic
bacteria contains only one of two types of
photosystems.
• Type II found in purple bacteria: having a cyclic
electron flow pathway; electrons activated from the
reaction center chlorophylls (bleached by 870 nm
light, thus P870) are first accepted by pheophytins
(脱镁叶绿素, chlorophylls lacking the central Mg2+)
causing charge separation; then to a quinone, before
being transferred back to P870 via cytochrome bc1
complex and Cyt c2.
• Structure of such a photosystem was determined
using X-ray crystallography and an electron flow
path was deduced (described above).
• Type I found in green sulfur bacteria, with a cyclic
electron pathway similar to the one in purple
bacteria and a linear electron flowing pathway
where electrons are transferred from H2S to NAD+
via a ferredoxin (a 2Fe-2S protein) and ferredoxinNAD reductase.
• The cytochrome bc1 complexes, being similar to the
complex III in mitochondria, pumps protons across
the plasma membranes.
The cyclic and noncyclic electron transferring path
found in photosynthetic bacteria.
Cyt c2
A deduced path of the
electron flow
(Photochemical
Reaction center)
QH2
The 3-D structure of the photoreaction center
in purple bacteria was determined!
24. Two photosystems (PSII and PSI)
work in tandem to move electrons
from H2O to NADP+ in higher plants
• The phototransduction machninery of higher plants
seem to be evolved from a combination of the two
types of photosystems found in bacteria.
• PSI (similar to the linear pathway found in green
sulfur bacteria) and PSII (similar to the one found in
purple bacteria) were revealed by quantum
efficiency studies (“red drop” at >680 nm and
“enhancement” at 680 nm + 700 nm for chloroplasts)
and bleaching studies (a temporary decrease in
absorption of light at a specific wavelength).
• The electrons are charged twice (at P680 and P700)
on their flowing way from H2O to NADP+.
• Pheophytin also acts as the first electron acceptor for
the excited chlorophyll molecules (the “special pair”)
in PSII resulting in charge separation.
• Plastoquinone, structurally similar to ubiquinone,
carries electrons from pheophytin to cytochrome b6f
complex.
• The cytochrome b6f complex (also similar to the
cytochrome bc1 complex for oxidative
phosphorylation) pumps H+ across the thylakoid
membrane.
• Plastocyanin (质体蓝素), a Cu-containing soluble
protein, carries electrons from the cytochrome b6f
complex to P700 of PSI.
• Plastocyanin plays a similar role as cytochrome c in
oxidative phosphorylation.
• The cytochrome b6f complex and cytochrome c act
in both oxidative phosphorylation and
phtophosphorylation in cyanobacteria (蓝藻细菌).
• PSII are often found only in the stacked regions of
the thylakoid membrane, with PSI and ATP synthase
often only in the unstacked region (thus having free
access to the NADP+ and ADP in the stroma) to
prevent exciton larceny.
Proton
gradient
The “ Z scheme” to
show the electron
flow from PSII to
PSI in plants
Flow of electrons from QH2 to
plastocyanin via cytochrome b6f
complex with H+ pumping
The cytochrome b6f complex
and cytochrome c act in both
oxidative phosphorylation
and phtophosphorylation in
cyanobacteria.
H+
H+
H+
H+
H+
H+
H+
25. P680+ in PSII extracts electrons
from H2O to form O2 via a Mncontaining oxygen-evolving complex
• P680+ first accepts electrons from a Tyr residue
(often designated as Z) of the D1 subunits of PSII,
producing a Tyrosyl radical (Tyr*).
• Tyr* then accepts electrons from the Mn complex in
the oxygen-evolving (or water-splitting) complex.
• The Mn complex is believe to serve as a charge
accumulator that enables O2 to be formed (from 2
splitting H2O) without generating hazardous partly
reduced intermediates, however with mechanism yet
to be elucidated.
• Two protons from the Mn complex are released into
the lumen of thylakoid when a pair of electrons are
donated from the water molecule, contributing to the
build-up of the proton gradient.
?
2 H2O
?
The Mn complex releases H+ to the thylakoid lumen
while transferring electrons from H2O to Tyr via a
mechanism yet to be revealed
26. ATP synthesis is driven by the H+
gradient across the thylakoid membrane
produced by electron flow
• ATP was found to be generated from ADP and Pi
during photosynthetic electron transfer in
illuminated spinash chloroplasts in 1954 (Arnon).
• The molecular mechanism for ATP synthesis in
chloroplasts and in mitochondria is believed to be
very similar.
• About 3 ATP can be synthesized with the production
of each O2 from H2O with the absorption of 4
photons.
27. Cyclic electron flow in PSI
produces ATP, but not NADPH and O2
• Electrons reaching the ferredoxin protein do not
continue to move to NADP+, but return to
cytochrome b6f complex, resulting in proton
pumping, but no NADPH and O2 production.
• This allows the chloroplasts to vary the ratio of
NADPH and ATP formed in photophosphorylation.
Cyclic electron
flow in PSI:
produces ATP
but no NADPH
and O2
28. Compounds other than water are
also used as electron donors in
photosynthetic bacteria
• Some obligate anaerobic photosynthetic bacteria use
other inorganic compounds as electron donors.
• Hydrogen sulfide is used by green sulfur bacteria,
generating elemental sulfur, instead of O2.
• Some photosynthetic bacteria use organic
compounds (e.g., lactate, malate, succinate) as
electron donors.
29. A single protein in halophilic
bacteria, bacteriorhodopsin, absorbs
light and pumps protons
• Illumination of the 26 kDa bacteriorhodopsin leads
to a photoisomerization of its prosthetic group, alltrans-retinal (the chromophore, 发色团） to form
13-cis-retinal (13-顺式-视黄醛).
• The restoration of all-trans-retinal results in an
outward pumping of protons across the plasma
membrane through a series of concerted proton
“hops” through a transmembrane proton path made
of a series of Asp, Glu residues and a series of
closely associated water molecules.
Protons “hops” across a path inside
bacteriorhodopsin under illumination
The proton path made of a series of Asp and Glu
Residues and water molecules in bacteriorhodopsin
Summary
• ATP is synthesized using the same strategy in
oxidative phosphorylation and photophosphorylation.
• Electrons collected in NADH and FADH2 are
released (at different entering points) and
transported to O2 via the respiratory chain, which
consists of four multiprotein complexes (I, II, III,
and IV) and two mobile electron carriers
(ubiquinone and cytochrome c).
• A proton gradient across the inner membrane of
mitochondria is generated using the electron motive
force generated by electron transferring through the
respiratory chain.
• The order of the many electron carriers on the
respiratory chain have been elucidated via various
studies, including measurements of the standard
reduction potential, oxidation kinetics of the electron
carriers, and effects of various respiratory chain
inhibitors.
• Electron transfer to O2 was found to be coupled to
ATP synthesis from ADP + Pi in isolated
mitochondria.
• The chemiosmotic theory explains the coupling of
electron flow and ATP synthesis.
• Isotope exchange experiments revealed that the
G`0 for ATP synthesis on purified F1 is close to
zero!
• ATP synthase comprises a proton channel (Fo) and a
ATPase (F1).
• The binding-change model was proposed to explain
the action mechanism of ATP synthase.
• The energy stored in the proton gradient can be used
to do other work.
• Electrons in NADH generated in cytosol is shuttled
into mitochondria to enter the respiratory chain.
• The pathways leading to ATP sysnthesis is
coordinately regulated.
• Photosynthetic organisms generate ATPs (and
NADPH) via photophosphorylation.
• It took a long time for humans to understand the
chemical process of photosynthesis.
• The major light absorbing pigments on thylakoid
membrane was revealed to be chlorophylls.
• Photons absorbed by many chlorophylls funnel into
one reaction center via exciton transfer.
• Two types of photochemical reaction centers have
been revealed in bacteria.
• Two photosystems (II and I) work in tandem to
move electrons from H2O to NADP+ in higher plants.
• P680+ in PSII extracts electrons from H2O to form
O2 via a Mn-containing oxygen-evolving complex.
• ATP synthesis is driven by the H+ gradient across
the thylakoid membrane, with a higher concentration
in the thylakoid lumen.
• Cyclic electron flow in PSI produces ATP, but not
NADPH and O2
• Compounds other than water are also used as
electron donors in photosynthetic bacteria.
• A single protein in halophilic bacteria,
bacteriorhodopsin, absorbs light and pumps protons