Download Bchem 253: Metabolism in health and diseases 1

BCHEM 253:
METABOLISM IN HEALTH AND
DISEASES 1
Lecture 7:
Photosynthesis
Lecturer:
Dr. Christopher Larbie
Introduction
• The vast majority of energy consumed by living organisms
stems from solar energy captured by phototrophic
organisms.
• 1.5 X 1022 kJ of energy produced by the sun reaches the
earth every day.
• Photosynthetic organisms convert 1 % of the solar energy into
chemical energy.
• This chemical energy is stored in the form of biomolecules, which
are harvested by the organisms that eat them forming food chains.
• The basic equation of photosynthesis is deceptively
simple.
• Water and carbon dioxide combine to form carbohydrates and
molecular oxygen.
• 6CO2 + 6H2O → C6H12O6 + 6O2
ΔGo’ = 2,798 kJ/mol
• The process of photosynthesis is a complex process
involving
• photoreceptors, reaction centres, protein complexes, electron
carriers, etc.
• By this complex process 1011 tons of carbon dioxide is
fixed globally every year.
• A diverse group of organisms are capable of
photosynthesis.
• From bacteria to the tallest trees, photosynthesis occurs in
membranes.
• In photosynthetic bacteria, the plasma membrane fills up
the cells interior.
• In eukaryotes, the photosynthetic membranes are
contained within an organelle called a chloroplast.
Basic Process in Photosynthesis
• Photosynthesis is a reduction process and the ultimate
reducing agent is water but there other photosynthetic
bacteria that use other reductants.
• Furthermore, a hexose itself is not the primary
carbohydrate product.
CO2 + 2 H2A
Organisms
Plants, algae,
cyanobacteria
Green sulphur,
bacteria
Purple sulphur
bacteria
light
(CH2O)n + H2O + 2A
Reductant
Water
Reaction
CO2 + H2O → (CH2O) + H2O + O2
H2S
CO2 + 2H2S → (CH2O) + H2O + 2S
HSO3-
CO2 + H2O + 2(HSO3-) → (CH2O) +
2(HSO4)
Hill’s Experiment
• Hill and his co-workers in 1939 discovered that when
isolated chloroplasts that lack CO2 are illuminated in the
presence of a variety of artificial electron acceptors like
ferricyanide (Fe(CN)63+), O2 is evolved with concomitant
reduction of the Fe3+ to Fe2+.
4Fe3+ + 2H2O → 4Fe2+ + 4H+ + O2
• A number of reactions involving different inorganic oxidants
or electron acceptors occur in photosynthesis.
• Hill reaction also demonstrated that CO 2 does not
participate directly in the O2 producing reaction.
• Light and dark reactions are separate processes.
• It was also discovered that the final electron acceptor in the
light reaction is NADP+ which is reduced to NADPH.
The Chloroplast
• The chloroplast has many similarities to the mitochondrion.
• It has a porous outer membrane, an intermembrane space
and an inner membrane that is impermeable to most
molecules.
• The inner membrane encloses the stroma which is
analogous to the matrix of the mitochondria.
• In the stroma are the soluble enzymes that utilize NADPH and ATP to
convert CO2 into carbohydrates.
• The stroma has the DNA of the chloroplast and the
machinery for replication, transcription and
translation.
• Chloroplasts are not autonomous; they require many
proteins encoded by the nuclear DNA.
• Within the stroma are membranous structures called
thylakoids which are flattened discs stacked to form
granum. Different grana are linked together by
stroma lamellae.
• The chloroplasts have three distinct membranes
• Outer (which encloses inter-membrane space),
inner (which encloses the stroma) and thylakoid
membrane (which encloses the thylakoid space or
the thylakoid lumen).
• The thylakoid membrane serves the following
functions
• is the site of oxidation-reduction reactions that generate a
proton motive force analogous to the cristae in the
mitochondria.
• contains the energy-transducing machinery that harvests
the energy of the sun.
• the pigments that absorb light, the reaction centers,
electron transport chains and ATP synthase
Light and Dark Reactions of the Thylakoid
membranes
• The light reactions occur within the thylakoid membrane.
• The dark reactions are the chemical reactions involved in
fixing CO2 for the synthesis of carbohydrates. These reactions
occur in the stroma.
• Dark reactions can occur in the dark or in the light. They are
called dark reactions because they are driven by the energy
provided by ATP and NADPH, not by photons of light.
Light Absorption
• Visible light is electromagnetic radiation of wavelengths
between 400 – 700 nm which is a very small part of the
electromagnetic spectrum
• When a photon of light is
absorbed, an electron in the
absorbing molecule is lifted
to a higher energy level. This
requires that the energy of
the photon be exactly equal
to the energy of the
electronic transition. A
molecule that has absorbed
a proton is in an excited
state. The excited molecule
will eventually return to the
ground state giving up the
energy as either heat,
electron transfer, exciton
transfer or as emission of a
photon of light at a longer
wavelength (Fluorescence).
Chlorophylls
• Chlorophylls are
magnesium containing
substitute tetrapyrroles
• Chlorophylls have
magnesium
coordinated to the
planar centre of the
conjugated ring.
• Chlorophylls contain a
long chain alcohol
called pytol which is
attached to the
tetrapyrole ring by an
ester linkage
• Chlorophylls are excellent light absorbers because of
their aromaticity.
• When a chlorophyll molecule absorbs a photon of light,
the excited electron has an enhanced potential for
transfer to a suitable electron acceptor.
• The loss of this high energy electron is an oxidation-
reduction reaction. The net result is the conversion of
light energy into chemical energy driving a redox
reaction.
• Chloroplasts of plants always contain both chlorophyll a
and chlorophyll b.
• Both chlorophylls have a green colour, but their
absorption spectra are slightly different allowing them to
complement each other’s range of light absorption in the
visible region.
• Typically plants contain twice as much chlorophyll a than
chlorophyll b.
• There are other pigments in photosynthetic organisms
that increase the probability of absorption of visible light.
• These pigments are called accessory light harvesting
pigments which absorb wavelengths of light not absorbed
by the chlorophylls.
• These accessory pigments such as carotenoids and
phycobilins are responsible for the beautiful colours of
autumn because these accessory pigments last a lot
longer than chlorophyll.
• Like chlorophyll, these pigments have conjugated double
bonds and absorb visible light
Exciton Transfer
• The light absorbing pigments and chlorophylls of the
thylakoid membranes are arranged into functional arrays
called photosystems.
• Each chloroplast contains approximately 200 molecules of
chlorophylls and about 50 molecules of cartenoids.
• Only a few of the chlorophylls are associated with a
photochemical reaction centre where the energy of the
absorbed light is transduced into chemical energy.
• All of the other chlorophyll and pigment molecules are
light harvesting or antenna molecules.
• They absorb photons of light energy and transfer the
energy by exciton transfer to the reaction centres.
Photosystems I and II
• Photosystem I optimally absorbs photons of a wavelength
of 700 nm.
• Photosystem II optimally absorbs photons of a
wavelength of 680 nm.
The Z-Scheme
Photosystem II
• Photosystem II transfers electrons from water to
plastoquinone and in the process generates a pH
gradient.
• Plastoquinone (PQ) carries the electrons from PSII to the
cytochrome bf complex.
• Plastoquinone can functions as a one or two electron
acceptor and donor. When it is fully reduced to PQH 2 it is
called plastoquinol.
• PSII is an integral membrane protein.
• The core of this membrane protein is formed by two subunits
D1 and D2. These two subunits span the membrane.
• PSII contains a lot more subunits and additional chlorophylls
to achieve a lot higher efficiency than bacterial systems
• There is a special pair of chlorophylls in PSII bound by D1
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and D2 that are in close proximity of each other.
The PSII special pair consists of 2 chlorophyll a molecules
that absorb light at an optimal wavelength of 680 nm.
This special pair of chlorophylls is called P680.
On excitation-either by the absorption of a photon or exciton
transfer-P680* rapidly transfers an electron to a nearby
pheophytin a.
Pheophytin a is a chlorophyll a molecule with the
Magnesium replaced by two protons
• The electron is then transferred to a tightly bound
plastoquinone at the QA site. The electron is then
transferred to an exchangeable plastoquinone located at
the QB site of the D2 subunit. The arrival of a second
electron to the QB site with the uptake of two protons from
the stroma produces plastoquinol, PQH2.
• When the electron is rapidly transferred from P680* to
pheophytin a, a positive charge is formed on the special
pair, P680+. P680+ is an incredibly strong oxidant which
extracts electrons from water molecules bound at the
manganese centre.
• The structure of this manganese centre includes 4
Manganese ions, a calcium ion, a chloride ion, and a tyrosine
radical.
• Manganese is the core of this redox centre because it has
four stable oxidation states (Mn2+, Mn3+,Mn4+ and Mn5+) and
coordinates tightly to oxygen containing species.
• Each time the P680 is excited and an electron is kicked out,
the positively charged special pair extracts an electron from
the manganese centre.
• PSII spans the thylakoid membrane.
Cytochrome bf
• The plastoquinol formed by PSII contributes its electrons
through an electron transport chain that terminates at PSI.
• The intermediary electron transfer complex between PSII
and PSI is cytochrome bf also known as cytochrome b6f.
• In this electron transfer complex electrons are passed one
at a time from plastoquinol to plastocyanin (Pc), a copper
protein of the thylakoid lumen.
PQH2 +2Pc(Cu2+) → 2Pc(Cu+) + 2H+
• The protons are released into the thylakoid lumen.
• Plastocyanin is a water soluble electron carrier found in the
thylakoid lumen of chloroplasts.
• It contains a single Copper atom coordinated to two
histidine residues and a cysteine residue in a distorted
tetrahedron. The molecule is intensely blue in the cupric
form. This mobile electron carrier carries electrons from
cytochrome bf to PSI.
Photosystem I
• The final stage of the light reactions is catalysed by PSI.
• This protein has two main components forming its core,
psaA and psaB.
• These two subunits are quite a bit larger that the core
components of PSII and the bacterial photosystem.
Nonetheless, the subunits are all homologous. The psaA and
psaB subunits are shown together with chlorophyll molecules
and the 3 4Fe-4S clusters.
• A special pair of chlorophyll a molecules lies at the centre of
the structure which absorbs light maximally at 700 nm.
• This special pair is denoted P700. Upon excitation-either by
direct absorption of a photon or exciton transfer- P700*
transfers an electron through a chlorophyll and a bound
quinone (QA) to a set of 4Fe-4S clusters.
• From these clusters the electron is transferred to ferredoxin
(Fd), a water soluble mobile electron carrier located in the
stroma which contains a 2Fe-2S cluster coordinated to 4
cysteine residues.
• The electron transfer produces a positive charge on the
special pair which is neutralized by the transfer of an
electron from a reduced plastocyanin.
• The overall reaction is shown below.
Pc(Cu+) + Fdox → Pc(Cu2+) + Fdred
Ferredoxin-NADP+ Reductase
• Ferredoxin is a strong reductant but can only function in one
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electron reductions.
NADP+ can only accept 2 electrons in the form of a hydride.
Clearly we need an intermediary to facilitate the electron
transfer.
The transfer of electrons from reduced ferredoxin to NADP+ it
catalysed by ferredoxin-NADP+ reductase which is flavoprotein.
This complex contains a tightly bound FAD which accepts the
electrons one at a time from ferredoxin.
The FADH2 then transfers a hydride to NADP+ to form NADPH.
This reaction takes place on the stromal side of the thylakoid
membrane. The uptake of a proton by NADP+ further
contributes to the pH gradient across the thylakoid membrane.
Chloroplast ATP Synthase
• The transport of electrons from water to NADP+ generated a
pH gradient across the thylakoid membrane. This proton
motive force is used to drive the synthesis of ATP.
• The pH gradient generated between the stroma and the
thylakoid lumen is possible because the thylakoid membrane is
impermeable to protons.
• When the chloroplast is illuminated the thylakoid lumen
becomes markedly acidic, pH ≈ 4. The pH of the stroma is
around 7.5. The light induced pH gradient is about 3.5 pH units.
• The trans-membrane electrical potential is not a significant
factor in the proton motive force in the chloroplast because the
thylakoid membrane is permeable to Cl- and Mg2+. Because of
this permeability, the thylakoid lumen remains electrically
neutral while the pH gradient is generated. A pH gradient of 3.5
pH units thus corresponds to a proton motive force of -20
kJ/mol.
• The ATP synthase of the chloroplast is called the CF1-CF0
complex where C stands for chloroplast and F1 and F0
relate to the homologous ATP synthase of the mitochondria.
• The mitochondrial and the chloroplast ATP synthase are
essentially identical with similar subunits and subunit
stoichiometries.
• The catalytic subunit is the β subunit of CF1.
• The CF1 complex lies in the stroma.
• The CF0 complex channels protons from the thylakoid
lumen to the stroma driving rotation of the 12 c subunits
which in turn drives ATP synthesis.
• The ATP formed is released into the stroma where it is
needed for the dark reactions of photosynthesis.
Summary of Electron Transporters
Cyclic Photophosphorylation
• There is an alternative pathway for the electrons arising from
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PSI giving photosynthesis versatility.
The electrons carried in reduced ferredoxin can be transferred
to the cytochrome bf complex rather than the ferredoxin-NADP+
reductase complex. The electrons then flow back through
cytochrome bf to reduce plastocyanin, which then reduces the
P700+ to complete the cycle.
The net outcome of this cyclic flow of electrons is the pumping
of protons across the thylakoid membrane by the cytochrome
bf complex, producing a pH gradient which then drives the
synthesis of ATP.
In addition PSII does not participate in cyclic
photophosphorylation, so O 2 is not generated during this
process.
Cyclic photophosphorylation only occurs when the NADP+
concentration becomes limiting, such is the case when there is
a very high ratio of NADPH/NADP+.
Overall Stoichiometries
• The overall reaction is:
• 8 Photons of light + 2H2O + 2NADP+ + 10H+ stroma → O2 + 2NADPH +
12H+lumen
• The FC0 complex has 12 c-subunits, so it takes 12 H+ to
produce one complete rotation. Each complete rotation
produces 3 ATP molecules. Therefore, it takes 4H + to
synthesize one ATP.
THE DARK REACTIONS OF PHOTOSYNTHESIS
• The light reactions of photosynthesis transform the energy
of light into high energy ATP and NADPH.
• The dark reactions reduced carbon dioxide to form
carbohydrates.
• These reactions are called dark reactions because these
reactions do not directly depend on photons of light.
• NADPH is the anabolic reducing currency of the cell.
The Calvin Cycle
• The Calvin Cycle is the process by which carbon dioxide
is fixed into a form that it is useful for many processes.
• The carbon dioxide that is fixed by this process will
become the nucleic acids, proteins, carbohydrates and
fats of the plant.
• The capacity to accumulate carbon atoms from carbon
dioxide for the net synthesis of carbohydrate distinguishes
the photoautotrophic from the heterotrophic.
• The Calvin Cycle takes place in the stroma of the
chloroplast.
• The Calvin cycle is composed of three parts.
• The fixation of CO2 to 1,5- bisphosphoribulose to form 2
molecules of 3-phosphoglycerate.
• The conversion of 3-phosphoglycerate to glyceraldehyde
3-phosphate which then goes on to form hexoses.
• The regeneration of ribulose 1,5-bisphosphoglycerate.
CO2 Fixation by Rubisco
• The CO2 acceptor is ribulose 1-5-bisphosphate (RuBP).
• The enzyme catalysing the fixation is ribulose bisphosphate
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carboxylase/oxygenase or Rubisco for short.
Rubisco is found on the stromal surface of the thylakoid
membrane.
The carboxylase/oxygenase ending reflects the two activities of
this enzyme.
Rubisco catalyzes the addition of CO2 or O2 to RuBP.
Rubisco constitutes more than 15% of the total chloroplast
protein. Due to prevalence of plants in the biosphere, Rubisco
is world’s most abundant protein.
Rubisco requires a bound divalent metal (Mg2+) for catalytic
activity. The metal ion activates the bound substrate molecules
and stabilizes the negative charge developed in the transition
state.
• The metal centre plays a crucial role in catalysis. Ribulose
1.5-bisphosphate coordinates to the metal through its keto
group and adjacent hydroxyl group. The complex is then
deprotonated to form an enediolate intermediate. The
enediolate reacts with a coordinated CO2 group forming a
new carbon-carbon bond. A molecule of H2O is then added
to form an intermediate that cleaves into two molecules of 3phosphoglycerate.
The Oxygenase Reaction of Rubisco
• Sometimes the Magnesium metal centre of Rubisco
coordinates to RuBP and oxygen instead of carbon dioxide.
Rubisco then catalyses an oxygenase reaction.
• Under normal conditions the rate of the carboxylase reaction
is 4 times faster than the oxygenase reaction.
Salvage pathway for glycolate
The reduction of 3-phosphoglycerate and the
Synthesis of Hexose Phosphates
glyceraldehyde 3-phosphate dehydrogenase
phosphoglycerokinase
Regeneration of Ribulose 1,5-bisphosphate
• First transketolase converts fructose 6- phosphate and
glyceraldehydes 3-phosphate into erythrose 4-phosphate
and xylulose 5-phosphate.
• Then Aldolase condenses erythrose 4-phosphate with
dihydroxyacetone phosphate to form sedoheptulose 1,7bisphosphate.
• Sedoheptulose 1,7- bisphosphatase removes the phosphate
group from the 1 position to form Sedoheptulose 7phosphate.
• Sedoheptulose 7-phosphate then reacts with transketolase
with a second molecule of glyceraldehydes 3-phosphate to
form ribose-5-phosphate and xylulose 5-phosphate
• Finally, ribose 5-phosphate is converted into ribulose 5-phosphate by
phosphopentose isomerase, and xylulose 5-phosphate is converted into
ribulose 5-phosphate by phosphopentose epimerase. The last step is the
phosphorylation of ribulose 5-phosphate to form Ribulose 1,5-bisphosphate.
The net reaction:
• 6CO2 + 18 ATP + 12 NADPH + 12H+ + 12 H2O → C6H12O6 + 18 ADP + 18 Pi
+12 NADP+
Regulation of the Calvin Cycle
• The enzymes of the Calvin cycle are indirectly regulated by
light.
• When light is available to generate ATP and NADPH, the Calvin cycle
enzymes are activated for carbon dioxide fixation.
• In the dark, when ATP and NADPH generation ceases, CO2 fixation
also ceases.
• Light induced changes in the chloroplast regulate key
enzymes in the Calvin cycle. These light induced changes
include:
• Change in the stromal pH.
• Generation of NADPH and reduced ferredoxin.
• Mg+2 efflux from the thylakoid lumen.
• Effect of increased pH of the stroma
• The stromal pH rises to around 8 pH units.
• At this pH, Lys201 reacts with CO2 to form the carbamylated lysine
that coordinates to the Magnesium ion.
• The activities of Fructose 1,6-bisphosphatase, ribulose-5phosphate kinase and glyceraldehyde 3-phosphate dehydrogenase
reach their maxima.
• The light driven pumping of protons from the stroma to the
thylakoid lumen occurs with the concomitant efflux of Mg2+
ions from the lumen to the stroma. The efflux of Mg2+
maintains electrical neutrality.
• Both rubisco and fructose 1,6-bisphosphatase are Mg2+
activated, stimulating CO2 fixation. The rate determining step
of the Calvin cycle is fructose 1,6-bisphosphatase which
make this enzyme a key enzyme in Calvin cycle regulation.
• Illumination of the chloroplasts
activates photosynthetic electron
transport, which generates reducing
power in the form of NADPH and
reduced ferredoxin.
• Several of the enzymes involved in
CO2 fixation are activated upon
reduction of disulphide bonds.
• The most notable enzymes are
• fructose 1,6-bisphosphatase,
• sedoheptulose 1,7-bisphosphatase,
• ribulose 5-phosphate kinase.
THE C-4 PATHWAY OF CO2 FIXATION
(HATCH-SLACK PATHWAY)
• Recall the oxygenase activity of Rubisco.
• Under normal conditions the rate of the carboxylase
reaction is 4 times faster than the oxygenase reaction.
• Normal conditions being P = 1 atm, T = 25 oC, [CO2] = 10μM and
[O2] = 250 μM.
• When the temperature increases the rate of the
oxygenase activity increases more rapidly than the
carboxylase activity.
• Plants that grow in hot climates need a mechanism to
minimize the wasteful oxygenase activity. The plants
adapted to hot climates overcome this problem by
creating a high local concentration of CO2 in the stroma of
the chloroplasts.
• These plants use four carbon compounds C4 such as
aspartate and malate to carry CO2 from mesophyll cells,
which are the cells in contact with the air, to the bundle
sheath cells, which are the major sites of photosynthesis.
• The decarboxylation of the C4 compound in the bundle
sheath creates a high local concentration of CO 2 at the
site of the Calvin cycle.
• The decarboxylation creates a 3 carbon compound such
as pyruvate which can return to the mesophyll cell and
become recarboxylated.
Pyruvate-Pi dikinase