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Photosynthesis
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time. To see the images in their full glory, make sure you
come to the lecture!!
Introduction
Photosynthesis is undoubtedly one of the most important processes on
the planet. Arguably second to DNA replication (since DNA came first
and drives photosynthesis) but the operation which defines the chemical
nature of the whole planetary surface.
In summary, photosynthesis is the process by which plants use the
energy of light quanta to split water H2O into hydrogen and oxygen.
The oxygen bubbles off as a gas while the hydrogen reacts with CO2 to
produce sugars, specifically the simple hexose sugar glucose
6CO2 + 6H2O = C6H12O6 + 6O2 (needs 2870 kJ mole-1)
This reaction is plausibly believed to have started around 3.8 billion
years ago (give or take a generous margin for uncertainty), and certainly
changed the whole nature of our planet.
About 3800 million years ago…
The earth had oceans, life, but the air was CO2/CH4 without
oxygen. The oceans contained vast amounts of dissolved Iron II
(blue-back). The earliest fossils are of stromatolites.
Age of planet earth, billions of years
4.5
4
3.5
3
2. 5
2
1.5
1
Photosynthesis
Oxygen builds up in air, removing
Iron 2 from oceans as rust
0.5
0
Animals
Dinosaurs
Stromatolites
These are our oldest fossils but can still be found (notably Shark Bay in
Australia). Basically they are mounds made by photosynthetic algae
which entrap sediment so accrete, and can become quite large (a metre or
so) in shallow water. Humble, but we owe our existence to their slow
diligent production of oxygen. Recent DNA work suggests that modern
cyanophyta (blue-green algae) are relatively newly evolved, implying
that the earliest stromatolites were made by unknown ancestral algae.
The key player in modern photosynthesis is an organelle called the
chloroplast. We met this before under our ‘plant cells’ lecture, but
need to revisit the details.
You will recall that chloroplasts are derived from cyanobacteria by
endosymbiosis. This bizarre process allows one organism to live
inside the cell of another, gradually becoming subsumed and
dependent on its larger host.
(What I didn’t tell you is that this has happened hierarchically: The
photosynthetic ‘dual cell’ can itself be subsumed by a larger cell,
giving a chloroplast with 3 outer membranes. In the marine algae
called dinoflagellates the process has occurred a 3rd time, so that their
chloroplasts have 4 membranes! Almost as bizarre, many animals
contain algae in their cells and rely on them partially or wholly for
energy: Hydra, Cassiopeia, reef-forming corals, the giant clam
Tridacna, a flatworm Convoluta.)
Chloroplasts contain many stacked light-capturing plates
called thylakoids, stacked into structures discovered in
Victorian times and called grana (singular granum). Grana
number 10-50 in chloroplasts, and are connected together by
a dense network of membranes called stroma.
Granum
= stack of thylakoids
Contain chlorophyll
Connected by lamellae
The cytosol is called the
stroma
Ribosomes
DNA
Starch often stored here
Our understanding of photosynthesis derives in good part from
the use of radioactive tracers. For example it is possible to
supply plants with water or CO2 in which the oxygen is 18O.
Heavy water H2 18O
6CO2 + 6H2 18O = C6H12O6 + 618O2
Heavy carbon dioxide C18O2
6C18O2 + 6H2O = C6H1218O6 + 6O2
What does this show?
Water is the source of the O2 released during photosynthesis.
Further work (using labelled CO2 fed to algae which were given a
flash of light then killed at various times afterwards) made it clear that
there were many steps in the procedure, some of which needed light
but some of which did not.
Light reaction: needs light energy, produces ATP, a reduced electron
carrier (NADPH), and H+. In order to understand this we must go
into the photoelectric effect, electron transport and proton pumps.
The Calvin (or Calvin-Benson) cycle, which uses ATP and NADPH
CO2 to produce glucose.
The light reaction
Here chlorophyll absorbs light energy and uses it to impart energy to an
electron.
This isn’t as odd as it might seem: light energy comes in packets or
parcels called quanta (sing. quantum). Long wavelengths (red<-> infrared) light is low energy and does little. (This is why you can put your
hands under a food heater without risk of sunburn). Short wavelength
(blue -> ultra violet) quanta pack more energy and can impart enough
energy to a molecule to increase its energy state to a new (quantised)
value. This energy state is unstable and tends to decay, with the excess
energy being carried away by various means, one of which is the
emission of an electron. (This is why it is the UV component of sunlight
that causes the most tissue damage in sunburn).
The electromagnetic spectrum,
focussing especially on the
visible spectrum.
High energy
Low energy
The photoelectric effect
One of Albert Einstein’s 3 great publications was his explanation of the
photoelectric effect. When you shine light on some materials – he used
the element Selenium, but plants use the molecule chlorophyll –
electrons are emitted. In the case of selenium they fly off as radiation and
can be measured directly (along with their energy). Chlorophyll is a bit
more tricky…
If you turn up the intensity of the light more electrons are emitted, but
they all have the same energy. If you make the light bluer the energy of
electrons increases.
This was explained as light hitting the surface as bullets (quanta), with 1
quantum displacing one electron. Bluer light is equivalent to higher
speed bullets.
Red photon
Low E, only
able to warm
up surface
Blue photon
High E, able
to displace an
electron
The photoelectric
effect
Electron
e
Selenium
Energy of
emitted
electron
Blue green red
Wavelength of light
Energy of photon
Chlorophyll behaves much the same way as selenium, except that
the critical wavelength above which no electrons are released is c
730nm, just beyond the visible into the IR.
Unlike selenium the efficiency of capture is not a simple function of
wavelength but has a 2-peaked distribution: c 680nm (red) and c
425nm (blue).
My Dad used to say that his
house plants always died because
they needed the UV that window
glass filtered out. For just the
once, he was wrong about this
(their morbidity reflecting
instead the level of care
received…): plants do not need
UV, indeed are damaged by it.
The chlorophyll molecules
At the heart of the chlorophyll molecule lies an atom of metal held in
a ring structure, much like the iron atom held in the haem group of
haemoglobin. Unlike haemoglobin, chlorophyll contains
magnesium. Note that there are 2 forms: Chlorophyll_a absorbs
more into the UV than Chlorophyll_b.
Haem ring
Hydrophobic tail, anchors
into phospholipid
membranes
Accessory molecules (and why
plants are green).
If plants relied ONLY on chlorophyll they would be largely unable to
use much of the visible EM spectrum. Natural selection has favoured
ways to capture more energy, which is done by means of Accessory
Pigments. These are other coloured chemicals, which absorb light in
the visible region and pass their energy onto adjacent molecules of
chlorophyll.
These accessory pigments take various chemical forms – the
commonest are carotenoids such as ß carotene, which helps capture
longer-wavelength blue light. Together this means that plants can use
energy from much of the visible spectrum. There is a dip in efficiency
in the green region, for a quirk of evolution that never seems to have
been explained (and probably lies in quantum physics). Why does this
explain the colour of plants?
(Because the colour we see is what reflects off unused.)
The net action spectrum for plant photosynthesis.
Antenna systems
Accessory pigments are held in a tight array surrounding a chlorophyll
molecule, and when they intercept a photon one of 2 things can happen:
1: Fluorescence: The energy is re-released as a photon of light. No
chemical change takes place. (Ponder the wavelength of the re-emitted
light: longer, same or shorter than before?)
Answer: Longer – some energy will have been lost in the conversion.
2: The energy is passed along to the local reaction centre, a chlorophyll
molecule. The energy is passed as an electron from molecules that
receive high-energy quanta to progressively lower energy molecules, and
the lowest energy energy chlorophyll molecule is the reaction centre.
Its job is to act as a reducing agent.
REDOX – mnemonics, just in case you
forgot…
Redox state concerns the presence/absence of free electrons. It is
measured in volts, but here all we need worry about is the movement
of electrons.
Loss of an electron = Oxidation
Gain of an electron = Reduction
Loss of an Electron = Oxidation
Gain of an Electron = Reduction
Think of a lion: LEO says GER
GER
Chemists prefer the mnemonic OILRIG. Work it out…
Chlorophyll donates an electron so is a reducing agent. As it
happens normal chlorophyll is not much of a reducing agent,
but excited chlorophyll is. Here I use * to indicate the
presence of a high-energy electron.
Chl* + A
Chl+ + A-
This redox reaction would not occur in the dark.
Once the electron has been transferred to an acceptor molecule, the
energy can be passed on to other molecules (always with some loss of
energy). In fact there turn out to be two separate pathways for electrons
to flow, making ADP -> ATP and NADP+ -> NADPH.
ADP, ATP: Adenine di/tri phosphate – the unit of energy
NADP – Nicotinamide adenine dinucleotide phosphate
These two molecules together are the raw materials needed to make the
Calvin cycle work.
Non-cyclic flow
Here water is split into oxygen, H+ and electrons. It is these
electrons which replace those emitted by chlorophyll and captured by
NADP+
2 different photosystems are used, called photosystem 1 and
photosystem 2. They have different reaction centres and different
absorbance maxima.
Name
Ps1
abs max
700nm
role
uses light energy to reduce
NADP+ to NADPH
Ps2
680nm
uses light to split water:
H2O -> ½ O2 + 2H+ + 2ε-
This stage pumps protons
across the thylakoid
membrane for making
ATP.
Energy
ε-
ε-
Ferrodoxin
Phaephytin I
2H+
O2
NADP
reductase
PS2
NADPH
PS1
H2O
ε-
Time
ε-
NADP+
This probably arose from an earlier system used by photosynthetic
bacteria which oxidise hydrogen sulphide to sulphur: instead of
making O2 gas they make solid S
Cyclic electron flow
The non-cyclic electron flow which transfers an electron
from water to NADP+ makes about equal amounts of ATP
and NADPH. In fact the Calvin cycle (the CO2-fixing
reaction, that takes place in the stroma) needs more ATP
than NADPH, and a second mode of photosynthesis takes
place in which electrons are cycled, simply making ATP.
Here the high energy electron from P700 is captured by
ferrodoxin and moved down a chain of cytochromes which
acts as a proton pump: the energy of the electron is used to
pump a proton into the thylakoid interior.
Cyclic electron flow only makes ATP (via a proton pump)
Energy
εFerrodoxin
Proton
pumping
chain
PS1
Proton pumps and ATP
manufacture
We need to explore the uses and operations of proton pumps: these are
crucial to understanding both chloroplasts and mitochondria.
Protons are simply H+, a hydrogen that has lost its outer electron. Well,
actually no, it is a subatomic particle, which is immediately captured by
any passing molecule. In fact in water when we say H+ we tend to mean
H3O+, which is a molecule of water that has absorbed an extra proton, a
hydroxonium ion.
H2O + H+ => H3O+
Almost always when one talks of H+ one means this hydroxonium ion.
Actually in this case it is indeed a proton that gets pumped across a
membrane as a way of storing energy (like pump-storage scheme dams),
although as soon as released into the solution it becomes H3O+.
Both in chloroplasts and in mitochondria, the only way that the protons
can diffuse down the steep concentration gradient is to go through an
ATP synthetase, and enzyme which performs the reaction
ADP + Pi => ATP
This is of course the basic unit of energy in the cell.
The same operation works in the christae of mitochondria and the
thylakoids of chloroplasts. The actual shape of the inner membranes are
very different but more importantly the topologies are different.
In mitochondria, H+ is pumped out of the innermost layer, while in
chloroplasts they are concentrated inside this layer.
H+
In a mitochondrion, H+ is pumped from
the inside of the double membrane to the
middle space.
+
H+ H
H+
H+
H+
H+
H+
H+Christae of
mitochondria,
Outer
Inner
membrane membrane
This charge gradient is maintained by
several different energy-consuming
enzymes (cytochrome c oxidase /
reductase, NADH-Q reductase), and
cannot be relieved by simple diffusion
because the phospholipid bilayer is
charge-impermeable, like a plastic sheet.
ADP + Pi
The protons CAN diffuse
down the charge gradient
(pulled by the Proton
Motive Force), but only
by giving up enough
energy to charge up a
molecule of ATP.
ATP
H+ H+H+
H+
H+
H+
H+
In a Chloroplast, H+ is pumped inside
the thylakoid membrane. Otherwise the
operation is exactly the same as in a
mitochondrion.
+
H+ H+ pH5
H+ H+ H
The pH differences can be sharp - in the
light pH falls to 5 inside and rises to 8
outside - a concentration difference of
how much?
Outer
Inner
membrane (thylakoid)
membrane
ADP + Pi
ATP
H+ H+H+
H+
H+
1000 = antilog10(3)
H+
A brief insight into the
advancement of science
This idea, about using a proton gradient to generate ATP, is known as the
chemi-osmotic hypothesis, and is an example of a maverick theory
becoming standard accepted dogma. When I first learnt about
chloroplasts and mitochondria (1979) we we told that the details were
not fully understood, and that there was this wacky idea about acid
pumps….
That’s how science develops, why science keeps changing, and why
science’s focus on the operation of things gets tighter and tighter.
Contrast this with religions, who are stuck with immutable words in
some book written long ago, however unlikely they look…
The Calvin cycle (making sugars from CO2)
Also known as the Calvin-Benson cycle. In this process high energy
molecules made by light (ATP + NADPH) are used to drive the reduction
of CO2. This is sometimes called the dark reaction, as light is not
directly used, but the ATP etc are very short lived and in practice this
cycle only happens in the light. (So don’t call it that)
Our understanding of it dates to work by Calvin & Benson in the 1950s
using labelled CO2 (14CO2), applied to algae in the dark, given a flash of
intense light then killed in boiling ethanol a few seconds later. If the
algae were killed 30s later the labelled 14C ended up in many chemicals.
If they were only killed 3s after the the flash all the label was in one
molecule: 3-phosphoglycerate (3PG).
O
H-O-C-C-C-O-P
3PG
Here I use the convention that single
hydrogens are omitted and P refers to a
phosphate group PO43-
The process has a similarity to the citric acid cycle, by which glucose is
oxidised releasing energy. In this case an initial CO2 acceptor is entered
into a cyclical reaction in which a larger molecule is made, and the initial
CO2 acceptor is reformed.
The initial CO2 acceptor is ribulose 1,5 bisphosphate, a 5 carbon
phospho-sugar. It accepts CO2 to make a 6 carbon sugar, which is then
split to make 2 molecules of 3PG.
This all happens in the stroma – the space outside the thylakoid
membranes, into which H+ is pumped.
CO2 +
O
P-O-C-C-C-C-C-O-P
rubisco
Cleaves here
C
P-O-C-C-C-C-C-O-P
ribulose 1,5 bisphosphate
2*
O
H-O-C-C-C-O-P 3PG
O2
Rubisco
The limiting factor on the rate of photosynthesis in the light is the
fixation of CO2. Specifically, the rate-limiting step is the catalysis by
the enzyme Rubisco (= Ribulose bisphosphate carboxylase/oxygenase)
of the uptake of CO2 by ribulose 1,5 BP. Apart from improving the
efficiency of rubisco (which is presumably at an local evolutionary
high point and is unlikely to improve much now), all that a plant can
do to speed up the reaction is to make MORE rubisco. And they do, in
fact it dominates their proteome. >20% of all the protein in plants is
rubisco, making it the commonest protein in the world. This seems
odd since this is an intangible globular protein – if asked most people
would have guessed that animal connective proteins (collagen/keratin)
would be the commonest.
Any deviation from
this position causes a
severe reduction in
efficiency
efficiency so will be
Configuration (AA sequence) selected against
The 3 parts of the Calvin-Benson
Cycle
This cycle can be divided up into 3 parts (this is more meaningful for
our visualisation of the process than it reflects reality in the plant).
1: Fixation of CO2 – catalysed by rubisco and generating 3PG.
This is an acid not a sugar.
2: Conversion of PG into a sugar, in this case a simple 3 carbon sugar
called glyceraldehyde 3-phosphate (confusingly abbreviated to G3P).
3: Regeneration of the CO2 acceptor, ribulose 1,5 bisphosphate.
The core of the Calvin-Benson cycle
CO2
ATP
Ribulose 1,5BP
A series of reactions (more
complex than we need to learn here
today) takes P3G and, with the
energy of ATP, makes ribulose.
3PG
ATP, NADPH
P3G
But this makes nothing – where is the output?
The answer is that NOT ALL the P3G is recycled to make Ribulose. A
portion is instead used in another set of reactions that make glucose,
thence other complex sugars. This makes sense if we look at the
numbers of carbon atoms at each stage in the cycle: Note that 6 carbons
enter and 6 are withdrawn as P3G.
6* CO2
6 * ATP
6* Ribulose 1,5BP
12 *3PG 12 * ATP +
NADPH
10 * C3 molecules enter here
12 * P3G
2 * C3
molecules taken
away to make
sugars
Rubisco and its problems
In addition to capturing CO2, rubisco cleaves oxygen (adding it to make
the aldehyde group in 3PG). There is in fact a direct competition
between oxygen and the Calvin-Benson cycle: a curious and apparently
non-adaptive reaction called photorespiration actually generates CO2
under high-oxygen levels. The factor which determines which reaction
dominates is the O2:CO2 balance inside the leaf. To make sugars, leaves
need high levels of CO2 and low oxygen.
Some plants have evolved a special pattern of cell differentiation in their
leaves to concentrate CO2 in cells using rubisco. (This is not easy as
CO2 is only c. 0.04% in the atmosphere).
When I learned this in 1979 CO2 was quoted as 330ppm. As I type
in 2002 the official level is approaching 400ppm, and in industrial
countries >400ppm is routine. This WILL affect your future.
C3 plants
Most plants are C3. This means the the CO2 is captured first by
incorporation into a 3 carbon molecule. What was it called again? These
plants mainly have chloroplasts in the epidermal and mesophyll cells –
few in the bundle sheath cells which surround the veins.
Upper epidermis
Mesophyll cells, with many
chloroplasts (hence rubisco)
Bundle sheath cells, with few
chloroplasts.
Sieve tube members of the vein
Lower epidermis
C4 plants
The trouble with being a C3 plant develops towards the end of a hot dry
sunny day (when stomata are closed to conserve water) the levels of
CO2 in the leaf fall very low, oxygen is high, and photosynthesis stops.
(Light energy captured is fed into the photorespiration reaction instead).
There is a select group of plants which have evolved to bypass this, and
carry on photosynthesising after long dry periods without
photorespiration cutting in. These are the C4 plants.
Their special technique is to capture CO2 into a different molecule,
generating a 4-carbon compound. This 4C molecule is then moved to (or
stored for use by) to the rubisco-containing cells where it gives up its
CO2, generating a high local concentration of the gas. This ensures that
rubisco works efficiently without switching to photorespiration.
The receptor molecule C4 plants is a 3C atom called
phosphoenol pyruvate (PEP), which becomes oxaloacetate
via the action of an enzyme called PEP carboxylase. PEP
carboxylase turns out to have advantages over rubisco:
PEP carboxylase has no oxygenase activity (no losses to
photorespiration), and will fix CO2 at very low
concentrations. Its use allows the plant to maintain rubisco at
a good CO2/O2 balance even when ambient CO2 is very low.
This means that towards the end of a sunny day C4 plants
will capture more energy that an equivalent C3 plant. A
small point, which apparently evolved quite recently (c.12
MYBP) in response to the long-term decline in CO2.
Dinosaurs breathed air 4* enriched in CO2 compared to us.
Leaf anatomy of a C4 plant
Chloroplasts containing PEP
carboxylase
Upper epidermis
Chloroplasts containing rubisco.
Mesophyll cells where the chloroplasts
make oxalo-acetate.
Bundle sheath cells, with many
chloroplasts. Also a decarboxylase
which releases CO2 from
oxaloacetate.
vein
Lower epidermis
C4 separation in time and space
(Re-iteration of what you already know:)
The possession of C4 chloroplasts allows plants to capture CO2
efficiently even at very low levels and store the gas as oxaloacetate.
This chemical is ‘equivalent’ to CO2 since it can readily be
decarboxylated to release CO2.
Some tropical grasses (maize, sorghum, crabgrass) use this trick to
carry on photosynthesising down to very low internal CO2
concentrations. Here the rubisco chloroplasts are separated from the
PEP carboxylase chloroplasts in space, being physically segregated in
the bundle sheath cells.
(This bit is new:)
Another group of plants use the same trick to ensure that they never
need open their stomata during daylight. Here the rubisco and PEP are
separated in time.
CAM plants
CAM plants are adapted to dry conditions. This has evolved several
times, and is named after the Crassulas (thick-leaved succulent dicots
preferring very dry conditions.)
CAM = Crassulacean acid metabolism.
In the UK we have a few native crassulacea - the ?
stonecrops Sedum! Houseleeks (Sempervivum) are another in this
family. The unrelated cacti also have CAM, as do pineapples.
Here the oxaloacetate path is used at night to
open the stomata. The point is that the
stomata are never opened by day, but open
instead at night when conditions are cooler.
In CAM plants The C4 compound oxaloacetate is stored as another
C4 compound malic acid, which is then decarboxylated during the
day by the normal photosynthetic processes.
Night
CO2
Day
Malic acid C4
using
stored ATP
etc
Stomata shut
Night
Photosynthesis
makes 3PG using
rubisco as normal,
also ATP.
Stomata shut
Stomata shut