The light-independent reaction
Do not call this the dark reaction, or fall into the trap of assuming that it occurs later on at night.
It follows directly after the light-dependent reaction.
It is helpful to keep track of the number of carbon atoms (and phosphate groups) in the various compounds. The chemical conversions are all part of a cycle - usually called the Calvin cycle
reacts with ribulose bisphosphate
to form two molecules of glycerate 3-phosphate (GP)
. This reaction is catalysed by the enzyme RuBisCo.
to triose phosphate
(3-C sugar with 1 phosphate), using ATP and reduced NADP from the light-dependent reaction.
Most of the triose phosphate (10 out of 12 molecules) is used in a multi-stage process to regenerate RuBP
, so as to repeat the process of accepting carbon dioxide.
The rest of the triose phosphate is the product of the light-independent reaction, and 2 molecules of it are produced for every 6 molecules of CO2
which enter the cycle.
This is converted to useful organic substances
required by the plant, e.g. glucose
(a 6-C sugar) which may be converted (by polymerisation) into other carbohydrates, e.g. cellulose for cell walls or stored as starch.
It can also be converted into other classes of organic compound, e.g. lipids, amino acids and then proteins and nucleic acids.
The Calvin cycle
Melvin Calvin (1911–1997) was an American biochemist at the University of California, Berkeley.
Along with Andrew Benson and James Bassham, Calvin discovered the plant metabolic cycle which bears his name, sometimes together with the others.
He (alone) was awarded the 1961 Nobel Prize in Chemistry.
Calvin used this 'lollipop apparatus' in which he grew the green alga Chlorella
in a liquid containing carbon dioxide labelled with radioactive 14
C and he took extracts after illuminating it for different (short) periods of time.
Two-dimensional chromatograms were prepared and these were covered with photographic paper and left (in the dark) for some time before being processed in the normal way to develop a photographic image - an autoradiogram. Dark splodges showed the position of radioactive organic compounds resulting from the reaction, which were then identified. Reducing the time (and increasing the intensity of light) allowed the sequence of products to be determined.
It is interesting to note that in addition to carbohydrate products, amino acids have been formed in the later sample.
What is meant by a two-dimensional chromatogram?
Chromatogram that is run in one direction, then dried, rotated through 90°, and run again, probably with a different solvent
What are its advantages?
To separate overlapping compounds
The LIR in more detail
The following events take place within the stroma
- a larger fluid-filled space outside the thylakoids which is surrounded by the two outer chloroplast membranes - each of which is itself a lipid bilayer, with embedded proteins. The lipid compositions of these membranes differ from one another and from most other cell membranes which are mostly phospholipid. In fact the outer membrane is about 48% phospholipids, 46% galactolipids and 6% sulpholipids, whereas the composition of the inner membrane is 16% phospholipids, 79% galactolipids and 5% sulpholipids. This is quite similar to composition of the thylakoid membrane.
The chemical reactions which take place in the stroma are a cycle of enzyme-controlled covalent conversions and do not rely directly on electron transfer
which necessitates a membrane-bound chain of redox carriers.
Carbon dioxide fixation
Ribulose 1,5 bisphosphate (RuBP)
is a double phosphate ester of the ketopentose sugar ribulose. This is similar to ribose - a 5-carbon sugar - but it has a >C=O keto group at carbon 2, and it is 'linear' not ring-shaped as it has two phosphate groups, one on each end of the molecule.
RuBP is carboxylated (it reacts with carbon dioxide) to form an unstable compound, 3-keto-2-carboxyarabinitol 1,5-bisphosphate.
The enzyme RuBisCO (Ribulose Bisphosphate CarbOxylase) which catalyses this reaction is said to be the most common protein in the world as it makes up 20-25% of the mass of protein in most leaves and it is produced at a rate of about 1000 kg/second on earth.
The diagrams above show molecules in zigzag skeletal format,
but the Carbon coming in from CO2 is shown as a C
The unstable 6-carbon, 2 phosphate compound splits in the middle to form 2 molecules of glycerate-3 phosphate - GP
- a 3-carbon, 1 phosphate compound similar to glycerol but with a carboxylic acid group - shown on the right as -COO-
(conjugate base of glyceric acid).
GP is further phosphorylated using ATP
to form glycerate 1,3 bisphosphate (G1,3BP)
The next stage is effectively the same as in glycolysis (steps 6&7), but in reverse.
G1,3BP is reduced by reacting with reduced NADP which provides protons and electrons.
The resulting products are glyceraldehyde 3-phosphate
(G3P, also abbreviated to PGA, PGAL or GALP and others!) and inorganic phosphate.
G3P is possibly best known as triose phosphate (TP)
, and some of this is converted into useful compounds for the plant.
Typically 2 x G3P out of 12 molecules in the cycle can be converted into fructose 1,6 bisphosphate, fructose 6 phosphate, then glucose 6 phosphate, then glucose.
Regeneration of RuBp
The rest of the triose phosphate (10 out of 12 molecules) undergoes molecular rearrangement - in 8 stages - to form ribulose monophosphate - ribulose 5-phosphate, Ru5P
. This is then phosphorylated to form ribulose 1,5 bisphosphate
again, so the cycle can continue.
Both RuBP and ADP have two phosphate groups.
Why do we write RuBP as ribulose bisphosphate, but ADP as adenosine diphosphate?
In ADP both phosphates are attached (in a row) to the same part of the molecule, in RuBP there are are 2 different parts - at opposite ends of the molecule
Photorespiration - the downside of RuBisCO
As well as catalysing the (carboxylation) reaction between RuBP and carbon dioxide, the enzyme RuBisCO can also catalyse the (oxygenation) reaction between RuBP and oxygen.
Presumably the molecules of CO2
are similar in size and shape so that both can fit into the active site of RuBisCO.
This reaction, known as photorespiration, is wasteful as it results in the production of 2-phosphoglycolate at well as glycerate 3-phosphate (2 molecules of which are normally produced). The 2-phosphoglycolate is processed via a different cycle before being recycled into RuBP to re-enter the Calvin cycle, and carbon dioxide is lost in the process.
This is a drawback to the normal ('C3
') photosynthesis, as described above. It is more marked in conditions of low carbon dioxide concentration - especially when stomata are closed due to drought - or higher temperature.
Some plants use alternative methods of trapping carbon dioxide, using the enzyme phosphoenolpyruvate carboxylase
, which produces 4-carbon
compounds that are exported to another part of the leaf with a low oxygen concentration where CO2
is released to be accepted by RuBisCO in the normal way, avoiding oxygenation.
Some of the world's most economically important (tropical, grasslike) crops - sweetcorn or maize (Zea mays
), sugarcane (Saccharum officinarum
), sorghum (Sorghum bicolor
), and millets (several species) - use this C4
Most succulent plants and cacti use another alternative: Crassulacean Acid Metabolism CAM
in which CO2
is captured at night when it is cooler and there is less water loss via open stomata, and it is converted into C4 compounds and malic acid which is stored in the vacuole.
During daytime they release CO2
from their reserves to perform photosynthesis when stomata are closed and water conservation mechanisms operate.
Evolution of photosynthesis
Early conditions on planet Earth were very different than today:
Many environments were anaerobic and reducing, rather like hydrothermal vents today.
There was practically no oxygen in the atmosphere, but a higher amount of carbon dioxide
This meant that there was no ozone layer and much more ultraviolet light.
Carbon dioxide dissolved to produce hydrogencarbonate ions.
Originally there must have been bacteria with an established respiration process involving movement of electrons between a number of redox molecules like haem and cytochromes. Within one of these a single photosystem evolved, as magnesium replaced iron in the porphyin ring of haem and the pigment chlorophyll was formed, and this absorbed energy from light.
This early photosystem probably took electrons from hydrogen sulphide or iron, and using chlorophyll passed them into the respiratory cycles enabling organic compounds like sugars to be produced from CO2
Another version of the photosystem emerged which probably took excited electrons from chlorophyll and used them to move protons across the membrane and create ATP before returning the electrons to chlorophyll. This is similar to cyclic photophosphorylation. Both of these systems would differ somewhat and evolved to be more efficient in different environments.
It is thought that (possibly following gene exchange) some bacteria had both systems, but they had a mechanism which allowed them to use only one at a time, avoiding unnecessary synthesis of intermediates: the first provided organic compounds for growth when hydrogen sulphide was available, and the second gave ATP for maintenance when it was unavailable. This is called the "redox switch hypothesis".
Nowadays, several groups of bacteria perform photosynthesis but most have only photosystem I or photosystem II; only cyanobacteria have both systems available, and in some species they switch off PS II when performing nitrogen fixation, which is inhibited by oxygen.
Conceivably depletion of raw materials meant that these bacteria were subject to selection pressure.
Manganese, which is quite common on the ocean floor today, absorbs ultraviolet radiation which could be destructive to organic molecules within cells, so it would act as a sort of antioxidant screen to this radiation. In doing so it emits electrons which would be absorbed by chlorophyll which has lost electrons as it absorbed light energy.
It is thought that cyanobacteria used manganese to split water, which is a much more widely available chemical resource.
Furthermore it is thought that bypassing the redox switch enabled the two systems to work in synchrony.
The resulting continuous electron flow could then be passed from one photosystem to the other, and used to produce both ATP and reduced NADP.
This double photosystem with the manganese-based oxygen-evolving complex became the standard combination when chloroplasts were formed by the coalescence of two cells, forming an early single-celled organism like an alga, from which many-celled plants presumably evolved. Cyanobacteria and higher plants have transformed Earth's atmosphere by adding oxygen obtained by splitting water, and this has allowed a number of aerobically respiring organisms to evolve.
See 'endosymbiont theory' (link below).
Most of the previous section dealt with the light-dependent reaction.
Several versions of the light-independent reaction (C3
, CAM) have been mentioned above, but earlier versions probably used respiration pathways (running in the opposite direction to 'normal').
For example, green sulphur bacteria are currently often found in deep waters, near to hydrothermal vents which emit a dull light as well as various mineral ions which provide reducing conditions resulting in an anoxic environment. Unsurprisingly, they are anaerobic.
They employ PS I for photosynthesis, using several bacteriochlorophylls which absorb light at 720-750 nm but the P840 chlorophylls at the reaction centre emit electrons which are passed to ferredoxin for reduction of NADP. Electrons are repaid following the oxidation of sulphide, resulting in the accumulation of elemental sulphur. The fixation of carbon dioxide is achieved by a reaction which is the complete reverse of the tricarboxylic acid (Krebs, citric acid) cycle, so they use a respiratory pathway.