Lecture on Photosynthesis

David Nelson

Chemolithotrophic bacteria, and organisms that derive their food chains from them, are the 
only life on the planet that can live truely free from the sun.  The rest of us are dependent 
on sunlight for our existence which is ultimately linked to photosynthesis.  Photosynthesis 
is the light driven fixation of CO2 into carbohydrate.  Photosynthesis is wide spread in 
nature.  It occurs in prokaryotes and eukaryotes.  In eukaryotes, photosynthesis is done in 
special organelles called chloroplasts that have evolved from cyanobacteria.  
Photosynthesis has a special place in the evolution of life on earth, because it changed the 
atmosphere by releasing oxygen from water by photolysis.

In prokaryotes and eukaryotes, photosynthesis is a two step process.  The first step is 
capture of light energy and its conversion into chemical energy in the form of ATP and 
NADPH.  These reactions are light dependent.  The second step is the conversion of CO2, 
ATP and NADPH into carbohydrate, specifically glucose.  These reactions do not require 
light.  These distinct stages in photosynthesis are called the light reactions and the dark 
reactions.  Each part has been the subject of Nobel Prize winning work.  Johann 
Deisenhofer, Harmut Michel and Robert Huber won in 1984 for solving the X-ray crystal 
structure of the light reaction center from a bacterium.  This was the first crystal structure of 
a membrane protein.  Melvin Calvin won the Nobel in 1961 for working out the Calvin-
Benson cycle, in one of the first studies to use a radioactive tracer to understand 
biochemical pathways.  

We will consider the light reactions and the dark reactions separately.

LIGHT REACTIONS

The light reactions are membrane dependent.  The proteins involved are found in 
complexes, similar to the electron transport complexes in mitochondria.  Some of them are 
completely analogous, such as the CF1CFO ATPase that is the chloroplast equivalent of the 
F1FO ATPase.  The reactions depend on compartmentation and impermeable membranes, 
just like the mitochondria.  To begin, let's look at the structure of a chloroplast and see how 
the membranes are arranged.

CHLOROPLAST STRUCTURE

See fig 22.1, p. 699 for a micrograph and 22.2, p. 700 for a cartoon of chloroplast 
structure.  Both chloroplasts and mitochondria evolved from bacteria, but they have 
significantly different structures.  Both have an outer and an inner membrane, but 
chloroplasts have in addition to that a third membrane called the thylakoid membrane.  This 
is inside the compartment of the inner membrane.  The space in this compartment is called 
the stroma and it is similar to the matrix of the mitochondria.  The thylakoid membrane 
forms another compartment called the thylakoid space or the thylakoid lumen.  The 
thylakoid membrane is organized into grana, where the membranes form stacks of discs, 
and lamellae that connect the grana.  The light reactions take place in the thylakoid 
membrane and the dark reactions take place in the stroma.

LIGHT REACTIONS REQUIRE CHROMOPHORES TO CAPTURE LIGHT ENERGY,
CHLOROPHYLLS, PHEOPHYTINS, CAROTENOIDS, ETC.

Chloroplasts have a variety of chromophores to capture light energy.  The most significant 
is chlorophyll.  (See fig. 22.5, p.702)  Chlorophyll is similar to heme, except it has Mg 
instead of iron at the center, and the Mg does not undergo oxidation changes during the 
light capturing process.  There are other differences in the structure including a long side 
chain.  In 1930, Han Fischer was given the Nobel Prize for work done on porphyrin 
chemistry, including hemin and chlorophyll.  In humans, a defect in alpha oxidation of the 
beta methyl group leads to build up of phytanic acid that eventually results in Refsum's 
disease.  YOU DO NOT NEED TO MEMORIZE THE STRUCTURE OF 
CHLOROPHYLL.

Because chlorophylls only absorb light in a fairly narrow range of wavelengths, there are 
accessory pigments that can absorb light in other regions and transfer that energy to the 
light harvesting system.  All pigments that absorb light result in an electron that is boosted 
to a higher energy level.  There are four ways the energy of absorbed light can be 
transmitted.  The electron may return to its resting state and the energy can be lost as heat.  
The energy may be given off as a quantum of light in fluorescence.  The energy may be 
transferred to an adjacent pigment molecule by resonance energy transfer, or the electron 
may move to a new carrier as in the electron transport chain.  Chloroplasts tend to use the 
last two methods for transferring the energy to the site where it will be used.

Fig 22.9, p. 705 shows a hypothetical pathway for light energy to take moving through an 
antenna complex.  The green disks represent chlorophyll molecules.  Experiments have 
shown that there are relatively few sites that acually use the light energy.  These are called 
reaction centers.  Chlorophyll molecules outnumber reaction centers about 300 to 1, so the 
majority of chlorophyll molecules serve as antennas to shuttle light energy to the reaction 
centers.  These chlorophylls are not free in the membranes, but they are attached to proteins 
and arranged in ways to be maximally efficient at gathering light energy.  They are tilted at 
about every angle.

The reaction centers are surrounded by light harvesting complexes (LHCs) that contain the 
chlorophylls.  The major LHC in green plants is LHC-II.  It structure is known.  It 
contains 13 chlorophylls and 2 carotenoids.  There are only three transmembrane helices 
which serve as a scaffold to mount the chlorophylls.

There are two types of reaction centers in chloroplasts.  These are located in photosystem I 
and photosystem II.  The actual reaction center in both photosystems is a special pair of 
chlorophylls that have a slightly lower energy excited state than their antenna chlorophylls.  
That way they can trap the energy feeding in from the antenna complexes in a slight energy 
well.  

PHOTOSYSTEM II, PHOTOSYSTEM I AND THE Z SCHEME

Photosystem I and photosystem II are distinct protein complexes like the electron transfer 
complexes. (see fig 22.13, p. 708)  They have different redox active centers and they do 
different things.  Photosystem II splits water to make oxygen.  It is present in 
cyanobacteria and chloroplasts, but it is not found in purple or green bacteria.  These 
bacteria have reaction centers that cannot split water and cannot evolve oxygen.  
Photosystem II is evolutionarily related to the light reaction center of purple bacteria, even 
though that system does not split water.  Because the light reaction center from purple 
bacteria has a known X-ray crystal structure, we have a pretty good idea what PSII looks 
like.  

Photosystem II has two extra subunits, not present in PSI, that carry antenna chlorophylls 
(about 50).  In contrast, PSI has its own antenna chlorophylls built in (about 100).  
Comparison of the sequences of the PSII antenna proteins with the Photosystem I proteins 
suggests that the N-terminal part of the Photosystem I proteins evolved from the separate 
antenna proteins seen in photosystem II.  This suggests that a fusion occurred between 
genes to form the photosystem I proteins..  

Photosystem I appears to be quite different from photosystem II.  It has 100 chlorophyll a 
molecules and 20 carotenoids to act as an antenna.  A special pair of chlorophyll a 
molecules form the light capturing chromophore called P700.  This is the reaction center of 
the complex.  There are two phylloquinones and 3 iron-sulfur centers.  A low resolution 
crystal structure is available at about 4.5 angstrom resolution.  There are 31 transmembrane 
helices.  

The two photosystems are linked because the excited electrons from PSII are eventually 
passed through an electron transport chain to the P700+ reaction center of PSI.  The PSI 
electron, also boosted by light goes on to form NADPH.  When it leaves, an electron hole 
is created (P700+) that is filled by an electron from PSII.  

The PSII reaction center called P680 also gives up an electron when it is activated.  This 
forms P680+ which represents an electron hole that is filled by electrons taken from a 
manganese containing active site.  When dark adapted chloroplasts are flashed with short 
pulses of light, oxygen is released with a periodicity of every 4th flash(see fig. 22.14, p. 
710).  Since each flash is assumed to excite one electron at photosystem II, the active site 
where the Mn is, must go through 5 different oxidation states called S0 to S4.  When four 
electrons have been taken, by four independent photons being absorbed, water is split and 
O2 is released.  The electrons that fill the P680+ hole initially come from the Mn active site, 
but the active site recovers them from water.  

The Two photosytems and the connecting electron transport chain are frequently drawn so 
the complexes and electron carriers are placed on a reduction potential scale.  This makes 
the whole system look like a Z, so it is called the Z scheme.  

STRUCTURES OF THE COMPLEXES IN THE Z SCHEME AND ELECTRON 
MOVEMENTS.

There are three membrane protein complexes involved in the Z scheme.  PSI, PSII and the 
cytochrome b6/cytochrome f complex.  The cytochrome b6/f complex is similar to the bc1 
complex in mitochondria, in that it receives electrons from a lipid soluble quinone called 
plastoquinone, and it shuttles electrons through Rieske iron-sulfur protein to cytochrome f 
that is analogous to cytochrome c1.  From here, electrons go to a soluble carrier called 
plastocyanin that is analogous to cytochrome c, though it has a copper rather than a 
cytochrome redox center.  The cytochrome b6/f complex does not do a Q cycle like 
complex III.  The b hemes are used in another process called cyclic photophosphorylation.
Electrons enter the complex at the b6 hemes.  They do not go out from the b hemes.
Plastoquinone is analogous to ubiquinone in mitochondria (see Fig. 22.15, p. 711)

As electrons are sent through the cytochrome b6/f complex, protons are pumped into the 
thylakoid lumen.  The ratio seems to be one proton per electron, not as efficient as in 
mitochondria.  If we consider the stroma to be similar to the matrix of the mitochondria, 
then this direction of pumping is similar to what is seen in mitochondria.  The thylakoid 
lumen is then analogous to the periplasmic space in mitochondria.  

There is a chloroplast ATP synthase that uses the proton gradient made by electron flow to 
form ATP.  The F1 part faces the stroma.  This process is called photophosphorylation.

STRUCTURE OF PHOTOSYSTEM II

Fig. 22.20, p. 714 summarizes the key features of photosystem II.  The thylakoid lumen 
has the Mn complex where water is bound.  Two subunits D1 and D2 hold the critical 
redox active centers.  P680 is the reaction center and it is composed of two chlorophylls 
very close together, near the Mn complex.  Farther into the membrane is a pair of 
pheophytin molecules.  Pheophytin is similar to chlorophyll except it has no Mg.  The Mg 
has been replaced by two protons instead.  Closer to the stroma is a pair of quinones called 
QA and QB and one central iron.  The electrons flow from this complex to plastoquinone.  
Plastoquinone feeds to the cytochrome b6/f complex that we already discussed.

As I mentioned earlier, PSII is structurally similar to the purple bacteria reaction center.  
This is shown in Fig 22.19, p. 713.  Though the two arms of the chromophores in the 
reaction center are nearly identical, all the electrons flow down one side and not the other.  
This probably happens in PSII also.  The iron does not become reduced in bacteria and it 
probably doesn't in chloroplasts either. 

THE STRUCTURE OF PSI

All the reaction centers, PSI, PSII and the bacterial reaction centers seem to be based on a 
similar plan.  In PSI there are two main subunits PsaA and PsaB that hold the special pair 
of chlorophylls and a series of intermediate electron carriers.  In PSI these include two 
more chlorophylls, instead of pheophytins, and 2 phyloquinones (Vitamin K1) and a 
central iron-sulfur.  One major difference in PSI is that the electron transport chain is 
surrounded by 100 chlorophylls that are part of the two large subunits.  PSII has its 
chlorophylls in extra subunits.  On the stromal side, there are two more iron-sulfur centers 
and a ferredoxin binding site.  Ferredoxin carries the electrons to Ferredoxin NADP+ 
reductase (one at a time, where NADP+ is reduced in a two electron reduction).  There is a 
shematic of PSI in fig. 22.21. p. 714.

CYCLIC PHOTOPHOSPHORYLATION

Some of the electrons from PSI can return to cytochrome b6/f complex and feed through 
that complex to plastocyanin and then back to P700+.  This is a cyclic pathway that can 
form a proton gradient and make ATP.  It is called cyclic photophosphorylation(see fig. 
22.24, p. 719).  The electrons enter the b6/f complex at b6.  Theoretically it takes 4 
photons to split water.  This sends four electrons to plastocyanin and eventually to P700+.  
That means that 4 electrons have to go through PSI so the electrons from PSII have 
someplace to go.  That adds up to 8 electrons (and 8 photons) per oxygen released.  In 
practice this number is more like 10, because cyclic photophosphorylation sends some of 
the electrons from P700+ back to fill that electron hole.  

HOW DO PURPLE BACTERIA DIFFER FROM CHLOROPLASTS?

Purple bacteria do not slpit water.  The electrons that are generated by light absorption at 
the special pair rapidly transfer to ubiquinone and then on to a bacterial bc1 complex.  This 
is the same as the bc1 complex in mitochondria, with a Q cycle included.  The electron 
flow pumps protons and feeds into a soluble cytochrome analogous to cytochrome c.  This 
carries the electrons back to the excited special pair to fill the electron hole.  There are only 
two complexes (and an F1F0 ATPase).  See fig. 22.18, p. 713. This process is similar to 
PSI cyclic photophosphorylation.  

RECENT EVIDENCE CONCERNING THE IMPORTANCE OF PHOTOSYSTEM I

Elias Greenbaum of the Oak Ridge National Lab in Tennessee has made a claim that PSI is 
not necessary to fix CO2 and the Z scheme is in need of revision.  He has isolated mutants 
defective in PSI and they still grow.  There is lots of skepticism from photosynthesis 
researchers who believe something is not right with the experiments.  The results have been 
published in Nature and Science (Science 273,310 and  364-367 1996)
They point out that some workers have measured a quantum requirement of 5-6 
photons/oxygen.  This is in disagreement with the minimum required by the Z scheme 
(8 photons/oxygen).  It is in agreement with a photosystem II reduction of NADP+ to be 
used in CO2 fixation.  However, this has not been shown.  There may be some new 
enzymes and new pathways to get to NADPH from PSII alone.  

THE DARK REACTIONS

CO2 is fixed into carbohydrate by a complex series of reactions that require ATP and 
NADPH made in the light reaction phase of photosynthesis.   The Calvin-Benson cycle is 
shown in Fig. 22.28, p. 724.  The CO2 fixation step is catalyzed by an enzyme called 
ribulose-1,5-bisphosphate carboxylase/oxygenase or rubisco for short.  Ribulose 1,5 
bisphosphate is the 5 carbon compound that reacts with CO2 to form a 6 carbon 
intermediate that hydrolyzes to form two molecules of 3-phosphoglycerate, an intermediate 
in glycolysis.  The reaction is shown in more detail in Fig 22.26, p. 721.  

Good, let's feed this product into the glycolysis pathway, run it back up through 
gluconeogenesis and be done you say, but it is not that simple.  It is necessary to make 
more of the 5 carbon compound ribulose-1,5-bisphosphate, or the pathway could not 
continue.  The rest of the reactions in fig. 22.28 are required to make one molecule of 
glucose and regenerate the ribulose-1,5-bisphosphate.  

The enzyme that catalyzes the first step, rubisco, is the most abundant protein on earth
It is found in the stroma of chloroplasts at about 15% of total chloroplast protein.  The 
protein is an alpha 8 beta 8 complex, with the smaller subunits enhancing the activity of the 
catalytic subunits by about 100 fold.  

Rubisco is activated by light and is regulated in a complex manner by covalent 
modifications and accessibilty to Mg.  The levels of Mg are also regulated by light and 
another regulatory protein called rubisco activase is activated by an increase in stromal pH 
that is regulated by light.  For rubisco to be active, lys 201 must be carbamylated by 
addition of CO2 to form NH-COO-.  This reaction is enhanced by alkaline pH.  pH in the 
stroma goes up in response to light due to proton pumping into the thylakoid space, so 
carbamylation increases in the light.  The carbamylated protein is still not active until it 
binds Mg.  Mg concentration in the stroma increases with light because Mg is ejected from 
the thylakoid space.  This tends to activate the rubisco.  

PHOTORESPIRATION

Rubisco can also react with O2 rather than CO2.  This wastes rubisco-1,5-bisphosphate, 
which is pretty hard to make.  The oxygenase reaction reduces the efficiency of carbon 
fixation by rubisco.  The consequences of this side reaction are shown in fig. 22.27, p. 
722.one molecule of 3-phosphoglycerate is formed, with one molecule of 
phosphoglycolate.  During metabolism of the glycolate, CO2 is formed.  Therefore, O2 is 
being consumed and CO2 is being released as in respiration, the enzyme involved is 
normally a photosynthetic enzyme, so this process is called photorespiration.  

THE CALVIN-BENSON CYCLE

By using radioactive CO2, Melvin Calvin was able to identify the label in the compounds 
shown in fig. 22.28 and deduce this cyclic pathway for production of glucose from CO2 
and regeneration of  the starting compound ribulose-1,5-bisphosphate.  Reactions 2-8 are 
just gluconeogenesis (fig. 21.1, p. 662), with the one exception that glyceraldehyde 3 
phosphate dehydrogenase is a different enzyme that uses NADPH instead of NADH.  
Step 9 is the reverse of the reaction shown in fig.21.42, p. 691, where a two carbon unit is 
transferred from fructose-6-P to glyceraldehyde-3-P to form a 5 carbon and a four carbon 
product.  The erythrose-4-P combines with dihydroxyacetone phosphate to form the 7 
carbon sedoheptulose-1,7- bisphosphate.  This is dephosphorylated by a plant specific 
enzyme to make sedoheptulose-7-P.  This is a donor of a two carbon fragment to 
glyceraldehyde-3-P to form two 5 carbon compounds.  This is the reverse of the reaction 
shown in fig. 21.41, p. 691.   The two 5 carbon compounds are converted by one 
epimerase(fig. 21.40, p. 690) and one isomerase(fig. 21.39, p. 690) to form ribulose-5-P, 
which is then phosphorylated by another plant specific enzyme, phosphoribulokinase to 
make the starting compound ribulose-1,5-bisphosphate.

This whole process takes 12 NADPH and 18 ATP.

REGULATION OF PHOTSYNTHETIC PATHWAYS BY LIGHT

We discussed how the rubisco enzyme was regulated by light.  Some of the other enzymes 
involved in photosynthesis are also regulated by light.   The mechanisms are indirect, such 
as pH changes, metal ion changes and changes in the level of reducing power in the 
stroma.  The photophosphorylation that accompanies the Z-scheme proceeds by proton 
pumping into the thylakoid lumen.  That means that the stroma will become more alkaline 
only in the presence of light.  This is the time when pathways like the Calvin cycle need to 
be activated.  Several enzymes in this pathway have an alkaline pH optimum, thus 
activating them in the presence of light.  

NADPH also accumulates during the Z-scheme events.  This produces a pool of reduced 
ferredoxin.  Other compounds that can exist in a reduced or oxidized state will also become 
reduced in this environment.  These might include glutathione, and a small protein called 
thioredoxin.  This protein serves to activate several enzymes of the Calvin cycle by 
reducing them.

Mg ions are required for many enzymes activity.  We already saw how Mg ions played a 
part in regulating rubisco.  Fructose-1,6-bisphosphatase is a second enzyme activated by 
Mg, whose concentration is affected by light.  

These multiple regulatory controls on the Calvin cycle are present to keep the Calvin cycle 
from operating in the dark.  In the dark, the plant cells use mitochondrial respiration to 
supply ATP.  It would be counterproductive to have both processes going on at the same 
time, since one makes carbohydrate from CO2 and the other makes CO2 from 
carbohydrate.

THE C4 PATHWAY

The use of O2 as a substrate by rubisco is wasteful, as we have already discussed.  The 
concentration of O2 and CO2 in combination with the Km values of the rubisco enzyme for 
both substrates dictates that CO2 will be favored over O2, but only 3:1 or 4:1.  This is not a 
great ratio.  Some plants have evolved ways to improve this ratio by two methods.  Both 
methods seek to separate the two substrates so the O2 concentration is less and it cannot 
compete with CO2 as well.  Both methods convert CO2 and phosphoenol pyruvate into a 
four carbon compound oxaloacetate.  This is where the two methods diverge.  In C4 
plants, the oxaloacetate is further modified and transported across cell membranes to the 
inner part of the plant.   Here CO2 is again released along with a three carbon fragment that 
returns to the  surface to be converted back to PEP for another round.  Inside the mesophyll 
cells the O2 concentration is less and rubisco can react with CO2 more often than at the 
surface.  

CRASSULACEAN ACID METABOLISM

The second method for lowering O2 relative to CO2 is time dependent rather than space 
dependent.  Desert succulent plants cannot open their stomata to bring in CO2 during the 
heat of the day.  They also cannot do photosynthesis at night.  The solution is to bring in 
the CO2 during the night and store it as malate until the day.  As the CO2 is released during 
the day, rubisco can use it even though the stomata are closed, and O2 concentration should 
be reduced.