The Biochemical Nature Of?light Detection And Emission Essay, Research Paper
In this essay I aim to describe the range of biochemical pathways and
mechanisms used by living organisms both to detect and to emit light.? I will discuss general principles employed,
and illustrate the range of different biochemistry involved by the use of many
specific examples.Light Detection ????
I will discuss the mechanism and function of light detection by five
groups of light detecting molecule.? The
biggest of these is the rhodopsin group of proteins, I will also look at the
role of phytochromes, cryptochromes, flavoproteins and porphirins in light
detection.????
Rhodopsins are found in a diverse array of organisms, all featuring a
retinoid prosthetic group linked to a an apo-protein, opsin via a protonated
schiff base linkage.? Electrons from the
schiff base lone pair occupy an extra orbital?
(the ?n orbital?), therefore electrons can undergo a n-p* transition as
well as a p-p* transition.? ?Retinal proteins were first discovered in 1876 by Bell, who
observed a reddish pigment that bleaches on exposure to light, which he called
visual purple.? Most rhodopsins contain
retinal as the prosthetic group, but some have one of the other chromophores as
shown below.?? For example freshwater
fish have a rhodopsin containing 3,4-didehydroretinal, which has a red shifted
UV absorption band.?? The opsins found
in all organisms show strong homology for one another.??
All rhodopsins seem to be involved in light detection, with the notable
exception of bacteriorhodopsin, which pumps protons using energy from light
photons in order to generate ATP in anaerobic conditions i.e. is not a light
sensing protein. ????
Halobacteria do however have two sensory rhodopsins.? Sensory rhodopsin I (archaeorhodopsin) has
all trans retinal as the prosthetic group in its native state.? It is photoisomerised by green-orange light
(lmax
= 587 nm) to the deprotonated 13-cis state (lmax = 370nm).? Reisomerisation to the all-trans state is
accelerated by absorption at 370nm.? A response is elucidated in the bacterium
by a pumping of protons by the rhodopsin.?
Sensory rhodopsin I causes the halobacteria to show a phototactic
response to green light (needed for bacteriorhodopsin function), and a
photophobic response to UV light (causes cell damage).?? Sensory rhodopsin II (photorhodopsin) also
has the retinal chromophore in the all-trans state.? Light absorption causes chloride ions to
be pumped across the membrane, triggering a photophobic response to blue-green
light. ???
???
Bovine rhodopsin is the most extensively studied of mammalian
rhodopsins.? It is a single polypeptide
of 348 amino acids which forms 7 TM helices and has a Mr of approximately
38kDa.? Upon absorption of light it
follows the photocycle pictured below.????
The retinal chromophore shows a bathochromic shift on attachment to an
opsin.? This can be explained by an
interaction with two carboxylate groups which act as counter ions, shifting the
lmax
from 440nm (in methanol) to 500nm (in rhodopsin).?? The different absorption maximums of the cone cells of the
retina can be explained by differing counter ion structure in their opsins.? Glu 113 has been determined as a counter ion
by site directed mutagenesis experiments. ????
The photocycles of rhodopsins have been studied using time resolved
laser spectroscopy.? The intermediates
have been isolated by low-temperature spectroscopy, i.e. rapid cooling thus
blocking the normal decay of the intermediates.? For example the photocycle of Octopus rhodopsin was
elucidated.?? It was found that
metarhodopsin is thermostable , thus doesn?t bleach in the retina.? FTIR data has suggested that the interaction
of the chromophore with opsin in the batho state is very different to bovine
rhodopsin.????
Fly visual sense cells have a sensitizing pigment ? 3-hydroxyretinol,
which binds non-covalently to the rhodopsin.??
The sensitizing pigment absorbs in the UV, then transfers the energy to
11-cis 3-hydroxyretinal via radiationless dipole-dipole interactions.?? This allows flys to receive visual
information from wavelengths in the UV (lmax = 350nm). ????
????
The physiological response to light absorption has been studied in
detail in higher animals.?? In mammals
the rhodopsin molecules are found in the membrane of the outer segment of the
retina?s rod (or cone) cells.? In the
dark sodium and calcium ions are able to enter the outer segment through cGMP
gated channels.? This inward movement
balances the outward flux of cations caused by the sodium-potassium pump. Upon
absorption of a photon and the isomerisation of retinal, the following
transduction cascade occurs. ??????????????????????????????????????????????????????????????????????????????????????
cGMP???????????????? inactive cGMP???????????????????????? active cGMP???????????????????????????????????? cation channnels phosphodiesterase??????????????????? phosphodiesterase??????????????????????????? close ???????????? ????????????????????????????????????????????????????????????????????????????????????
5` GMP ? ?????????????????????????????????????????????????????????????????????
??????????????????????????????????????hyperpolarised ???????????????????????????????????????????????????????????????????????????????????????????????????????????
electrical signal ? ?????????????????????????????????????????????????????????????????????????
????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????
slowing of neurotransmitter ??????????????????????????????????????????????????????????????????????????????????????????????????
release at synaptic terminal????
The cGMP phosphodiesterase is activated by the G-protein Transducin?s a subunit.
Transducin is activated by the binding of Metarhodopsin II (the photoexcited
state of rhodopsin).?? This transduction
cascade allows a large amplification of the original photon absorption into a
transmittable electrical signal. One Metarhodopsin II molecule can activate
many Tas before the retinal dissociates from the opsin apo-protein. One Ta will remove the
inhibition from one phosphodiesterase, which can hydrolyse up to 1000 cGMP
molecules per second. ????
Cryo-elcctron microscopy has delivered structural information about
rhodopsin and the intermediates of the photocycle that has allowed the changes
in structure on photoexcitation to be elucidated.? A motion of helix III relative to helix IV has been identified ?
this would mean a change in the conformation of the third cytoplasmic loop,
which is the region that interacts with Ta.? Retinal directly interacts with helix III in the region of
Glu121.? Isomerisation of retinal
results in a rearrangement in hydrogen bonding between Glu134, Tyr223, Trp265,
Lys296 and Tyr306.? Breakage of the salt
bridge between Lys296 and Glu113 allows activation to take place i.e.
metarhodopsin II can form.??
Metarhodopsin II is deactivated by phosphorylation and arrestin
binding.?? Arrestin binds to Ser334,
Ser338, Ser343 near the C terminus of opsin. Ta deactivates itself by its
own GTPase activity. ?Rhodopsin kinase
is inhibited by Ca2+ bound recoverin, so when the cytosolic [Ca2+]
decreases rhodopsin kinase becomes more active.? Phosphodiesterase recombines with its inhibitory subunits.??? The drop in cytosolic calcium
concentration from 0.5 to 0.1mM after a light flash stimulates guanylate cyclase which results in
the reopening of cation channels and the dissipation of the electrical
signal.??? The regeneration of rhodopsin
after photobleaching starts with the dissociation of all-trans retinal from opsin
and its conversion to all-trans retinol.?
An isomerase converts all-trans to 11-cis retinol, which is then
dehydrogenated to 11-cis retinal.?? This
mechanism would not be fast enough to maintain the rhodopsin content of the
membrane, so it only occurs occasionally.?
There is instead a fast light mediated interconversion between
metarhodopsin and rhodopsin, i.e. rhodopsin is regenerated by the absorption of
light by metarhodopsin and subsequent reisomerisation of retinal.?? In invertebrates the retinal does not
dissociate fron the opsin, an exchange of chromophore occurs between two
pigment systems, rhodopsin and retinochrome by a retinal binding protein.? Retinochrome is found associated with the
inner segment.? It consists of an
apo-protein of Mr 24000 and bound retinal (all-trans).? Absorbance of light (lmax = 496nm)
causes isomerisation of all-trans to 11-cis retinal. ???
There are two known retinal disorders related to rhodopsin, Retinis
pigmentosa and congenital night blindness.??
70 different mutations in the rhodopsin gene have been identified that
can cause retinis pigmentosa, either by producing a misfolded opsin or
producing one which is unable to bind retinal.?
Congenital night blindness is an inability of the retina to adapt to
dark conditions.? Two disease causing
mutations have been identified ? Ala292 to Glu and Gly90 to Asp. ???????????????????????????????????????????????????????????????????????????????
????
The phytochrome light detection and signaling pathway has a wide range
of physiological roles within plants including phototropism of seedlings, ion
fluxes, leaf orientation, intracellular movements and day length dependent
processes. The phytochrome protein has a Mr 0f 120,000 an exists as a dimer.? Little sequence homology is seen between
phytochromes in different plants, for example only 65% homology between oat and
zucchini. However the hydropathy profiles between different phytochromes are
very similar.? Light absorption by the
tetrapyrrole chromophore causes structural changes in the chromophore which are
transmitted to the surrounding apo-protein.?
CD studies carried out in the UV spectrum have revealed that large
conformational changes occur near the N-terminus upon phototransformation of Pr
to Pfr and vice versa.? Absorption in
the red band of the spectrum (lmax = 666nm) converts the inactive Pr to the physiologically active
Pfr.? Absorption in the far red (lmax = 730nm)
will reconvert the phytochrome.????? ??? Pr???
??????????????????Lumi-R? ????Meta-Ra? ?????Meta-Rc?????????Pfr????????????????? ?????????response Biosynthesis???????????????????????????????????? ????????????????????????????????Degradation ?? ????
Absorption at 666nm causes the isomerisation of the C15-C16 bond from
cis to trans.? The structures of the two
forms of the tetrapyrrole chromophore are shown below. ??? The
chromophore is linked to the protein via a thioester linkage, although the
nature of the overall chromophore-protein interaction is still unclear, it is
thought that hydrophobic interactions might be important.?? The apo-protein and chromophore synthesis
are regulated separately-only Pr is synthesised and Pfr is degraded 100x faster
than Pr, thus functioning as a mechanism of replenishing Pr. The biochemical
mechanism for Pfr elucidating its response is not known, but a kinase activity
has been found in phytochrome preparations, so it could be by phosphorylation.? ?It
is thought that Pfr binds to operators on the DNA sequence and effects the rate
of transcription.?? Pfr thus regulates
gene expression in a tissue specific manner.?
It can also elicit a response by regulating enzyme activity.? ????
The physiological response could be under control of one of a range of
light factors measured by phytochrome; light quality (spectral distribution),
light quantity, direction of light, duration of light and polarisation of
light. ????
It is likely that phytochrome regulates enzymes by phosphorylating them,
for example NTPase activity can be shown to be light controlled.? Intracellular movement is regulated by the
Ca2+ gradient across the cell, which in turn is generated by the Pr/Pfr
gradient across the cell.? Phytochrome
is oriented in the membrane, and can therefore cause a response to the direction
of light.? The direction of light
falling on a leaf will cause a specific Pr/Pfr gradient to be set up across the
cell, which will effect actin/myosin such that the leaf is directed at 90o
to the plane of light.????
Porphyrins are derivatives of porphin such as haem or uroporphirnogen
VII.Evidence for their participation in
photobiological phenomena relies on the similarity in spectral nature between
the absorption spectra of the porphyrin and the action spectra of the biological
response.?? The spectral nature or a
particular porphyrin depends on the side chain protonation of N atoms and the chelation
of metal of metal ions.? They typically
have a strong absorption band in the far violet called the ?Soret band?.