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’.
