Meghan Goudy
Chem 444
Dr. R. S. Murphy
Current Critical Review
Introduction
Rhodopsin is an important protein that plays a major role in the mechanism of vision. Without it, there
would be no visual information present. Rhodopsin, a receptor that binds with a G-protein, is located in
specialized cells called rod cells. Rod cells are located in our eyes (1). The human vision system is capable of
distinguishing between one photon per second and over one million photons per second and it is able to absorb
light between approximately 400 nanometers to 780 nanometers (2). Rod cells are designed to collect photons
in low light scenarios so that the organism can receive visual information. Its chromophore retinal, also known
as vitamin A, is situated in the centre of rhodopsin and bound by a protonated Schiff’s base. It has two isomers
– the 11-cis retinal and the all-trans retinal.
The 11-cis retinal transforms into the alltrans retinal once rhodopsin has absorbed a
photon of light via its chromophore retinal
(1). The chromophore retinal absorbs light
very quickly and this has been measured at
200 femtoseconds (3). The retinal is made
up of a polyene chain which has several
conjugated double bonds (4). Rhodopsin is
made up of seven alpha-helices and the
retinal is packed close together with the
Figure 1: The isomerisation of 11-cis retinal to all-trans
retinal upon the absorption of a photon (3).
alpha-helices. The retinal’s binding site is
therefore constrained by two of the helices
and is located on the inactive or dark side of
rhodopsin (5).
11-cis retinal’s conversion to the all-trans isomer is very efficient upon absorbing a photon. Its quantum
yield has been measured at approximately 0.65 (6). Many different computational techniques have been
employed to study the chromophore retinal including quantum mechanics/molecular mechanics (QM/MM) and
quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD). More recently, new methods
have become more popular with theorists. These new techniques are called Quantum Monte Carlo (QMC)
methods. Experimental techniques such as time-resolved spectroscopy have also been used to try and
understand the reaction mechanism of the chromophore retinal.
There is lots of evidence on why evolution chose retinal as the chromophore to absorb light. In humans,
the conjugated end of retinal which is bonded to rhodopsin creates a red shift towards the visible range of light.
Visible light is usually in the same range as the sun’s peak wavelength, which is approximately 500 nm. The
majority of other photoreceptors absorb in the ultraviolet region of the light spectrum. There is a significant
change from 11-cis retinal to the all-trans retinal. Depending upon the absorption of a photon, rhodopsin is able
to determine whether it is in the dark or whether it is in the light (7).
The Mechanism of Vision
The mechanism of vision starts with the 11-cis retinal
molecule absorbing a photon of light. Since the chromophore is
located in a tight space, as soon as it absorbs a photon and
transforms into the all-trans retinal it can no longer fit within its
space. This causes rhodopsin to become activated almost
immediately after a photon is absorbed (7). The retinal rotates
around the carbon-11 and carbon-12 double bond (4). The
carbon-13 and the carbon-14 bond also is rotated (1) and this
conformational change causes a change in the protein’s entire
structure, which begins G-protein binding to transducin.
Transducin is a type of G-protein (4). This results in a series of
reactions that culminate in the visual nerve becoming excited.
The retinal undergoes hydrolysis which leaves behind the
rhodopsin molecule with its binding space and the retinal
molecule, not bound to one another. The retinal isomerizes
Figure 2: The reaction mechanism for
the activation of rhodopsin (1).
again to its 11-cis form and another rhodopsin protein binds to it, which ends the cycle of rhodopsin (3). When
the visual nerve is stimulated, it sends an electrical signal to the brain and thus a visual picture is seen.
Shown on the previous page is the reaction pathway for rhodopsin in the visual cycle. When the photon
of light is first absorbed, rhodopsin transforms into a short-lived intermediate known as photorhodopsin. This
intermediate quickly is relaxed into the next intermediate called bathorhodopsin (4, 1). The retinal remains in
its all-trans isomer in both photorhodopsin and bathorhodopsin. This is due to the short reaction time and
therefore the amino acids that are bound to these intermediates do not have time to change their positions in the
close binding space. Bathorhodopsin stores a certain amount of energy from the light and this energy is
discharged as bathorhodopsin decays into other known intermediate structures including lumirhodopsin and
metarhodopsin I (1). When metarhodopsin I is converted into metarhodopsin II, its enthalpy increases and as a
result there is a big increase in entropy. There is a huge structural change that takes place during this conversion
(1). Metarhodopsin II’s lifetime determines how many transducin molecules can be activated to form the noncyclic structure. Its lifetime is longer in a rod cell and therefore it has a bigger response to light and activity
with the transducin is done before hydrolization occurs at the Schiff base (8).
Theoretical Aspects
Quantum Mechanics, Molecular Mechanics and Classical Molecular Dynamics
There are many computational studies that have been done in order to study the different properties of
rhodopsin. This includes vibrational excitation of the chromophore, the mechanism which rhodopsin utilizes in
order to complete its transformation from 11-cis retinal to all-trans retinal, 11-cis retinal’s geometry in the
inactive state (also known as its dark state) and the entire photoreaction. Many of these studies employ quantum
mechanical methods, lots of times combined with molecular mechanics and occasionally classical molecular
dynamics have been used.
One such study used the CASSCF/AMBER QM/MM theory to optimize rhodopsin’s ground state
configuration and the chromophore as well as the end CH2 group were included in the calculation (3). It then
went on to determine the photodynamics of the entire reaction. The study found that a non-adiabatic QM/MM
approach was the best method to simulate the fast and efficient photoreaction. It also calculated the various
state energies that led to the formation of the all-trans retinal (3).
Another study chose to use QM/MM-MD
methods to study how rhodopsin’s chromophore
behaves. The data showed that once the
chromophore absorbs a photon of light, it becomes
excited into the S1 state after 10 femtoseconds.
The S1 state declines in energy at this point while
the energy of the singlet ground state gains a big
amount of energy. This is due to the relaxation of
the long chain on the retinal molecule and it
begins to shift into the all-trans form from the cis
configuration (4). During this relaxation period,
Figure 3: The path that the chromophore takes when a
photon of light is absorbed (4).
the energies of the S1 and S0 states decrease and
increase respectively, though the decrease of the S1 state is not as sharp as the increase of the S0 state. Once the
two energies meet, the rest of the reaction proceeds in the singlet ground state until 200 femtoseconds have
passed (4). At this point, the all-trans isomer of retinal is fully formed. This article provides many known facts
about the activation of rhodopsin while at the same time it explains the findings and also compares these
findings to experimental results.
Ground state energies and excitation energies have been extensively studied in the theoretical realm.
One particular study used different ground-state
geometries of 11-cis retinal and then calculated
the excitation energies for those ground states.
The graph on the right demonstrates that the
excitation energy for 11-cis retinal changes
when using different ground state
configurations. It is therefore important to
choose the ground-state carefully in order to get
good results for the excitation energy of the
chromophore. The researchers determined that
Figure 4: A graph depicting the excitation energies of
11-cis retinal and the different methods used to
calculate the ground-state configuration of 11-cis
retinal (9).
the CASPT2/S-IPEA and NEVPT2 methods
had the best results. The angles in the ring of
retinal and the bond lengths also affect the excitation energies (9). Computations were also done for the alltrans isomer to compare the energies of the excited state. Stated at the end of the results section is the
conclusion that the excitation energies of both the 11-cis and the all-trans retinal are very close in value and that
this shows that the excitation energy is not affected by the different isomers (9).
Of late, many
computational chemists
have been using QMC
techniques such as
Variation Monte Carlo
(VMC) to figure out the
optimal ground state
configuration for the
retinal molecule in two
different scenarios – one
in the natural
environment of
Figure 5: A graph that depicts the different bond lengths between each conjugated
carbon atom in retinal. The black line represents retinal in a gas environment and the
blue line represents retinal in its natural rhodopsin environment (6).
rhodopsin and one in
the gas phase. This study also compares the bond lengths in the chromophore to experimental nuclear magnetic
resonance (NMR) data. It concludes that the VMC computations agree with the experimental data and that this
technique is a good method for figuring out the chromophore’s configuration in both scenarios (6).
The graph above shows the various bond lengths between each double bonded carbon in the
chromophore in two different situations – in a gas phase environment and in the protein environment. Both
were calculated with VMC. In the gas phase, there is very little difference between the carbon-13 and carbon14 bond and the carbon-14 and carbon-15 double bond. The researchers suggest that this is caused by a
delocalized positive charge across the chain but centered on the nitrogen atom. In its natural rhodopsin
environment, there is no delocalized positive charge but rather a localized positive charge on the nitrogen which
is stable due to the negatively charged glutamic acid that is bonded to the retinal.
Critical Review
There have been many studies on the role of the chromophore retinal in the visual mechanism, both
theoretical and experimental. Most of the studies presented in this review are computational in nature but there
are a few that discuss different experiments for how the chromophore is involved in vision.
One article titled Photochemical Reaction Dynamics of the Primary Event of Vision is an example of a
computational study. The researchers mainly focused on the first reaction in the visual cycle in order to
simulate the fast reaction rate of 200 femtoseconds (4) but they also state that calculations were made for the
entire reaction cycle as well. The group used ab initio QM/MM-MD computations for their experiment. It was
determined that the quick reaction time is due to a perturbation from the protein environment and because of
motions in the excited state of retinal. There were a few reasons mentioned as to why the reaction proceeds as
fast as it does. Two of the more dominant reasons were due to the relatively uniform dynamics of the reaction
and that the excited state of retinal and its complex were unaffected by changes in rhodopsin`s temperature. The
relatively uniform reaction dynamics is because the protein environment does not interfere much with the
excited state dynamics of retinal. Hydrogen-out-of-plane (HOOP) vibrations are also mentioned repeatedly
throughout the article and the computations showed big vibrational excitation of these HOOP modes along with
big low-frequency shifts that follow once the S1 state transitions into the S0 state (4). All of this is laid out in
detail but there are only a few comparisons to experimental techniques that have been used to figure out the
reaction mechanism. It also does not have hardly any discussion on future experiments or questions that still
need to be resolved in order to fully understand the visual mechanism.
The article does mention that lots of time-resolved spectroscopy experiments have been used for this
purpose. New ultrafast spectroscopy is now being employed because it is able to show the molecular vibrations
that are in sync with other retinal molecules that have been excited. HOOP frequency shifts have been seen in
timed-dependant Ramon spectroscopy and this article suggests that the reason for seeing these shifts in
experiment could be due to the transition from S1 to S0 (4).
Another study entitled Relationship between the Excited State Relaxation Paths of Rhodopsin and
Isorhodopsin carried out theoretical QM/MM calculations for an analogue of rhodopsin called isorhodopsin
(10). While this study states that isorhodopsin and rhodopsin have comparable photoisomerization mechanisms,
this article is not really relevant to this critical review because the calculations were done on isorhodopsin data.
However, these calculations are compared to calculations at the same level of theory for rhodopsin
photodynamics. It also mentions some questions that are left unanswered by the study and these questions are
also shared by experimentalists.
The scientific article called Modelling vibrational coherence in the primary rhodopsin photoproduct uses
MD computations for the excited state of
retinal. The theoretical results for the
oscillations of photorhodopsin were a bit higher
than those observed experimentally (3). This
article highlights its computational results and
compares them with data from experimental
studies. One graph shown in the article is
illustrating the energy differences between
various states that lead to the formation of the
Figure 6: A graph depicting the differences in energies
between the S2 - S1 and S1 - S0 states in retinal that lead
to the all-trans isomer (3).
final all-trans retinal photoproduct. This graph
demonstrates the change in energy as time
moves forward. Ultrafast femtosecond
ultraviolet experiments provide more evidence for the values in the graph (3). This article is very informative
for the first part of the reaction mechanism but it does not go much further than that.
As stated earlier, QMC theoretical techniques have recently become popular in studies with retinal. Two
of the articles studied for this review deal with QMC techniques for calculating the excitation configuration of
the retinal into its first singlet excited state. Both research groups are in agreement that the QMC methods are
superior to older methods that couldn`t quite compute the geometries for excited state retinal (9, 6). However,
the work of Valsson, Angeli and Filippi mainly focuses on the excitation energies for the chromophore without
dealing with the actual photoisomerization mechanism. Although the first step of this mechanism is very
important to the cycle, more should be said about the rest of the steps in the process.
Many experiments have been performed to figure out how the visual cycle works. Only a few of the
articles are experimental rather than theoretical but they are important nonetheless. The article titled Location
of the Retinal Chromophore in the Activated state of Rhodopsin deals with NMR methods used to discover the
movement of the different carbon atoms during the transition from the 11-cis retinal to the intermediate known
as metarhodopsin II. It also tells about the location of the retinal molecule in the metarhodopsin II intermediate
(5). The NMR data below shows the result of tagging rhodopsin and metarhodopsin II with 13C methionine. In
the rhodopsin data, there are no peaks between the methionine and the C5 or C18 atoms of the retinal but once it
was changed into metarhodopsin II,
there are peaks between the
methionine and those same carbon
atoms of the retinal molecule (5).
Lots of the NMR study is
written in a complicated way that few
readers would be able to understand.
It does go into too much detail about
the NMR data and the NMR
techniques.
Figure 7: The NMR results for rhodopsin and metarhodopsin II.
Rhodopsin`s result is in black while metarhodopsin II`s is in red (5).
Palczewski`s article provides a sufficient amount of background information of the visual cycle and its
mechanism. It is heavily based on the biology of the eye rather than the photochemistry that takes place in the
conversion from 11-cis to all-trans retinal. There is no mention of any experimental techniques as this article`s
main objective is to get information out to its readers, much like a textbook. Everything that is known about the
visual mechanism is in this article.
According to many of the articles studied, there are many disagreements between computational
chemists as to which method is the best to calculate the excitation energies of the retinal. Some of them prefer
to use QM/MM methods while newer studies prefer the QMC methods or the NEVPT2 perturbation approach.
There is also a debate about what the relationship is between dissociation and optical absorption (9). A lot of
the theoretical data about the visual mechanism is supported by experiments such as ultraviolet femtosecond
spectroscopy and time-resolved spectroscopy techniques.
Visual Diseases
There are many visual diseases that humans can get and one of the harsher diseases is Autosomal
Dominant Retinitis Pigmentosa (ADRP). Many causes of ADRP do not occur in the rhodopsin complex but
sometimes mutations do occur in rhodopsin. Rhodopsin mutations occur in approximately 30 percent of ADRP
patients (1). These mutations seem to weaken the rhodopsin structure and cause it to malfunction, causing
issues for seeing in the dark and therefore affecting the visual cycle. Later, more opsins in the cone cells may
become affected and then loss of light vision will occur (1).
Conclusion
Much is known about the role of the chromophore retinal in the protein rhodopsin. The visual cycle is
repeated an uncountable number of times throughout the day. Rhodopsin is located in the rod cells in the
human eye and is responsible for low light scenarios. The excitation energies of retinal and the ground state
geometries have been calculated theoretically and supported by experimental data. Studies have been done to
determine how the retinal rotates within its binding space to form its all-trans isomer when it is hit by a photon
of light. After a photon is absorbed, the transition from the original rhodopsin protein to the first intermediate
photorhodopsin takes approximately 200 femtoseconds, which is one of the fastest reactions in nature.
Theoretical computations have shown this timescale and this is also supported by experiments.
However, there are still many aspects of the visual cycle and the role of the chromophore that are not yet
understood. So far, not a single theoretical study can successfully reproduce the intermediate photorhodopsin
(10) but this intermediate has been observed experimentally. Another aspect that is not understood currently is
the function of water in the activation of rhodopsin. There is experimental evidence of an enthalpy increase and
an entropy increase that happens when metarhodopsin I changes into metarhodopsin II. Chemists believe that
this could be a result of water being released from the system (1). It is also not completely understood how the
G-protein attaches itself to the rhodopsin molecule and subsequently activates it. More research in both the
theoretical and experimental realms needs to be done to be able to fully explain how the entire visual cycle
works.
References
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309-328.
2. Palczewski, K. Chemistry and Biology of Vision. Biol. Chem 2012, 287(3), 1612-1619.
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Chem. Phys 2012, 137, 22A523, 10.1063/1.4742814.
4. Hayashi, S.; Tajkhorshid, E.; Schulten, K. Photochemical Reaction Dynamics of the Primary Event of
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