G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor

We have shown previously that phosphoinositide 3-kinase in the retina is activated in vivo through light-induced tyrosine phosphorylation of the insulin receptor (IR). The light effect is localized to photoreceptor neurons and is independent of insulin secretion (Rajala, R. V., McClellan, M. E., Ash, J. D., and Anderson, R. E. (2002) J. Biol. Chem. 277, 43319-43326). These results suggest that there exists a cross-talk between phototransduction and other signal transduction pathways. In this study, we examined the stage of phototransduction that is coupled to the activation of the IR. We studied IR phosphorylation in mice lacking the rod-specific alpha-subunit of transducin to determine if phototransduction events are required for IR activation. To confirm that light-induced tyrosine phosphorylation of the IR is signaled through bleachable rhodopsin, we examined IR activation in retinas from RPE65(-/-) mice that are deficient in opsin chromophore. We observed that IR phosphorylation requires the photobleaching of rhodopsin but not transducin signaling. To determine whether the light-dependent activation of IR is mediated through the rod or cone transduction pathway, we studied the IR activation in mice lacking opsin, a mouse model of pure cone function. No light-dependent activation of the IR was found in the retinas of these mice. We provide evidence for the existence of a light-mediated IR pathway in the retina that is different from the known insulin-mediated pathway in nonneuronal tissues. These results suggest that IR phosphorylation in rod photoreceptors is signaled through the G-protein-coupled receptor rhodopsin. This is the first study demonstrating that rhodopsin can initiate signaling pathway(s) in addition to its classical phototransduction.     

Illuminated rhodopsin is required for strong activation of retinal guanylate cyclase by guanylate cyclase-activating proteins

We have recently shown that activation of retinal guanylate cyclase (retGC) by GC-activating proteins (GCAPs) is much stronger than that previously reported and that preincubation of photoreceptor outer segment homogenates with ATP or its analogue, adenylyl imidodiphosphate (AMP-PNP), is required for the strong activation [Yamazaki, A., Yu, H., Yamazaki, M., Honkawa, H., Matsuura, I., Usukura, J., and Yamazaki, R. K. (2003) J. Biol. Chem. 278, 33150-33160]. Here we show that illuminated rhodopsin is essential for development of the AMP-PNP incubation effect. This was demonstrated by illumination of dark homogenates and treatments of illuminated homogenates with 11-cis-retinal and hydroxylamine prior to the AMP-PNP incubation and by measurement of the GCAP2 concentration required for 50% activation. We also found that the AMP-PNP incubation effect was not altered by addition of guanosine 5'-O-(3-thiotriphosphate), indicating that transducin activation is not required. It is concluded that illuminated rhodopsin is involved in retGC activation in two ways: to initiate the ATP incubation effect for preparation of retGC activation as shown here and to reduce the Ca2+ concentrations through cGMP phosphodiesterase activation as already known. These two signal pathways may be activated in a parallel and perhaps proportional manner and finally converge for strong activation of retGC by Ca2+-free GCAPs.     

Characterization of rhodopsin congenital night blindness mutant T94I

The Thr94 --> Ile mutation in the second transmembrane segment of rhodopsin has been reported to be associated with a congenital night blindness phenotype in a large Irish pedigree. Previously, two other known rhodopsin mutants that cause congenital night blindness, A292E and G90D, have been shown in vitro to constitutively activate the G protein transducin in the absence of a chromophore. The proposed mechanism of constitutive activation of these two mutants is an electrostatic disruption of the active site salt bridge between Glu113 and Lys296 that contributes to stabilization of the protein in the inactive state. Here, the T94I rhodopsin mutant is characterized and compared to the two other known rhodopsin night blindness mutants. The T94I mutant opsin is shown also to constitutively activate transducin. The T94I mutant pigment (with a bound 11-cis-retinal chromophore), like the other known rhodopsin night blindness mutants, is not active in the dark and has wild-type activity upon exposure to light. Similar to the Gly90 --> Asp substitution, position 94 is close enough to the Schiff base nitrogen that an Asp at this position can functionally substitute for the Glu113 counterion. However, in contrast to the other night blindness mutants, the T94I MII intermediate decays with a half-life that is approximately 8-fold slower than in the wild-type MII intermediate. Thus, the one phenotype shared by all congenital night blindness mutants that is different from the wild-type protein is constitutive activation of the apoprotein.     

Identification of regions of arrestin that bind to rhodopsin

Arrestin facilitates phototransduction inactivation through binding to photoactivated and phosphorylated rhodopsin (RP). However, the specific portions of arrestin that bind to RP are not known. In this study, two different approaches were used to determine the regions of arrestin that bind to rhodopsin: panning of phage-displayed arrestin fragments against RP and cGMP phosphodiesterase (PDE) activity inhibition using synthetic arrestin peptides spanning the entire arrestin protein. Phage display indicated the predominant region of binding was contained within amino acids 90-140. A portion of this region (residues 95-140) expressed as a fusion protein with glutathione S-transferase is capable of binding to rhodopsin regardless of the activation or phosphorylation state of the receptor. Within this region, the synthetic peptide of residues 109-130 was shown to completely inhibit the binding of arrestin to rhodopsin with an IC50 of 1.1 mM. The relatively high IC50 of this competition suggests that this portion of the molecule may be only one of several regions of binding between arrestin and RP. A survey of synthetic arrestin peptides in the PDE assay indicated that the two most effective inhibitors of PDE activity were peptides of residues 111-130 and 101-120. These results indicate that at least one of the principal regions of binding between arrestin and RP is contained within the region of residues 109-130.     

Rhodopsin-stimulated activation-deactivation cycle of transducin: kinetics of the intrinsic fluorescence response of the alpha subunit

The intrinsic tryptophan fluorescence of the alpha subunit of transducin (alpha T) has been shown to be sensitive to the binding of guanine nucleotides, with the fluorescence being enhanced by as much as 2-fold upon the binding of GTP or nonhydrolyzable GTP analogues [cf. Phillips and Cerione (1988) J. Biol. Chem. 263, 15498-15505]. In this work, we have used these fluorescence changes to analyze the kinetics for the activation (GTP binding)-deactivation (GTPase) cycle of transducin in a well-defined reconstituted phospholipid vesicle system containing purified rhodopsin and the alpha T and beta gamma T subunits of the retinal GTP-binding protein. Both the rate and the extent of the GTP-induced fluorescence enhancement are dependent on [rhodopsin], while only the rate (and not the extent) of the GTP gamma S-induced enhancement is dependent on the levels of rhodopsin. Comparisons of the fluorescence enhancements elicited by GTP gamma S and GTP indicate that the GTP gamma S-induced enhancements directly reflect the GTP gamma S-binding event while the GTP-induced enhancements represent a composite of the GTP-binding and GTP hydrolysis events. At high [rhodopsin], the rates for GTP binding and GTPase are sufficiently different such that the GTP-induced enhancement essentially reflects GTP binding. A fluorescence decay, which always follows the GTP-induced enhancement, directly reflects the GTP hydrolytic event. The rate of the fluorescence decay matches the rate of [32P]Pi production due to [gamma-32P]GTP hydrolysis, and the decay is immediately reversed by rechallenging with GTP. The GTP-induced fluorescence changes (i.e., the enhancement and ensuing decay) could be fit to a simple model describing the activation-deactivation cycle of transducin. The results of this modeling suggest the following points: (1) the dependency of the activation-deactivation cycle on [rhodopsin] can be described by a simple dose response profile; (2) the rate of the rhodopsin-stimulated activation of multiple alpha T(GDP) molecules is dependent on [rhodopsin] and when [alpha T] greater than [rhodopsin], the activation of the total alpha T pool may be limited by the rate of dissociation of rhodopsin from the activated alpha T(GTP) species; and (3) under conditions of optimal rhodopsin-alpha T coupling (i.e., high [rhodopsin]), the cycle is limited by GTP hydrolysis with the rate of Pi release, or any ensuing conformational change, being at least as fast as the hydrolytic event.     

Functional interaction between bovine rhodopsin and G protein transducin

To elucidate the mechanisms of specific coupling of bovine rhodopsin with the G protein transducin (G(t)), we have constructed the bovine rhodopsin mutants whose second or third cytoplasmic loop (loop 2 or 3) was replaced with the corresponding loop of the G(o)-coupled scallop rhodopsin and investigated the difference in the activation abilities for G(t), G(o), and G(i) among these mutants and wild type. We have also prepared the Galpha(i) mutants whose C-terminal 11 or 5 amino acids were replaced with those of Galpha(t), Galpha(o), and Galpha(q) to evaluate the role of the C-terminal tail of the alpha-subunit on the specific coupling of bovine rhodopsin with G(t). Replacement of loop 2 of bovine rhodopsin with that of the scallop rhodopsin caused about a 40% loss of G(t) and G(o) activation, whereas that of loop 3 enhanced the G(o) activation four times with a 60% decrease in the G(t) activation. These results indicated that loop 3 of bovine rhodopsin is one of the regions responsible for the specific coupling with G(t). Loop 3 of bovine rhodopsin discriminates the difference of the 6-amino acid sequence (region A) at a position adjacent to the C-terminal 5 amino acids of the G protein, resulting in the different activation efficiency between G(t) and G(o). In addition, the binding of region A to loop 3 of bovine rhodopsin is essential for activation of G(t) but not G(i), even though the sequence of the region A is almost identical between Galpha(t) and Galpha(i). These results suggest that the binding of loop 3 of bovine rhodopsin to region A in Galpha(t) is one of the mechanisms of specific G(t) activation by bovine rhodopsin.     

Structural constraints imposed by a non-native disulfide cause reversible changes in rhodopsin photointermediate kinetics

Suspensions of bovine rhodopsin in 2% lauryl maltoside detergent were treated with Cu(phen)(3)(2+) to form a disulfide bridge between cysteines 140 and 222 which occur naturally in the bovine rhodopsin sequence. Absorption difference spectra were collected after excitation with a pulse of 477 nm light on the time scale from 1 micros to 690 ms, and the results were analyzed using global exponential fitting. Only two exponentials could be fit to data from the Cu(phen)(3)(2+)-treated rhodopsin, while three exponentials were needed to fit data either from untreated rhodopsin or from Cu(phen)(3)(2+)-oxidized rhodopsin after further dithiothreitol reduction. Dithiothreitol treatment of rhodopsin which had not been previously oxidized with Cu(phen)(3)(2+) had no effect on the observed kinetics. Since the 140-222 disulfide has previously been shown to block transducin activation, its effects on rhodopsin activation are of considerable interest. Cu(phen)(3)(2+) treatment favors formation of the meta I(380) intermediate relative to meta I(480) and slows formation of meta II from meta I(380). This suggests that the protein change involved in meta I(380) formation is similar to the structural constraint introduced by the 140-222 disulfide. These results show that formation of disulfides in rhodopsin has potential as a tool for discriminating between the three isochromic, 380 nm absorbing intermediates involved in rhodopsin activation and for gaining insight into how their structures differ.     

Rhodopsin/transducin interactions. II. Influence of the transducin-beta gamma subunit complex on the coupling of the transducin-alpha subunit to rhodopsin.

In these studies we have investigated the role of the beta gamma T subunit complex in promoting the rhodopsin-stimulated guanine nucleotide exchange reaction (i.e. the activation event) of the alpha T subunit. The results of these studies demonstrate that although the beta gamma T subunit complex increases the association of the alpha T subunit with lipid vesicles that lack the photoreceptor, the beta gamma T complex is not necessary for the binding of alpha T to lipid vesicles containing rhodopsin, provided sufficient amounts of rhodopsin are present. The rhodopsin-promoted GDP/guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) exchange reaction, within the rhodopsin-alpha T complex, then results in the dissociation of the alpha TGTP gamma S species from the rhodopsin-containing phospholipid vesicles. A second line of evidence for the occurrence of rhodopsin/alpha T interactions, in the absence of beta gamma T, comes from phosphorylation studies using the beta 1 isoform of protein kinase C. The phosphorylation of the alpha T subunit by protein kinase C is inhibited by beta gamma T, both in the absence and in the presence of rhodopsin, but is enhanced by rhodopsin in the absence of beta gamma T. These rhodopsin-alpha T complexes also appear to be capable of undergoing a rhodopsin-stimulated guanine nucleotide exchange event. When the guanine nucleotide exchange is allowed to occur prior to the addition of protein kinase C, the phosphorylation of the alpha T subunit is inhibited. Although beta gamma T is not absolutely required for the rhodopsin/alpha T interaction, it appears to increase the apparent affinity of the alpha T subunit for rhodopsin, both when rhodopsin was inserted into phosphatidylcholine vesicles and when soluble lipid-free preparations of rhodopsin were used. This results in a significant kinetic advantage for the rhodopsin-stimulated guanine nucleotide exchange event, such that the addition of beta gamma T causes a 10-fold promotion of the rhodopsin-stimulation [35S]GTP gamma S binding to alpha T after 1 min but provides less than a 20% promotion of the rhodopsin-stimulated binding after 1 h. The ability of beta gamma T to increase the association of alpha T with the lipid vesicle surface does not appear to contribute significantly to the ability of rhodopsin to couple functionally to alpha T subunits, and there appears to be no requirement for beta gamma T in the alpha T activation event, once the rhodopsin-alpha T complex has formed.     

A spectrally silent transformation in the photolysis of octopus rhodopsin: a protein conformational change without any accompanying change of the chromophore's absorption

A spectrally silent transformation in the photolysis of octopus rhodopsin was detected by the time-resolved transient grating method. Our results showed that at least two photointermediates, which share the same chromophore absorption spectrum, exist after the final absorption changes. Previously, mesorhodopsin was thought to decay to the final photoproduct, acid metarhodopsin with a lifetime of 38 micros at 15 degrees C, but the present results show that there is at least one intermediate species (called transient acid metarhodopsin) with a lifetime of 180 micros at 15 degrees C, before forming acid metarhodopsin. This indicates that the parts of the protein distant from the chromophore are still changing even after the changes in microenvironment around the chromophore are over. From the signal intensity detected by the transient grating method, the volume change of the spectrally silent transformation was found to be DeltaV = 13 ml/mol. The activation energy of the spectrally silent transformation is much lower than those of other transformations of octopus rhodopsin. Since stable acid metarhodopsin has not been shown to activate the G protein, this transient acid metarhodopsin may be responsible for G protein activation.     

Calculation of diffusion-limited kinetics for the reactions in collision coupling and receptor cross-linking

Both enzyme (e.g., G-protein) activation via a collision coupling model and the formation of cross-linked receptors by a multivalent ligand involve reactions between two molecules diffusing in the plasma membrane. The diffusion of these molecules is thought to play a critical role in these two early signal transduction events. In reduced dimensions, however, diffusion is not an effective mixing mechanism; consequently, zones in which the concentration of particular molecules (e.g., enzymes, receptors) becomes depleted or enriched may form. To examine the formation of these depletion/ accumulation zones and their effect on reaction rates and ultimately the cellular response, Monte Carlo techniques are used to simulate the reaction and diffusion of molecules in the plasma membrane. The effective reaction rate at steady state is determined in terms of the physical properties of the tissue and ligand for both enzyme activation via collision coupling and the generation of cross-linked receptors. The diffusion-limited reaction rate constant is shown to scale with the mean square displacement of a receptor-ligand complex. The rate constants determined in the simulation are compared with other theoretical predictions as well as experimental data.     

Light-induced decreases in cGMP concentration precede changes in membrane permeability in frog rod photoreceptors

This study examines whether changes in cGMP concentration initiated by illumination of frog rod photoreceptors occur rapidly enough to implicate cGMP as an intermediate between rhodopsin activation in the disc membrane and permeability changes in the plasma membrane. Previous studies using whole retinas or isolated outer segments have provided conflicting evidence on the role of cGMP in the initial events of phototransduction. The rod photoreceptor preparation employed in this work consists of purified suspensions of outer segments still attached to the mitochondria-rich ellipsoid portion of the inner segment. These photoreceptors are known to retain normal electrophysiological responses to illumination and have cGMP levels comparable to those measured in the intact retina. When examined under several different conditions, changes in cGMP concentrations were found to occur as rapidly or more rapidly than the suppression of the membrane dark current. Subsecond changes in cGMP concentration were analyzed with a rapid quench apparatus and confirmed by comparison with a rapid freezing technique. In a 1 mM Ca2+ Ringer's solution, cGMP levels decrease to 65% of their final extent within 200 ms after bright illumination; changes in membrane dark current follow a similar time course. When the light intensity is decreased to 8000 rhodopsins bleached per rod per s, the light-induced cGMP decrease is completed within 50 ms, with 7 X 10(5) cGMP molecules hydrolyzed per rhodopsin bleached. During this time the dark current has not yet begun to change. Thus, under physiological conditions it is clear that changes in cGMP concentration precede permeability changes at the plasma membrane. The correlation of rapid changes in cGMP levels with changes in membrane current leave open the possibility that changes in cGMP concentration may be an obligatory step in the reaction sequence linking rhodopsin activation by light and the resultant decrease in sodium permeability of the plasma membrane.     

Effect of phospholipid removal on the kinetics of the metarhodopsin I to metarhodopsin II reaction.

The kinetics of the metarhodopsin (meta) I → metarhodopsin II reaction have been studied by flash photolysis in two different types of preparations of bovine rhodopsin: (i) digitonin-solubilized rod outer segment (ROS) membranes with a molar ratio of phospholipid to rhodopsin of approximately 90, and (ii) digitonin-solubilized phospholipid-free rhodopsin with a molar ratio of phospholipid to rhodopsin of less than 0.2. At 20 °C the kinetics in both preparations are multiexponential, but four terms are required to fit the data with the solubilized membranes, whereas only two are required with the phospholipid-free preparation. Thus, phospholipid removal simplifies the kinetics of the meta I → meta II reaction, but the resulting preparation still does not show first-order kinetics. The ratio of the time constants of these two components with detergent-solubilized phospholipid-free rhodopsin was nearly equal to the values found with ROS particles, rhodopsin-phospholipid recombinants and intact rabbit eyes. This suggests a common origin for these two components in all these preparations and appears to exclude heterogeneity in bound phospholipid as the basis of these two-component kinetics.     

Involvement of rhodopsin and ATP in the activation of membranous guanylate cyclase in retinal photoreceptor outer segments (ROS-GC) by GC-activating proteins (GCAPs): a new model for ROS-GC activation and its link to retinal diseases

Membranous guanylate cyclase in retinal photoreceptor outer segments (ROS-GC), a key enzyme for the recovery of photoreceptors to the dark state, has a topology identical to and cytoplasmic domains homologous to those of peptide-regulated GCs. However, under the prevailing concept, its activation mechanism is significantly different from those of peptide-regulated GCs: GC-activating proteins (GCAPs) function as the sole activator of ROS-GC in a Ca²⁺-sensitive manner, and neither reception of an outside signal by the extracellular domain (ECD) nor ATP binding to the kinase homology domain (KHD) is required for its activation. We have recently shown that ATP pre-binding to the KHD in ROS-GC drastically enhances its GCAP-stimulated activity, and that rhodopsin illumination, as the outside signal, is required for the ATP pre-binding. These results indicate that illuminated rhodopsin is involved in ROS-GC activation in two ways: to initiate ATP binding to ROS-GC for preparation of its activation and to reduce [Ca²⁺] through activation of cGMP phosphodiesterase. These two signal pathways are activated in a parallel and proportional manner and finally converge for strong activation of ROS-GC by Ca²⁺-free GCAPs. These results also suggest that the ECD receives the signal for ATP binding from illuminated rhodopsin. The ECD is projected into the intradiscal space, i.e., an intradiscal domain(s) of rhodopsin is also involved in the signal transfer. Many retinal disease-linked mutations are found in these intradiscal domains; however, their consequences are often unclear. This model will also provide novel insights into causal relationship between these mutations and certain retinal diseases.     

Fourier transform IR spectroscopy study for new insights into molecular properties and activation mechanisms of visual pigment rhodopsin

Fourier transform IR (FTIR) spectroscopy has been successfully applied in recent years to examine the functional and structural properties of the membrane protein rhodopsin, a prototype G protein coupled receptor. Unlike UV-visible spectroscopy, FTIR spectroscopy is structurally sensitive. It may give us both global information about the conformation of the protein and very detailed information about the retinal chromophore and all other functional groups, even when these are not directly related to the chromophore. Furthermore, it can be successfully applied to the photointermediates of rhodopsin, including the active receptor species, metarhodopsin II, and its decay products, which is not expected presently or even in the near future from crystallographic approaches. In this review we show how FTIR spectroscopy has significantly contributed to the understanding of very different aspects of rhodopsin, comprising both structural properties and the mechanisms leading to receptor activation and deactivation.     

Fourier transform infrared spectroscopy indicates a major conformational rearrangement in the activation of rhodopsin.

The study of the structural differences between rhodopsin and its active form (metarhodopsin II) has been carried out by means of deconvolution analysis of infrared spectra. Deconvolution techniques allow the direct identification of the spectral changes that have occurred, which results in a significantly different view of the conformational changes occurring after activation of the receptor as compared with previous difference spectroscopy analysis. Thus, a number of changes in the bands assigned to solvent-exposed domains of the receptor are detected, indicating significant decreases in extended (beta) sequences and in reverse turns, and increases in irregular/aperiodic sequences and in helices with a non-alpha geometry, whereas there is no decrease in alpha-helices. In addition to secondary structure conversions, qualitative alterations within a given secondary structure type are detected. These are seen to occur in both reverse turns and helices. The nature of this spectral change is of great importance, since a clear alteration in the helices bundle core is detected. All these changes indicate that the rhodopsin --> metarhodopsin II transition involves not a minor but a major conformational rearrangement, reconciling the infrared data with the energetics of the activation process.     

Phosphorylation of rhodopsin: Most rhodopsin molecules are not phosphorylated

Phosphorylation of rod membrane proteins is a light-dependent reaction. Most rhodopsin molecules, however, are not phosphorylated. The protein that is highly phosphorylated (>3 moles phosphate per mole phosphorylated protein) appears to be a rhodopsin species that is different from the rest or is located in different parts of the rod membrane system.     

Chemical modification of rhodopsin and its effect on regeneration and G protein activation

The studies reported are concerned with the functional consequences of the chemical modifications of the lysines and carboxyl-containing amino acids of bovine rhodopsin. The 10 non-active-site lysine residues of rhodopsin can be completely dimethylated and partially acetimidated (8-9 residues) with no loss in the ability of the proteins to activate the G protein when photolyzed or to regenerate with 11-cis-retinal. These modifications do not alter the net charge on the protein. Surprisingly, heavy acetylation of these lysines (eight to nine residues) with acetic anhydride, which neutralizes the positive charges of the lysine residues, yields a modified rhodopsin fully capable of activating the G protein and being regenerated. It is concluded that the non-active-site lysine residues of rhodopsin are not importantly and directly involved in interactions with the G protein during photolysis. However, this is not to say that they are unimportant in maintaining the tertiary structure of the protein because heavy modification of these residues by succinylation and trinitrophenylation produces proteins incapable of G protein activation, although the succinylated protein still regenerated. The active-site lysine of rhodopsin was readily modified and prevented from regenerating with 11-cis-retinal and with o-salicylaldehyde and o-phthalaldehyde/mercaptoethanol, two sterically similar aromatic aldehyde containing reagents which react by entirely different mechanisms. It is suggested that rhodopsin contains an aromatic binding site within its active-site region. Monoethylation, but not monomethylation, of the active-site lysine also prevented regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interplay o light and heat in bleaching rhodopsin

Rhodopsin, the pigment of the retinal rods, can be bleached either by light or by high temperature. Earlier work had shown that when white light is used the bleaching rate does not depend on temperature, and so must be independent of the internal energy of the molecule. On the other hand thermal bleaching in the dark has a high temperature dependence from which one can calculate that the reaction has an apparent activation energy of 44 kg. cal. per mole. It has now been shown that the bleaching rate of rhodopsin becomes temperature-dependent in red light, indicating that light and heat cooperate in activating the molecule. Apparently thermal energy is needed for bleaching at long wave lengths where the quanta are not sufficiently energy-rich to bring about bleaching by themselves. The temperature dependence appears at 590 mµ. This is the longest wave length at which bleaching by light proceeds without thermal activation, and corresponds to a quantum energy of 48.5 kg. cal. per mole. This value of the minimum energy to bleach rhodopsin by light alone is in agreement with the activation energy of thermal bleaching in the dark. At wave lengths between 590 and 750 mµ, the longest wave length at which the bleaching rate was fast enough to study, the sum of the quantum energy and of the activation energy calculated from the temperature coefficients remains between 44 and 48.5 kg. cal. This result shows that in red light the energy deficit of the quanta can be made up by a contribution of thermal energy from the internal degrees of freedom of the rhodopsin molecule. The absorption spectrum of rhodopsin, which is not markedly temperature-dependent at shorter wave lengths, also becomes temperature-dependent in red light of wave lengths longer than about 570 to 590 mµ. The temperature dependence of the bleaching rate is at least partly accounted for by the temperature coefficient of absorption. There is some evidence that the temperature coefficient of bleaching is somewhat greater than the temperature coefficient of absorption at wave lengths longer than 590 mmicro;. This means that the thermal energy of the molecule is a more critical factor in bleaching than in absorption. It shows that some of the molecules which absorb energy-deficient quanta of red light are unable to supply the thermal component of the activation energy needed for bleaching, so bringing about a fall in the quantum efficiency. The experiments show that there is a gradual transition between the activation of rhodopsin by light and the activation by internal energy. It is suggested that energy can move freely between the prosthetic group and the protein moiety of the molecule. In this way a part of the large amount of energy in the internal degrees of freedom of rhodopsin could become available to assist in thermal activation. Assuming that the minimum energy required for bleaching is 48.5 kg. cal., an equation familiar in the study of unimolecular reaction has been used to estimate the number of internal degrees of freedom, n, involved in supplying the thermal component of the activation energy when rhodopsin is bleached in red light. It was found that n increases from 2 at 590 mµ to a minimum value of 15 at 750 mµ. One wonders what value n has at 1050 mµ, where vision still persists, and where rhodopsin molecules may supply some 16 kg. cal. of thermal energy per mole in order to make up for the energy deficit of the quanta.     

Thermal activation and photoactivation of visual pigments

A visual pigment molecule in a retinal photoreceptor cell can be activated not only by absorption of a photon but also "spontaneously" by thermal energy. Current estimates of the activation energies for these two processes in vertebrate rod and cone pigments are on the order of 40-50 kcal/mol for activation by light and 20-25 kcal/mol for activation by heat, which has forced the conclusion that the two follow quite different molecular routes. It is shown here that the latter estimates, derived from the temperature dependence of the rate of pigment-initiated "dark events" in rods, depend on the unrealistic assumption that thermal activation of a complex molecule like rhodopsin (or even its 11-cis retinaldehyde chromophore) happens through a simple process, somewhat like the collision of gas molecules. When the internal energy present in the many vibrational modes of the molecule is taken into account, the thermal energy distribution of the molecules cannot be described by Boltzmann statistics, and conventional Arrhenius analysis gives incorrect estimates for the energy barrier. When the Boltzmann distribution is replaced by one derived by Hinshelwood for complex molecules with many vibrational modes, the same experimental data become consistent with thermal activation energies that are close to or even equal to the photoactivation energies. Thus activation by light and by heat may in fact follow the same molecular route, starting with 11-cis to all-trans isomerization of the chromophore in the native (resting) configuration of the opsin. Most importantly, the same model correctly predicts the empirical correlation between the wavelength of maximum absorbance and the rate of thermal activation in the whole set of visual pigments studied.