The pKa of the protonated Schiff bases of gecko cone and octopus visual pigments.

A visual pigment is composed of retinal bound to its apoprotein by a protonated Schiff base linkage. Light isomerizes the chromophore and eventually causes the deprotonation of this Schiff base linkage at the meta II stage of the bleaching cycle. The meta II intermediate of the visual pigment is the active form of the pigment that binds to and activates the G protein transducin, starting the visual cascade. The deprotonation of the Schiff base is mandatory for the formation of meta II intermediate. We studied the proton binding affinity, pKa, of the Schiff base of both octopus rhodopsin and the gecko cone pigment P521 by spectral titration. Several fluorinated retinal analogs have strong electron withdrawing character around the Schiff base region and lower the Schiff base pKa in model compounds. We regenerated octopus and gecko visual pigments with these fluorinated and other retinal analogs. Experiments on these artificial pigments showed that the spectral changes seen upon raising the pH indeed reflected the pKa of the Schiff base and not the denaturation of the pigment or the deprotonation of some other group in the pigment. The Schiff base pKa is 10.4 for octopus rhodopsin and 9.9 for the gecko cone pigment. We also showed that although the removal of Cl- ions causes considerable blue-shift in the gecko cone pigment P521, it affects the Schiff base pKa very little, indicating that the lambda max of visual pigment and its Schiff base pKa are not tightly coupled.     

Rapid release of retinal from a cone visual pigment following photoactivation

As part of the visual cycle, the retinal chromophore in both rod and cone visual pigments undergoes reversible Schiff base hydrolysis and dissociation following photobleaching. We characterized light-activated release of retinal from a short-wavelength-sensitive cone pigment (VCOP) in 0.1% dodecyl maltoside using fluorescence spectroscopy. The half-time (t(1/2)) of release of retinal from VCOP was 7.1 s, 250-fold faster than that of rhodopsin. VCOP exhibited pH-dependent release kinetics, with the t(1/2) decreasing from 23 to 4 s with the pH decreasing from 4.1 to 8, respectively. However, the Arrhenius activation energy (E(a)) for VCOP derived from kinetic measurements between 4 and 20 °C was 17.4 kcal/mol, similar to the value of 18.5 kcal/mol for rhodopsin. There was a small kinetic isotope (D(2)O) effect in VCOP, but this effect was smaller than that observed in rhodopsin. Mutation of the primary Schiff base counterion (VCOP(D108A)) produced a pigment with an unprotonated chromophore (λ(max) = 360 nm) and dramatically slowed (t(1/2) ~ 6.8 min) light-dependent retinal release. Using homology modeling, a VCOP mutant with two substitutions (S85D and D108A) was designed to move the counterion one α-helical turn into the transmembrane region from the native position. This double mutant had a UV-visible absorption spectrum consistent with a protonated Schiff base (λ(max) = 420 nm). Moreover, the VCOP(S85D/D108A) mutant had retinal release kinetics (t(1/2) = 7 s) and an E(a) (18 kcal/mol) similar to those of the native pigment exhibiting no pH dependence. By contrast, the single mutant VCOP(S85D) had an ~3-fold decreased retinal release rate compared to that of the native pigment. Photoactivated VCOP(D108A) had kinetics comparable to those of a rhodopsin counterion mutant, Rho(E113Q), both requiring hydroxylamine to fully release retinal. These results demonstrate that the primary counterion of cone visual pigments is necessary for efficient Schiff base hydrolysis. We discuss how the large differences in retinal release rates between rod and cone visual pigments arise, not from inherent differences in the rate of Schiff base hydrolysis but rather from differences in the properties of noncovalent binding of the retinal chromophore to the protein.     

A nonbleachable rhodopsin analogue with a slow photocycle

Archaeal rhodopsins, e.g. bacteriorhodopsin, all have cyclic photoreactions. Such cycles are achieved by a light-induced isomerization step of their retinal chromophores, which thermally re-isomerize in the dark. Visual pigment rhodopsins, which contain in the dark state an 11-cis retinal Schiff base, do not share such a mechanism, and following light absorption, they experience a bleaching process and a subsequent release of the photo-isomerized all-trans chromophore from the binding pocket. The pigment is eventually regenerated by the rebinding of a new 11-cis retinal. In the artificial visual pigment, Rh(6.10), in which the retinal chromophore is locked in an 11-cis geometry by the introduction of a six-member ring structure, an activated receptor may be formed by light-induced isomerization around other double bonds. We have examined this activation of Rh(6.10) by UV-visible and FTIR spectroscopy and have revealed that Rh(6.10) is a nonbleachable pigment. We could further show that the activated receptor consists of two different subspecies corresponding to 9-trans and 9-cis isomers of the chromophore. Both subspecies relax in the dark via separate pathways back to their respective inactive states by thermal isomerization presumably around the C(13)=C(14) double bond. This nonbleachable pigment can be repeatedly photolyzed to undergo identical activation-relaxation cycles. The rate constants of these photocycles are pH-dependent, and the half-times vary between several hours at acidic pH and about 1.5 min at neutral to alkaline pH, which is several orders of magnitude longer than for bacteriorhodopsin.     

Photoreactions of metarhodopsin III

Meta III is an inactive intermediate thermally formed following light activation of the visual pigment rhodopsin. It is produced from the Meta I/Meta II photoproduct equilibrium of rhodopsin by a thermal isomerization of the protonated Schiff base C=N bond of Meta I, and its chromophore configuration is therefore all-trans 15-syn. In contrast to the dark state of rhodopsin, which catalyzes exclusively the cis to trans isomerization of the C11=C12 bond of its 11-cis 15-anti chromophore, Meta III does not acquire this photoreaction specificity. Instead, it allows for light-dependent syn to anti isomerization of the C15=N bond of the protonated Schiff base, yielding Meta II, and for trans to cis isomerizations of C11=C12 and C9=C10 of the retinal polyene, as shown by FTIR spectroscopy. The 11-cis and 9-cis 15-syn isomers produced by the latter two reactions are not stable, decaying on the time scale of few seconds to dark state rhodopsin and isorhodopsin by thermal C15=N isomerization, as indicated by time-resolved FTIR methods. Flash photolysis of Meta III produces therefore Meta II, dark state rhodopsin, and isorhodopsin. Under continuous illumination, the latter two (or its unstable precursors) are converted as well to Meta II by presumably two different mechanisms.     

E113 is required for the efficient photoisomerization of the unprotonated chromophore in a UV-absorbing visual pigment

Protonation of the retinal Schiff base chromophore is responsible for the absorption of visible light and is stabilized by the counterion residue E113 in vertebrate visual pigments. However, this residue is also conserved in vertebrate UV-absorbing visual pigments (UV pigments) which have an unprotonated Schiff base chromophore. To elucidate the role played by this residue in the photoisomerization of the unprotonated chromophore in UV pigments, we measured the quantum yield of the E113Q mutant of the mouse UV cone pigment (mouse UV). The quantum yield of the mutant was much lower than that of the wild type, indicating that E113 is required for the efficient photoisomerization of the unprotonated chromophore in mouse UV. Introduction of the E113Q mutation into the chicken violet cone pigment (chicken violet), which has a protonated chromophore, caused deprotonation of the chromophore and a reduction in the quantum yield. On the other hand, the S90C mutation in chicken violet, which deprotonated the chromophore with E113 remaining intact, did not significantly affect the quantum yield. These results suggest that E113 facilitates photoisomerization in both UV-absorbing and visible light-absorbing visual pigments and provide a possible explanation for the complete conservation of E113 among vertebrate UV pigments.     

Constitutive activity of a UV cone opsin

Vertebrate visual pigment proteins contain a conserved carboxylic acid residue in the third transmembrane helix. In rhodopsin, Glu113 serves as a counterion to the positively charged protonated Schiff base formed by 11-cis retinal attached to Lys296. Activation involves breaking of this ion pair. In UV cone pigments, the retinyl Schiff base is unprotonated, and hence such a salt bridge is not present; yet the pigment is inactive in the dark. Mutation of Glu108, which corresponds to rhodopsin's Glu113, to Gln yields a pigment that remains inactive in the dark. The apoproteins of both the wild-type and mutant, however, are constitutively active with the mutant being of significantly higher activity. Thus, one important role for preserving the negatively charged glutamate in the third helix of UV pigments is to maintain a less active opsin in a manner similar to rhodopsin. Ligand binding itself in the absence of a salt bridge is sufficient for deactivation.     

Formation of a long-lived photoproduct with a deprotonated Schiff base in proteorhodopsin, and its enhancement by mutation of Asp227

Proteorhodopsin, a retinal protein of marine proteobacteria similar to bacteriorhodopsin of the archaea, is a light-driven proton pump. Absorption of a light quantum initiates a reaction cycle (turnover time of ca. 50 ms), which includes photoisomerization of the retinal from the all-trans to the 13-cis form and transient deprotonation of the retinal Schiff base, followed by recovery of the initial state. We report here that in addition to this fast cyclic conversion, illumination at high pH results in accumulation of a long-lived photoproduct absorbing at 362 nm. This photoconversion is much more efficient in the D227N mutant in which the anionic Asp227, which together with Asp97 constitutes the Schiff base counterion, is replaced with a neutral residue. Upon illumination at pH 8.5, most of the D227N pigment is converted to the 362 nm species, with a quantum efficiency of ca. 0.2. The pK(a) for this transition in the wild type is 9.6, but decreased to 7.5 after mutation of Asp227. The short wavelength of the absorption maximum of the photoproduct indicates that it has a deprotonated Schiff base. In the dark, this photoproduct is converted back to the initial pigment with a time constant of 30 min (in D227N, at pH 8.5), but it can be reconverted more rapidly by illumination with near-UV light. Experiments with "locked" retinal analogues which selectively exclude rotation around either the C9=C10, C11=C12, or C13=C14 bond show that formation of the 362 nm species involves isomerization around the C13=C14 bond. In agreement with this, retinal extraction indicates that the 362 nm photoproduct is 13-cis whereas the initial state is predominantly all-trans. A rapid shift of the pH from 8.5 to 4 greatly accelerates thermal reconversion of the 362 nm species to the initial pigment, suggesting that its recovery involving the thermal isomerization of the chromophore is controlled by ionizable residues, primarily the Schiff base and Asp97. The transformation to the long-lived 362 nm photoproduct is apparently a side reaction of the photocycle, a response to high pH, caused by alteration of the normal reprotonation and reisomerization pathway of the Schiff base.     

Molecular dynamics study of the nature and origin of retinal's twisted structure in bacteriorhodopsin

The planarity of the polyene chain of the retinal chromophore in bacteriorhodopsin is studied using molecular dynamics simulation techniques and applying different force-field parameters and starting crystal structures. The largest deviations from a planar structure are observed for the C(13)==C(14) and C(15)==N(16) double bonds in the retinal Schiff base structure. The other dihedral angles along the polyene chain of the chromophore, although having lower torsional barriers in some cases, do not significantly deviate from the planar structure. The results of the simulations of different mutants of the pigment show that, among the studied amino acids of the binding pocket, the side chain of Trp-86 has the largest impact on the planarity of retinal, and the mutation of this amino acid to alanine leads to chromophore planarity. Deletion of the methyl C(20), removal of a water molecule hydrogen-bonded to H(15), or mutation of other amino acids to alanine did not show any significant influence on the distortion of the chromophore. The results from the present study suggest the importance of the bulky residue of Trp-86 in the isomerization process, in both ground and excited states of the chromophore, and in fine-tuning of the pK(a) of the retinal protonated Schiff base in bacteriorhodopsin. The dark adaptation of the pigment and the last step of the bacteriorhodopsin photocycle imply low barriers against the rotation of the double bonds in the Schiff base region. The twisted double bonds found in the present study are consistent with the proposed mechanism of these ground state isomerization events.     

Serine 85 in transmembrane helix 2 of short-wavelength visual pigments interacts with the retinylidene Schiff base counterion.

Short-wavelength cone visual pigments (SWS1) are responsible for detecting light from 350 to 430 nm. Models of this class of pigment suggest that TM2 has extensive contacts with the retinal binding pocket and stabilizes interhelical interactions. The role of TM2 in the structure-function of the Xenopus SWS1 (VCOP, lambda(max) = 427 nm) pigment was studied by replacement of the helix with that of bovine rhodopsin and also by mutagenesis of highly conserved residues. The TM2 chimera and G78D, F79L, M81E, P88T, V89S, and F90V mutants did not produce any significant spectral shift of the dark state or their primary photointermediate formed upon illumination at cryogenic temperatures. The mutant G77R (responsible for human tritanopia) was completely defective in folding, while C82A and F87T bound retinal at reduced levels. The position S85 was crucial for obtaining the appropriate spectroscopic properties of VCOP. S85A and S85T did not bind retinal. S85D bound retinal and had a wild-type dark state at room temperature and a red-shifted dark state at 45 K and formed an altered primary photointermediate. S85C absorbed maximally at 390 nm at neutral pH and at 365 nm at pH >7.5. The S85C dark state was red shifted by 20 nm at 45 K and formed an altered primary photointermediate. These data suggest that S85 is involved in a hydrogen bond with the protonated retinylidene Schiff base counterion in both the dark state and the primary photointermediate.     

The Physical Mechanism for Retinal Discrete Dark Noise: Thermal Activation or Cellular Ultraweak Photon Emission?

For several decades the physical mechanism underlying discrete dark noise of photoreceptors in the eye has remained highly controversial and poorly understood. It is known that the Arrhenius equation, which is based on the Boltzmann distribution for thermal activation, can model only a part (e.g. half of the activation energy) of the retinal dark noise experimentally observed for vertebrate rod and cone pigments. Using the Hinshelwood distribution instead of the Boltzmann distribution in the Arrhenius equation has been proposed as a solution to the problem. Here, we show that the using the Hinshelwood distribution does not solve the problem completely. As the discrete components of noise are indistinguishable in shape and duration from those produced by real photon induced photo-isomerization, the retinal discrete dark noise is most likely due to ‘internal photons’ inside cells and not due to thermal activation of visual pigments. Indeed, all living cells exhibit spontaneous ultraweak photon emission (UPE), mainly in the optical wavelength range, i.e., 350–700 nm. We show here that the retinal discrete dark noise has a similar rate as UPE and therefore dark noise is most likely due to spontaneous cellular UPE and not due to thermal activation.     

Role of the retinal hydrogen bond network in rhodopsin Schiff base stability and hydrolysis

Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin. We report here our investigation into this process focusing on the role of amino acids involved in a hydrogen bond network around the retinal Schiff base. We find conservative mutations in this network (T94I, E113Q, S186A, E181Q, Y192F, and Y268F) increase the activation energy (E(a)) and abolish the concave Arrhenius plot normally seen for Schiff base hydrolysis in dark state rhodopsin. Interestingly, two mutants (T94I and E113Q) show dramatically faster rates of Schiff base hydrolysis in dark state rhodopsin, yet slower hydrolysis rates in the active MII form. We find deuterium affects the hydrolysis process in wild-type rhodopsin, exhibiting a specific isotope effect of approximately 2.5, and proton inventory studies indicate that multiple proton transfer events occur during the process of Schiff base hydrolysis for both dark state and MII forms. Taken together, our study demonstrates the importance of the retinal hydrogen bond network both in maintaining Schiff base integrity in dark state rhodopsin, as well as in catalyzing the hydrolysis and release of retinal from the MII form. Finally, we note that the dramatic alteration of Schiff base stability caused by mutation T94I may play a causative role in congenital night blindness as has been suggested by the Oprian and Garriga laboratories.     

Chemical kinetic analysis of thermal decay of rhodopsin reveals unusual energetics of thermal isomerization and hydrolysis of Schiff base

The thermal properties of rhodopsin, which set the threshold of our vision, have long been investigated, but the chemical kinetics of the thermal decay of rhodopsin has not been revealed in detail. To understand thermal decay quantitatively, we propose a kinetic model consisting of two pathways: 1) thermal isomerization of 11-cis-retinal followed by hydrolysis of Schiff base (SB) and 2) hydrolysis of SB in dark state rhodopsin followed by opsin-catalyzed isomerization of free 11-cis-retinal. We solve the kinetic model mathematically and use it to analyze kinetic data from four experiments that we designed to assay thermal decay, isomerization, hydrolysis of SB using dark state rhodopsin, and hydrolysis of SB using photoactivated rhodopsin. We apply the model to WT rhodopsin and E181Q and S186A mutants at 55 °C, as well as WT rhodopsin in H(2)O and D(2)O at 59 °C. The results show that the hydrogen-bonding network strongly restrains thermal isomerization but is less important in opsin and activated rhodopsin. Furthermore, the ability to obtain individual rate constants allows comparison of thermal processes under various conditions. Our kinetic model and experiments reveal two unusual energetic properties: the steep temperature dependence of the rates of thermal isomerization and SB hydrolysis in the dark state and a strong deuterium isotope effect on dark state SB hydrolysis. These findings can be applied to study pathogenic rhodopsin mutants and other visual pigments.     

Regulation of phototransduction in short-wavelength cone visual pigments via the retinylidene Schiff base counterion.

Short-wavelength visual pigments (SWS1) have lambda(max) values that range from the ultraviolet to the blue. Like all visual pigments, this class has an 11-cis-retinal chromophore attached through a Schiff base linkage to a lysine residue of opsin apoprotein. We have characterized a series of site-specific mutants at a conserved acidic residue in transmembrane helix 3 in the Xenopus short-wavelength sensitive cone opsin (VCOP, lambda(max) approximately 427 nm). We report the identification of D108 as the counterion to the protonated retinylidene Schiff base. This residue regulates the pK(a) of the Schiff base and, neutralizing this charge, converts the violet sensitive pigment into one that absorbs maximally in the ultraviolet region. Changes to this position cause the pigment to exhibit two chromophore absorbance bands, a major band with a lambda(max) of approximately 352-372 nm and a minor, broad shoulder centered around 480 nm. The behavior of these two absorbance bands suggests that these represent unprotonated and protonated Schiff base forms of the pigment. The D108A mutant does not activate bovine rod transducin in the dark but has a significantly prolonged lifetime of the active MetaII state. The data suggest that in short-wavelength sensitive cone visual pigments, the counterion is necessary for the characteristic rapid production and decay of the active MetaII state.     

Thermal Stability of Rhodopsin and Progression of Retinitis Pigmentosa: COMPARISON OF S186W AND D190N RHODOPSIN MUTANTS

Over 100 point mutations in the rhodopsin gene have been associated with retinitis pigmentosa (RP), a family of inherited visual disorders. Among these, we focused on characterizing the S186W mutation. We compared the thermal properties of the S186W mutant with another RP-causing mutant, D190N, and with WT rhodopsin. To assess thermal stability, we measured the rate of two thermal reactions contributing to the thermal decay of rhodopsin as follows: thermal isomerization of 11-cis-retinal and hydrolysis of the protonated Schiff base linkage between the 11-cis-retinal chromophore and opsin protein. We used UV-visible spectroscopy and HPLC to examine the kinetics of these reactions at 37 and 55 °C for WT and mutant rhodopsin purified from HEK293 cells. Compared with WT rhodopsin and the D190N mutant, the S186W mutation dramatically increases the rates of both thermal isomerization and dark state hydrolysis of the Schiff base by 1–2 orders of magnitude. The results suggest that the S186W mutant thermally destabilizes rhodopsin by disrupting a hydrogen bond network at the receptor''s active site. The decrease in the thermal stability of dark state rhodopsin is likely to be associated with higher levels of dark noise that undermine the sensitivity of rhodopsin, potentially accounting for night blindness in the early stages of RP. Further studies of the thermal stability of additional pathogenic rhodopsin mutations in conjunction with clinical studies are expected to provide insight into the molecular mechanism of RP and test the correlation between rhodopsin''s thermal stability and RP progression in patients.     

Mechanism of isomerization of 11-cis-retinal in lipid dispersions by aromatic amines

It has previously been shown that retinotoxic, primary aromatic amines catalyze the isomerization of 11-cis-retinal to its all-trans congener after Schiff base formation [Bernstein, P.S., Fulton, B.S., & Rando, R.R. (1986) Biochemistry 25, 3370-3377]. This process led to the short-circuiting of the visual cycle and the observed retinotoxicity when it occurred in vivo. The catalysis was also observed to occur in vitro in phosphatidylcholine-based vesicles but not in hydrocarbon solutions. The rate of isomerization of an aromatic amine Schiff base of 11-cis-retinal in the phospholipid vesicles was typically 10(3)-fold more rapid than in hydrocarbon solutions. In this article, the mechanistic basis of this apparently membrane-specific catalysis is described. It was found that the rate enhancement effect observed was independent of the lipid used. Moreover, a bilayer structure was not important because rate enhancements were also observed in micelles. The rapid isomerization rates observed in lipid dispersions appear not be free radical initiated because free radical quenching agents, such as alpha-tocopherol and beta-carotene, had little effect on the isomerization rates. It was further found that aliphatic amines, such as n-dodecylamine, could be substituted for the aromatic amines in phospholipid. Finally, and most importantly, it was found that the isomerization of the aromatic amine retinal Schiff bases in phospholipid vesicles was acid-catalyzed. It is concluded that the rate enhancements observed for the isomerization of 11-cis-retinal-aromatic amine Schiff bases in lipid dispersions over that in hydrocarbon solvents are due to the occurrence of acid-base catalysis in the former.     

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.     

Schiff base protonation changes in Siberian hamster ultraviolet cone pigment photointermediates

Molecular structure and function studies of vertebrate ultraviolet (UV) cone visual pigments are needed to understand the molecular evolution of these photoreceptors, which uniquely contain unprotonated Schiff base linkages between the 11-cis-retinal chromophore and the opsin proteins. In this study, the Siberian hamster ultraviolet cone pigment (SHUV) was expressed and purified in an n-dodecyl-β-D-maltoside suspension for optical characterization. Time-resolved absorbance measurements, over a spectral range from 300 to 700 nm, were taken for the purified pigment at time delays from 30 ns to 4.64 s after photoexcitation using 7 ns pulses of 355 nm light. The resulting data were fit globally to a sum of exponential functions after noise reduction using singular-value decomposition. Four exponentials best fit the data with lifetimes of 1.4 μs, 210 μs, 47 ms, and 1 s. The first photointermediate species characterized here is an equilibrated mixture similar to the one formed after rhodopsin's Batho intermediate decays into equilibrium with its successor, BSI. The extremely large red shift of the SHUV Batho component relative to the pigment suggests that SHUV Batho has a protonated Schiff base and that the SHUV cone pigment itself has an unprotonated Schiff base. In contrast to SHUV Batho, the portion of the equilibrated mixture's spectrum corresponding to SHUV BSI is well fit by a model spectrum with an unprotonated Schiff base. The spectra of the next two photointermediate species revealed that they both have unprotonated Schiff bases and suggest they are analogous to rhodopsin's Lumi I and Lumi II species. After decay of SHUV Lumi II, the correspondence with rhodopsin photointermediates breaks down and the next photointermediate, presumably including the G protein-activating species, is a mixture of protonated and unprotonated Schiff base photointermediate species.     

Thermal properties of rhodopsin: insight into the molecular mechanism of dim-light vision

Rhodopsin has developed mechanisms to optimize its sensitivity to light by suppressing dark noise and enhancing quantum yield. We propose that an intramolecular hydrogen-bonding network formed by ～20 water molecules, the hydrophilic residues, and peptide backbones in the transmembrane region is essential to restrain thermal isomerization, the source of dark noise. We studied the thermal stability of rhodopsin at 55 °C with single point mutations (E181Q and S186A) that perturb the hydrogen-bonding network at the active site. We found that the rate of thermal isomerization increased by 1-2 orders of magnitude in the mutants. Our results illustrate the importance of the intact hydrogen-bonding network for dim-light detection, revealing the functional roles of water molecules in rhodopsin. We also show that thermal isomerization of 11-cis-retinal in solution can be catalyzed by wild-type opsin and that this catalytic property is not affected by the mutations. We characterize the catalytic effect and propose that it is due to steric interactions in the retinal-binding site and increases quantum yield by predetermining the trajectory of photoisomerization. Thus, our studies reveal a balancing act between dark noise and quantum yield, which have opposite effects on the thermal isomerization rate. The acquisition of the hydrogen-bonding network and the tuning of the steric interactions at the retinal-binding site are two important factors in the development of dim-light vision.     

The Effect of Protonation and Electrical Interactions on the Stereochemistry of Retinal Schiff Bases

Based on quantumchemical MNDOC calculations it is shown that the ground-state properties of a retinal Schiff base depend sensitively on its protonation state and charge environment. This is exemplified for the equilibrium geometry, for the distribution of partial charges and, in particular, for the thermal isomerization barriers around the π-bonds. It is demonstrated that a protein, by protonating the retinal Schiff base and by providing one or two negative ions in its environment, can reduce double-bond isomerization barriers from 50 kcal/mol for the unprotonated compound to ~ 5 kcal/mol and can increase single bond barriers from 5 kcal/mol to ~ 20 kcal/mol. Thereby, the specific location of the ions relative to the polyene chain of the protonated retinal Schiff base determines the barrier heights. The results explain the ground-state isomerization reactions of retinal observed in bacteriorhodopsin and in squid retinochrome.     

Correlation between absorption maxima and thermal isomerization rates in bacteriorhodopsin

The reported rates of thermal 13-cis to all-trans isomerization of the protonated Schiff base of retinal (PSBR) in solution and in bacteriorhodopsin (BR) are shown to be correlated with the red shift in the absorption maximum of the chromophore, though the linear fit is different for BR and for a model PSBR in solution. Because the red shift in the absorption has been previously shown to be correlated with π-electron delocalization in the chromophore, this suggests that the thermal isomerization rate is largely regulated by the amount of double bond character in the chromophore. Because the linear fit of isomerization rates with absorption maxima is different for BR and the model PSBR, specific interactions of the protein with the chromophore must also be a factor in determining thermal isomerization rates in BR. A model of the later steps in the photocycle of BR is presented in which the 13-cis to all-trans thermal isomerization occurs during the O intermediate.