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The structure and function of the light-driven proton pump bacteriorhodopsin appear to be determined by the exact geometrical conformation of specific groups in the retinal binding site, including bound water molecules. This applies to the pKa values of the protonated Schiff base, which links the retinal chromophore to Lys216, and to Asp85. In the present work we show that the geometrical constraints imposed by the ring structures of several synthetic retinals can induce substantial changes in the pKa values of the Schiff base and of Asp85. Thus, the artificial pigments derived from 13-demethyl-11,14-epoxyretinal (2) and 13-demethyl-9,12-epoxyretinal (3) show protonated Schiff base pKa values of 8.2 +/- 0.1 and 9.1 +/- 0.1, respectively, as compared with 13.3 in the native (all-trans-retinal) pigment. We also suggest that in both systems the pKa of Asp85 increases from 3.2 in the native bR to above 9. Analogous, though smaller, effects are obtained for artificial bR pigments derived from 12,14-ethanoretinal (4), 11,13-propanoretinal (5), 11,13-ethanoretinal (6), and p-(CH3)2N-C6H4-HC = CH-C(CH3) = CH-CHO 7. The effects of geometry on the pKa values (those on Asp85 being more pronounced) are attributed to the disruption of the original, well-defined, structure in which the Schiff base and its Asp85 counterion are bridged by bound water molecules. These results are the first to show that it is possible to modify the pKa values of the Schiff base and Asp85 in appropriate artificial pigments, without inducing intrinsic pKa changes in the chromophore or introducing a mutation in the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-Isorhodopsin, an artificial visual pigment with a 9-cis-4,5-dehydro-5,6-dihydro(alpha)retinal chromophore, was photolyzed at low temperatures and absorption difference spectra were collected as the sample was warmed. A bathorhodopsin (Batho)-like intermediate absorbing at ca 495 nm was detected below 55 K,a blue-shifted intermediate (BSI)-like intermediate absorbing at ca 453 nm was observed when the temperature was raised to 60 K and a lumirhodopsin (Lumi)-like intermediate absorbing at ca 470 nm was found when the sample was warmed to 115 K. Photointermediates from this pigment were compared to those of native rhodopsin and 5,6-dihydroisorhodopsin. As in native rhodopsin, Batho is the first intermediate detected in alpha-isorhodopsin, though unlike native rhodopsin at low temperatures BSI is observed prior to Lumi formation. Alpha-Isohodopsin behaves similarly to 5,6-dihydroisorhodopsin, with the same early intermediates observed in both artificial visual pigments lacking the C5-C6 double bond. The transition temperature for BSI formation is higher in alpha-isorhodopsin, suggesting an interaction involving the chromophore ring in BSI formation. The transition temperature for Lumi formation is similar for these two pigments as well as for native rhodopsin, suggesting comparable changes in the protein environment in that transition.

A system is described that allows for the delineation of the factors that effect the complexation of retinal to the apoprotein of bacteriorhodopsin. This complexation is investigated in various states of hydration, in H2O and D2O, at a variety of pH levels, with mutant membranes and labeled retinals. The complexation reaction was also investigated using absorption spectroscopy and vibrational spectra using difference Fourier transform infrared spectroscopy. The results demonstrate the crucial role of water in controlling the protein conformations that lead to protein/ligand binding reactions and begin to shed new light on the protein control of a reaction that normally cannot take place in an aqueous medium.

Nanosecond laser photolysis measurements were conducted on the cone-type visual pigment P521 in digitonin extracts of Tokay gecko (Gekko gekko) retina containing physiological chloride ion levels and also on samples which had been chloride depleted or which contained high levels (4 M) of chloride. Absorbance difference spectra were recorded at a sequence of time delays from 30 ns to 60 microseconds following excitation with a pulse of either 532- or 477-nm actinic light. Global analysis showed the kinetic decay data for gecko pigment P521 to be best fit by two exponential processes under all chloride conditions. The initial photoproduct detected had a broad spectrum characteristic of an equilibrated mixture of a Batho P521 intermediate with its blue-shifted intermediate (BSI P521) decay product. The first exponential process was assigned to the decay of this mixture to the Lumi P521 intermediate. The second exponential process was identified as the decay of Lumi P521 to Meta I P521. The initial photoproduct's spectrum exhibited a strong dependence on chloride concentration, indicating that chloride affects the composition of the equilibrated mixture of Batho P521 and BSI P521. These results suggest that the affinity for chloride is reduced approximately 5-fold in the Batho P521 intermediate and approximately 50-fold in the BSI P521 intermediate. Chloride concentration also affects the apparent decay rate of the equilibrated mixture. When the apparent decay rate is corrected for the composition of the equilibrated mixture, a relatively invariant microscopic rate constant is obtained for BSI decay (k = 1/55 ns-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine 57 is one of the residues present in the retinal binding pocket and is conserved in all the halophilic retinal proteins. We have studied mutants of bacteriorhodopsin, expressed in Halobacterium salinarium, in which tyrosine 57 is replaced by an asparagine (Y57N) or phenylalanine (Y57F). In Y57N the photocycle proceeds only up to the L intermediate; no M is formed at neutral pH. The lifetime of L intermediate is extremely long, ca. 500 ms. Proton release is severely affected in both the mutants which suggests that Y57 is associated with the proton release pathway. By comparing the pH-induced absorption changes in the UV in Y57N and Y57F with those in the wild-type (WT), we determined that the pKa of Y57 is 10.2. In Y57F, which shows M formation, the rate constant of the L-->M transition is pH dependent (pKa 8.7) suggesting that Y57 is probably not the residue that normally controls the transition into the alkaline photocycle. Y57 is either part of the counterion complex or in close proximity to D85 since its mutation influences the pKa of Asp85. In Y57F the pKa of D85 is approximately 4.9 (compared to approximately 2.9 in the WT). The Y57N mutant shows two pKa's in the purple to blue transition, approximately 3.8 and < 1. In the presence of hydroxylamine, at neutral pH, Y57N is stable in the dark but bleaches very rapidly upon illumination compared to the WT. Since the lifetime of L intermediate is long in Y57N, we suggest that the Schiff base becomes accessible to hydroxylamine in this state.

The structure and the photocycle of bacteriorhodopsin (bR) containing 13-cis,15-syn retinal, so-called bR548, has been studied by means of molecular dynamics simulations performed on the complete protein. The simulated structure of bR548 was obtained through isomerization of in situ retinal around both its C13-C14 and its C15-N bond starting from the simulated structure of bR568 described previously, containing all-trans,15-anti retinal. After a 50-ps equilibration, the resulting structure of bR548 was examined by replacing retinal by analogues with modified beta-ionone rings and comparing with respective observations. The photocycle of bR548 was simulated by inducing a rapid 13-cis,15-anti-->all-trans,15-syn isomerization through a 1-ps application of a potential that destabilizes the 13-cis isomer. The simulation resulted in structures consistent with the J, K, and L intermediates observed in the photocycle of bR548. The results offer an explanation of why an unprotonated retinal Schiff base intermediate, i.e., an M state, is not formed in the bR548 photocycle. The Schiff base nitrogen after photoisomerization of bR548 points to the intracellular rather than to the extracellular site. The simulations suggest also that leakage from the bR548 to the bR568 cycle arises due to an initial 13-cis,15-anti-->all-trans,15-anti photoisomerization.

The picosecond dynamics of the photoreaction of an artificial bacteriorhodopsin (BR) pigment containing a retinal in which a five-membered ring spans the C-12 to C-14 positions of the polyene chain (BR5.12) is examined by using time-resolved absorption and fluorescence and resonance Raman spectroscopy. The ring within the retinal chromophore of BR5.12 blocks the C-13 = C-14 isomerization proposed to be a primary step in the energy storage/transduction mechanism in the BR photocycle. Relative to the native BR pigment (BR-570), the absorption spectrum of BR5.12 is red-shifted by 8 nm. The fluorescence spectrum of BR5.12 closely resembles that of BR-570 although the relative fluorescence yield is higher (approximately 10-fold). Picosecond transient absorption (4-ps pulses, 568-662 nm) measurements reveal an intermediate absorbing to the red side of BR5.12. Kinetic fits show that the red-absorbing intermediate appears within < 3 ps and decays with a time constant of 17 +/- 1 ps to form only BR5.12. No emission in the 650- to 900-nm region can be attributed to the red-absorbing species. Since rotation around C-12 - C-13 and isomerization around C-13 = C-14 are prevented in BR5.12, these results demonstrate that motion in these regions of the retinal is (i) necessary to form the K-like intermediate observed in the native BR-570 photocycle and (ii) not necessary to form a red-absorbing intermediate that has spectral and kinetic properties analogous to those of J-625 in the native BR photocycle. Discussions of the excited and ground electronic state assignments for the intermediate observed in the BR5.12 photoreaction are presented.

The M stage in the photocycle of bacteriorhodopsin (bR), a key step in its light-induced proton pump mechanism, is studied in water/glycerol suspensions over the temperature range between 20 and -60 degrees C. The biexponential decay of M is analyzed for wild-type (WT) bR and for its D96N, Y185F, and D115N mutants, at various pH values, according to the scheme: bR-->(hv) L-->M<-->(k1, k-1) N-->(k2) bR. The analysis leads to the conclusion that the N state is generated, with analogous rate parameters, in all cases, including the D96N mutant. Another approach involves probing the M state, generated by steady-state illumination at -60 degrees C, by fast cooling to -180 degrees C. Subsequent irradiation with blue light, followed by gradual warming up, induces the M-->(hv) M'-->bR'-->bR sequence of reactions. On the basis of characteristic difference spectra and transition temperatures observed for the M'-->bR' process, it is concluded that the initially observed M state at -60 degrees C, denoted as (M)a, is composed of three (or four) equilibrated substrates, MI, MII, MIII, and MIV. During the M-->N equilibration, which corresponds to the fast phase of the M decay, (M)a transforms into a second state, (M)b, in which MIII has been replaced by a fifth M substate, denoted as MV. MV is identified as the protein state in which an appropriate structural change allows reprotonation of the Schiff base, generating the N state. The low-temperature heterogeneity in M is discussed in terms of the two M states (M1 and M2) previously postulated [Váró, G., & Lanyi, J. K. (1990) Biochemistry 29, 2241] for the room temperature photocycle. The following conclusions are derived for both low and room temperature photocycles: (a) The M population is highly heterogeneous and pH dependent. (b) At least three transitions are observed between the initially formed M state and the M state that is equilibrated with N. These are assigned to protein conformational changes and to water molecule rearrangements. (c) In an aqueous suspension of WT bR at room temperature, the Schiff base reprotonation is controlled by D96. However, our results show that the formation and stability of the N state do not require the D96 residue. Moreover, at low temperatures, the (M)a-->(M)b protein structural transformation, which has not yet been resolved at room temperature, becomes the rate-determining step in the protonation of the Schiff base.

The active site of an ion pump must communicate alternately with the two opposite membrane surfaces. In the light-driven proton pump, bacteriorhodopsin, the retinal Schiff base is first the proton donor to D85 (with access to the extracellular side), and then it becomes the acceptor of the proton of D96 (with access to the cytoplasmic side). This "reprotonation switch" has been associated with a protein conformation change observed during the photocycle. When D85 is replaced with asparagine, the pKa value of the Schiff base is lowered from above 13 to about 9. We determined the direction of the loss or gain of the Schiff base proton in unphotolyzed and in photoexcited D85N, and the D85N/D96N and D85N/D96A double mutants, in order to understand the intrinsic and the induced connectivities of the Schiff base to the two membrane surfaces. The influence of D96 mutations on proton exchange and on acceleration of proton shuttling to the surface by azide indicated that in either case the access of the Schiff base on D85N mutants is to the cytoplasmic side. In the wild-type protein (but with the pKa of the Schiff base lowered by 13-trifluoromethyl retinal substitution) the results suggested that the Schiff base can communicate also with the extracellular side. Raising the pH without illumination of D85N so as to deprotonate the Schiff base caused the same, or nearly the same, change of X-ray scattering as observed when the Schiff base deprotonates during the wild-type photocycle. The results link the charge state of the active site to the global protein conformation and to the connectivity of the Schiff base proton to the membrane surfaces. Their relationship suggests that the conformation of the unphotolyzed wild-type protein is stabilized by coulombic interaction of the Schiff base with its counter-ion. A proton is translocated across the membrane after light-induced transfer of the Schiff base proton to D85, because the protein assumes an alternative conformation that separates the donor from the acceptor and opens new conduction pathways between the active site and the two membrane surfaces.

The light-induced proton pumping activity of bacteriorhodopsin (bR) is based on the photocycle of its light-adapted all-trans-retinal protein pigment. The photocycle of the 13-cis pigment lacks the M intermediate (which carries a deprotonated retinal Schiff base, characteristic of the all-trans photocycle) and is not associated with proton release and uptake. Aiming at establishing the reasons for the lack of light-induced Schiff base deprotonation and proton pumping in 13-cis-bR, we carried out pulsed-laser and continuous excitation experiments with artificial 13-cis-bR pigments derived from 13-demethylretinal, 13-demethyl-14-fluoro-bR, and 13-demethyl-12,14-difluoro-bR. Pulsed-laser photolysis shows that both M formation and proton pumping are restored in 13-cis-13-demethyl-bR by raising the pH to 8.5-9. M formation, but not proton pumping, is restored at neutral pH by 14-fluorine substitution. Continuous-illumination experiments lead, in all cases, to the generation of extremely long-lived (minutes to hours) M photoproducts. We show that such species are due to secondary photoreactions of late intermediates of the primary photolysis. Feasible mechanisms accounting for Schiff base deprotonation in the all-trans photocycle, but not in that of 13-cis-bR, are considered. Our findings favor a mechanism which attributes the lack of light-induced Schiff base deprotonation of 13-cis-bR to an insufficient change in the relative pKa of the donor (Schiff base) and acceptor (probably Asp-85) groups and/or to a high activation barrier for the proton transfer. The required change in relative pKas may be achieved either by deprotonation of a protein moiety (YH) with pKa approximately 8.5 or by fluorine substitution at position 14. Similarly, both YH titration and 14-fluorine substitution may reduce the barrier for proton transfer by affecting H-bonding interactions in the vicinity of the Schiff base linkage. The lack of proton release and uptake in the photocycle of 13-cis-13-demethyl-14-fluoro-bR, despite the presence of an M intermediate, is discussed. It appears that Schiff base deprotonation does not essentially imply proton release and uptake. Our conclusions bear on the molecular mechanism of the photocycle and of proton pumping in all-trans-bR.

Bacteriorhodopsin contains all-trans-retinal linked via a protonated Schiff base to K216. The proton transport in this pump is initiated by all-trans to 13-cis photoisomerization of the retinal and the ensuing transfer of the Schiff base proton to D85. Changed geometrical relationship of the Schiff base and D85 after the photoisomerization is a possible reason for the proton transfer. We introduced small volume/shape changes with site-specific mutagenesis of residues V49 and A53 that contact the side chain of K216, in order to force the Schiff base into somewhat different positions relative to D85. Earlier [Zimányi, L., Váró, G., Chang, M., Ni, B., Needleman, R., & Lanyi, J. K. (1992) Biochemistry 31, 8535-8543] we had described the kinetics of absorbance changes in the microsecond to millisecond time range after photoexcitation with the scheme L<-->M1<-->M2 + H+ (where the first equilibrium is the internal proton transfer and the second is proton release on the extracellular surface). Testing it at various pH values with mutants, where selected rate constants are changed, now confirms the validity of this scheme. The kinetics of the M state thus allowed examination of the transient equilibrium that develops in the L<-->M1 reaction and represents the redistribution of the proton between the Schiff base and D85. From the structure of the protein, the V49A and V49M residue replacements were both predicted to cause decreased alignment of the Schiff base and D85, and indeed we found that they both changed the equilibrium toward the protonated Schiff base. In contrast, the residue replacements A53V and A53G were predicted to move the Schiff base in opposite directions, away from and closer to alignment with D85, respectively. The former indeed changed the equilibrium toward the protonated Schiff base and the latter toward the deprotonated Schiff base. In addition, the hydroxyl stretch band of a bound water in the L state was affected by all mutations that disfavor proton transfer to D85. We conclude that the geometry of the proton donor and acceptor in the Schiff base-D85 pair, mediated by bound water, is a determinant of the proton transfer equilibrium.

The second order nonlinear polarizability and dipole moment changes upon light excitation of light-adapted bacteriorhodopsin (BR), dark-adapted BR, blue membrane, and acid purple membrane have been measured by second harmonic generation. Our results indicate that the dipole moment changes of the retinal chromophore, delta mu, are very sensitive to both the chromophore structure and protein/chromophore interactions. Delta mu of light-adapted BR is larger than that of dark-adapted BR. The acid-induced formation of the blue membrane results in an increase in the delta mu value, and formation of acid purple membrane, resulting from further reduction of pH to 0, returns the delta mu to that of light-adapted BR. The implications of these findings are discussed.

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.

The photoreactions of rhodopsin regenerated with three 9-cis retinal analogs, modified at or in the vicinity of the beta-ionone ring (namely 5,6-epoxy, 7,8-diH, diethyl-acyclic) have been investigated by UV-vis and FTIR difference spectroscopy. In parallel, the ability to catalyze the GDP-->GTP exchange of G-protein (transducin) has been monitored by time-dependent fluorescence spectroscopy. The first photoproduct obtained with all three pigments at liquid nitrogen temperature is a blue-shifted intermediate (BSI), followed by a lumi-like intermediate at 170 K. For the 5,6-epoxy-ISO and 7,8-diH-ISO pigment we obtain two further intermediates similar to the META-I and META-II states of native RHO. For the diethyl-acyclic-ISO pigment only one further intermediate can be stabilized at 280 K. As compared to META-II the respective photoproduct exhibits striking differences. The latter two pigments have also been investigated in the solubilized lipid-free state (detergent: dodecyl maltoside) at 280 K. For the 5,6-epoxy-ISO pigment, the UV-vis, FTIR, and activation data agree with the formation of a META-II-like photoproduct (81% activation). Less META-II formation is observed for the 7,8-dihydro-ISO pigment in membranes (65% activation), but full formation in detergent (100% activation). Neither the membrane-bound nor the solubilized diethyl-acyclic-ISO pigment forms a META-II-like intermediate (18% and 0% activation, respectively). Therefore, we conclude that the substitution of the beta-ionone ring by two ethyl groups abolishes steric interactions with the protein, which are essential for META-II formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Following light absorption, at neutral pH the bacteriorhodopsin mutant Y57N does not show Schiff base deprotonation (no M intermediate) or proton pumping activity. We reasoned that this might be due to improper delta pKa between the proton-donating Schiff base and the proton-accepting Asp-85 after light absorption. To test this, we reduced the intrinsic pKa of the protonated Schiff base in the pigment (and thus in the photointermediates) by replacing the retinal chromophore with an analogue, 14-F retinal. This substitution restores light-induced M formation, strongly suggesting that light-induced Schiff base deprotonation is accomplished by lowering its pKa during the photochemical cycle. Thus, while it is generally accepted that the Schiff base deprotonation during the photocycle takes place because of the light-induced reduction in its pKa, we provide here the first experimental evidence of this phenomenon.

The structure of bacteriorhodopsin based on electron microscopy (EM) studies, as provided in Henderson et al. (1990), is refined using molecular dynamics simulations. The work is based on a previously refined and simulated structure which had added the interhelical loops to the EM model of bR. The present study applies an all-atom description to this structure and constraints to the original Henderson model, albeit with helix D shifted. Sixteen waters are then added to the protein, six in the retinal Schiff base region, four in the retinal-Asp-96 interstitial space, and six near the extracellular side. The root mean square deviation between the resulting structure and the Henderson et al. (1990) model measures only 1.8 A. Further simulations of retinal analogues for substitutions at the 2- and 4-positions of retinal and an analogue without a beta-ionone ring agree well with observed spectra. The resulting structure is characterized in view of bacteriorhodopsin's function; key features are (1) a retinal Schiff base-counterion complex which is formed by a hydrogen bridge network involving six water molecules, Asp-85, Asp-212, Tyr-185, Tyr-57, Arg-82, and Thr-89, and which exhibits Schiff base nitrogen-Asp-85 and -Asp-212 distances of 6 and 4.6 A; (2) retinal assumes a corkscrew twist as one views retinal along its backbone; and (3) a deviation from the usual alpha-helical structure of the cytoplasmic side of helix G.

Results are presented demonstrating that the backbone of the active site lysine of bacteriorhodopsin undergoes light-induced structural alterations during bacteriorhodopsin-mediated light-induced proton pumping. This conclusion is based on difference Fourier transform infrared spectroscopy of isotopically labeled bacteriorhodopsin. The data demonstrate that the backbone carbonyl of lysine achieves an extremely low vibrational frequency during M412 intermediate formation. This is preceded by a structural transition in the lysine backbone that leads to an active site lysine carbonyl with the observed low vibrational frequency, probably due to a high degree of solvation.

Bacteriorhodopsin pigments lacking the retinal-Lys-216 covalent bond were prepared by reconstituting the K216G mutant protein with retinal alkylamine Schiff bases. The procedure follows the approach of Zhukovsky et al. [Zhukovsky, E., Robinson, P., & Oprian, D. (1991) Science 251, 558-560] in the case of visual (rhodopsin) pigments. Reconstitution leads to a mixture of three pigments. One of them, bR(K216G)/566a, absorbs (pH = 6.9) at 566 nm. Its absorption is pH-dependent, exhibiting a purple to blue transition. The pigment's laser-induced photocycle patterns are similar to those of wild-type all-trans-bR. A second component, bR(K216G)/566b, exhibits an independent photocycle reminiscent of that of wild-type 13-cis-bR. A third pigment component, bR(K216G)/630, absorbs around 630 nm. Experiments in the presence of a pH dye indicator show that illumination of bR(K216G)/566 produces a detectable proton gradient. It is concluded that a covalent bond between the retinal chromophore and the protein backbone is not a prerequisite for the basic structure and photochemical features of bR or for its proton pump activity.

The picosecond molecular dynamics in an artificial bacteriorhodopsin (BR) pigment containing a structurally modified all-trans retinal chromphore with a six-membered ring bridging the C11=C12-C13 positions (BR6.11) are measured by picosecond transient absorption and picosecond time-resolved fluorescence spectroscopy. Time-dependent intensity and spectral changes in absorption in the 570-650-nm region are monitored for delays as long as 5 ns after the 7-ps, 573-nm excitation of BR6.11. Two intermediates, J6.11 and K6.11/1, both with enhanced absorption to the red (> 600 nm) of the BR6.11 spectrum are observed within approximately 50 ps. The J6.11 intermediate decays with a time constant of 12 +/- 3 ps to form K6.11/1. The K6.11/1 intermediate decays with an approximately 100-ps time constant to form a third intermediate, K6.11/2, which is observed through diminished 650-nm absorption (relative to that of K6.11/1). No other transient absorption changes are found during the remainder of the initial 5-ns period of the BR6.11 photoreaction. Fluorescence in the 650-900-nm region is observed from BR6.11, K6.11/1, and K6.11/2, but no emission assignable to J6.11 is found. The BR6.11 fluroescence spectrum has a approximately 725-nm maximum which is blue-shifted by approximately 15 nm relative to that of native BR-570 and is 4.2 +/- 1.5 times larger in intensity (same sample optical density). No differences in the profile of the fluorescence spectra of BR6.11 and the intermediates K6.11/1 and K6.11/2 are observed. Following ground-state depletion of the BR6.11 population, the time-resolved fluroescence intensity monitored at 725 nm increases with two time constants, 12 +/- 3 and approximately 100 ps, both of which correlate well with changes in the picosecond transient absorption data. The resonance Raman spectrum of ground-state BR6.11, measured with low-energy, 560-nm excitation, is significantly different from the spectrum of native BR-570, thus confirming that the picosecond transient absorption and picosecond time resolved fluorescence data are assignable to BR6.11 and its photoreaction alone and not to BR-570 reformed during there constitution process (<5% of the BR6.11 sample could be attributed to native BR-570).The J6.11 and K6.11 absorption and fluorescence data presented here are generally analogous to those measured for native J-625 and K-590, respectively, and therefore, the primary events in the BR6.11 photoreaction can be correlated with those in the native BR photocycle. The BR6.11 photoreaction, however, exhibits important differences including slower formation rates for J and K intermediates as well as the presence of a second K intermediate. These results demonstrate that the restricted motion in the C11=C12-C13 region of retinal found in BR6.11 does not greatly change the overall photoreaction mechanism,but does alter the rates at which processes occur.

The Schiff base linkage bond configuration of bacteriorhodopsin was studied using model compounds consisting of all-trans- and 13-cis-retinal-protonated Schiff bases bearing C = N anti and syn bond configurations. The C = N configuration was analyzed using a combination of Fourier transform infrared spectroscopy and isotopically labeled chromophores. It was found that, in the model compounds, the coupling between the C14--C15 stretching frequency and the N--H rock is weak in the all-trans-retinal-protonated Schiff base in both the anti and syn C = N configurations. However, this coupling is relatively strong in the 13-cis-retinal-protonated Schiff base in both the anti and syn C = N configurations. Thus, it is concluded that, in model compounds, the C14--C15 mode can serve as a marker for the C13 = C14 bond configuration but not for the C = N. A different situation may prevail in bacteriorhodopsin due to different conformations of the retinal chromophore in the protein binding site and in solution. This difference suggests that the C14--C15/NH coupling in retinal-protonated Schiff bases is affected by the retinal conformation.

Artificial bovine rhodopsin pigments derived from synthetic retinal analogues carrying electron-withdrawing substituents (fluorine and chlorine) were prepared. The effects of the electron withdrawing substituents on the pKa values of the pigments and on the corresponding Schiff bases in solution were analyzed. The data suggest that the apparent pKa of the protonated Schiff base is above 16. However, the alternative possibility that the retinal Schiff base linkage in bovine rhodopsin is not accessible for titration from the aqueous bulk medium cannot be definitely ruled out.

We have applied low temperature difference FTIR spectroscopy to investigate intermediates produced from the M intermediate upon blue light excitation (<480 nm). In agreement with an earlier report by Balashov and Litvin (1981), who studied these intermediates with low temperature visible absorption spectrophotometry, we have observed at least three stages in this backphotoreaction. The initial photoproduct is stable at 100 K, and two products of subsequent thermal reactions are observed upon raising the temperature to 130 and 160 K, respectively.The alterations in the C=N stretching mode of the Schiff base have been identified by isotopically labeling the retinal chromophore, and changes in C=O stretching modes of amino acid residues with acidic side chains have been investigated. Analysis of the C=N stretching mode shows that the Schiff base remains unprotonated after the photochemical reaction at 100 K. Moreover, there are two types of Schiff bases, presumably associated with different bR species, that become thermally reprotonated at 130 and 160 K, respectively. Bands associated with the C=O stretching modes suggest that Asp 85 rather than Asp 96 reprotonates the Schiff base during the M to bR backphotoreaction. This conclusion is consistent with earlier observations that the polarity of electrical signals during this photochemical back reaction is reversed as compared to the thermal regeneration of bR from M.

Bacteriorhodopsin (bR) has been biosynthetically prepared with lysine deuterated at its alpha carbon (C alpha--H). The labeled membranes containing bR were investigated by difference Fourier transform infrared (FTIR) spectroscopy. It has been derived from K/bR and M/bR difference spectra (K and M are photocycle intermediates) that several bands previously assigned to the retinal chromophore are coupled to the C alpha--H. The vibrational modes that exhibit this coupling are principally associated with C15--H and N--H vibrations. [C alpha--2H]Lysine-labeled bR was fragmented enzymatically, and bR structures were regenerated with the C alpha--2H label either on lysine-216 and -172 or on the remaining five lysine residues of the protein. FTIR studies of the regenerated bR system, together with methylation of all lysines except the active-site lysine, reveal that the changes observed due to backbone labeling arise from the active-site lysine. The intensity of the C15--H out-of-plane wag is interpreted as a possible indication of a twist around the C15 = N bond.

The back photoreaction of the M intermediate in the photocycle of bacteriorhodopsin is investigated both for the native pigment and its D96N mutant. The experimental setup is based on creating the M intermediate by a first pulse, followed by a (blue) laser pulse which drives the back photoreaction of M. Experiments are carried out varying the delay between the two pulses, as well as the temperature over the -25 degrees C-20 degrees C range. It is found that the kinetic patterns of the M back photoreaction change with time after the generation of this intermediate. The data provide independent evidence for the suggestion of a photocycle mechanism based on two distinct M intermediates. They are thus in keeping with the consecutive model of Varo and Lanyi (Biochemistry 30, 5016-5022; 1991), although they cannot exclude other models such as those based on branched or parallel cycles. More generally, we offer a "photochemical" approach to discriminating between intermediate stages in the photocycle which does not depend on spectroscopic and/or kinetic data. While markedly affecting the rate of the M --> N transition in the photocycle, the rate of the thermal step in back photoreaction of M, at both room and low temperatures, is not significantly affected by the D96N mutation. It is proposed that while Asp 96 is the Schiff-base protonating moiety in the M --> N transition, another residue (most probably Asp 85) reprotonates the Schiff base following light absorption by M.

In variance with chlorophyll-based photosynthetic pigments, the triplet states of rhodopsins, either visual or photosynthetic, have not been observed experimentally. This is due to the ultrafast crossing from S1 to S0, which effectively competes with intersystem crossing to the triplet (T1) state. In order to populate T1 indirectly, laser photolysis experiments are performed with model protonated Schiff bases of retinal in solution, in which both inter- and intramolecular energy transfer to the polyene chromophore are carried out from an appropriate triplet energy donor. The experiments are then extended to bacteriorhodopsin (bR) by detaching the native retinal chromophore from the protein-binding site and replacing it by an analogous (synthetic) protonated Schiff base polyene, attached in a nonconjugated way to a naphthone triplet donor. Pulsed laser excitation of the latter moiety led, for the first time, to the observation of the triplet state of a rhodopsin. Possible locations and roles of the T1 state in bR and in visual pigments are discussed briefly.

Factors affecting the C = N stretching frequency of protonated retinal Schiff base (RSBH+) were studied with a series of synthetic chromophores and measured under different conditions. Interaction of RSBH+ with nonconjugated positive charges in the vicinity of the ring moiety or a planar polyene conformation (in contrast to the twisted retinal conformation in solution) shifted the absorption maxima but did not affect the C = N stretching frequency. The latter, however, was affected by environmental perturbations in the vicinity of the Schiff base linkage. Diminished ion pairing (i.e., of the positively charged nitrogen to its anion) achieved either by substituting a more bulky counteranion or by designing models with a homoconjugation effect lowered the C = N stretch energy. Decreasing solvation of the positively charged nitrogen leads to a similar trend. These effects in the vicinity of the Schiff base linkage also perturb the deuterium isotope effect observed upon deuteriation of the Schiff base. The results are interpreted by considering the mixing of the C = N stretching and C = N-H bending vibration. The C = N mode is shifted due to electrostatic interaction with nonconjugated positive charges in the vicinity of the Schiff base linkage, an interaction that does not influence the isotope effect. Weak hydrogen bonding between the Schiff base linkage in bacteriorhodopsin (bR) and its counteranion or, alternatively, poor solvation of the positively charged Schiff base nitrogen can account for the C = N stretching frequency of 1640 cm-1 and the deuterium isotope effect of 17 cm-1 observed in this pigment.(ABSTRACT TRUNCATED AT 250 WORDS)

The absorption maximum (568 nm) of light-adapted bacteriorhodopsin bR568 undergoes reversible changes after acidification. At pH 2.9, the absorption shifts to 605 nm (forming bR605) and it blue shifts to 565 nm, after further acidification to pH approximately 0.5 (forming bR565). Molecular models accounting for such acid-induced changes are relevant to the structure and function of bacteriorhodopsin. In the present study we approached the problem by applying artificial bR pigments based on selectively modified synthetic retinals. This may allow direct identification of the specific regions in the retinal binding site where the above changes in the protein-retinal interactions take place. We investigated the spectroscopic effects of acid in a variety of artificial pigments, including cyaninelike retinals, retinals bearing bulky groups at C4, short polyenes, and retinals in which the beta-ionone ring was substituted by aromatic rings. The results provide direct evidence for the hypothesis that the generation of bR605 is due to changes in polyene-opsin interactions in the vicinity of the Schiff base linkage. The second transition (to bR565) was not observed in artificial pigments bearing major changes in the ring structure of the retinal. Two approaches accounting for this observation are presented. One argues that the generation of bR565 is associated with acid-induced changes in retinal-protein interactions in the vicinity of the retinal ring. The second involves changes in polyene-opsin interactions in the vicinity of the Schiff base linkage. For both bRw and bRN5, our results do not discriminate between the direct titration of negative or dipolar protein groups in the binding site and changes in the retinal-protein interactions induced by changes in the protein structure outside of the binding site.

A number of retinoid derivatives have been synthesized for use as labels for cellular retinol-binding protein. Introduction of substituents abolished the binding of the derivatives to the protein, except in the case of the photo-reactive derivative, 4-azidoretinol. This compound was found to compete successfully with all-trans-retinol for binding to cellular retinol-binding protein, with a high relative binding affinity. Irradiation of a complex of 4-azidoretinol and a semi-purified preparation of cellular retinol-binding protein from liver resulted in a firm attachment stable to SDS-gel electrophoresis. It is therefore suggested that the irradiated product is held together covalently. A method for the synthesis of 4-azidoretinol is described.

1. 1 alpha-Hydroxy[7-3H]cholecalciferol (specific radioactivity of 2-Ci/mmol) was synthesized, and its metabolism in chicks studied. 2. 1 alpha-Hydroxy[7-3H]cholecalciferol was metabolized very rapidly in the chick to 1 alpha,25-dihydroxy[7-3H]cholecalciferol and to a metabolite less polar than 1 alpha-hydroxycholecalciferol. Intestine exhibited highest accumulation of 1 alpha-25-dihydroxy[7-3H]cholecalciferol, and liver exhibited highest accumulation of the non-polar metabolite. 3. Tissue uptake of 1 alpha-hydroxy[7-3H]cholecalciferol and its metabolites in chicks that were dosed continuously for 16 days with 1 alpha-hydroxy[7-3H]cholecalciferol did not exceed by very much that observed in tissues obtained from chicks that were dosed with a single injection of 1 alpha-hydroxy[7-3H]cholecalciferol 24 h before killing, except for liver and kidney. 4. Lowest accumulation of metabolites was noted in muscle and bone, and for the latter, highest uptake of 1 alpha,25-dihydroxy[7-3H]cholecalciferol was noted in the epiphysial periosteum and the metaphysis. 5. Formation of 1 alpha,24,25-trihydroxy[7-3H]cholecalciferol was not observed in the chicks that were dosed continuously with 1 alpha-hydroxy[7-3H]cholecalciferol, despite the fact that plasma calcium and phosphorus were normal and despite the presence of renal 24-hydroxylase activity. 6. The vitamin D status of the chicks did not appear to affect the metabolic profile of the administered 1 alpha-hydroxy[7-3H]cholecalciferol.

The photolysis intermediates of an artificial bovine rhodopsin pigment, cis-5,6-dihydro-isorhodopsin (cis-5,6,-diH-ISORHO, lambda max 461 nm), which contains a cis-5,6-dihydro-9-cis-retinal chromophore, are investigated by room temperature, nanosecond laser photolysis, and low temperature irradiation studies. The observations are discussed both in terms of low temperature experiments of Yoshizawa and co-workers on trans-5,6-diH-ISORHO (Yoshizawa, T., Y. Shichida, and S. Matuoka. 1984. Vision Res. 24: 1455-1463), and in relation to the photolysis intermediates of native bovine rhodopsin (RHO). It is suggested that in 5,6-diH-ISORHO, a primary bathorhodopsin intermediate analogous to the bathorhodopsin intermediate (BATHO) of the native pigment, rapidly converts to a blue-shifted intermediate (BSI, lambda max 430 nm) which is not observed after photolysis of native rhodopsin. The analogs from lumirhodopsin (LUMI) to meta-II rhodopsin (META-II) are generated subsequent to BSI, similar to their generation from BATHO in the native pigment. It is proposed that the retinal chromophore in the bathorhodopsin stage of 5,6-diH-ISORHO is relieved of strain induced by the primary cis to trans isomerization by undergoing a geometrical rearrangement of the retinal. Such a rearrangement, which leads to BSI, would not take place so rapidly in the native pigment due to ring-protein interactions. In the native pigment, the strain in BATHO would be relieved only on a longer time scale, via a process with a rate determined by protein relaxation.

Artificial bacteriorhodopsin pigments based on synthetic retinal analogues carrying an electron-withdrawing CF3 substituent group were prepared. The effects of CF3 on the spectra, photocycles, and Schiff base pKa values of the pigments were analyzed. A reduction of 5 units in the pKa of the Schiff base is observed when the CF3 substituent is located at the C-13 polyene position, in the vicinity of the protonated Schiff base nitrogen. The results lead to the unambiguous characterization of the (direct) titration of the Schiff base in bacteriorhodopsin and to the conclusion that the deprotonation rate of the Schiff base during the photocycle (i.e., the generation of the M412 intermediate) is determined by a structural change in the protein.