The effects of octanol on the late photointermediates of rhodopsin.

Membrane suspensions of unperturbed rhodopsin and rhodopsin perturbed with 2.5 mM octanol were photolyzed with 477 nm laser pulses at 20 degrees C and 35 degrees C. Changes in absorbance were monitored at times ranging from 1 microsecond to 80 ms after excitation. The data were analyzed using singular value decomposition, global exponential fitting and kinetic modeling. A recently proposed model involving the photointermediate Meta-I380 (T. E. Thorgeirsson, J. W. Lewis, S. E. Wallace-Williams, and D. S. Kliger, Biochemistry 32, 13861-13872, 1993) fits data for samples with and without octanol. Comparison of the microscopic rates shows this alcohol accelerates the formation of Meta-II via Meta-I380. Activation and equilibrium thermodynamic parameters obtained from Arrhenius plots suggest that octanol reduces the entropy increase in forming both Meta-I380 and Meta-II. It also lowers the enthalpy of Meta-I380 relative to Lumi and of Meta-II relative to Meta-I480. To help determine whether octanol affects the protein directly or indirectly through the lipid bilayer, similar experiments were conducted using rhodopsin solubilized in 0.13% dodecyl maltoside with and without octanol. Spectral shifts in the presence of octanol suggest that a direct protein interaction exists in addition to previously reported effects dependent on membrane free volume.

Proton transfer reactions linked to rhodopsin activation.

Purified bovine rhodopsin solubilized in dodecyl maltoside was photolyzed at 20 degreesC with 477 nm light, and difference spectra were collected at time delays ranging from 10 micros to 10 ms after photolysis. Bromocresol purple was added to the samples to detect pH changes in the aqueous environment due to changes in the protonation state of rhodopsin. The data were analyzed using singular value decomposition and global exponential fitting, which revealed three exponential processes indicating the presence of at least four intermediates. Spectral changes of the indicator dye were separated from those of rhodopsin, and proton release and uptake rates were analyzed within the framework of rhodopsin photoreaction kinetics. Proton release occurred during Lumi decay to Meta-I380 followed by uptake upon Meta-I380 decay and by a more significant proton uptake with the time course of Meta-I480 decay. On the basis of the estimated number of protons released and taken up in each step of the rhodopsin photoreaction, we concluded that two forms of Meta-II are present. The two forms of Meta-II, Meta-IIa' and Meta-IIb, differ in protonation state from one another as do both from the earlier, 380 nm absorbing form, Meta-I380.

Effects of pH on rhodopsin photointermediates from lumirhodopsin to metarhodopsin II.

Time-resolved absorption difference spectra of membrane suspensions of bovine rhodopsin at pH 5, 6, 7, 8, 9, and 10 were collected in the time range from 1 micro s to 200 ms after laser photolysis with 7-ns pulses of 477-nm light. The data were analyzed using singular value decomposition (SVD) and global exponential fitting. At pH 7 the data agree well with previously obtained data (Thorgeirsson et al. (1993) Biochemistry 32, 13861-13872) with fits improved at all pH's by inclusion of a small component due to an absorbance change caused by rotational diffusion which is detectable even at magic angle polarization. A "square scheme" suggested to best explain the previous data, which involves two branches following decay of the lumi intermediate with pathways (1) lumi --> MI480 right harpoon over left harpoon MII and (2) lumi right harpoon over left harpoon MI380 --> MII, could be confirmed throughout the entire pH range. However, to account for the increased rate of the MII --> MI480 reaction in path 1 for rising pH values, we propose that the MII in the square scheme consists of deprotonated MII and protonated MIIH+ forms in rapid equilibrium with each other, resulting in an extended square scheme and increasing the number of 380-nm products from two to three. In addition to the kinetic processes described by the extended square scheme, above pH 8 fast ( approximately 10 micro s) and slow ( approximately 50 ms) components were found. The fast component was assigned to the decay of a blue-shifted lumi intermediate, and the slow component, resolvable only at pH 10, was assigned to formation of a 450 nm absorbing photoproduct.

Low-temperature trapping of early photointermediates of alpha-isorhodopsin.

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.