Nanosecond photolysis of rhodopsin: evidence for a new blue-shifted intermediate

Abstract

Early photolysis intermediates of native bovine rhodopsin (RHO) are investigated by nanosecond laser photolysis near physiological temperature. Absorption difference spectra are collected after excitation with 477-, 532-, and 560-nm laser pulses of various energies and with 477-nm laser excitation at 5, 12, 17, 21, and 32 degrees C. The data are analyzed by using singular-value decomposition (SVD) and a global exponential fitting routine. Two rate constants associated with distinct spectral changes are observed during the time normally associated with the decay of bathorhodopsin to lumirhodopsin. Various models consistent with this observation are considered. A sequential model in which there is a reversible step between a bathorhodopsin intermediate and a new intermediate (BSI), which is blue-shifted relative to lumirhodopsin, is shown to best fit the data. The temperature dependence of the observed and calculated rate constants leads to linear Arrhenius plots. Extrapolation of the temperature dependence suggests that BSI should not be observable after rhodopsin photolysis at temperatures below -100 degrees C. The results are discussed with regard to the artificial visual pigments cis-5,6-dihydroisorhodopsin and 13-demethylrhodopsin. It is proposed that the rate of the BATHO to BSI transition is limited by the relaxation of the strained all-trans-retinal chromophore within a tight protein environment. The transition to LUMI involves chromophore relaxation concurrent with protein relaxation. While the first process is strongly affected by changes in the chromophore, the second transition seems to be determined more by protein relaxation.

Chromophore structural changes in rhodopsin from nanoseconds to microseconds following pigment photolysis

“…1a) could be fit to Scheme II and agreed in max values (Fig. 1d, Table 1) as well as in time constants (Table 2) with the intermediates of native rho measured previously under identical conditions (11). The difference spectra of mutant E113D are shown in Fig.…”

“…The new intermediate, blue-shifted intermediate of rho (bsi), is entropically favored and thus not trapped at low temperatures (11).…”

“…The data were analyzed using global exponential fitting following singular value decomposition as described elsewhere (11,19,20). This procedure fits the entire set of absorbance data at all wavelengths and delay times simultaneously.…”

“…One way to characterize the intermediates is to trap them at low temperatures. This approach led to the classical ''bleaching'' scheme ( max values in nanometers): rho (498) 3 bathorhodopsin (batho) (542) 3 lumirhodopsin (lumi) (497) 3 meta I (480) 3 meta II (380) Scheme I However, at more physiologically relevant temperatures, time-resolved measurements up to 1 msec after photolysis reveal a new intermediate and a different kinetic scheme (3,11). The following reaction scheme was proposed to occur during the nanosecond-to-microsecond time regime (3): rho (498) 3 batho (529) ª bsi (477) 3 lumi (492) 3 .…”

Vertebrate ultraviolet visual pigments: Protonation of the retinylidene Schiff base and a counterion switch during photoactivation

“…Again, the E108Q BLI max is blue-shifted compared to wild type. BLI appears analogous to the previously observed intermediate in chicken blue opsin (25) and BSI in rhodopsin (26). However, there may be slight differences between the cone (BLI) and rod intermediates, because rhodopsin's BSI cannot be trapped at low temperature (26).…”