Primary photochemical event in vision: proton translocation.

Abstract

Picosecond studies of rhodopsin in low-temperature glasses have been carried out in order to observe directly the risetime of prelumirhodopsin, the first intermediate in the visual pathway. Only at 20 K or below can the risetime of this intermediate be resolved and even at 4 K it is astoundingly rapid, about 36 psec. An examination of the Arrhenius dependence on temperature of the rate of formation of prelumirhodopsin shows a strong deviation from linearity at low temperatures, i.e., non-Arrhenius behavior. This marked nonlinear behavior is characteristic of a quantum mechanical tunneling event such as the translocation of hydrogen. An excellent candidate for the tunnelling process is the hydrogen of the protonated Schiff base formed between opsin and its retinal chromophore. Deuterium-exchanged rhodopsin, in which the Schiff base hydrogen is replaced by a deuterium, also shows a marked non-Arrhenius temperature dependence at low temperatures, consistent with tunneling. The rate of formation of prelumirhodopsin in deuterium-exchanged samples is much slower and a deuterium isotope effect kH/kD t 7 is observed. The data support a model in which the formation of prelumirhodopsin involves translocation of a roton toward the Schiff base nitrogen of the retinal chromopfiore.The primary process in visual excitation is initiated by a photochemical event, the absorption of a photon by the photoreceptor, rhodopsin, resulting in the formation of a new species, prelumirhodopsin. The characterization of this new species has been carried out by photostationary studies in low-temperature glasses (1, 2) and by picosecond kinetic studies near room temperatures (3, 4). Prelumirhodopsin is formed within 6 X 10-12 sec (6 psec) following excitation of rhodopsin and has an absorption maximum at 543 nm which is bathochromically shifted compared to that of rhodopsin. This event has been classically described as the isomerization of the 11-cis-retinal chromophore of rhodopsin to the all-trans-retinal form (5, 6). That full isomerization of a bulky chromophore could occur within this time scale has been questioned (3, 7), and speculation still exists as to the nature of this photochemical event.The time resolution for most ultrafast kinetic studies is about 6 psec, which is slower than the actual risetime of prelumirhodopsin at room temperature. To overcome this restriction, we have excited rhodopsin in low-temperature glasses and monitored the formation rate of prelumirhodopsin. The kinetics are sufficiently slow at 20 K or below to allow us to measure directly the formation of prelumirhodopsin. Our picosecond data suggest that the initial photochemical step in the visual process is not cis-trans isomerization, but rather proton translocation. Plausible models are presented and discussed. METHODOLOGY Rod outer segments were isolated from frozen bovine retinas (G. Hormel Co.) and were solubilized in 0.3 M Ammonyx LO The costs of publication of this article were defrayed in part by the payment of page charges from funds made avai...

Bathorhodopsin intermediates from 11-cis-rhodopsin and 9-cis-rhodopsin.

“…RESULTS Rhodopsin samples were purified to remove free unbound retinal that might interfere with visual pigment absorption and were solubilized for high optical clarity to minimize light scattering. The rate of production of bathorhodopsin (2,7,8), the low temperature spectra (1,(8)(9)(10)(11), and the quantum yield of bleaching (12)(13)(14) are the same whether rhodopsin is solutTo whom reprint requests should be addressed. …”

Development of Ultrashort Pulse Lasers for Ultrafast Spectroscopy

“…Elucidation of ultrafast dynamics in various systems accelerates development in various applications like photosensors [3][4][5], ultrafast optical switches [6][7][8], and ultrafast optical memories [9][10][11]. Many interesting ultrafast dynamics in biological systems including photosynthesis [12][13][14] and vision processes [15][16][17], were studied by ultrafast spectroscopy using the visible or UV pulse lasers. In these experiments, sometimes the intensities of the generated pulses are not stable due to the various nonlinearities in the generation process.…”

Ultra‐fast Laser Spectroscopy of Visual Pigments

“…By excitation of cattle rhodopsin at very low temperature (4-35 K), Peters et al (1977) observed the formation of a bathochromic photoproduct which was first assigned as a lowest excited state of rhodopsin and later as a ground state intermediate, named "Batho"' or "prebathorhodopsin" (Honig et al, 1979;Peters and Leonitis, 1982;Ottolenghi, 1982). It decayed to the next bathochromic intermediate (presumably bathorhodopsin) with a time constant of 36 ps at 4 K. Kobayashi (1980) also observed the bathochromic photoproduct [first assigned as an excited state and later as a ground state intermediate named "primerhodopsin" (Ohtani et al, 1988)] by excitation of cattle rhodopsin at room temperature.…”