The M intermediate of Pharaonis phoborhodopsin is photoactive

The retinal protein phoborhodopsin (pR) (also called sensory rhodopsin II) is a specialized photoreceptor pigment used for negative phototaxis in halo bacteria, Upon absorption of light, the pigment is transformed into a short -wavelength intermediate, M, that most likely is the signaling state (or it s precursor) that triggers the motility response of the cell. The M interme diate thermally decays into the initial pigment, completing the cycle of tr ansformations. In this study we attempted to determine whether M can be con verted into the initial state by light. The M intermediate was trapped by t he illumination of a water glycerol suspension of phoborhodopsin from Natro nobacterium pharaonis called pharaonis phoborhodopsin (ppR) with yellow lig ht (>450 nm) at -50 degrees C. The M intermediate absorbing at 390 nm is st able in the dark at this temperature. We found, however, that M is converte d into the initial (or spectrally similar) state with an absorption maximum at 501 nm upon illumination with 380-nm light at -60 degrees C. The revers ible transformations ppR <-> M are accompanied by the perturbation of trypt ophan(s) and probably tyrosine(s) residues, as reflected by changes in the UV absorption band. Illumination at lower temperature (-160 degrees C) reve als two intermediates in the photoconversion of M, which we termed M' (or M '(404)) and ppR' (or ppR'(496)). A third photoproduct, ppR'(504) is formed at -110 degrees C during thermal transformations of M'(404) and PPR'(496). The absorption spectrum of M'(404) (maximum at 404 nm) consists of distinct vibronic bands at 362, 382, 404, and 420 nm that are different from the vi bronic bands of M at 348, 368, 390, and 415 nm. ppR'(496) has an absorption band that is shifted to shorter wavelengths by 5 nm compared to the initia l ppR, whereas ppR'(504) is redshifted by at least 3 nm. As in bacteriorhod opsin, photoexcitation of the M intermediate of ppR and, presumably, photoi somerization of the chromophore during the M --> M' transition result in a dramatic increase in the proton affinity of the Schiff base, followed by it s reprotonation during the M' --> ppR' transition. Because the latter react ion occurs at very low temperature, the proton is most likely taken from th e counterion (Asp(75)) rather than from the bulk. The phototransformation o f M reveals a certain heterogeneity of the pigment, which probably reflects different populations of M or its photoproduct M'. Photoconversion of the M intermediate provides a possible pathway for photoreception in halobacter ia and a useful tool for studying the mechanisms of signal transduction by phoborhodopsin (sensory rhodopsin II).