title: Photo-induced bleaching of sensory rhodopsin II (phoborhodopsin) from Halobacterium salinarum by hydroxylamine: identification of the responsible intermediates.
abstract: Sensory rhodopsin II from Halobacterium salinarum (HsSRII) is a retinal protein in which retinal binds to a specific lysine residue through a Schiff base. Here, we investigated the photobleaching of HsSRII in the presence of hydroxylamine. For identification of intermediate(s) attacked by hydroxylamine, we employed the flash-induced bleaching method. In order to change the concentration of intermediates, such as M- and O-intermediates, experiments were performed under varying flashlight intensities and concentrations of azide that accelerated only the M-decay. We found the proportional relationship between the bleaching rate and area under the concentration-time curve of M, indicating a preferential attack of hydroxylamine on M. Since hydroxylamine is a water-soluble reagent, we hypothesize that for M, hydrophilicity or water-accessibility increases specifically in the moiety of Schiff base. Thus, hydroxylamine bleaching rates may be an indication of conformational changes near the Schiff base. We also considered the possibility that azide may induce a small conformational change around the Schiff base. We compared the hydroxylamine susceptibility between HsSRII and NpSRII (SRII from Natronomonas pharaonis) and found that the M of HsSRII is about three times more susceptible than that of the stable NpSRII. In addition, long illumination to HsSRII easily produced M-like photoproduct, P370. We thus infer that the instability of HsSRII under illumination may be related to this increase of hydrophilicity at M and P370.
authors: College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime, Japan.
authors: Jun
authors: J
authors: Tamogami
authors: Takashi
authors: T
authors: Kikukawa
authors: Yoichi
authors: Y
authors: Ikeda
authors: Makoto
authors: M
authors: Demura
authors: Toshifumi
authors: T
authors: Nara
authors: Naoki
authors: N
authors: Kamo
mesh_terms: D019617:Halobacterium salinarum / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects*; D057927:Hydrophobic and Hydrophilic Interactions; D019811:Hydroxylamine / Q000737:chemistry* / Q000494:pharmacology; D008027:Light; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012545:Schiff Bases / Q000737:chemistry; D025603:Sensory Rhodopsins / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D014867:Water / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D012545:Schiff Bases; D025603:Sensory Rhodopsins; D014867:Water; D019811:Hydroxylamine