title: Proton release and uptake of pharaonis phoborhodopsin (sensory rhodopsin II) reconstituted into phospholipids.
abstract: pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a photo-receptor for negative phototaxis in Natronobacterium pharaonis. During the photoreaction cycle (photocycle), ppR exhibits intraprotein proton movements, resulting in proton pumping from the cytoplasmic to the extracellular side, although it is weak. In this study, light-induced proton uptake and release of ppR reconstituted with phospholipid were analyzed using a SnO(2) electrode. The reconstituted ppR exhibited properties in proton uptake and release that are different from those of dodecyl maltoside solubilized samples. It showed fast proton release before the decay of ppR(M) (M-photointermediate) followed by proton uptake, which was similar to that of bacteriorhodopsin (BR), a light-driven proton pump. Mutant analysis assigned Asp193 to one (major) of the members of the proton-releasing group (PRG). Fast proton release was observed only when the pH was approximately 5-8 in the presence of Cl(-). When Cl(-) was replaced with SO(4)(2-), the reconstituted ppR did not exhibit fast proton release at any pH, suggesting Cl(-) binding around PRG. PRG in BR consists of Glu204 (Asp193 in ppR) and Glu194 (Pro183 in ppR). Replacement of Pro183 by Glu/Asp, a negatively charged residue, led to Cl(-)-independent fast proton release. The transducer binding affected the properties of PRG in ppR in the ground state and in the ppR(M) state, suggesting that interaction with the transducer extends to the extracellular surface of ppR. Differences and similarities in the molecular mechanism of the proton movement between ppR and BR are discussed.
authors.affiliation: Laboratory of Biomolecular Systems, Center for Advanced Science and Technology, Hokkaido University, Sapporo 001-0021, Japan. iwamoto@cast.hokudai.ac.jp
authors.forename: Masayuki
authors.initials: M
authors.lastname: Iwamoto
authors.forename: Chisa
authors.initials: C
authors.lastname: Hasegawa
authors.forename: Yuki
authors.initials: Y
authors.lastname: Sudo
authors.forename: Kazumi
authors.initials: K
authors.lastname: Shimono
authors.forename: Tsunehisa
authors.initials: T
authors.lastname: Araiso
authors.forename: Naoki
authors.initials: N
authors.lastname: Kamo
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001436:Bacteriorhodopsins / Q000737:chemistry; D002264:Carboxylic Acids / Q000737:chemistry; D002338:Carotenoids / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D025602:Halorhodopsins; D006863:Hydrogen-Ion Concentration; D008027:Light; D019633:Natronobacterium / Q000737:chemistry* / Q000235:genetics; D010713:Phosphatidylcholines / Q000737:chemistry*; D010782:Photolysis; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics; D011392:Proline / Q000235:genetics; D011522:Protons; D025603:Sensory Rhodopsins
chemical_list: D019843:Archaeal Proteins; D002264:Carboxylic Acids; D025602:Halorhodopsins; D010713:Phosphatidylcholines; D020283:Photoreceptors, Microbial; D011522:Protons; D025603:Sensory Rhodopsins; C061508:sensory rhodopsin II protein, archaeal; D001224:Aspartic Acid; D002338:Carotenoids; D018698:Glutamic Acid; D001436:Bacteriorhodopsins; D011392:Proline