title: Photochemical characterization of actinorhodopsin and its functional existence in the natural host.
abstract: Actinorhodopsin (ActR) is a light-driven outward H+pump. Although the genes of ActRs are widely spread among freshwater bacterioplankton, there are no prior data on their functional expression in native cell membranes. Here, we demonstrate ActR phototrophy in the native actinobacterium. Genome analysis showed that Candidatus Rhodoluna planktonica, a freshwater actinobacterium, encodes one microbial rhodopsin (RpActR) belonging to the ActR family. Reflecting the functional expression of RpActR, illumination induced the acidification of the actinobacterial cell suspension and then elevated the ATP content inside the cells. The photochemistry of RpActR was also examined using heterologously expressed RpActR in Escherichia coli membranes. The purified RpActR showed λmaxat 534nm and underwent a photocycle characterized by the very fast formation of M intermediate. The subsequent intermediate, named P620, could be assigned to the O intermediate in other H+pumps. In contrast to conventional O, the accumulation of P620remains prominent, even at high pH. Flash-induced absorbance changes suggested that there exists only one kind of photocycle at any pH. However, above pH7, RpActR shows heterogeneity in the H+transfer sequences: one first captures H+and then releases it during the formation and decay of P620, while the other first releases H+prior to H+uptake during P620formation.
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan. Electronic address: kikukawa@sci.hokudai.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kikukawa
authors.affiliation: College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Tamogami
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Masakatsu
authors.initials: M
authors.lastname: Kamiya
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Aizawa
authors.affiliation: Research Institute for Limnology, University of Innsbruck, Mondsee, Austria.
authors.forename: Martin W
authors.initials: MW
authors.lastname: Hahn
authors.affiliation: Center for Gene Research, Nagoya University, Nagoya, Japan.
authors.forename: Kunio
authors.initials: K
authors.lastname: Ihara
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Kamo
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Demura
mesh_terms: D039903:Actinobacteria / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D004734:Energy Metabolism / Q000528:radiation effects*; D004735:Energy Transfer; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016680:Genome, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D010782:Photolysis; D052817:Phototrophic Processes / Q000528:radiation effects*; D011487:Protein Conformation; D011522:Protons; D025604:Rhodopsins, Microbial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects*; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
chemical_list: D011522:Protons; D025604:Rhodopsins, Microbial; D000255:Adenosine Triphosphate
keywords: Actinorhodopsin; Flash photolysis; Light-driven proton pump; Microbial rhodopsin; Photocycle; Phototrophy