Journal Article Photochemical characterization of actinorhodopsin and its functional existence in the natural host

Nakamura, Shintaro  ,  Kikukawa, Takashi  ,  Tamogami, Jun  ,  Kamiya, Masakatsu  ,  Aizawa, Tomoyasu  ,  Hahn, Martin W.  ,  Ihara, Kunio  ,  Kamo, Naoki  ,  Demura, Makoto

1857 ( 12 )  , pp.1900 - 1908 , 2016-12 , Elsevier
ISSN:0005-2728
Description
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 lambda(max) at 534 nm and underwent a photocycle characterized by the very fast formation of M intermediate. The subsequent intermediate, named P-620, could be assigned to the 0 intermediate in other H+ pumps. In contrast to conventional 0, the accumulation of P620 remains prominent, even at high pH. Flash-induced absorbance changes suggested that there exists only one kind of photocycle at any pH. However, above pH 7, RpActR shows heterogeneity in the H+ transfer sequences: one first captures H+ and then releases it during the formation and decay of P-650, while the other first releases H+ prior to H+ uptake during P-620 formation. (C) 2016 Elsevier B.V. All rights reserved.
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