Role of Asn112 in a Light-Driven Sodium Ion-Pumping Rhodopsin.
Light-driven outward sodium-pumping rhodopsin (NaR) was recently found in marine bacteria. Krokinobacter eikastus rhodopsin 2 (KR2) actively transports sodium and lithium ions in NaCl and LiCl, respectively, while it pumps protons in KCl. NaR has a conserved NDQ (N112, D116, and Q123 in KR2) motif, and previous studies suggested an important role for N112 in the function of KR2. Here we replaced N112 with 19 different amino acids and studied the molecular properties of the mutants. All mutants exhibited absorption bands from a protonated Schiff base in the λmaxrange from 508 to 531 nm upon heterologous expression in Escherichia coli, whose ion-pumping activity was measured using pH electrodes. The function of these mutants was classified into three phenotypes: wild-type (WT)-like Na+/H+compatible pump, exclusive H+pump, and no pump. Among the 19 mutants, only N112D, -G, -S, and -T showed light-driven Na+pump activity, N112A, -C, -P, -V, -E, -Q, -I, -L, -M, -F, and -W were exclusively H+pumps, and N112H, -K, -Y, and -R exhibited no pump activity. The mutants of the no pump function lack a blue-shifted M intermediate, indicating that Schiff base deprotonation is a prerequisite for Na+and H+pumps. In contrast, the subsequent red-shifted O intermediate was observed for WT and N112V but absent for N112T and N112A, suggesting that observation of this intermediate depends on kinetics. Although N112D, -G, -S, and -T are able to pump Na+, they also pump H+in NaCl, where Na+and H+pumps compete with each other because of the decreased Na+uptake efficiency. From these facts, an exclusive Na+pump in NaCl exists only in WT. We conclude that N112 is one of the functional determinants of NaR.
Department of Life Science and Applied Chemistry, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.
Rei
R
Abe-Yoshizumi
Department of Life Science and Applied Chemistry, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.|OptoBioTechnology Research Center, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.|Frontier Research Institute for Material Science, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.|PRESTO, Japan Science and Technology Agency , 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
Keiichi
K
Inoue
Department of Biological Sciences, Graduate School of Science, The University of Tokyo , 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Hideaki E
HE
Kato
Department of Biological Sciences, Graduate School of Science, The University of Tokyo , 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Osamu
O
Nureki
Department of Life Science and Applied Chemistry, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.|OptoBioTechnology Research Center, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.
Hideki
H
Kandori
D001216:Asparagine / Q000737:chemistry*; D008027:Light; D009154:Mutation; D012243:Rhodopsin / Q000737:chemistry*; D012964:Sodium / Q000737:chemistry*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D001216:Asparagine; D012243:Rhodopsin; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase