Photochemistry of Acetabularia Rhodopsin II from a Marine Plant, Acetabularia acetabulum Takashi Kikukawa, † Kazumi Shimono, ‡,§ Jun Tamogami, †,§ Seiji Miyauchi, §,∥ So Young Kim, ⊥ Tomomi Kimura-Someya, ‡ Mikako Shirouzu, ‡ Kwang-Hwan Jung, ⊥ Shigeyuki Yokoyama,* ,‡,# and Naoki Kamo* ,§ † Faculty of Life Science, Hokkaido University, Sapporo 060-0810, Japan ‡ RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan § College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime 790-8578, Japan ∥ Graduate School of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan ⊥ Department of Life Science and Institute of Biological Interfaces, Sogang University, Seoul 121-742, Korea # Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan ABSTRACT: Acetabularia rhodopsins are the first microbial rhodopsins discovered in a marine plant organism, Acetabularia acetabulum. Previously, we expressed Acetabularia rhodopsin II (ARII) by a cell-free system from one of two opsin genes in A. acetabulum cDNA and showed that ARII is a light-driven proton pump [Wada, T., et al. (2011) J. Mol. Biol. 411, 986−998]. In this study, the photochemistry of ARII was examined using the flash- photolysis technique, and data were analyzed using a sequential irreversible model. Five photochemically defined intermediates (P i ) were sufficient to simulate the data. Noticeably, both P 3 and P 4 contain an equilibrium mixture of M, N, and O. Using a transparent indium tin oxide electrode, the photoinduced proton transfer was measured over a wide pH range. Analysis of the pH-dependent proton transfer allowed estimation of the pK a values of some amino acid residues. The estimated values were 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values were assigned as the pK a of Asp81 (Asp85 BR ) in the dark, Asp92 (Asp96 BR ) at N, Glu199 (Glu204 BR ) at M, Glu199 in the dark, an undetermined proton- releasing residue at the release, and the pH to start denaturation, respectively. Following this analysis, the proton transfer of ARII is discussed. R hodopsin is a membrane protein in which retinal as a chromophore binds to the lysine residue of the opsin (apoprotein) via a Schiff base. There are two types of rhodopsins. 1 One is type 2 rhodopsin, which is found in the eyes of animals, and the other is type 1 rhodopsin, which is now also called microbial rhodopsin. Originally, type 1 rhodopsin was found in haloarchaea in the early 1970s in the form of a light-driven proton pump, bacteriorhodopsin (BR). 2,3 Later, homologues with different functions were discovered, including halorhodopsin (HR), 4−6 sensory rhodopsin I (SRI), 7−9 and sensory rhodopsin II (SRII, also called phoborhodopsin). 10−14 These proteins have similar structural folds composed of seven helices and retinal binding to the conserved lysine residue of the last helix, whereas the function is different when essential amino acid residues are optimized. BR and HR are ion pumps, and SRI and SRII are photoreceptors. Type 1 rhodopsins have linear cyclic photochemical reactions called photocycles. The illumination of the pigment protein leads to the excited state, which is relaxed thermally to the original pigment via various photochemical intermediates. The best-studied rhodopsin is BR. BR at the ground state and intermediates K−O have been researched with various spectroscopic methods and X-ray crystallography. 2,3 The photocycle comprises stepwise reactions of the thermal reisomerization of the photoisomerized 13-cis- retinal to the initial all-trans-retinal, and the proton is transferred toward the higher-pK a residue accompanied by pK a changes during the photocycle. Type 1 rhodopsins have been found not only in archaea but also in eubacteria, fungi, and algae, and thus, type 1 rhodopsins are classified as microorganisms belonging to all three biological domains. 1 Many type 1 rhodopsins function as either ion pumps with fast photocycles or photoreceptors with slow photocycles. In addition, a new type, a photogated ion channel, was added recently to the family of type 1 rhodopsins on the basis of a study of Chlamydomonas. 15−17 Thus, the world of the type 1 rhodopsin (microbial rhodopsin) continues to expand. 1 As early as 1968, Schilde 18 reported a fast light-induced transmembrane voltage change from a giant unicellular marine alga, Acetabularia acetabulum, and on the basis of these results suggested that rhodopsin acts as a photoreceptor. In 2004, Received: June 28, 2011 Revised: September 5, 2011 Published: September 12, 2011 Article pubs.acs.org/biochemistry © 2011 American Chemical Society 8888 dx.doi.org/10.1021/bi2009932 | Biochemistry 2011, 50, 8888−8898