Cyanobacterial Light-Driven Proton Pump, Gloeobacter Rhodopsin: Complementarity between Rhodopsin-Based Energy Production and Photosynthesis

Gloeobacter Rhodopsin: Complementarity between Rhodopsin- Based Energy Production and Photosynthesis. PLoS ONE 9(10): e110643. doi:10.1371/journal.pone.0110643 Cyanobacterial Light-Driven Proton Pump, Gloeobacter Rhodopsin: Complementarity between Rhodopsin-Based Energy Production and Photosynthesis Ah Reum Choi 0 Lichi Shi 0 Leonid S. Brown 0 Kwang-Hwan Jung 0 Wolfgang R. Hess, University of Freiburg, Germany 0 1 Department of Life Science and Institute of Biological Interfaces, Sogang University , Seoul , Korea , 2 Department of Physics, University of Guelph , Ontario , Canada A homologue of type I rhodopsin was found in the unicellular Gloeobacter violaceus PCC7421, which is believed to be primitive because of the lack of thylakoids and peculiar morphology of phycobilisomes. The Gloeobacter rhodopsin (GR) gene encodes a polypeptide of 298 amino acids. This gene is localized alone in the genome unlike cyanobacterium Anabaena opsin, which is clustered together with 14 kDa transducer gene. Amino acid sequence comparison of GR with other type I rhodopsin shows several conserved residues important for retinal binding and H+ pumping. In this study, the gene was expressed in Escherichia coli and bound all-trans retinal to form a pigment (lmax = 544 nm at pH 7). The pKa of proton acceptor (Asp121) for the Schiff base, is approximately 5.9, so GR can translocate H+ under physiological conditions (pH 7.4). In order to prove the functional activity in the cell, pumping activity was measured in the sphaeroplast membranes of E. coli and one of Gloeobacter whole cell. The efficient proton pumping and rapid photocycle of GR strongly suggests that Gloeobacter rhodopsin functions as a proton pumping in its natural environment, probably compensating the shortage of energy generated by chlorophyll-based photosynthesis without thylakoids. - Archaeal rhodopsins are light-responsive seven transmembrane proteins that bind all-trans-retinal as chromophore. They are type I rhodopsins, in contrast to 11-cis-retinal-based type II rhodopsins, such as the animal visual pigments [1,2]. Bacteriorhodopsin (BR) and halorhodopsin (HR) are light-driven proton and chloride pumps. They enable Halobacterium salinarum to grow phototrophically by establishing electrochemical ion gradients across the cell membrane to provide cellular energy and pH balance in the extreme ionic environmental conditions. Sensory rhodopsins (SRs) I and II, however, act as attractants for orange (SRI) and repellent photoreceptors for UV-blue (SRI) and green light (SRII), respectively. The covalent attachment of the chromophore (via a protonated Schiff base to a lysine residue in the helix G) and interactions with selected amino acids within the binding pocket allow a tuning of the absorption wavelength over a wide spectral range. Light-induced trans-cis isomerization of the chromophore triggers a photocycle that includes Schiff base deprotonation (in BR and SRs) and protein conformational changes, the former evident from a strong photochromic shift of this intermediate species [1]. In recent years, evidence has emerged that the occurrence of type I rhodopsins has spread beyond the borders of the Archaea domain [35] Type I archaeal rhodopsins are found in phylogenetically diverse microorganisms, including halo-archaea, proteobacteria, cyanobacteria, fungi and algae [1,610]. Among those, cyanobacteria are known to use light as a source of energy. For example, Anabaena rhodopsin is photosensory receptor that is spectrally tuned to relay information to the cell regarding the intensity and color of light in the environment, possibly to adjust a composition of the light-harvesting machinery [9,11,12]. While the exact origin of oxygenic photosynthetic organisms is not known, we are aware of continuity/similarity of reaction center complexes in photosynthetic bacteria [13]. Also, we do not know the origin of type I rhodopsins, which represent a very simple energy production system and various sensory receptors for the light-dependent signal relay. Since the discovery of a type I rhodopsin in the early seventies, only recently the co-existence of a proton transporting rhodopsin and photosynthetic pigments has been reviewed in [3,5]. One can try to understand the emergence of two kinds of energy production system in one organism, either through lateral gene transfer or the last universal common ancestor. The appearance of oxygenic photosynthetic organisms determined the direction of global biological evolution through an increase in the oxygen concentration of the Earth atmosphere and developing a more efficient energy production through photosynthetic machinery. There will be many challenges to understanding the discontinuity of the presence of rhodopsins using the photosynthetic microbes in which both systems coexist. One possible approach is to search an organism that has retained primitive properties, such as photosynthetic protein complexes, their localization, reaction processes, and so on. It was the adherence to this approach that led us to focus on the cyanobacterium Gloeobacter violaceus. G. violaceus is a rod-shaped unicellular cyanobacterium isolated from calcareous rock in Switzerland [14]. According to phylogenetic analysis based on the 16S rRNA sequence, it diverges very early from the common cyanobacterial phylogenetic branch, suggesting that it may retain some of the primitive properties of early cyanobacteria [1517]. However, it has several differences from typical cyanobacteria. They lack thylakoid membrane development [14,18], having its photosynthetic and respiratory systems located in the cell membranes instead of thylakoid membranes, where the machinery is found in other cyanobacteria [14]. This means that components facing the lumen in the cytoplasm in other cyanobacteria are exposed to periplasm in Gloeobacter, thus the photosynthetic electron transfer system should co-exist in the cytoplasmic membrane with a respiratory system by sharing some components [19]. The morphology of phycobilisomes is distinct from other cyanobacteria as well. Phycobiliproteins form rod-shaped elements and these elements form bundle-shaped aggregates, which attach to the cell membranes from the cytoplasmic side [16], and oxygen evolution is thus mediated in the periplasmic space. A gene encoding new rhodopsin with high sequence homology to the previously described type I rhodopsins of Archaea and Eucarya was found in this cyanobacterium through genome sequencing [19]. In this study, we identified, heterologously expressed, and functionally characterized the gene product that acts as a light-driven proton pump in G. violaceus PCC7421. The pigment has been named GR (Gloeorhodopsin). While this manuscript was in preparation, two FTIR studies [20,21] were published, reporting on other spectroscopic features consistent with the proton-pumping function of GR, in full agreement with the present study. In addition, here we report th (...truncated)