Spectroscopic and Photochemical Characterization of a Deep Ocean Proteorhodopsin

A second group of proteorhodopsin-encoding genes (blue-absorbing proteorhodopsin, BPR) differing by 20 – 30% in predicted primary structure from the first-discovered green-absorbing (GPR) group has been detected in picoplankton from Hawaiian deep sea water. Here we compare BPR and GPR absorption spectra, photochemical reactions, and proton transport activity. The photochemical reaction cycle of Hawaiian deep ocean BPR in cells is 10-fold slower than that of GPR with very low accumulation of a deprotonated Schiff base intermediate in cells and exhibits mechanistic differences, some of which are due to its glutamine residue rather than leucine at position 105. In contrast to GPR and other characterized microbial rhodopsins, spectral titrations of BPR indicate that a second titratable group, in addition to the retinylidene Schiff base counterion Asp-97, modulates the absorption spectrum near neutral pH. Mutant analysis confirms that Asp-97 and Glu-108 are proton acceptor and proton donor, respectively, in retinylidene Schiff base proton transfer reactions during the BPR photocycle as previously shown for GPR, but BPR contains an alternative acceptor evident in its D97N mutant, possibly the same as the second titratable group modulating the absorption spectrum. BPR, similar to GPR, carries out outward light-driven proton transport in Escherichia coli vesicles but with a reduced translocation rate attributable to its slower photocycle. In energized E. coli cells at physiological pH, the net effect of BPR photocycling is to generate proton currents dominated by a triggered proton influx, rather than efflux as observed with GPR-containing cells. Reversal of the proton current with the K-ionophore valinomycin supports that the influx is because of voltagegated channels in the E. coli cell membrane. These observations demonstrate diversity in photochemistry and mechanism among proteorhodopsins. Calculations of photon fluence rates at different ocean depths show that the difference in photocycle rates between GPR and BPR as well as their different absorption maxima may be explained as an adaptation to the different light intensities available in their respective marine environments. Finally, the results raise the possibility of regulatory (i.e. sensory) rather than energy harvesting functions of some members of the proteorhodopsin family. Microbial rhodopsins, seven-helix integral membrane proteins that use retinal as a chromophore, comprise a large family of photoactive proteins present in archaeal, bacterial, and eukaryotic microorganisms (1–3). Among the most abundant and widely distributed are the proteorhodopsins, the first of which was identified in the ubiquitous uncultured SAR86 group of marine proteobacteria in plankton from Monterey Bay coastal waters (4). Nearly identical genes detected in picoplankton from the Southern Ocean (1), Mediterranean, and Red Sea (5) define a phylogenetic group of Monterey Bay-type pigment genes (clade I). The Monterey Bay pigment, a green light-absorbing proteorhodopsin (max 525 nm, hence referred to as “GPR” 1 (5)), has been demonstrated to function as a light-driven proton pump (4), and its transport, spectroscopic, and photochemical reactions have been characterized by a number of laboratories in Escherichia coli expressed forms (1, 4 –10). In particular, its rapid photocycle, characteristic of transport rhodopsins, in E. coli membranes (4) and in its native membranes from Monterey Bay plankton (1) provides strong evidence that GPR functions as a light-driven proton pump photoenergizing SAR86 cells in their natural environment.