Different structural changes occur in blue- and green-proteorhodopsins during the primary photoreaction.

Proteorhodopsins (PRs) are members of the microbial rhodopsin family. Since their initial discovery in Monterey Bay (1), over 4000 different variants have been discovered using environmental genome sequencing techniques (2–4). It is estimated that PR containing bacteria account for 13% of the microorganism species in the ocean photic zone (5). The widespread distribution of PR in worldwide oceanic waters indicates that PR phototrophy is a significant oceanic microbial process (5, 6). Similar to BR and other microbial rhodopsins, PRs consist of 7 transmembrane alpha-helices containing a retinylidene chromophore covalently bound to a lysine (Lys231) via a protonated Schiff base (1). Light absorption by PR results in an all-trans to 13-cis isomerization of the retinal which then drives a series of reaction steps that result in a proton being pumped from the cytoplasm to the extracellular medium (7, 8). There are two major subgroups of proteorhodopsins characterized by their visible absorption maxima. The green-absorbing proteorhodopsin (GPR) subgroup absorbs maximally at ~520 nm and function as a light driven proton pump similar to bacteriorhodopsin (BR) (1, 7–9). A second subgroup called blue-absorbing proteorhodopsin (BPR), absorbs near 490 nm (6). The different variants of PRs are thought to have evolved through Darwinian selection (10). One difference between GPR and BPR which was found to be a key determinant of their different properties is the identity of residue 105 (The numbering scheme for BPR is offset by one from GPR due to an extra glycine at residue 2 in BPR. Throughout, when referring to the amino acid positions in proteorhodopsins, the GPR numbering system is used). In GPR residue 105 is a leucine while in BPR it is a glutamine (11–14). In a related paper, we show using resonance Raman spectroscopy that this residue is involved in tuning the visible absorption via interaction with the Schiff base and the C13 methyl of the retinal chromophore (15). BPR also pumps protons; however it has a 10-fold slower photocycle than GPR. It has been suggested that the slower photocycle may indicate that BPR functions primarily as a photoregulator rather than an energy harvesting pigment (14, 16, 17). Fourier transform infrared (FTIR) difference spectroscopy has been successfully used to elucidate many of the details of the proton transport mechanism in BR (18–21) and GPR (7, 8, 22–26) as well to characterize the mechanisms of a variety of other microbial rhodopsins (27, 28). This study examines the initial events in the photocycle of BPR using static low-temperature FITR difference spectroscopy. The 80 K light induced difference spectra reflect the formation of a red shifted K-like photoproduct similar to the K-intermediates in GPR and light-adapted BR. Furthermore, the C=C and C-C retinal chromophore ethylenic and fingerprint stretching modes show that the primary photoreaction involves an all-trans → 13-cis isomerization of the retinal chromophore, although the difference spectrum also may exhibit small contributions from a 13-cis photocycle. Several prominent bands near 1700 cm−1 are assigned to structural changes of Asn230 and Gln105 side chains on the basis of site-directed mutations indicating that these residues are perturbed in the earliest events of the BPR photocycle. In addition, a pair of bands at 1551 (−) and 1537 (+) cm−1 are assigned to an amide II downshift on the basis of total-15N isotope labeling. Evidence is presented on the basis of assignment of bands to water molecule vibrations with H218O that there are two different sites for water molecules near the active site in BPR and GPR, respectively. In the Q105L mutant of BPR, which has a GPR-like visible absorption spectrum, both waters are present.