Exploring the Active Site Structure of a Photoreceptor Protein by Raman Optical Activity

We have developed a near-infrared excited Raman optical activity (ROA) spectrometer and report the first measurement of near-infrared ROA spectra of a light-driven proton pump, bacteriorhodopsin. Our results demonstrate that a near-infrared excitation enables us to measure the ROA spectra of the chromophore within a protein environment. Furthermore, the ROA spectra of the <i>all</i>-<i>trans</i>, 15-<i>anti</i> and 13-<i>cis</i>, 15-<i>syn</i> isomers differ significantly, indicating a high structural sensitivity of the ROA spectra. We therefore expect that future applications of the near-infrared ROA will allow the experimental elucidation of the active site structures in other proteins as well as reaction intermediates

Photoinduced Proton Release in Proteorhodopsin at Low pH: The Possibility of a Decrease in the p<i>K</i>_a of Asp227

Proteorhodopsin (PR) is one of the microbial rhodopsins that are found in marine eubacteria and likely functions as an outward light-driven proton pump. Previously, we [Tamogami, J., et al. (2009) <i>Photochem. Photobiol.</i> <i>85</i>, 578–589] reported the occurrence of a photoinduced proton transfer in PR between pH 5 and 10 using a transparent ITO (indium–tin oxide) or SnO₂ electrode that works as a time-resolving pH electrode. In the study presented here, the proton transfer at low pH (<4) was investigated. Under these conditions, Asp97, the primary counterion to the protonated Schiff base, is protonated. We observed a first proton release that was followed by an uptake; during this process, however, the M intermediate did not form. Through the use of experiments with several PR mutants, we found that Asp227 played an essential role in proton release. This residue corresponds to the Asp212 residue of bacteriorhodopsin, the so-called secondary Schiff base counterion. We estimated the p<i>K</i>_a of this residue in both the dark and the proton-releasing photoproduct to be ∼3.0 and ∼2.3, respectively. The p<i>K</i>_a value of Asp227 in the dark was also estimated spectroscopically and was approximately equal to that determined with the ITO experiments, which may imply the possibility of the release of a proton from Asp227. In the absence of Cl^–, we observed the proton release in D227N and found that Asp97, the primary counterion, played a key role. It is inferred that the negative charge is required to stabilize the photoproducts through the deprotonation of Asp227 (first choice), the binding of Cl^– (second choice), or the deprotonation of Asp97. The photoinduced proton release (possibly by the decrease in the p<i>K</i>_a of the secondary counterion) in acidic media was also observed in other microbial rhodopsins with the exception of the <i>Anabaena</i> sensory rhodopsin, which lacks the dissociable residue at the position of Asp212 of BR or Asp227 of PR and halorhodopsin. The implication of this p<i>K</i>_a decrease is discussed

Influence of Halide Binding on the Hydrogen Bonding Network in the Active Site of <i>Salinibacter</i> Sensory Rhodopsin I

In nature, organisms are subjected to a variety of environmental stimuli to which they respond and adapt. They can show avoidance or attractive behaviors away from or toward such stimuli in order to survive in the various environments in which they live. One such stimuli is light, to which, for example, the receptor sensory rhodopsin I (SRI) has been found to respond by regulating both negative and positive phototaxis in, e.g., the archaeon <i>Halobacterium salinarum</i>. Interestingly, to date, all organisms having SRI-like proteins live in highly halophilic environments, suggesting that salt significantly influences the properties of SRIs. Taking advantage of the discovery of the highly stable SRI homologue from <i>Salinibacter ruber</i> (<i>Sr</i>SRI), which maintains its color even in the absence of salt, the importance of the chloride ion for the color tuning and for the slow M-decay, which is thought to be essential for the phototaxis function of SRIs, has been reported previously [Suzuki, D., et al. (2009) <i>J. Mol. Biol.</i> <i>392</i>, 48–62]. Here the effects of the anion binding on the structure and structural changes of SRI during its photocycle are investigated by means of Fourier transform infrared (FTIR) spectroscopy and electrochemical experiments. Our results reveal that, among other things, the structural change and proton movement of a characteristic amino acid residue, Asp102 in <i>Sr</i>SRI, is suppressed by the binding of an anion in its vicinity, both in the K- and M-intermediate. The presence of this anion also effects the extent of chromophore distrotion, and tentative results indicate an influence on the number and/or properties of internal water molecules. In addition, a photoinduced proton transfer could only be observed in the absence of the bound anion. Possible proton movement pathways, including the residues Asp102 and the putative Cl binding site His131, are discussed. In conclusion, the results show that the anion binding to SRI is not only important for the color tuning, and for controlling the photocycle kinetics, but also induces some structural changes which facilitate the observed properties

Formation of M‑Like Intermediates in Proteorhodopsin in Alkali Solutions (pH ≥ ∼8.5) Where the Proton Release Occurs First in Contrast to the Sequence at Lower pH

Proteorhodopsin (PR) is an outward light-driven proton pump observed in marine eubacteria. Despite many structural and functional similarities to bacteriorhodopsin (BR) in archaea, which also acts as an outward proton pump, the mechanism of the photoinduced proton release and uptake is different between two H⁺-pumps. In this study, we investigated the pH dependence of the photocycle and proton transfer in PR reconstituted with the phospholipid membrane under alkaline conditions. Under these conditions, as the medium pH increased, a blue-shifted photoproduct (defined as M_a), which is different from M, with a p<i>K</i>_a of ca. 9.2 was produced. The sequence of the photoinduced proton uptake and release during the photocycle was inverted with the increase in pH. A p<i>K</i>_a value of ca. 9.5 was estimated for this inversion and was in good agreement with the p<i>K</i>_a value of the formation of M_a (∼9.2). In addition, we measured the photoelectric current generated by PRs attached to a thin polymer film at varying pH. Interestingly, increases in the medium pH evoked bidirectional photocurrents, which may imply a possible reversal of the direction of the proton movement at alkaline pH. On the basis of these findings, a putative photocycle and proton transfer scheme in PR under alkaline pH conditions was proposed