Chromophore−Anion Interactions in Halorhodopsin from Natronobacterium pharaonis Probed by Time-Resolved Resonance Raman Spectroscopy

Abstract

Halorhodopsin of Natronobacterium pharaonis which acts as a light-driven chloride pump is studied by time-resolved resonance Raman spectroscopy. In single-beam experiments, resonance Raman spectra were obtained of the parent state HR578 and the first thermal intermediate HR520. The parent state is structural heterogeneous including ca. 80% all-trans and 20% 13-cis isomers. The resonance Raman spectra indicate that the all-trans conformer exhibits essentially the same chromophoric structure as in the parent states of bacteriorhodopsin or halorhodopsin from Halobacterium salinarium. Special emphasis of the resonance Raman spectroscopic analysis was laid on the C=C and C=N stretching region in order to probe the interactions between the protonated Schiff base and various bound anions (chloride, bromide, iodide). These investigations were paralleled by spectroscopic studies of retinal Schiff base model complexes in different solvents in an attempt to determine the various parameters which control the C=C and C=N stretching frequencies. From these data, it was concluded that in the parent state the anion is not involved in hydrogen bonding interactions with the Schiff base proton but is presumably bound to a nearby (positively charged) amino acid residue. On the other hand, the anion still exerts an appreciable effect on the chromophore structure which is, for instance, reflected by the variation of the isomer composition in the presence of different anions and in the anion-depleted form. In contrast to the parent state, the intermediate HR520 reveals frequency shifts of the C=N stretching in the presence of different anions. These findings indicate a closer proximity of the bound anion to the Schiff base proton which is sufficient for hydrogen bonding interactions. These changes of the anion-chromophore interaction upon transition from HR578 to HR520 may be related to the coupling of the chromophore movement with the anion translocation.

Resonance Raman Study of an Anion Channelrhodopsin: Effects of Mutations near the Retinylidene Schiff Base

“…25 It is also interesting to note that HRs from Halobacterium salinarum ( Hs HR) and from Natronobacterium pharaonis ( Np HR) both exhibit C=N frequencies near 1632 cm −1 with much smaller H/D-induced downshifts of approximately 10–12 cm −1 . 41,42 …”

“…Second, the magnitude and direction of υ C=C and visible λ max shifts for D234N indicate that Asp234 does not strongly interact with the PSB in an ionized form. In fact, replacement of an ionized Asp234 with a neutral Asn234 is expected on the basis of a simple point charge model 42,71 to cause a decrease in υ C=C and an increase in visible λ max , while the opposite response is observed for D234N (Figure 6 and Figure S5). …”

A Photochromic Histidine Kinase Rhodopsin (HKR1) That Is Bimodally Switched by Ultraviolet and Blue Light

“…The significant frequency difference between these two bands, which shift down to 1621 cm Ϫ1 upon H/D exchange (Fig. 2, inset), can at least partly be related to different hydrogen bonding or electrostatic interactions with the counter ion (21). For the Rh-UV state, however, there is no indication for structural heterogeneity of the chromophore (Fig.…”

Microbial and Animal Rhodopsins: Structures, Functions, and Molecular Mechanisms

“…The hydrogen bond of the RSB is strengthened in the L intermediate, but the hydrogen-bonding acceptor is not the chloride ion, but most likely a water molecule (Figure 15). 306,312 Rearrangement of the water-containing hydrogen-bonding network most likely opens the valve to the cytoplasmic region, and the chloride ion is released to the cytoplasmic side during the transition to the O intermediate. 313,314 …”