Chromophore-anion interactions in halorhodopsin from Natronobacterium pharaonis probed by time-resolved resonance Raman spectroscopy

  • Gerscher S
  • Mylrajan M
  • Hildebrandt P
 et al. 
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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.

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  • Sandra Gerscher

  • Muthusamy Mylrajan

  • Peter Hildebrandt

  • Marie Hélène Baron

  • Renate Müller

  • Martin Engelhard

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