CHROMOPHORE-ANION INTERACTIONS IN HALORHODOPSIN FROM NATRONOBACTERIUM-PHARAONIS PROBED BY TIME-RESOLVED RESONANCE RAMAN-SPECTROSCOPY

Halorhodopsin of Natranobacterium pharaonis which acts as a light-driv en chloride pump is studied by time-resolved resonance Raman spectrosc opy. In single-beam experiments, resonance Raman spectra were obtained of the parent state HR578 and the first thermal intermediate HR520. T he parent state is structural heterogeneous including ca. 80% all-tran s and 20% 13-cis isomers. The resonance Raman spectra indicate that th e all-trans conformer exhibits essentially the same chromophoric struc ture as in the parent states of bacteriorhodopsin or halorhodopsin fro m Halobacterium salinarium. Special emphasis of the resonance Raman sp ectroscopic 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 investigation s were paralleled by spectroscopic studies of retinal Schiff base mode l complexes in different solvents in an attempt to determine the vario us parameters which control the C=C and C=N stretching frequencies, Fr om these data, it was concluded that in the parent state the anion is not involved in hydrogen bonding interactions with the Schiff base pro ton but is presumably bound to a nearby (positively charged) amino aci d residue, On the other hand, the anion still exerts an appreciable ef fect 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 stretchin g in the presence of different anions. These findings indicate a close r proximity of the bound anion to the Schiff base proton which is suff icient for hydrogen bonding interactions. These changes of the anion-c hromophore interaction upon transition from HR578 to HR520 may be rela ted to the coupling of the chromophore movement with the anion translo cation.