The role of double bond isomerization in light induced activation of Halorhodopsin

Sansa Dutta, Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel

Halorhodopsin (HR), a member of archaeal rhodopsins contains retinal chromophore bound via a protonated Schiff base to the ɛ-amino group of a lysine residue. The photoisomerization reaction around the C13=C14 double bond triggers a series of conformational changes by which a chloride ion is transported. The HR homologs from Halobacterium salinarum (shR) and Natronomonas pharaonis (phR) investigated so far differs in many aspects. As for example, the two retinal isomers coexist in the light-adapted state of shR.  On the contrary, the light-adapted state of phR contains the retinal isomer with an all-trans configuration as the major component. In the present work we have checked if light absorption by the over-expressed mutant of halorhodopsin from Natronomonas pharaonis can induce protein conformational changes that are not associated with double bond isomerization. In this regard we performed kinetic study of hydroxyl amine reactions in dark and under illumination with the native protein well as with different noniosmerizable artificial pigments.

We have observed faster reaction kinetics in case of the light adapted phR compared to the dark adapted state for the native phR. The catalytic effect of light is also observed in case of the artificial pigments which are derived from tailored retinal analogs bearing a rigid cyclic structures at the C13=C14 position. HPLC analysis of the artificial pigments confirmed that light did not cause any double bond isomerization. It can be concluded that light absorption may trigger conformational alterations which are not associated with double bond isomerization but rather via light induced dipole developed in the retinal chromophore. Interestingly we have also observed an unusual decreasing trend in rate constants with rise in temperature both for native and artificial pigments which was not reported for other retinal proteins.