Internal water molecules of the proton-pumping halorhodopsin in the presence of azide

In the FTIR study of rhodopsins, we have so far found that strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm-1) are only present in the proteins exhibiting proton-pumping activity. Halorhodopsin (HR) is a light-driven chloride pump in haloarchaea, which does not possess such water molecules. On the other hand, it is known that addition of azide converts HR into a proton pump. Although the mechanism has not been understood, we observed strongly hydrogen-bonded water molecules in the azide-bound HR of Natronobacterium pharaonis (pHR). This finding is consistent with the previous results, implying that the presence of strongly hydrogen-bonded water molecules is requested for the proton-pumping function of rhodopsins.