Halorhodopsin (HR) is a light-driven chloride pump in haloarchaea. Our previous low-temperature Fourier-transform infrared (FTIR) study of pharaonis HR reported that the chloride binding site is destabilized in the L-1 intermediate because of the changes in its polar environment. The present FTIR spectroscopy of the wild-type and mutant pharaonis HR revealed that Glu234 becomes deprotonated in the L-2 intermediate. This suggests that appearance of a negative charge at the extracellular surface prevents a chloride ion from translocation toward the extracellular side. (c) 2006 Elsevier B.V. All rights reserved.