Effects of Ca2+ binding on the states of aspartic acid residues near the retinal pocket and the secondary structure of bacteriorhodopsin (bR) have been studied by infrared spectroscopy in combination with the Fourier self-deconvolution technique. The band at 1754 cm(-1) of deionized bR is assigned to the C=O stretch of the COOH group of protonated Asp-85, and the band at 1405 cm(-1) of metal-ion-bound bR to the symmetric stretch of the COO- group of deprotonated Asp-85, on the basis of the infrared spectra of a bR mutant in which Asp-85 is replaced with Asn. The binding of Ca2+ to the second high affinity site causes deprotonation of the Asp-85 COOH group and the blue to purple color change. The Ca2+ binding causes changes in the secondary structure of bR.