Infrared difference spectroscopy analysis of the purified melibiose permease of Escherichia coli reconstituted into liposomes was carried out as a function of the presence of the two symporter substrates (Na(+), melibiose) in either H(2)O or in D(2)O media. Essentially, the data first show that addition of Na(+) induces appearance of peaks assigned to changes in the environment and/or orientation of alpha-helical domains of purified melibiose permease. Likewise, melibiose addition in the presence of Na(+) produces peaks corresponding to additional changes of alpha-helix environment or tilt. In addition to these changes, a pair of peaks (1599 (+) cm(-1)/1576 (-) cm(-1)) appearing in the Na(+)-induced difference spectrum is assigned to the antisymmetric stretching of COO(-) groups, since they show practically no shift upon H/D exchange. It is proposed that these acidic groups participate in Na(+) co-ordination. A corresponding pair of peaks, again fairly insensitive to H/D substitution (1591 (-) cm(-1)/1567 (+) cm(-1)), appear in the melibiose-induced difference spectra, and may again be assigned to COO(-) groups. The latter carboxyl groups may correspond to part or all of the acidic residues interacting with Lys or Arg in the resting state that become free upon melibiose binding.