Halocyanin, a blue copper protein from Natronobacterium pharaonis was studied by Raman spectroscopic techniques. Near-infrared Fourier transform Raman spectra, which for the first time have been employed to study copper proteins in both oxidation states, display the Raman bands of the protein and, in the oxidized state, also the preresonance-enhanced bands of the copper center. The frequency of the amide I band at 1676 cm-1 indicates a predominant beta-sheet protein structure, which is typical for small blue copper proteins. The Raman spectra provide no evidence for major redox-linked changes of the secondary structure. Resonance Raman spectra of the oxidized protein obtained upon excitation close to the 600-nm absorption band were measured in the pH range between 7.7 and 4.5. The vibrational band pattern in the Cu-cysteine stretching region is closely related to that of azurin, indicating far-reaching similarities of the coordination geometry of the copper center in both proteins. Significantly lower frequencies, however, are noted for the Cu-histidine stretches, which appear as a closely spaced doublet at ca. 260 cm-1. Lowering the pH to 4.5 leads to an increase of this band splitting with one component shifting down to 247 cm-1. This downshift is attributed to the rupture of a hydrogen bond between one of the histidine ligands and a nearby carboxyl group, which becomes protonated at such a low pH. On the other hand, no major changes in the Cu-cysteine stretching region are noted at pH 4.5, implying that the coordination geometry remains largely unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)