FTIR spectra of native Scapharca homodimeric hemoglobin (HbI) and of the Phe97-->Ile mutant have been measured in the region 2400-2700 cm(-1) where the absorption of the sulfhydryl groups can be observed. In native HbI, the two Cys92 residues give rise to a relatively intense band centered at 2559 cm(-1) that is shifted to 2568 cm(-1) and strongly quenched upon ligand binding. In the Phe97-->Leu mutant, such ligand-linked changes are not observed and the strong peak at around 2560 cm(-1) persists in the liganded derivatives. In native HbI, the observed changes have been attributed to the decrease in polarity of the interface due to the ligand-induced extrusion of the Phe97 phenyl ring from the heme pocket to the interface and the subsequent release of several water molecules that are clustered in the vicinity of Cys92. In contrast, in the Phe97-->Leu mutant, the Leu residue does not leave the heme pocket upon ligand binding and the interface is unaltered. The Cys92/S-H infrared band, therefore, represents a sensitive probe of the structural rearrangements that take place in the intersubunit interface upon ligand binding to HbI. The heterotetrameric Scapharca hemoglobin HbII contains, in addition to the Cys92 residues in the interfaces, two extra sulfhydryl groups per tetramer (Cys9 in the B chain) that are exposed to solvent in the A helix. The frequency of the Cys9/S-H stretching vibration occurs at 2582 cm(-1) in the unliganded and at 2586 cm(-1) in the liganded derivative, pointing to the involvement of the A helix in the ligand-linked polymerization characteristic of HbII.