Resonance Raman (RR) spectra of oxyhemocyanins (oxyHcs) from an arthropod (Limulus polyphemus) and two mollusks (Busycon canaliculatum and Octopus dofleini) exhibit their peroxide-like O-O stretching mode at the unusual low frequency of similar to 745 cm(-1). This low energy appears to be indicative of a mu-eta(2):eta(2) coordination geometry in which a side-on peroxide bridges two Cu(II) ions, as has been observed in the crystal structure of Limulus oxyHc (Magnus, K. A.; et al. Proteins, in press) and a model compound (Kitajima, N.; et al. J. Am. Chem. Sec. 1992, 114, 1277-1291). We have now identified a Cu-peroxide stretch, nu(as)(Cu2O2), at 542 cm(-1) (519 cm(-1) in O-18(2)) and its first overtone, 2 nu(as)(CU2O2), at 1085 cm(-1) (1039 cm(-1) in O-18(2)) in Octopus oxyHc. When the protein is oxygenated with (OO)-O-16-O-18, only a single nu(as)((Cu2OO)-O-16-O-18) mode appears at 529 cm(-1). These results provide definitive evidence that the peroxo group is symmetrically bound to the two Cu atoms, as expected for a mu-eta(2):eta(2) geometry. Similar nu(as)(Cu2O2) and 2 nu(as)(Cu2O2) vibrations are detected in the RR spectra of Busycon and Limulus Hcs. These findings make it likely that the proteins from both phyla have the same mu-eta(2):eta(2) copper peroxide structure. In addition, oxyHcs from both phyla have a set of eight distinct vibrational modes between 170 and 370 cm(-1). Through the use of normal coordinate analysis, these peaks can be assigned to nu(s)(Cu2O2) and nu(as)(Cu2O2) modes coupled with Cu-N(His) stretching vibrations. The extensive coupling with imidazole modes is supported by Cu-65- and D-substitution data for Busycon oxyHc. The similarity of the vibrational patterns between the two phyla suggests that molluskan Hcs also have six terminal His ligands at the dinuclear Cu site. The greatest RR intensity is associated with the Cu-N(axial His) stretching mode at similar to 280 cm(-1) because the axial His ligands are the most affected by changes in copper oxidation state. This mode may be sensitive to R- and T-state conformations and, thus, serve as an indicator of oxygen affinity.