COMMON OXYGEN-BINDING SITE IN HEMOCYANINS FROM ARTHROPODS AND MOLLUSKS - EVIDENCE FROM RAMAN-SPECTROSCOPY AND NORMAL-COORDINATE ANALYSIS

Resonance Raman (RR) spectra of oxyhemocyanins (oxyHcs) from an arthro pod (Limulus polyphemus) and two mollusks (Busycon canaliculatum and O ctopus dofleini) exhibit their peroxide-like O-O stretching mode at th e unusual low frequency of similar to 745 cm(-1). This low energy appe ars to be indicative of a mu-eta(2):eta(2) coordination geometry in wh ich a side-on peroxide bridges two Cu(II) ions, as has been observed i n the crystal structure of Limulus oxyHc (Magnus, K. A.; et al. Protei ns, 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 stret ch, 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, on ly a single nu(as)((Cu2OO)-O-16-O-18) mode appears at 529 cm(-1). Thes e results provide definitive evidence that the peroxo group is symmetr ically bound to the two Cu atoms, as expected for a mu-eta(2):eta(2) g eometry. Similar nu(as)(Cu2O2) and 2 nu(as)(Cu2O2) vibrations are dete cted 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):e ta(2) copper peroxide structure. In addition, oxyHcs from both phyla h ave 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 i s supported by Cu-65- and D-substitution data for Busycon oxyHc. The s imilarity of the vibrational patterns between the two phyla suggests t hat molluskan Hcs also have six terminal His ligands at the dinuclear Cu site. The greatest RR intensity is associated with the Cu-N(axial H is) stretching mode at similar to 280 cm(-1) because the axial His lig ands 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.