RESONANCE RAMAN STRUCTURAL CHARACTERIZATION AND THE MECHANISM OF FORMATION OF LACTOPEROXIDASE COMPOUND-III

Lactoperoxidase compound III (LPO-III) is characterized by resonance Raman spectroscopy with both the Soret and Q-band excitations. The identical spectral patterns observed for LPO-III prepared by the addition of 100-fold hydrogen peroxide to ferric enzyme and by the oxygenation of the ferrous enzyme confirm the formulation of LPO-III as a low-spin dioxygen adduct. The Fe-O2 stretching vibration (identified at 531 cm-1 for the O-16(2) adduct and 513 cm-1 for the O-18(2) derivative) is the lowest yet reported for an oxygenated heme protein. The anomalous isotopic shift of the nu(Fe-O2) mode is attributed to vibrational coupling of the Fe-O-18(2) stretching mode with a heme mode located at 508 cm-1. In addition, the Fe-O-O bending vibration is also observed at 491 cm-1. The study of the nu(Fe-O2) mode of LPO-III, prepared by different combinations of isotopically labeled peroxides, establishes unambiguously that the ferryl oxygen atom of LPO-II is displaced and the dioxygen fragment of LPO-III is derived from the hydrogen peroxide.