DISTINCT HEME ACTIVE-SITE STRUCTURE IN LACTOPEROXIDASE REVEALED BY RESONANCE RAMAN-SPECTROSCOPY

Low-frequency resonance Raman spectra of the cyanide and carbon monoxi de adducts of lactoperoxidase are obtained with Soret excitation. The nu(Fe-CN) and delta(Fe-C-N) modes are detected at 360 and 453 cm-1, re spectively. Upon the isotopic substitution of (CN)-C-13-N-14, (CN)-C-1 2-N-15, and (CN)-C-13-N-15, the band at 453 cm-1 in the natural abunda nce adduct shifts to 448, 452, and 445 cm-1, while the 360-cm-1 peak s hifts to 358, 357, and 356 cm-1, respectively. The 360-cm-1 band is sh ifted to 355 cm-1 when the pH is changed from 7.0 to 10.5. On the basi s of a previous normal-mode analysis of the cyanoferric adduct of myel operoxidase, a bent Fe-C-N linkage is suggested for the cyanide adduct of lactoperoxidase. The nu(Fe-CN) (374 cm-1) and delta(Fe-C-N) (480 c m-1) modes are observed for the cyanide adduct of reduced lactoperoxid ase. For the carbon monoxide adduct, the nu(Fe-CO) (533 cm-1) and delt a(Fe-C-O) (578 cm-1) modes at pH 7.0 are observed to shift to 498 and 570 cm-1 as the pH is raised from 7.0 to 10.0. The strong intensity of delta(Fe-C-O) at both acid and alkaline pHs, along with a suggested b ent structure of the Fe-C-N moiety, implies a narrow heme pocket for l actoperoxidase.