The critical role of the proximal calcium ion in the structural propertiesof horseradish peroxidase

The extent to which the structural Ca2+ ions of horseradish peroxidase (HRP C) are a determinant in defining the heme pocket architecture is investigat ed by electronic absorption and resonance Raman spectroscopy upon removal o f one Ca2+ ion. The Fe(III) heme states are modified upon Ca2+ depletion, w ith an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca2+-depleted HRPC forms complexes with benzohydroxamic acid and CO which display spectra very similar to those of native HRPC, indicating t hat any changes to the distal cavity structural properties upon Ca2+ deplet ion are easily reversed. Contrary to the native protein, the Ca2+-depleted ferrous form displays a low-spin bis-histidyl heme state and a small propor tion of high-spin heme. Furthermore, the v(Fe-Im) stretching mode downshift s 27 cm(-1) upon Ca2+ depletion revealing a significant structural perturba tion of the proximal cavity near the histidine ligand. The specific activit y of the Ca2+-depleted enzyme is 50% that of the native form. The effects o n enzyme activity and spectral features observed upon Ca2+ depletion are re versible upon reconstitution. Evaluation of the present and previous data f irmly favors the proximal Ca2+ ion as that which is lost upon Ca2+ depletio n and which likely plays the more critical role in regulating the heme pock et structural and catalytic properties.