High-resolution proton nuclear magnetic resonance spectroscopy of chloride peroxidase: identification of new forms of the enzyme.

Chloride peroxidase from the mold Caldariomyces fumago in the native high-spin iron(III) and low-spin cyanoiron (III) states has been subjected to high-field proton nuclear magnetic resonance spectroscopic measurements. Signals shifted well outside the diamagnetic envelope by the paramagnetic iron(III) center are surprisingly insensitive to pH changes over the range from pH 3 to pH 7. The previously identified major form of chloride peroxidase (form A) and the minor form (B) show very similar chemical shift patterns. Of greatest significance, however, is the discovery that each of the separable forms of the enzyme exhibits splitting of porphyrin ring methyl resonances. The appearance of two sets of signals in both native and cyanide-complexed enzyme is best explained by the existence of two additional forms of the A and B isoenzymes. Structural differences for the newly identified forms of chloride peroxidase must be located in the vicinity of the heme prosthetic group.