A mass spectrometric investigation of native and oxidatively inactivated chloroperoxidase.

The enzyme chloroperoxidase (CPO) found in Caldariomyces fumago is able to catalyze several stereoselective oxidation reactions by using a clean oxidant, usually hydrogen peroxide (H(2)O(2)), without the need for expensive cofactor generation. CPO's lack of operational stability, however, is a major limitation for its commercial use. In the present study, a capillary-LC on-line trypsin-digestion system combined with reversed-phase chromatography and mass spectrometric detection was optimized for studying the primary sequence of CPO. Samples containing native CPO, CPO treated with H(2)O(2), and CPO oxidatively inactivated by the use of indole and H(2)O(2) were analyzed and compared. Three oxidized peptides were found in the samples treated with H(2)O(2). Two additional oxidized peptides were found in the CPO samples that were completely inactivated, one of which contained an oxidized cysteine residue, Cys50, which is an essential amino acid due to its function as the axial ligand to the iron in the heme--the prosthetic group in CPO. In addition, the heme group was absent in the inactivated samples but was readily detected in other samples.