Oxidative modification of tryptophan 43 in the heme vicinity of the F43W/H64L myoglobin mutant

The F43W/H64L myoglobin mutant was previously constructed to investigate th e effects of electron-rich tryptophan residue in the heme vicinity on the c atalysis, where we found that Trp-43 in the mutant was oxidatively modified in the reaction with m-chloroperbenzoic acid (mCPBA). To identify the exac t structure of the modified tryptophan in this study, the mCPBA-treated F43 W/H64L mutant has been digested stepwise with Lys-C achromobacter and tryps in to isolate two oxidation products by preparative fast protein liquid chr omatography. The close examinations of the H-1 NMR spectra of peptide fragm ents reveal that two forms of the modified tryptophan must have 2,6-disubst ituted indole substructures. The C-13 NMR analysis suggests that one of the modified tryptophan bears a unique hydroxyl group in stead of the NH2 grou p at the amino-terminal. The results together with mass spectrometry (MS)/M S analysis (30 Da increase in mass of Trp-43) indicate that oxidation produ cts of Trp-43 are 2,6-dihydro-2,6-dioxoindole and 2,6-dihydro-2-imino-6-oxo indole derivatives. Our finding is the first example of the oxidation of ar omatic carbons by the myoglobin mutant system.