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J. Biol. Chem., Vol. 276, Issue 39, 36067-36070, September 28, 2001

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ACCELERATED PUBLICATION
Oxidative Modification of Tryptophan 43 in the Heme Vicinity of the F43W/H64L Myoglobin Mutant^*

Isao Hara, Takafumi Ueno^§¶, Shin-ichi Ozaki, Shinobu Itoh^**, Keonil Lee, Norikazu Ueyama, and Yoshihito Watanabe^§¶§§

From the  Department of Structural Molecular Science, The Graduate University for Advanced Studies, Myodaiji, Okazaki 444-8585, Japan, the ^§ Center for Integrative Bioscience, Myodaiji, Okazaki 444-8585, Japan, the  Faculty of Education, Yamagata University, Kojirakawa, Yamagata 990-8560, Japan, the ^** Department of Chemistry, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan, the  Department of Macromolecular Science, Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka, Osaka 560-0043, Japan, and the ^¶ Institute for Molecular Science, Myodaiji, Okazaki 444-8585, Japan

The F43W/H64L myoglobin mutant was previously constructed to investigate the effects of electron-rich tryptophan residue in the heme vicinity on the catalysis, where we found that Trp-43 in the mutant was oxidatively modified in the reaction with m-chloroperbenzoic acid (mCPBA). To identify the exact structure of the modified tryptophan in this study, the mCPBA-treated F43W/H64L mutant has been digested stepwise with Lys-C achromobacter and trypsin to isolate two oxidation products by preparative fast protein liquid chromatography. The close examinations of the ¹H NMR spectra of peptide fragments reveal that two forms of the modified tryptophan must have 2,6-disubstituted indole substructures. The ¹³C NMR analysis suggests that one of the modified tryptophan bears a unique hydroxyl group in stead of the NH₂ group at the amino-terminal. The results together with mass spectrometry (MS)/MS analysis (30 Da increase in mass of Trp-43) indicate that oxidation products of Trp-43 are 2,6-dihydro-2,6-dioxoindole and 2,6-dihydro-2-imino-6-oxoindole derivatives. Our finding is the first example of the oxidation of aromatic carbons by the myoglobin mutant system.

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