Initial O2 Insertion Step of the Tryptophan Dioxygenase Reaction Proposed by a Heme-Modification Study

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• 作者：Ryu Makino ; Eiji Obayashi ; Hiroshi Hori ; Tetsutaro Iizuka ; Keisuke Mashima ; Yoshitsugu Shiro ; Yuzuru Ishimura
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：June 16, 2015
• 年：2015
• 卷：54
• 期：23
• 页码：3604-3616
• 全文大小：566K
• ISSN：1520-4995

文摘

l-Tryptophan 2,3-dioxygenase (TDO) is a protoheme-containing enzyme that catalyzes the production of N-formylkynurenine by inserting O₂ into the pyrrole ring of l-tryptophan. Although a ferrous鈥搊xy form (Fe²⁺鈥揙₂) has been established to be an obligate intermediate in the reaction, details of the ring opening reaction remain elusive. In this study, the O₂ insertion reaction catalyzed by Pseudomonas TDO (PaTDO) was examined using a heme-modification approach, which allowed us to draw a quantitative correlation between the inductive electronic effects of the heme substituents and the substituent-induced changes in the functional behaviors of the ferrous鈥搊xy form. We succeeded in preparing reconstituted PaTDO with synthetic hemes, which were different with respect to the inductive electron-withdrawing nature of the heme substituents at positions 2 and 4. An increase in the electron-withdrawing power of the heme substituents elevated the redox potential of reconstituted PaTDO, showing that the stronger the electron-withdrawing ability of the heme substituents, the lower the electron density on the heme iron. The decrease in the electron density of the heme iron resulted in a higher frequency shift of the C鈥揙 stretch of the heme-bound CO and enhanced the dissociation of O₂ from the ferrous鈥搊xy intermediate. This result was interpreted as being due to weaker 蟺 back-donation from the heme iron to the bound CO or O₂. More importantly, the reaction rates of the ferrous鈥搊xy intermediate to oxidize l-Trp were increased with the electron-withdrawing ability of the heme substituents, implying that the more electron-deficient ferrous鈥搊xy heme is favored for the PaTDO-catalyzed oxygenation. On the basis of these results, we propose that the initial step of the dioxygen activation by PaTDO is a direct electrophilic addition of the heme-bound O₂ to the indole ring of l-Trp.