title: Initial O₂ Insertion Step of the Tryptophan Dioxygenase Reaction Proposed by a Heme-Modification Study.
abstract: 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-oxy form (Fe²⁺-O₂) 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-oxy 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-O stretch of the heme-bound CO and enhanced the dissociation of O₂ from the ferrous-oxy intermediate. This result was interpreted as being due to weaker π back-donation from the heme iron to the bound CO or O₂. More importantly, the reaction rates of the ferrous-oxy intermediate to oxidize L-Trp were increased with the electron-withdrawing ability of the heme substituents, implying that the more electron-deficient ferrous-oxy 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.
authors.affiliation: †Department of Life Science, College of Science, Rikkyo University, Nishi-ikebukuro 3-34-1, Toshima-ku, Tokyo 171-8501, Japan.
authors.forename: Ryu
authors.initials: R
authors.lastname: Makino
authors.affiliation: ‡Department of Biochemistry, Shimane University School of Medicine, Izumo, Shimane 693-8501, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Obayashi
authors.affiliation: §Center for Quantum Science and Technology under Extreme Conditions, Osaka University, Toyonaka, Osaka 560-8531, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hori
authors.affiliation: ∥RIKEN Harima Institute/Spring 8, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Tetsutaro
authors.initials: T
authors.lastname: Iizuka
authors.affiliation: †Department of Life Science, College of Science, Rikkyo University, Nishi-ikebukuro 3-34-1, Toshima-ku, Tokyo 171-8501, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Mashima
authors.affiliation: ∥RIKEN Harima Institute/Spring 8, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Shiro
authors.affiliation: ⊥Department of Biochemistry, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Ishimura
mesh_terms: D000107:Acetylation; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000378:metabolism; D002245:Carbon Dioxide / Q000737:chemistry / Q000378:metabolism; D020605:Delftia acidovorans / Q000201:enzymology; D003905:Deuteroporphyrins / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D007737:Kynurenine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008652:Mesoporphyrins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*; D014366:Tryptophan Oxygenase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003905:Deuteroporphyrins; D008024:Ligands; D008652:Mesoporphyrins; D009211:Myoglobin; C007772:N'-formylkynurenine; D002245:Carbon Dioxide; C029198:deuteroheme; D007737:Kynurenine; D006418:Heme; C031468:mesoporphyrin IX; D014364:Tryptophan; D014366:Tryptophan Oxygenase; D019475:Camphor 5-Monooxygenase; D010100:Oxygen