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Abstract
The critical role of the ferryl intermediate in catalyzing the oxygen chemistry of monooxygenases, oxidases, or peroxidases has been known for decades. In contrast, its involvement in heme-based dioxygenases, such as human indoleamine 2,3-dioxygenase (hIDO), was not recognized until recently. In this study, H(2)O(2) was used as a surrogate to generate the ferryl intermediate of hIDO. Spectroscopic data demonstrate that the ferryl species is capable of oxidizing azinobis(3-ethylbenzothiazoline-6-sulfonic acid) but not L-Trp. Kinetic studies reveal that the conversion of the ferric enzyme to the ferryl intermediate facilitates the L-Trp binding rate by >400-fold; conversely, L-Trp binding to the enzyme retards the peroxide reaction rate by ∼9-fold, because of the significant elevation of the entropic barrier. The unfavorable entropic factor for the peroxide reaction highlights the scenario that the structure of hIDO is not optimized for utilizing H(2)O(2) as a co-substrate for oxidizing L-Trp. Titration studies show that the ferryl intermediate possesses two substrate-binding sites with a K(d) of 0.3 and 440 μM and that the electronic properties of the ferryl moiety are sensitive to the occupancy of the two substrate-binding sites. The implications of the data are discussed in the context of the structural and functional relationships of the enzyme.
Highlights
It is generally accepted that for heme proteins to carry out oxygen chemistry, the relatively inert dioxygen has to be activated first
This study demonstrates that the reaction of the ferric hIDO with H2O2 leads to the formation of the compound II (Cmpd-II) type of ferryl adducts
With respect to the ligand-free ferric enzyme, the ferryl intermediate exhibits ϳ400-fold faster binding rate (Ͼ2 ϫ 106 MϪ1 sϪ1 versus 5.5 ϫ 103 MϪ1sϪ1) and 3000-fold higher affinity (Kd of 0.3 versus 900 M) toward L-Trp, manifesting the fact that ligand binding in hIDO introduces conformational changes to the protein matrix, thereby promoting L-Trp binding
Summary
It is generally accepted that for heme proteins to carry out oxygen chemistry, the relatively inert dioxygen has to be activated first. In monooxygenases, such as P450s, dioxygen is acti-. The requirement of the reductive activation of dioxygen in P450s is confirmed by the observation that the reactions can be bypassed by reacting the ferric enzymes with H2O2 (the so-called “peroxide shunt”). In contrast to the P450 reactions, the catalytic cycle of IDO and TDO is initiated by the binding of O2 and L-Trp to the ferrous enzyme to generate the ternary complex, which turns over to produce the product, NFK, leaving the heme iron in the active ferrous state that is ready for a new turnover (see Reaction 2). Unlike monooxygenase reactions, the dioxygenase reaction does not consume any electrons and protons (see Ref. 1 and references therein)
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