Identification of the Fe–O2 and the Fe=O Heme Species for Indoleamine 2,3-Dioxygenase during Catalytic Turnover

Abstract

Resonance Raman spectroscopy has been applied to two distinct temporal species of indoleamine 2,3-dioxygenase during catalytic turnover. We have identified two oxygen-isotopesensitive Raman modes at 569 and 798 cm ¹1 for the two respective species. The O analysis of the 798 cm ¹1 band indicates the existence of a ferryl-oxo heme, which is inconsistent with the previously proposed reaction mechanism. The present study thus provides a physical basis for the structures of the possible reaction intermediates.Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase 1 localized in several different mammalian tissues, with the exception of liver.2 This enzyme catalyzes a dioxygenation reaction for incorporation of two oxygen atoms from O 2 into L-tryptophan (Trp) in the production of N-formylkynurenine, which is the main catabolic pathway of Trp. The absorption spectra of the two distinct species of IDO are shown in Figure S1.6 Figure S1A 6 was obtained for the reduced IDO samples after addition of dioxygen. The spectrum exhibits absorption maxima at 413, 542, and 577 nm and is consistent with the reported spectra for oxygenated IDO. 7 The spectrum of Figure S1B 6 was obtained for oxygenated IDO samples after addition of Trp and exhibits absorption maxima at 410, 544, and 577 nm with decreased intensities relative to the absorption maxima of the oxygenated state. This spectrum also has a shoulder at 593 nm, and the intermediate represented in this spectrum, which has not hitherto been reported, is hereafter referred to as the "593 nm species." The absorption band at 593 nm is not seen in the absorption spectra of ferric and ferrous IDO (not shown), and the molar extinction coefficient at 593 nm is not known at present. If we assume that it is comparable to those of the ¡ and ¢ absorption bands of ferric and ferrous IDO, it is estimated that the fractional population of the 593 nm species seen in Figure S1B 6 is not smaller than 30%. We measured the time-dependent change of the concentrations of the 593 nm species and N-formylkynurenine during turnover. The results are shown in Figure S2. 6 When the time course of the absorption change at 321 nm ( Figure S2B 6 ) is compared with the time course of the absorption change at 593 nm ( Figure S2C 6

NADH Oxidase Activity of Indoleamine 2,3-Dioxygenase

“…Although Shimizu et al (27) noted in passing that IDO exhibits a peroxidase activity, no further consideration of the interaction of IDO with peroxide was reported until Poljak et al (54) demonstrated that exposure of IDO to hydrogen peroxide (Ն10 M) decreases the dioxygenase activity of the enzyme and oxidizes up to five of the eight Cys residues to sulfinic or sulfonic acid to result in changes to the structure of the enzyme. Following the detection of ferryl heme intermediates by resonance Raman spectroscopy during turnover of IDO (63,85,86), Lu and Yeh (87) The docking simulations conducted in this study suggest that binding of NADH at the active site of IDO is possible, and the ability to elute IDO bound to Cibacron blue affinity resin with either NADH or L-Trp is consistent with such interaction (88,89). Nevertheless, we were unable to detect any change in the electronic absorption spectrum of IDOFe 3ϩ or in the fluorescence emission spectrum of NADH upon mixing these two components anaerobically, so no simple means of quantifying or even verifying the affinity of the distal side of the heme cavity for NADH is available.…”

Ferryl Derivatives of Human Indoleamine 2,3-Dioxygenase

“…In particular, the 2 / 3 modes shift to 1579/1503 cm Ϫ1 (with the intensity of the 3 mode considerably enhanced), the 8 mode shifts to 332 cm Ϫ1 , and the ␦ vinyl mode splits into two bands at 423 and 433 cm Ϫ1 . In addition, various in-plane asymmetric modes and outof-plane modes are activated, indicating that L-Trp binding to the ferryl species causes the reduction of the in-plane symmetry, thereby introducing out-of-plane distortion of the porphyrin macrocycle of the heme (29,49 Orelated modes, respectively. Intriguingly, the substrate-free spectrum shows two positive peaks at 786 and 810 cm Ϫ1 , in conjunction with a single negative peak at 752 cm Ϫ1 (Fig.…”

A Specific Interaction of l-Tryptophan with CO of CO-Bound Indoleamine 2,3-Dioxygenase Identified by Resonance Raman Spectroscopy

“…This band was identified for the ferric cyanide complex but was not identified when the enzyme is in the reduced state (15). It is also seen for the ferryl-oxo intermediate and the oxygenated intermediate in the presence of L-Trp (330-332 cm -1 ) (34) but not for the oxygenated intermediate in the absence of L-Trp (11). Thus, the band at 328 cm -1 could serve as a marker for the structure of the Fe-ligand moiety when L-Trp is bound.…”

“…Thus, one might consider that the ratelimiting step could be different as a result of the intramolecular reaction in IDO and could represent an electron transfer step between the reductant and the mediator or between the mediator and IDO. However, since both the oxygenated and the ferryl-oxo intermediates are detectable when the reaction is initiated using IDO reduced with Na 2 S 2 O 4 (11), it is natural to consider that the rate-limiting step of N-formylkynurenine formation is represented by the rate of decay of either the oxygenated intermediate or the ferryl-oxo intermediate.…”

“…Laser power of 125 μW at the sample was chosen to minimize photodissociation of CO. The detector was a liquid nitrogen-cooled CCD (Roper Scientific, Spec-10: 400B/LN) attached to a single polychromator (Chromex, 500IS) as described previously (11). The spectral slit width was 8 cm -1 .…”