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

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