hIDO1 catalyzes the addition of molecular oxygen (O2) to free L-tryptophan (L-Trp) to form N-formyl-L-kynurenine (NFK) via the heme-centered dioxygenation reaction. In the published literature, ferryl species have been detected by Raman spectroscopy during L-Trp conversion to NFK. Also, monooxygenated tryptophan derivatives have been identified by mass spectrometry in the quenched assay mixtures of hIDO1. These reports led to the revision of hIDO1 catalytic mechanism, supporting a stepwise process for insertion of O2 into L-Trp. Here we present results on quantifying the activity of hIDO1 by independent monitoring of O2 consumption and NFK formation. We observe that during L-Trp dioxygenation a stoichiometric amount of O2 is consumed for each equivalent of L-Trp. However, a sub-stoichiometric amount of NFK is detected when the reaction is monitored by stopped-flow spectrophotometry. Additionally, formation of a neutral ferryl derivative of hIDO1, (Cpd II)●L-Trp, which is not part of the steady-state distribution of the catalytically-active enzyme species, is observed during steady-state formation of NFK. We determine the concentration of (Cpd II)●L-Trp derivative during the NFK formation and show that NFK and (Cpd II)●L-Trp have markedly different kinetics. Taken together, these observations suggest that the monooxygenase activity of hIDO1 towards L-Trp occurs simultaneously with (yet independently of) its normal turnover as a dioxygenase.

Is ferryl a side-product or an intermediate in catalysis of L-tryptophan dioxygenation by human indoleamine 2,3-dioxygenase (hIDO1)?

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.