The heme enzyme indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the first reaction of l-tryptophan oxidation along the kynurenine pathway. IDO1 is a central immunoregulatory enzyme with important implications for inflammation, infectious disease, autoimmune disorders, and cancer. Here we demonstrate that IDO1 is a mammalian nitrite reductase capable of chemically reducing nitrite to nitric oxide (NO) under hypoxia. Ultraviolet–visible absorption and resonance Raman spectroscopy showed that incubation of dithionite-reduced, ferrous–IDO1 protein (FeII–IDO1) with nitrite under anaerobic conditions resulted in the time-dependent formation of an FeII–nitrosyl IDO1 species, which was inhibited by substrate l-tryptophan, dependent on the concentration of nitrite or IDO1, and independent of the concentration of the reductant, dithionite. The bimolecular rate constant for IDO1 nitrite reductase activity was determined as 5.4 M–1 s–1 (pH 7.4, 23 °C), which was comparable to that measured for myoglobin (3.6 M–1 s–1; pH 7.4, 23 °C), an efficient and biologically important mammalian heme-based nitrite reductase. IDO1 nitrite reductase activity was pH-dependent but differed with myoglobin in that it showed a reduced proton dependency at pH >7. Electron paramagnetic resonance studies measuring NO production showed that the conventional IDO1 dioxygenase reducing cofactors, ascorbate and methylene blue, enhanced IDO1’s nitrite reductase activity and the time- and IDO1 concentration-dependent release of NO in a manner inhibited by l-tryptophan or the IDO inhibitor 1-methyl-l-tryptophan. These data identify IDO1 as an efficient mammalian nitrite reductase that is capable of generating NO under anaerobic conditions. IDO1’s nitrite reductase activity may have important implications for the enzyme’s biological actions when expressed within hypoxic tissues.

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Formation of alpha‐ketobutyrate (KB) from methionine involves catalysis that is facilitated by covalently bound coenzyme pyridoxal phosphate of methionine gamma lyase deaminase (Mgld). In this study we used molecularly cloned, overexpressed, and purified Mgld from Porphyromonas gingivalis. Sequence comparison of Mgld amino acids between organism revealed conserved residues of active site pockets. Based on pH and temperature responses of the purified enzyme we speculated that up until the release of the final product KB, the participation of active site residues on catalysis must be present. Deuterium labeled methionine substrates at alpha and beta position showed significant decrease in the reaction rate. This revealed that there are multiple rate‐limiting steps, with abstraction of alpha‐proton and gamma cleavage being the most rate limiting steps. From overall steady‐state catalytic reactions monitored by UV‐VIS spectroscopy, the heavily populated 478 nm absorbing species of L‐Met, L‐ethionine, L‐methionine sulfone and L‐homoserine was assigned to a late crotonate intermediate. In addition, this is the first report to show the more red‐shifted (498 nm) species observed during L‐homoserine‐lactone reaction with Mgld, which we assigned to a quinonoid species from DFT and time‐dependent self‐consistent field (TD‐SCF) calculations. The kinetic parameters differed significantly for the various substrates. Highest catalytic efficiency was observed for L‐vinylglycine (kcat/Km = 6455 s‑1 M‑1), exceeding that of L‐Met (kcat/Km = 4211 s‑1 M‑1). L‐Met sulfone and L‐Hse were markedly weaker binders (Km » 30 mM) while L‐Met sulfone displayed the largest turnover number (kcat = 1638 min‐1). A linear semi‐logarithmic correlation between experimental kcat values for substrates and DFT‐calculated g‐cleavage Gibbs activation energies was determined. Lower calculated activation energies for gamma elimination were associated with larger kcat values, suggesting that the gamma cleavage step in the reaction pathway is strongly rate‐determining.