The heme environment of recombinant human indoleamine 2,3-dioxygenase. Structural properties and substrate-ligand interactions.

Indoleamine 2,3-dioxygenase is a heme enzyme that catalyzes the oxidative degradation of L-Trp and other indoleamines. We have used resonance Raman spectroscopy to characterize the heme environment of purified recombinant human indoleamine 2,3-dioxygenase (hIDO). In the absence of L-Trp, the spectrum of the Fe(3+) form displayed six-coordinate, mixed high and low spin character. Addition of L-Trp triggered a transition to predominantly low spin with two Fe-OH(-) stretching modes identified at 546 and 496 cm(-1), suggesting H-bonding between the NH group of the pyrrole ring of L-Trp and heme-bound OH(-). The distal pocket of Fe(3+) hIDO was explored further by an exogenous heme ligand, CN(-); again, binding of L-Trp introduced strong H-bonding and/or steric interactions to the heme-bound CN(-). On the other hand, the spectrum of Fe(2+) hIDO revealed a five-coordinate and high spin heme with or without L-Trp bound. The proximal Fe-His stretching mode, identified at 236 cm(-1), did not shift upon L-Trp addition, indicating that the proximal Fe-His bond strength is not affected by binding of the substrate. The high Fe-His stretching frequency suggests that Fe(2+) hIDO has a strong "peroxidase-like" Fe-His bond. Using CO as a structural probe for the distal environment of Fe(2+) hIDO revealed that binding of L-Trp in the distal pocket converted IDO to a peroxidase-like enzyme. Binding of L-Trp also caused conformational changes to the heme vinyl groups, which were independent of changes of the spin and coordination state of the heme iron. Together these data indicate that the strong proximal Fe-His bond and the strong H-bonding and/or steric interactions between l-Trp and dioxygen in the distal pocket are likely crucial for the enzymatic activity of hIDO.