EPR and Mössbauer spectroscopy show inequivalent hemes in tryptophan dioxygenase.

Tryptophan 2,3-dioxygenase (TDO) is an essential enzyme in the pathway of NAD biosynthesis and important for all living organisms. TDO catalyzes oxidative cleavage of the indole ring of L-tryptophan (L-Trp), converting it to N-formylkynurenine (NFK). The crystal structure of TDO shows a dimer of dimer quaternary structure of the homotetrameric protein. The four catalytic sites of the protein, one per subunit, contain a heme that catalyzes the activation and insertion of dioxygen into L-Trp. Because of the alpha(4) structure and because only one type of heme center has been identified in previous spectroscopic studies, the four hemes sites have been presumed to be equivalent. The present work demonstrates that the heme sites of TDO are not equivalent. Quantitative interpretation of EPR and Mössbauer spectroscopic data indicates the presence of two dominant inequivalent heme species in reduced and oxidized states of the enzyme, which is consistent with a dimer of dimer protein quaternary structure that now extends to the electronic properties of the hemes. The electronic properties of the hemes in the reduced state of TDO change significantly upon L-Trp addition, which is attributed to a change in the protonation state of the proximal histidine to the hemes. The binding of O(2) surrogates NO or CO shows two inequivalent heme sites. The heme-NO complexes are 5- and 6-coordinate without L-Trp, and both 6-coordinate with L-Trp. NO can be selectively photodissociated from only one of the heme-NO sites and only in the presence of L-Trp. Cryoreduction of TDO produces a novel diamagnetic heme species, tentatively assigned as a reduced heme-OH complex. This work presents a new description of the heme interactions with the protein, and with the proximal His, which must be considered during the general interpretation of physical data as it relates to kinetics, mechanism, and function of TDO.