Generation of High-Spin Iron(I) in a Protein Environment Using Cryoreduction

High-spin Fe¹⁺ sites are potentially important in iron–sulfur proteins but are rare in synthetic compounds and unknown in metalloproteins. Here, we demonstrate a spectroscopically characterized example of high-spin non-heme Fe¹⁺ in a protein environment. Cryoreduction of Fe²⁺-substituted azurin at 77 K with ⁶⁰Co γ radiation generates a new species with a <i>S</i> = ³/₂ (high-spin) Fe¹⁺ center having <i>D</i> > 0 and <i>E</i>/<i>D</i> ∼ 0.25. This transient species is stable in a glycerol–water glass only up to ∼170 K. A combination of electron paramagnetic resonance and Mössbauer spectroscopies provides a powerful means of identifying a transient high-spin Fe¹⁺ site in a protein scaffold

Probing the Ternary Complexes of Indoleamine and Tryptophan 2,3-Dioxygenases by Cryoreduction EPR and ENDOR Spectroscopy

We have applied cryoreduction/EPR/ENDOR techniques to characterize the active-site structure of the ferrous-oxy complexes of human (hIDO) and <i>Shewanella oneidensis</i> (sIDO) indoleamine 2,3-dioxygenases, <i>Xanthomonas campestris</i> (<i>Xc</i>TDO) tryptophan 2,3-dioxygenase, and the H55S variant of <i>Xc</i>TDO in the absence and in the presence of the substrate l-Trp and a substrate analogue, l-Me-Trp. The results reveal the presence of multiple conformations of the binary ferrous-oxy species of the IDOs. In more populated conformers, most likely a water molecule is within hydrogen-bonding distance of the bound ligand, which favors protonation of a cryogenerated ferric peroxy species at 77 K. In contrast to the binary complexes, cryoreduction of all of the studied ternary [enzyme-O₂-Trp] dioxygenase complexes generates a ferric peroxy heme species with very similar EPR and ¹H ENDOR spectra in which protonation of the basic peroxy ligand does not occur at 77 K. Parallel studies with l-Me-Trp, in which the proton of the indole nitrogen is replaced with a methyl group, eliminate the possibility that the indole NH group of the substrate acts as a hydrogen bond donor to the bound O₂, and we suggest instead that the ammonium group of the substrate hydrogen-bonds to the dioxygen ligand. The present data show that substrate binding, primarily through this H-bond, causes the bound dioxygen to adopt a new conformation, which presumably is oriented for insertion of O₂ into the C₂−C₃ double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by “shielding” it from water