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 Fe ²⁺-substituted azurin at 77 K with ⁶⁰Co γ radiation generates a new species with a S = ³/₂ (high-spin) Fe ¹⁺ center having D > 0 and E/D ∼ 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.