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