Generation of high-spin iron(I) in a protein environment using cryoreduction

High-spin Fe(1+) 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 Fe(1+) in a protein environment. Cryoreduction of Fe(2+)-substituted azurin at 77 K with (60)Co 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 Fe(1+) site in a protein scaffold.