Application of radiation and electron spin resonance spectroscopy to the study of ferryl myoglobin

Abstract

Addition of an excess of hydrogen peroxide to aqueous metmyoglobin (Fe³⁺—OH₂) followed by rapid freezing gave a species having no detectable e.s.r. features assignable to iron. However, on exposure to ⁶⁰Co γ-rays at 77 K, e.s.r. features characteristic of a low-spin Fe^III centre grew-in. On annealing above 77 K a modified centre with a larger spread of g values was identified as hydroxy-myoglobin. On further annealing this finally changed to normal high-spin Hb⁺ characterised by an intense feature at g= 6. These results strongly support the ‘ferryl’ structure, (FeO²⁺) previously assigned to the major product formed from hydrogen peroxide.

We were unable to detect e.s.r. features assignable to the expected (Fe³⁺—OOH) intermediate even at relatively low H₂O₂ concentrations. This species has been previously identified by e.s.r. spectroscopy as an intermediate in the formation of (Fe³⁺H₂O) following electron addition to oxymyoglobin (Fe²⁺O₂). This result shows that, although the hydroperoxide derivative, (Fe³⁺—OOH), is probably an essential intermediate in the route to the ferryl derivative, it is never present in concentrations high enough to be detected by e.s.r. spectroscopy.