Fe(II)‐substituted horse liver alcohol dehydrogenase, a model for non‐heme iron enzymes:Various states of iron‐dioxygen interaction investigated by Mössbauer and EPR spectroscopy

The catalytic Zn(II) ion of horse liver alcohol dehydrogenase (EE isozyme) was replaced by Fe(II), providing a novel iron protein with the unusual coordination of Fe(II) to two cysteines residues, one histine residue and water. The electronic structure of iron in this system was characterized by Mössbauer spectroscopy at various temperatures as well as applied magnetic fields and analysed in terms of the spin Hamiltonian formalism. The novelty we found is an unusually weak spin coupling (|J| < 0.1 cm⁻¹) of a paramagnet (S= 1) with iron (S= 2). From EPR and biochemical studies we conclude that the corresponding chemical species is triplet oxygen (O₂). The quantitative determination of the coupling energy was possible utilizing the competition between Zeemann interaction and spin coupling at weak magnetic fields and low temperature. Oxidation experiments followed by Mössbauer spectroscopy showed that the spin‐coupled system is an outer‐sphere Fe(II) · (O₂)_aq complex occurring as an intermediate during a Fe(II)‐catalyzed dioxygen activation. We observed two additional Fe(II) species after treatment with O₂ and dithionite. The spin Hamiltonian parameters of iron in the coupled system are presented. The results are compared with those of iron in other non‐heme iron proteins.