Dioxygen‐activating iron center in putidamonooxin

The mononuclear non‐haem iron center is the dioxygen‐binding site of putidamonooxin which is the dioxygen‐activating component of the 4‐methoxybenzoate monooxygenase. Replacement of dioxygen by nitrosyl leads to the formation of a rather stable Fe 3+ · NO − complex which is characterized by electron spin resonance (ESR) at g · 4 and g · 2. The ESR features can be composed by two spectral components which are characterized by different tetragonal distortions of the axial symmetry. Binding of 4‐hydroxybenzoate, which is the product of the enzymatic reaction, leads to the formation of an ESR spectrum with pure axial symmetry. After binding of 4‐methoxybenzoate, i.e. the physiological substrate of the monooxygenase, only one spectral component, i.e. that with a small tetragonal distortion, is observed. Binding of substrate analogues, like 4‐aminobenzoate and 4‐trifluoromethylbenzoate, leads to a spectral heterogeneity with variable amounts of the ESR component with a large tetragonal distortion. Benzoate induces an ESR spectrum with only that spectral component with large tetragonal distortion. The iron‐depleted substrate‐free form of the enzyme, ligated with NO, also shows ESR heterogeneity, i.e. both spectral components overlap, with 60% of the component with large tetragonal distortion. Binding of 4‐methoxybenzoate leads to the occurrence of a pure spectrum, i.e. with small tetragonal distortion, whereas binding of benzoate leads to a pure spectrum with large tetragonal distortion. Thus, the structural heterogeneity is removed by binding of both the ligand (NO) and substrate. The Fe 3+ · NO − complex is discussed as an analogue of the native oxy complex Fe 3+ · O − 2 .