Mössbauer studies of the redox state of the ferric uptake regulator [2Fe–2S]2+ cluster in Escherichia coli

Abstract

The Ferric uptake regulator (Fur) proteins from Haemophilus influenzae and Escherichia coli overexpressed in E. coli cells (MC4100) grown in M9 medium supplemented with ⁵⁷Fe were studied with Mössbauer spectroscopy. Previous studies have shown that Fur proteins from H. influenzae and E. coli bind a [2Fe–2S]²⁺ cluster in response to elevation of intracellular free iron content. Here we find that when the [2Fe–2S]²⁺ clusters in purified Fur proteins are reduced with dithionite, the reduced clusters are quickly decomposed, forming compounds with two distinct spectral signatures of high spin Fe(II) in tetrahedral and octahedral coordination, respectively. The instability of the reduced [2Fe-2S]¹⁺ cluster in Fur is unique, as the [2Fe–2S]²⁺ clusters in many other proteins can reversibly undergo one-electron reduction-oxidation. The Mössbauer spectra of whole E. coli cells overexpressing Fur proteins show a quadrupole doublet with the isomer shift of δ₁ = 0.28 mm/s and ΔE_Q1 = 0.52 mm/s, typical for oxidized [2Fe-2S]²⁺ clusters and identical with that in the purified Fur protein. The corresponding spectra in large applied magnetic fields show the diamagnetic pattern that unambiguously reveals an exchange-coupled system with a diamagnetic electronic ground state, which confirms its assignment to the oxidized [2Fe-2S]²⁺ cluster clusters from Fur. No reduced [2Fe-2S]¹⁺ clusters of Fur are observed in the whole-cell E. coli spectra. The Mössbauer spectra of the whole-cell E. coli without the Fur expression do not contain the components associated with the [2Fe–2S]²⁺ cluster of Fur.