<p>NO
and O<sub>2</sub> are detoxified in many organisms using flavodiiron proteins
(FDPs). The exact coordination of the iron centre in the active site of these
enzymes remains unclear despite numerous structural studies. Here, we used <sup>57</sup>Fe
nuclear resonance vibrational spectroscopy (NRVS) to probe the iron-ligand
interactions in <i>Escherichia coli</i> FDP. This data combined with density
functional theory (DFT) and <sup>57</sup>Fe Mössbauer spectroscopy indicate
that the oxidised form of FDP contains a dihydroxo-diferric Fe(III)–(µOH<sup>–</sup>)<sub>2</sub>–Fe(III)
active site, while its reduction gives rise to a monohydroxo-diferrous
Fe(II)–(µOH<sup>–</sup>)–Fe(II) site upon elimination of one bridging OH<sup>–</sup>
ligand, thereby providing an open coordination site for NO binding. Prolonged
NRVS data collection of the oxidised FDP resulted in photoreduction and
formation of a partially reduced diiron center with two bridging hydroxyl
ligands. These results have crucial implications for studying and understanding
the mechanism of FDP as well as other non-haem diiron enzymes.</p>