Hydroxo-bridged active site of flavodiiron NO reductase revealed by NRVS and DFT

The mechanisms of NO and O2 detoxification in many organisms involve flavodiiron proteins (FDPs). The active site of these enzymes comprises a diiron cofactor, whose exact non amino acid ligands coordination remains debated in view of controversies between crystallographic and spectroscopic/theoretical studies. Here, we used 57Fe nuclear resonance vibrational spectroscopy (NRVS) to elucidate the vibrational fingerprint of the flavodiiron protein from Escherichia coli in the as-isolated diferric and reduced diferrous forms. These data combined with Mössbauer measurements and density functional theory (DFT) calculations show the diferric form of FDP bearing a dihydroxo Fe(III)–(µOH–)2–Fe(III) active site, while its reduction results in a monohydroxo Fe(II)–(µOH–)–Fe(II) center, undergoing elimination of one bridging OH– ligand. Our study provides detailed new insights into the coordination of the diiron site of E. coli FDP and emphasizes the importance of careful evaluation of NRVS data from synchrotron radiation, as undesired photoreduction processes can take place even using low incident photon fluxes.