Hydroxo-Bridged Active Site of Flavodiiron NO Reductase Revealed by Spectroscopy and Computations

09 September 2020, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


NO and O2 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 57Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the iron-ligand interactions in Escherichia coli FDP. This data combined with density functional theory (DFT) and 57Fe Mössbauer spectroscopy indicate that the oxidised form of FDP contains a dihydroxo-diferric Fe(III)–(µOH)2–Fe(III) active site, while its reduction gives rise to a monohydroxo-diferrous Fe(II)–(µOH)–Fe(II) site upon elimination of one bridging OH 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.


NO reductase
Mössbauer spectroscopy
DFT analyses
Catalytic Cycle Intermediates
Diiron Complex


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