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Folgosa_ChemRxiv.pdf (4.71 MB)
Hydroxo-Bridged Active Site of Flavodiiron NO Reductase Revealed by Spectroscopy and Computations
submitted on 08.09.2020, 13:33 and posted on 09.09.2020, 12:09by Filipe Folgosa, Vladimir Pelmenschikov, Matthias Keck, Christian Lorent, Yoshitaka Yoda, James A. Birrell, Martin Kaupp, Miguel Teixeira, Kenji Tamasaku, Christian Limberg, Lars Lauterbach
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.
This work was financially supported by the Portuguese Fundação para a Ciência e Tecnologia (FCT), grant PTDC/BIA-BQM/27959/2017, and by ITQB MOSTMICRO (LISBOA-01-0145-FEDER-007660) Research Unit funded by FCT, through National funds, and by FEDER under the PT2020 Partnership Agreement. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement Nº 810856. The work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy – EXC 2008 – 390540038 – UniSysCat. J.A.B. acknowledges the Max Planck Society and DFG Priority Programme “Iron-Sulfur for Life: Cooperative Function of Iron-Sulfur Centers in Assembly, Biosynthesis, Catalysis and Disease” (SPP 1927) Project BI 2198/1-1 for funding.