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

preprint
submitted on 08.09.2020 and posted on 09.09.2020 by Filipe Folgosa, Vladimir Pelmenschikov, Matthias Keck, Christian Lorent, Yoshitaka Yoda, James A. Birrell, Martin Kaupp, Miguel Teixeira, Kenji Tamasaku, Christian Limberg, Lars Lauterbach

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.

Funding

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.

History

Email Address of Submitting Author

lars.lauterbach@tu-berlin.de

Institution

Technische Universität Berlin

Country

Germany

ORCID For Submitting Author

0000-0002-6601-6473

Declaration of Conflict of Interest

There are no conflicts to declare.

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