Redox-dependent vibrational fingerprints of the diiron active site. Nuclear resonance vibrational spectroscopy and quantum chemical calculations reveals distinct iron–ligand vibrational patterns for the oxidised (red) and reduced (blue) states of the flavodiiron NO reductase. (Credit: Lars Lauterbach, RWTH)
An international research team including scientists from RWTH Aachen University, TU Berlin, ITQB NOVA University of Lisbon (Portugal), SPring8 (Japan) and PETRA III at DESY have revealed, in atomic detail, how bacteria detoxify the reactive signalling molecule nitric oxide which the human body produces to combat bacterial infection. Their findings may contribute to the development of novel antibiotics and therapeutic strategies.
Nitric oxide (NO) is a potent molecule produced by the human immune system and is highly toxic for bacteria. As part of their infection strategy, many microorganisms have evolved a way to neutralise NO using specialised enzymes known as flavodiiron proteins (FDPs). However, how exactly the enzymes achieve this neutralisation has been a long-standing open question in enzymology.
More precisely, despite extensive investigations over the past two decades, the precise structure and behaviour of the enzyme’s iron-based active site – the pocket that binds NO – remained unresolved. Now, researchers have revealed how the active centres of these enzymes rearrange between oxidised and reduced states forming hydroxo bridges, which were previously undetected. They found that reduction, i.e. the gain of electrons, triggers a structural rearrangement of these hydroxo bridges resulting in activation the enzymes. Activated FDPs can now bind nitric oxide and convert it to harmless nitrous oxide (N2O). For their study, which was published in the Proceedings of the National Academy of Sciences (PNAS), the authors used a unique multimodal approach that combines quantum modelling with spectroscopic techniques allowing them to probe the active site in exquisite detail. Part of the nuclear resonance vibrational spectroscopy measurements was performed at the PETRA III beamline P01. The redox-dependent structural switch explains how flavodiiron proteins convert NO to N2O and resolves inconsistencies between earlier models.
“Active sites with two irons occur in many diverse enzymes involved in methane oxidation, DNA synthesis and fatty acid metabolism, so these findings really have broad relevance for biology, chemistry and biotechnology”, says Lars Lauterbach, corresponding author of the study and professor at RWTH Aachen University.
The work also delivers an important methodological insight: The authors used synchrotron radiation as part of their structural investigations and found that even low-intensity radiation can induce changes in sensitive metal centres during measurements. This finding has implications well beyond FDPs and is relevant for the structural analysis of many enzymes that have metals as co-factors, highlighting the need for radiation-aware experimental strategies.
The study was led by Filipe Folgosa (ITQB NOVA), Vladimir Pelmenschikov (TU Berlin) and Giorgio Caserta (TU Berlin) as co-first authors. The work was funded in part by the German Research Foundation (DFG) through Germany’s Excellence Strategy, including the Clusters of Excellence, Fuel Science Center (FSC) and UniSysCat.
(Partly from DESY News / RWTH Aachen University's Press Release)
Reference:
F. Folgosa, V. Pelmenschikov, G. Caserta, M. Keck, C. Lorent, K. Laun, Y. Yoda, L.B. Gee, M. Kaupp, K. Tamasaku, J.A. Birrell, I. Sergueev, C. Limberg, M. Teixeira, & L. Lauterbach, Hydroxo-bridged active site of flavodiiron NO reductase revealed by NRVS and DFT, Proc. Natl. Acad. Sci. U.S.A. 123 (2) e2512429123, DOI: 10.1073/pnas.2512429123
