New insights into the bacterial machinery that disarms antimicrobials
Oeiras, 13th January 2026
When the human immune system fights infection, it releases antimicrobials. One of these is nitric oxide, which harms microbial cells. To survive, many microorganisms rely on flavodiiron enzymes that eliminate nitric oxide by converting it into the harmless nitrous oxide. Scientists have studied these proteins for years, but the exact structure of the site where this reaction happens, its catalytic site, was not fully established.
Researchers from ITQB NOVA's Functional Biochemistry of Metalloenzymes Laboratory, led by Miguel Teixeira, co-corresponding author, together with several international laboratories, have now uncovered crucial key details about how that process works. In a study now published in PNAS – Proceedings of the National Academy of Sciences, the team revealed the detailed structure of the catalytic site of a flavodiiron protein from the model bacterium Escherichia coli, present in the human gut. The study of these enzymes, namely from human pathogens, has been a major research theme of this research group, with strong in-house (particularly the Macromolecular Crystallography Unit) and international collaborations.
To accomplish this study, the researchers used advanced spectroscopic methods, which examine how molecules interact with electromagnetic radiation. “Each molecule produces a unique vibrational pattern, similar to a fingerprint, allowing to determine its structure”, explains Filipe Folgosa, from ITQB NOVA, and co-first author of the study.
The team combined the experimental data with comprehensive computational tools to understand how the enzyme rearranges itself as it switches between resting and active states. They found that the protein’s active site, which is constituted by two iron atoms, has two small chemical species derived from water, called hydroxo, bridging the iron atoms responsible for the reaction and contributing to hold them in place. When the enzyme is activated by receiving electrons, one of those species is released, giving the iron atoms the ability to bind nitric oxide in the first step of the mechanism. “This feature had not been directly observed before and gives us a new look into the site where nitric oxide reacts”, adds the researcher.
These results give a much clearer understanding of one of the processes by which microbes protect themselves from the immune system, which is essential if we want to find new ways to fight infection. The study also provides broader insights into how enzymes' metal catalytic sites change shape as they work. Such knowledge may inform future efforts to study, inhibit, or replicate these reactions in controlled laboratory and biomedical contexts.
Some of the past and current member of the Functional Biochemistry of Metalloenzymes group involved in the study of flavodiiron enzymes.
Original paper:
PNAS Journal | doi.org/10.1073/pnas.2512429123
Hydroxo-bridged active site of flavodiiron NO reductase revealed by NRVS and DFT
Filipe Folgosa, Vladimir Pelmenschikov, Giorgio Caserta, Matthias Keck, Christian Lorent, Konstantin Laun, Yoshitaka Yoda, Leland B. Geee, Martin Kaupp, Kenji Tamasaku, James A. Birrell, Ilya Sergueev, Christian Limberg, Miguel Teixeira, and Lars Lauterbach
This study was developed in collaboration with the Institut für Chemie, Technische Universität zu Berlin, the Department of Chemistry, Humboldt-Universität zu Berlin, the Deutsches Elektronen-Synchrotron DESY, Hamburg, and the Institute of Applied Microbiology, RWTH Aachen University (Germany); the JASRI and SPring-8 Center, Sayo-gun, Hyogo (Japan); the National Accelerator Laboratory, Menlo Park, California (USA); and the School of Life Sciences, University of Essex, Wivenhoe Park (United Kingdom).
Over the years, this project was financially supported by several grants from Fundação para a Ciência e Tecnologia and also European Union (TIMB3 – Grant agreement ID: 810856).
