Molecular bases of cluster release and destabilizing effects of nitric oxide

Abstract

Funding Information: Cindy Vallières was supported by Marie Skłodowska-Curie Actions MSCA-IF-2020 (FungiFeS/ID 101030584 ). Deborah Grifagni was supported by European Molecular Biology Organization (EMBO) through Grant Application Number 9373 and by PHC Galileo PROJECT N° 48929TH. L. B., F. C. and M. P. acknowledges the support by the Italian Ministry for University and Research (FOE funding) to the CERM/CIRMMP Italian Centre of Instruct-ERIC, a ESFRI Landmark. OA Fees have been covered with the support of the European Cooperation in Science and Technology ( COST ) Action CA21115. Funding Information: This article is based on work from COST Action FeSImmChemNet (CA21115) supported by COST (European Cooperation in Science and Technology). We acknowledge the financial support from the Agence Nationale de la Recherche (ANR-21-CE44-0016) and from the European Union - NextGenerationEU-National Recovery and Resilience Plan, Mission 4 Component 2-Investment 1.5-THE-Tuscany Health Ecosystem-ECS00000017-CUP B83C22003920001. This work was funded also by national funds through FCT–Fundação para a Ciência e a Tecnologia , I.P. (FCT), Project MOSTMICRO-ITQB with refs UIDB/04612/2020 and UIDP/04612/2020 , and LS4FUTURE Associated Laboratory ( LA/P/0087/2020 ). The authors are grateful to Inês B. Trindade for helpful discussions on the electrochemical data. Publisher Copyright: © 2024 The AuthorsThe NEET proteins, an important family of iron-sulfur (Fe-S) proteins, have generated a strong interest due to their involvement in diverse diseases such as cancer, diabetes, and neurodegenerative disorders. Among the human NEET proteins, CISD3 has been the least studied, and its functional role is still largely unknown. We have investigated the biochemical features of CISD3 at the atomic and in cellulo levels upon challenge with different stress conditions i.e., iron deficiency, exposure to hydrogen peroxide, and nitric oxide. The redox and cellular stability properties of the protein agree on a predominance of reduced form of CISD3 in the cells. Upon the addition of iron chelators, CISD3 loses its Fe-S clusters and becomes unstructured, and its cellular level drastically decreases. Chemical shift perturbation measurements suggest that, upon cluster oxidation, the protein undergoes a conformational change at the C-terminal CDGSH domain, which determines the instability of the oxidized state. This redox-associated conformational change may be the source of cooperative electron transfer via the two [Fe2S2] clusters in CISD3, which displays a single sharp voltammetric signal at −31 mV versus SHE. Oxidized CISD3 is particularly sensitive to the presence of hydrogen peroxide in vitro, whereas only the reduced form is able to bind nitric oxide. Paramagnetic NMR provides clear evidence that, upon NO binding, the cluster is disassembled but iron ions are still bound to the protein. Accordingly, in cellulo CISD3 is unaffected by oxidative stress induced by hydrogen peroxide but it becomes highly unstable in response to nitric oxide treatment.publishersversionpublishe