doi:10.1039/d6dt00171hengSerra, IleniaSchmidt, DanielFurtmüller, Paul G.González, Pablo J.Obinger, ChristianVan Doorslaer, SabineGarcía-Rubio, InésNitrite binding modes in ferric heme proteins probed by HYSCORE spectroscopyART-2026-149145Nitrite plays a fundamental role in the environmental nitrogen cycle and various biochemical reactions. Heme proteins such as globins and peroxidases, often participate in nitrite-mediated pathways, sparking interest in the coordination geometry of nitrite to the heme iron. In most cases, nitrite binds the ferric heme iron via the nitrogen atom (N-nitro mode), while for myoglobin and hemoglobin a less common O-nitrito ligation through one oxygen atom was reported. Our previous study on nitrite binding to the heme-containing enzyme chlorite dismutase (Cld) using continuous-wave electron paramagnetic resonance and crystal-field theory, supported by molecular dynamics simulations, suggested the coexistence of both O-nitrito and N-nitro ligation modes. Here, we present an in-depth hyperfine sublevel correlation (HYSCORE) analysis of NO2-ligated ferric horse heart myoglobin, a Clade-II Cld from Cyanothece sp. PCC7425 and a Clade-I Cld from Magnetospirillum sp. 15N-labelled nitrite was used to discriminate the signals ascribed to the nitrogen nucleus of nitrite from the endogenous N nuclei. The O-nitrito and N-nitro modes can be distinguished based on the nitrite nitrogen hyperfine coupling. Moreover, we describe a distinct HYSCORE spectral fingerprint for the O-nitrito binding mode which can be used as direct evidence of the ligation mode without further detailed analysis. Together, these results provide a generally applicable EPR/HYSCORE-based tool for (bio)inorganic nitrite coordination chemistry of heme systems, enabling more reliable interpretation of nitrite reactivity and mechanism in heme-based catalysts and nitrite-processing enzymes.2026http://zaguan.unizar.es/record/17104510.1039/d6dt00171hhttp://zaguan.unizar.es/record/171045oai:zaguan.unizar.es:171045info:eu-repo/grantAgreement/ES/DGA/E09-23Rinfo:eu-repo/grantAgreement/EC/H2020/813209/EU/Paramagnetic Species in Catalysis Research. A Unified Approach Towards Heterogeneous, Homogeneous and Enzyme Catalysis/PARACATThis project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 813209-PARACATinfo:eu-repo/grantAgreement/ES/MICINN/PID2021-127287NB-I00info:eu-repo/grantAgreement/ES/MICIU/CEX2023-001286-SDalton Transactions 55, 14 (2026), 5769-5779byhttps://creativecommons.org/licenses/by/4.0/deed.esinfo:eu-repo/semantics/openAccess