Nitrite binding modes in ferric heme proteins probed by HYSCORE spectroscopy

Nitrite 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.