Abstract. Paramagnetic NMR spectroscopy and iron-sulfur (Fe–S) proteins have maintained a synergic relationship for decades. Indeed, the hyperfine shifts with their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues have been extensively used as a fingerprint of the type and of the oxidation state of the Fe–S cluster within the protein frame. The identification of NMR signals from residues surrounding the metal cofactor is crucial for understanding the structure-function relationship in Fe–S proteins, but it is generally impaired in standard NMR experiments by paramagnetic relaxation enhancement due to the presence of the paramagnetic cluster(s). On the other hand, the availability of systems of different size and stability has, over the years, stimulated NMR spectroscopists to exploit iron-sulfur proteins as paradigmatic cases to develop experiments, models and protocols. Here, the cluster binding properties of human mitoNEET have been investigated by one-dimensional and two-dimensional 1H diamagnetic and paramagnetic NMR, in its oxidized and reduced states. The NMR spectra of both oxidation states of mitoNEET appeared to be significantly different from those reported for previously investigated [Fe2S2]2+/+ proteins. We show how the use of 1D NOE experiments, 13C direct-detected experiments, and the optimization of NMR experiments for paramagnetic systems significantly reduce the blind sphere of the protein around the paramagnetic cluster. The application of this approach provided a detailed description of the unique electronic properties of mitoNEET, that are responsible for its biological function. Indeed, the NMR properties suggested that the specific electronic structure of the cluster possibly drives the functional properties of different [Fe2S2] proteins.
IR and NMR properties of N-base:PH2F:BeX2 ternary and corresponding binary complexes stabilised by pnicogen and beryllium bonds