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Nuclear Magnetic Resonance (NMR) spectroscopy is the ideal tool to address the structure, reactivity and dynamics of both inorganic and biological substances. The knowledge of nuclear spin interaction and spin dynamics is increasingly consolidated, and this allows for tailoring pulse sequences. When dealing with paramagnetic systems, several decades of research have led to the development of rule-of-the-thumb criteria for optimizing the experiments, allowing for the detection of nuclei that are in very close proximity to the metal center. In turn, the observation of these systems, coupled with the development of robust and accessible quantum chemical methods, is promising to provide a link between the spectra and the structural features through the interpretation of the electronic structure. In this review, we list the challenges encountered and propose solutions for dealing with paramagnetic systems with the greatest satisfaction. In our intentions, this is a practical toolkit for optimizing acquisition and processing parameters for routine experiments aimed at detecting signals influenced by the hyperfine interaction. The implications of paramagnetic shift and line broadening are examined. With this endeavor, we wish to encourage non-expert users to consider the application of paramagnetic NMR to their systems.

In paramagnetic metalloproteins, longitudinal relaxation rates of 13C′ and 13Cα nuclei can be measured using 13C detected experiments and converted into electron spin-nuclear spin distance restraints, also known as Paramagnetic Relaxation Enhancement (PRE) restraints. 13C are less sensitive to paramagnetism than 1H nuclei, therefore, 13C based PREs constitute an additional, non-redundant, structural information. We will discuss the complementarity of 13C PRE restraints with 1H PRE restraints in the case of the High Potential Iron Sulfur Protein (HiPIP) PioC, for which the NMR structure of PioC has been already solved by a combination of classical and paramagnetism-based restraints. We will show here that 13C R1 values can be measured also at very short distances from the paramagnetic center and that the obtained set of 13C based restraints can be added to 1H PREs and to other classical and paramagnetism based NMR restraints to improve quality and quantity of the NMR information.