Transient iron coordination sites in proteins: Exploiting the dual nature of paramagnetic NMR

We provide here an historical perspective of NMR applied to iron-containing proteins. At first, the field developed using paramagnetic NMR: the 1H NMR spectra of heme and FeS proteins were used as clear spectroscopic fingerprints of the electronic structure of the metal ion and its inner and outer coordination spheres. Starting 1994, NMR of metalloproteins was focused more on the protein part and the determination of the 3D structures in solution of small proteins was achieved; paramagnetic NMR observables were exploited as non-conventional NMR constraints. With time, NMR has gained attention as a methodology to monitor protein–protein interactions, becoming a unique tool to learn about the interaction surfaces in weak transient complexes. The recent interest in the understanding of metal ion homeostatis and metal cofactor assembly has led to a renewed interest in the application of NMR: (i) as a spectroscopic tool, to characterize the novel binding site of metal centers where the metal is no more an integral part of the protein but rather a chemical entity that has to be transferred along trafficking pathways or a cofactor that has to undergo specific reactions and (ii) as a structural method for the definition of the metalmediated protein–protein interactions. The reported case examples cover the bacterial heme acquisition system, the cytosolic FeS protein assembly and mitochondrial FeS cluster assembly machineries and the eukaryotic iron-storage ferritin.