magnetochemistry Review Paramagnetic NMR Spectroscopy Is a Tool to Address Reactivity, Structure, and Protein–Protein Interactions of Metalloproteins: The Case of Iron–Sulfur Proteins Mario Piccioli Magnetic Resonance Center and Department of Chemistry, University of Florence, 50019 Sesto Fiorentino, Italy; [email protected]fi.it Received: 30 August 2020; Accepted: 22 September 2020; Published: 26 September 2020 Abstract: The study of cellular machineries responsible for the iron–sulfur (Fe–S) cluster biogenesis has led to the identification of a large number of proteins, whose importance for life is documented by an increasing number of diseases linked to them. The labile nature of Fe–S clusters and the transient protein–protein interactions, occurring during the various steps of the maturation process, make their structural characterization in solution particularly difficult. Paramagnetic nuclear magnetic resonance (NMR) has been used for decades to characterize chemical composition, magnetic coupling, and the electronic structure of Fe–S clusters in proteins; it represents, therefore, a powerful tool to study the protein–protein interaction networks of proteins involving into iron–sulfur cluster biogenesis. The optimization of the various NMR experiments with respect to the hyperfine interaction will be summarized here in the form of a protocol; recently developed experiments for measuring longitudinal and transverse nuclear relaxation rates in highly paramagnetic systems will be also reviewed. Finally, we will address the use of extrinsic paramagnetic centers covalently bound to diamagnetic proteins, which contributed over the last twenty years to promote the applications of paramagnetic NMR well beyond the structural biology of metalloproteins. Keywords: iron–sulfur proteins; paramagnetic NMR; iron–sulfur cluster biogenesis; solution structures; metalloproteins 1. Introduction Paramagnetic nuclear magnetic resonance (NMR) has been, over the last twenty years, one of more lively and active branches of biomolecular NMR, widely used to characterize metalloproteins. Indeed, metalloproteins represent a wide percentage of the entire proteome and a large share of metalloproteins is paramagnetic. The first solution structure of a paramagnetic metalloprotein was solved in 1994 [1]. Since then, many protein structures of paramagnetic systems were obtained in solution in different oxidation states [2–6]. The quest for novel methodological advancements flourished, redox dependent effects were investigated [7], and a number of paramagnetism-based NMR restraints were proposed [8]. Likewise, the main programs for solution structures calculations were revisited and complemented with routines able to tackle paramagnetism-derived NMR restraints and to combine them with conventional NMR restraints [9,10]. Over the last decade, the popularity of NMR-based structural biology has been shadowed by a number of factors: the “mild” success of NMR as a high-throughput method for protein structure determination, the increasing performances of structure prediction approaches, the appearance into the scene of the brilliant and raising star of cryo-electron microscopy, that is replacing NMR for an increasing number of applications in proteomics and interactomics and is integrated with NMR to obtain structure determination of very large complexes at an atomic resolution [11,12]. Nevertheless, many methodological developments have been proposed Magnetochemistry 2020, 6, 46; doi:10.3390/magnetochemistry6040046 www.mdpi.com/journal/magnetochemistry Magnetochemistry 2020, 6, x FOR PEER REVIEW 2 of 20 determination of very large complexes at an atomic resolution [11,12]. Nevertheless, many Magnetochemistrymethodological2020 developments, 6, 46 have been proposed that have contributed to expand the range2 of 21of applications [13–18]; within this scenario, the exploitation of the hyperfine interaction has been one of the most exciting aspects. that haveIn paramagnetic contributed tometalloproteins, expand the range the of hyperfine applications interaction [13–18]; withinbetween this electron scenario, spin the and exploitation nuclear ofspins the can hyperfine be a tool interaction to: (i) elucidate has been catalytic one of mechanisms the most exciting in metalloenzymes aspects. and provide a molecular pictureIn paramagneticof the currently metalloproteins, known protein–protein the hyperfine interaction interaction networks between involving electron metalloproteins; spin and nuclear (ii) spinsuse small can beand a toolstable to: (i)metalloproteins elucidate catalytic as test mechanisms systems to in develop metalloenzymes novel experiments and provide and a molecularto obtain pictureadditional of the NMR currently restraints known that protein–protein could eventually interaction be used networks to study involving larger metalloproteins;and unstable proteins. (ii) use smallHowever, and stableprobably metalloproteins the most intriguing as test systems aspect tois developthe use of novel metal-based experiments spin and labels to obtain as an additional NMRsource restraints of structural that could constraints eventually in bediamagnetic used to study proteins. larger andThis unstable succeeded proteins. to extend However, the probablyrange of thesystems most that intriguing can be aspectstudied is thevia useparamagnetic of metal-based NMR: spin extrinsic labels para as anmagnetic additional centers source contributed of structural to constraintspromote the in applications diamagnetic of proteins. paramagnetic This succeeded NMR also to beyond extend thestructural range of biology systems in that solution can be [19–25]. studied via paramagneticOne of the aspects NMR: to extrinsic which paramagnetic paramagnetic NMR centers has contributed substantially to promotecontributed the is applications the discovery of paramagneticof molecular machineries NMR also beyond devoted structural to the biogenesis biology in of solution iron sulfur [19– 25proteins]. and the study of cellular traffickingOne of of the metal aspects cofactors. to which I will paramagnetic briefly overview NMR the has contribution substantially of contributedNMR studies is for the elucidating discovery ofaspects molecular of iron–sulfur machineries proteins devoted biogenesis, to the biogenesis where the of ironunderstanding sulfur proteins at a andmolecular the study level of provide cellular trasnapshotsfficking of metalprotein–protein cofactors. Iinteractions, will briefly overviewwhich are the crucial contribution for the biomedical of NMR studies aspects. for Then, elucidating I will aspectspresent ofhere iron–sulfur a summary proteins of the biogenesis, recent developments where the understanding in NMR methodologies at a molecular for levelparamagnetic provide snapshotsproteins and of protein–proteinshow how they can interactions, be used within which solution are crucial structure for the calculations. biomedical aspects.Finally, I Then, will briefly I will presentoverview here how a summary paramagnetic of the NMR recent came developments under the in sp NMRotlights methodologies when extrinsic for paramagnetic paramagnetic proteins agents andhave show been howattached they to can biomolecules be used within and solution used as structure a source calculations.of paramagnetism Finally, based I will NMR briefly restraints. overview how paramagnetic NMR came under the spotlights when extrinsic paramagnetic agents have been attached2. Paramagnetic to biomolecules NMR and used as a source of paramagnetism based NMR restraints. 2. ParamagneticThe theory NMRof the hyperfine interaction between electron spins and nuclear spins and its consequences on the nuclear relaxation properties have been exhaustively reviewed [2,26]. For the The theory of the hyperfine interaction between electron spins and nuclear spins and its ease of the reader, I will recap here the terms that are of major use in paramagnetic systems. The consequences on the nuclear relaxation properties have been exhaustively reviewed [2,26]. For the ease hyperfine shift, i.e., the contribution to the chemical shift arising from the hyperfine interaction, can of the reader, I will recap here the terms that are of major use in paramagnetic systems. The hyperfine be factorized into a contact (CS) and pseudo-contact (PCS) contributions, according to Equations (1)– shift, i.e., the contribution to the chemical shift arising from the hyperfine interaction, can be factorized (3) into a contact (CS) and pseudo-contact (PCS) contributions, according to Equations (1)–(3) ∂ = ∂ + ∂ obs CS PCS (1) @obs = @CS + @PCS (1) μ + A ggµBBSS(S(S+ 1)1) ∂@ == (2)(2) CSCS 3γγ kT 3 II kT 1 para 2 3 para 2 @PCS = Dχax (3 cos θMI 1) + Dχ (sin θMI cos 2'MI) (3) 121πr3 − 23 rh ∂ = MI Δχ para (3cos2 θ −1) + Δχ para (sin 2 θ cos 2φ ) (3) PCS π 3 ax MI rh MI MI where A is the hyperfine12 couplingrMI constant, which is proportional2 to the electron spin density at the nucleus and can be anisotropic due to electron orbital contributions; g is the average g value along where A is the hyperfine coupling constant, which is proportional to the electron spin density at the the principal directions of the contact coupling, when the latter is anisotropic; µ is the electron Bohr nucleus and can be anisotropic due to electron orbital contributions; g is the averageB g value along magneton; S is the electron spin number; γI is the gyromagnetic ratio of a generic I nucleus; k is the principal directions of the contact coupling, when the latter is anisotropic; μB is the