Paramagnetic NMR Spectroscopy and Density Functional Calculations in the Analysis of the Geometric and Electronic Structures of Iron−Sulfur Proteins

Abstract

Paramagnetic NMR spectroscopy has been underutilized in the study of metalloproteins. One difficulty of the technique is that paramagnetic relaxation broadens signals from nuclei near paramagnetic centers. In systems with low electronic relaxation rates, this makes such signals difficult to observe or impossible to assign by traditional methods. We show how the challenges of detecting and assigning signals from nuclei near the metal center can be overcome through the combination of uniform and selective 2H, 13C, and 15N isotopic labeling with NMR experiments that utilize direct one-dimensional (2H, 13C, and 15N) and two-dimensional (13C-X) detection. We have developed methods for calculating NMR chemical shifts and relaxation rates by density functional theory (DFT) approaches. We use the correspondence between experimental NMR parameters and those calculated from structural models of iron-sulfur clusters derived from X-ray crystallography to validate the computational approach and to investigate how structural differences are manifested in these values. We have applied this strategy to three iron-sulfur proteins: Clostridium pasteurianum rubredoxin, Anabaena [2Fe-2S] ferredoxin, and human [2Fe-2S] ferredoxin. Provided that an accurate structural model of the iron-sulfur cluster and surrounding residues is available from diffraction data, our results show that DFT calculations can return NMR observables with excellent accuracy. This suggests that it might be possible to use calculations to refine structures or to generate structural models of active sites when crystal structures are unavailable. The approach has yielded insights into the electronic structures of these iron-sulfur proteins. In rubredoxin, the results show that substantial unpaired electron spin is delocalized across NH...S hydrogen bonds and that the reduction potential can be changed by 77 mV simply by altering the strength of one of these hydrogen bonds. In reduced [2Fe-2S] ferredoxins, hyperfine shift data have provided quantitative information on the degree of valence trapping. The approach described here for iron-sulfur proteins offers new avenues for detailed studies of these and other metalloprotein systems.

NMR Spectroscopy of Paramagnetic Metalloproteins

“…This shift contains structural information; however, it is hidden inside the particular mechanisms of unpaired-electron delocalization. No general protocols are available for solution structure determination, but several attempts can be found in the literature [36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] for the use of such effects in specific cases. Examples are the use of contact shifts on the b-CH 2 protons of cysteine residues coordinated to iron(ii)/iron(iii) ions in ironsulfur proteins to provide dihedral-angle information through Karplus type relationships (Figure 4 A) [37] or the use of contact shifts (or combinations of contact and pseudocontact shifts, see below) of methyl protons in heme proteins containing low-spin iron(iii) ions to determine the orientation of the axial ligand(s) (Figure 4 B).…”

“…A remarkably good agreement with the experiments was obtained. [46,53] The paramagnetism has a strong effect on the nuclei close to the iron center, thereby leading to extreme line broadening and very large hyperfine shifts. [55] Often, the electron magnetic moment is anisotropic, that is, it takes up different values for different orientations of the protein in the external magnetic field.…”

Predicting the NMR Spectra of Paramagnetic Molecules by DFT: Application to Organic Free Radicals and Transition‐Metal Complexes

“…The approach has been extended to more complex molecules, such as porphyrins, [23] models of blue copper [24] and iron-sulfur [25] proteins. Calculated contact shifts have also been employed in the elucidation of the structure of solidphase polymorphs, [26] chromium-based catalysts, [27] and complexes of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) with perfluorocarbons.…”

Exclusively Heteronuclear NMR Experiments to Obtain Structural and Dynamic Information on Proteins

“…Therefore, experiments based on 13 C direct detection that only rely on heteronuclei (protonless NMR) [1,10] proved particularly useful for characterization of paramagnetic proteins also in regions where 1 H resonances are broadened beyond detection. [11][12][13][14][15][16][17][18] For similar reasons, namely, the favourable heteronuclear relaxation properties, 13 C direct-detection NMR experiments were also proposed for studying large macromolecules [19][20][21][22][23][24][25] and/or proteins in micelles, [26] where 2 H isotopic enrichment is necessary to reduce line widths at the expense of the amount of information that can be obtained through 1 H NMR spectroscopy. Carbon-13 direct-detection experiments are also very useful for studying systems that lack a stable 3D structure, where drastic reduction of the chemical-shift dispersion causes severe problems of overlap that are less severe for heteronuclei.…”