Paramagnetic NMR spectroscopy and density functional calculations in the analysis of the geometric and electronic structures of iron-sulfur proteins.

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