RRC ID 52859
Author Iwasaki T, Fukazawa R, Miyajima-Nakano Y, Baldansuren A, Matsushita S, Lin MT, Gennis RB, Hasegawa K, Kumasaka T, Dikanov SA.
Title Dissection of hydrogen bond interaction network around an iron-sulfur cluster by site-specific isotope labeling of hyperthermophilic archaeal Rieske-type ferredoxin.
Journal J Am Chem Soc
Abstract The electronic structure and geometry of redox-active metal cofactors in proteins are tuned by the pattern of hydrogen bonding with the backbone peptide matrix. In this study we developed a method for selective amino acid labeling of a hyperthermophilic archaeal metalloprotein with engineered Escherichia coli auxotroph strains, and we applied this to resolve the hydrogen bond interactions with the reduced Rieske-type [2Fe-2S] cluster by two-dimensional pulsed electron spin resonance technique. Because deep electron spin-echo envelope modulation of two histidine (14)N(δ) ligands of the cluster decreased non-coordinating (15)N signal intensities via the cross-suppression effect, an inverse labeling strategy was employed in which (14)N amino acid-labeled archaeal Rieske-type ferredoxin samples were examined in an (15)N-protein background. This has directly identified Lys45 N(α) as providing the major pathway for the transfer of unpaired electron spin density from the reduced cluster by a "through-bond" mechanism. All other backbone peptide nitrogens interact more weakly with the reduced cluster. The extension of this approach will allow visualizing the three-dimensional landscape of preferred pathways for the transfer of unpaired spin density from a paramagnetic metal center onto the protein frame, and will discriminate specific interactions by a "through-bond" mechanism from interactions which are "through-space" in various metalloproteins.
Volume 134(48)
Pages 19731-8
Published 2012-12-5
DOI 10.1021/ja308049u
PMID 23145461
PMC PMC3515699
MeSH Binding Sites Escherichia coli / genetics Ferredoxins / chemistry* Hydrogen Bonding Iron / chemistry* Isotope Labeling Models, Molecular Oxidation-Reduction Pyrodictiaceae / chemistry Substrate Specificity Sulfolobus solfataricus / chemistry Sulfur / chemistry*
IF 14.695
Times Cited 14
DNA material YM138 (RDB15943) MS1 (RDB15944).