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Dissection of Hydrogen Bond Interaction Network around an Iron–Sulfur Cluster by Site-Specific Isotope Labeling of Hyperthermophilic Archaeal Rieske-Type Ferredoxin

Iwasaki, Toshio, Fukazawa, Risako, Miyajima-Nakano, Yoshiharu, Baldansuren, Amgalanbaatar, Matsushita, Shinichi, Lin, Myat T., Gennis, Robert B., Hasegawa, Kazuya, Kumasaka, Takashi, Dikanov, Sergei A.
Journal of the American Chemical Society 2012 v.134 no.48 pp. 19731-19738
Escherichia coli, electron paramagnetic resonance spectroscopy, histidine, hydrogen bonding, isotope labeling, ligands, metalloproteins, nitrogen, stable isotopes
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 ¹⁴Nδ ligands of the cluster decreased non-coordinating ¹⁵N signal intensities via the cross-suppression effect, an inverse labeling strategy was employed in which ¹⁴N amino acid-labeled archaeal Rieske-type ferredoxin samples were examined in an ¹⁵N-protein background. This has directly identified Lys45 Nα 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.