Dissection of hydrogen bond interaction network around an iron-sulfur cluster by site-specific isotope labeling of hyperthermophilic archaeal rieske-type ferredoxin

Toshio Iwasaki, Risako Fukazawa, Yoshiharu Miyajima-Nakano, Amgalanbaatar Baldansuren, Shinichi Matsushita, Myat T. Lin, Robert B. Gennis, Kazuya Hasegawa, Takashi Kumasaka, Sergei A. Dikanov

Research output: Contribution to journalArticle

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 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.

Original languageEnglish (US)
Pages (from-to)19731-19738
Number of pages8
JournalJournal of the American Chemical Society
Volume134
Issue number48
DOIs
StatePublished - Dec 5 2012

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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