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 journalArticlepeer-review

    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. © 2012 American Chemical Society.

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

    Keywords

    • Binding Sites
    • Escherichia coli
    • Ferredoxins
    • Hydrogen Bonding
    • Iron
    • Isotope Labeling
    • Models, Molecular
    • Oxidation-Reduction
    • Pyrodictiaceae
    • Substrate Specificity
    • Sulfolobus solfataricus
    • Sulfur
    • Journal Article
    • Research Support, N.I.H., Extramural
    • Research Support, Non-U.S. Gov't
    • Research Support, U.S. Gov't, Non-P.H.S.

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