title: Dissection of hydrogen bond interaction network around an iron-sulfur cluster by site-specific isotope labeling of hyperthermophilic archaeal Rieske-type ferredoxin.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi, Tokyo 113-8602, Japan. tiwasaki@nms.ac.jp
authors.forename: Toshio
authors.initials: T
authors.lastname: Iwasaki
authors.forename: Risako
authors.initials: R
authors.lastname: Fukazawa
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Miyajima-Nakano
authors.forename: Amgalanbaatar
authors.initials: A
authors.lastname: Baldansuren
authors.forename: Shinichi
authors.initials: S
authors.lastname: Matsushita
authors.forename: Myat T
authors.initials: MT
authors.lastname: Lin
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
authors.forename: Kazuya
authors.initials: K
authors.lastname: Hasegawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Kumasaka
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Dikanov
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry*; D006860:Hydrogen Bonding; D007501:Iron / Q000737:chemistry*; D007553:Isotope Labeling; D008958:Models, Molecular; D010084:Oxidation-Reduction; D041523:Pyrodictiaceae / Q000737:chemistry; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000737:chemistry; D013455:Sulfur / Q000737:chemistry*
chemical_list: D005288:Ferredoxins; D013455:Sulfur; D007501:Iron