Dissection of Hydrogen Bond Interaction Network around an Iron-sulfur Cluster by Site-specific Isotope Labeling of Hyperthermophilic Archaeal Rieske-type Ferredoxin
| Autor: | Kazuya Hasegawa, Risako Fukazawa, Robert B. Gennis, Amgalanbaatar Baldansuren, Sergei A. Dikanov, Yoshiharu Miyajima-Nakano, Shinichi Matsushita, Takashi Kumasaka, Myat T. Lin, Toshio Iwasaki |
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| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: |
Models
Molecular Iron Iron–sulfur cluster Biochemistry Catalysis Article law.invention Substrate Specificity chemistry.chemical_compound Colloid and Surface Chemistry law Metalloprotein Escherichia coli Electron paramagnetic resonance Ferredoxin Histidine chemistry.chemical_classification Binding Sites Chemistry Hydrogen bond Hydrogen Bonding General Chemistry Pyrodictiaceae Amino acid Crystallography Unpaired electron Isotope Labeling Sulfolobus solfataricus Ferredoxins Oxidation-Reduction Sulfur |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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