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

Journal of the American Chemical Society (Impact Factor: 12.11). 11/2012; 134(48). DOI: 10.1021/ja308049u
Source: PubMed


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 applied this to resolve the hydrogen bond interactions with the reduced Rieske-type [2Fe-2S] cluster by two-dimensional pulsed electron spin resonance (EPR) 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 back-ground. 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.

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Available from: Amgalanbaatar Baldansuren, Jan 24, 2015
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