Spectroscopic and Computational Evaluation of the Structure of the High-Spin Fe(IV)-Oxo Intermediates in Taurine: r-Ketoglutarate Dioxygenase from Escherichia coli and Its His99Ala Ligand Variant Sebastian Sinnecker, ² Nina Svensen, ‡ Eric W. Barr, ‡ Shengfa Ye, ²,§ J. Martin Bollinger, Jr.,* ,‡,| Frank Neese,* ,²,§ and Carsten Krebs* ,‡,| Contribution from the Max-Planck Institut fu ¨r Bioanorganische Chemie, D-45470 Mu ¨lheim an der Ruhr, Germany, Department of Biochemistry and Molecular Biology, The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802, Institut fu ¨r Physikalische und Theoretische Chemie, UniVersita ¨t Bonn, D-53115 Bonn, Germany, and Department of Chemistry, The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802 Received November 9, 2006; E-mail: jmb21@psu.edu; neese@thch.uni-bonn.de; ckrebs@psu.edu Abstract: The Fe(II)- and R-ketoglutarate (RKG)-dependent dioxygenases activate O2 for cleavage of unactivated C-H bonds in their substrates. The key intermediate that abstracts hydrogen in the reaction of taurine:RKG dioxygenase (TauD), a member of this enzyme family, was recently characterized. The intermediate, denoted J, was shown to contain an iron(IV)-oxo unit. Other important structural features of J, such as the number, identity, and disposition of ligands in the Fe(IV) coordination sphere, are not yet understood. To probe these important structural features, a series of models for J with the Fe(IV) ion coordinated by the expected two imidazole (from His99 and His255), two carboxylate (succinate and Asp101), and oxo ligands have been generated by density functional theory (DFT) calculations, and spectroscopic parameters (Mo ¨ ssbauer isomer shift, quadrupole splitting, and asymmetry parameter, 57 Fe hyperfine coupling tensor, and zero field splitting parameters, D and E/D) have been calculated for each model. The calculated parameters of distorted octahedral models for J, in which one of the carboxylates serves as a monodentate ligand and the other as a bidentate ligand, and a trigonal bipyramidal model, in which both carboxylates serve as monodentate ligands, agree well with the experimental parameters, whereas the calculated parameters of a square pyramidal model, in which the oxo ligand is in the equatorial plane, are inconsistent with the data. Similar analysis of the Fe(IV) complex generated in the variant protein with His99, the residue that contributes the imidazole ligand cis to the oxo group, replaced by alanine suggests that the deleted imidazole is replaced by a water ligand. This work lends credence to the idea that the combination of Mo ¨ ssbauer spectroscopy and DFT calculations can provide detailed structural information for reactive intermediates in the catalytic cycles of iron enzymes. 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