Inorganic Chemistry 1994, 33, 83-87
One- and Two-Dimensional 1H NMR Studies of the Active Site of Iron(II) Superoxide Dismutase from Escherichia coli
Ming, L.-J.*
Department of Chemistry and Institute for
Lynch, J. B.; Holz, R. C.; Que, L., Jr.*
Department of Chemistry
Abstract:
The iron(II) superoxide dismutase
(FeSOD) from Escherichia coli exhibits
relatively sharp well-resolved paramagnetically
shifted NMR signals. These signals can be associated with the endogenous ligands characterized by X-ray crystallography for the
oxidized form (FeSOD). Our results demonstrate that
the active site remains intact upon reduction of the Fe(III)
site, retaining the same coordination modes for the three N-epsilon-coordinated
His residues (presumably His-26, His-75, and His-162) and the Asp residue
(presumably Asp-158). The N-H resonances of the coordinated histidines
are found at 88, 43, and 37 ppm, while the signals at 24.5 (E), 19 (G), and 15
(J) ppm are assigned to the Asp residue by the observation of nuclear Overhauser effects (NOE) and bond correlations among the
resonances. Distances of 1.9 and 2.4 Angstrom can be
estimated between the protons E and G and the protons E and J, respectively,
attributable to the -CbH2-CaH < spin system of the Asp residue. The Asp assignments are
corroborated by the observation of interresidue
interactions between the CaH's of Asp-158 and Trp-160. The resonances
of the latter residue are identified by NOE and scalar connectivities,
the most critical assignment being the NaH at 22 ppm (F). According to the
crystal structure of the corresponding enzyme from Psedumonas
ovalis (Stoddard, B. L.; Howell, P. L.; Ringe, D.; Petsko, G. A, Biochemistry 1990, 29, 8885-8893),
this proton is hydrogen-bonded to the free carboxylate
oxygen of Asp-158. This interaction thus rationalizes the paramagnetic shift
observed for signal F and its very slow exchange with solvent which occurs only
upon long-term standing.
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