Taurine/α-ketoglutarate (αKG) dioxygenase, or TauD, catalyzes the conversion of taurine (2-aminoethanesulfonic acid) to sulfite and aminoacetaldehyde in the presence of O₂, αKG, and Fe(II) as shown in Scheme S1 (1). This Escherichia coli protein is a member of a rapidly expanding enzyme superfamily that utilizes mononuclear Fe(II) active sites to catalyze a diverse range of chemical transformations, usually coupled to the oxidative decarboxylation of an α-keto acid (2–4). Other family members include enzymes that modify protein side chains (5, 6), repair alkylation-damaged DNA (7), degrade compounds in the environment (8–11), and synthesize antibiotics (12–14), plant metabolites (15, 16), or other small molecules (17, 18). Most representatives carry out specific hydroxylation reactions, but examples of enzymes catalyzing desaturations, ring closures, and ring expansion reactions have also been documented (19). Regardless of their overall chemistry, these enzymes generally are thought to form a high-valent iron-oxo intermediate; however, such an intermediate has never been directly observed.

TauD active site. The crystal structure of taurine-αKG-Fe(II)TauD (26) reveals that the metal ion is coordinated by His-99, Asp-101, and His-255 of the protein. The binding positions of the cosubstrates are shown with taurine near, but not coordinated to, the metal ion and αKG bound as a chelate. The positions of two Tyr residues located <10 Å from the metal ion also are shown.

Effect of O₂ on the absorption spectra of selected Fe(II)TauD states. Anaerobically prepared Fe(II)TauD samples were incubated with 100% oxygen in the headspace for at least 2 h and spectroscopically characterized. The samples included O₂-treated Fe(II)TauD (solid trace; 0.6 mM TauD subunit, 0.5 mM ferrous ammonium sulfate, and 25 mM Tris buffer, pH 8.0) and similarly treated enzyme with 3 mM taurine (dot-dot-dash line), 3 mM αKG plus 3 mM taurine (dotted line), or 3 mM succinate (dashed line). For comparison, the spectrum is shown (dot-dash line) for a sample of αKG-Fe(II)TauD mixed with an equal volume of 100% oxygen-saturated buffer after a 2-h incubation. This spectrum is adjusted in intensity so that the final enzyme and metal concentrations are equivalent to the other samples.

Effect of H₂O₂ addition on the absorption spectra of selected Fe(II)TauD states. Anaerobically prepared succinate-Fe(II)TauD (0.6 mM TauD subunit, 0.5 mM ferrous ammonium sulfate, 3 mM succinate, and 25 mM Tris buffer, pH 8.0) was adjusted to 1.5 mM H₂O₂ and allowed to react for 20 min (dashed line). An analogous sample was prepared with the additional presence of 100 mM bicarbonate (solid trace).

Interconversion of Fe(III)-catecholate chromophores. Succinate-Fe(II)TauD was mixed with 100% O₂-saturated buffer (so the final enzyme concentration is half that shown in Fig. 2) and allowed to react for 3 h (dark solid trace). The sample was adjusted to 100 mM bicarbonate and monitored (gray traces) up to 14 h (dashed line).

Resonance Raman spectra (λ_ex 632.8 nm) of (A) the ≈700-nm species generated by H₂O₂ treatment of succinate-Fe(II)TauD (2.5 mM subunit) and (B) the 550-nm species generated by the addition of 100 equivalents of bicarbonate to A.

UV/visible spectroscopic analysis of the reaction of succinate-bound TauD variants with oxygen. Spectra were obtained 2 h after mixing anaerobic succinate-Fe(II)TauD samples (3 mM succinate, 0.6 mM subunit, 0.5 mM ferrous ammonium sulfate in 25 mM Tris buffer, pH 8.0) with equal volumes of 100% O₂-saturated buffer (so the final enzyme concentrations are half those shown in Fig. 2). Spectra are presented for wild-type TauD (dot-dash trace) and the Y256I (dashed line), Y256F (dotted line), Y73I (dot-dot-dash), and Y73S (solid line) variants.

The spectral shift between the 720-nm and 550-nm chromophores can be understood in terms of the nature of the sixth ligand bound to the metal ion, assuming that both spectroscopic species arise from Fe(III) centers coordinated by two catecholate oxygens and the aspartyl and two histidinyl side chain ligands (26). We propose that a water molecule likely occupies the sixth coordination site in the 720-nm chromophore, whereas bicarbonate (and not CO₂) fills this site in the 550-nm chromophore (Scheme S2). The replacement of a neutral ligand with the anionic bicarbonate ligand reduces the Lewis acidity of the metal center and elicits the observed blue shift in the catecholate chromophore [as well as the downshift of the Fe-O4 (dihydroxyphenylalanine) vibration in the resonance Raman spectrum]. In agreement with this explanation, catecholate complexes of other 2-His-1-carboxylate enzymes, such as phenylalanine hydroxylase and tyrosine hydroxylase, exhibit absorption maxima near 700 nm (34–36), whereas synthetic iron-catecholate complexes experience blue shifts in their ligand-to-metal charge-transfer bands as neutral ligands are replaced by carboxylates (35). Thus, the fact that the catecholate chromophore observed initially in the reaction of αKG-Fe(II)TauD with O₂ has an absorbance maximum at 550 nm and a resonance Raman spectrum similar to that derived from succinate-Fe(II)TauD + H₂O₂ + bicarbonate suggests that an anion becomes bound at the sixth ligand position subsequent to oxidative decarboxylation of αKG and hydroxylation of Tyr-73. We propose that this anion is the bicarbonate derived from αKG oxidation, which dissociates slowly from the active site in the absence of taurine (Scheme S2).