| Abstract
| - Taurine/α-ketoglutarate dioxygenase (TauD), a non-heme mononuclear Fe(II) oxygenase,liberates sulfite from taurine in a reaction that requires the oxidative decarboxylation of α-ketoglutarate(αKG). The lilac-colored αKG-Fe(II)TauD complex (λmax = 530 nm; ε530 = 140 M-1·cm-1) reacts withO2 in the absence of added taurine to generate a transient yellow species (λmax = 408 nm, minimum of1600 M-1·cm-1), with apparent first-order rate constants for formation and decay of ∼0.25 s-1 and ∼0.5min-1, that transforms to yield a greenish brown chromophore (λmax = 550 nm, 700 M-1·cm-1). Thelatter feature exhibits resonance Raman vibrations consistent with an Fe(III) catecholate species presumedto arise from enzymatic self-hydroxylation of a tyrosine residue. Significantly, 18O labeling studies revealthat the added oxygen atom derives from solvent rather than from O2. The transient yellow species, identifiedas a tyrosyl radical on the basis of EPR studies, is formed after αKG decomposition. Substitution of twoactive site tyrosine residues (Tyr73 and Tyr256) by site-directed mutagenesis identified Tyr73 as thelikely site of formation of both the tyrosyl radical and the catechol-associated chromophore. The involvementof the tyrosyl radical in catalysis is excluded on the basis of the observed activity of the enzyme variants.We suggest that the Fe(IV) oxo species generally proposed (but not yet observed) as an intermediate forthis family of enzymes reacts with Tyr73 when substrate is absent to generate Fe(III) hydroxide (capableof exchanging with solvent) and the tyrosyl radical, with the latter species participating in a multistepTauD self-hydroxylation reaction.
|
