Web Release Date: January 31,
O2- and 
-Ketoglutarate-Dependent Tyrosyl Radical Formation in TauD, an -Keto
Acid-Dependent Non-Heme Iron Dioxygenase
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Departments of Microbiology and Molecular Genetics, Biochemistry and Molecular Biology, and Chemistry, Michigan State University, East Lansing, Michigan 48824, and Department of Chemistry and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 55455
Received September 11, 2002
Revised Manuscript Received December 18, 2002
Abstract:
Taurine/-ketoglutarate dioxygenase (TauD), a non-heme mononuclear Fe(II) oxygenase,
liberates sulfite from taurine in a reaction that requires the oxidative decarboxylation of -ketoglutarate
(KG). The lilac-colored KG-Fe(II)TauD complex (
max = 530 nm; 
530 = 140 M-1·cm-1) reacts with
O2 in the absence of added taurine to generate a transient yellow species (max = 408 nm, minimum of
1600 M-1·cm-1), with apparent first-order rate constants for formation and decay of ~0.25 s-1 and ~0.5
min-1, that transforms to yield a greenish brown chromophore (max = 550 nm, 700 M-1·cm-1). The
latter feature exhibits resonance Raman vibrations consistent with an Fe(III) catecholate species presumed
to arise from enzymatic self-hydroxylation of a tyrosine residue. Significantly, 18O labeling studies reveal
that the added oxygen atom derives from solvent rather than from O2. The transient yellow species, identified
as a tyrosyl radical on the basis of EPR studies, is formed after KG decomposition. Substitution of two
active site tyrosine residues (Tyr73 and Tyr256) by site-directed mutagenesis identified Tyr73 as the
likely site of formation of both the tyrosyl radical and the catechol-associated chromophore. The involvement
of 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 for
this family of enzymes reacts with Tyr73 when substrate is absent to generate Fe(III) hydroxide (capable
of exchanging with solvent) and the tyrosyl radical, with the latter species participating in a multistep
TauD self-hydroxylation reaction.
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