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O₂- and α-Ketoglutarate-Dependent Tyrosyl Radical Formation in TauD, an α-Keto Acid-Dependent Non-Heme Iron Dioxygenase^†

Matthew J. Ryle,^‡^§ Aimin Liu,^# Rajendra Bose Muthukumaran, Raymond Y. N. Ho, Kevin D. Koehntop, John McCracken, Lawrence Que, Jr., and Robert P. Hausinger*^‡^§

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

Biochemistry, 2003, 42 (7), pp 1854–1862

DOI: 10.1021/bi026832m

Publication Date (Web): January 31, 2003

†

These studies were supported by the National Institutes of Health (Postdoctoral Fellowship GM20196 to M.J.R. and Grants GM063584 to R.P.H., GM33162 to L.Q., and GM54065 to J.M.).

‡

Department of Microbiology and Molecular Genetics, Michigan State University.

Department of Biochemistry and Molecular Biology, Michigan State University.

University of Minnesota.

Current address: Department of Biohemistry, University of Mississippi Medical Center.

Department of Chemistry, Michigan State University.

To whom correspondence should be addressed. Tel: 517-355-6463 ext 1610. Fax: 517-353-8957. E-mail: hausinge@msu.edu.

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 (λ_max = 530 nm; ε₅₃₀ = 140 M^-¹·cm^-¹) reacts with O₂ in the absence of added taurine to generate a transient yellow species (λ_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 (λ_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 αKG 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.