Interconversion of two oxidized forms of taurine/α-ketoglutarate dioxygenase, a non-heme iron hydroxylase: Evidence for bicarbonate binding

doi:10.1073/pnas.0636740100

Interconversion of two oxidized forms of taurine/α-ketoglutarate dioxygenase, a non-heme iron hydroxylase: Evidence for bicarbonate binding

^*Departments of Microbiology and Molecular Genetics and of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-4320; and ^†Center for Metals in Biocatalysis and Department of Chemistry, University of Minnesota, Minneapolis, MN 55455

Edited by JoAnne Stubbe, Massachusetts Institute of Technology, Cambridge, MA, and approved January 31, 2003 (received for review November 5, 2002)

Abstract

Taurine/α-ketoglutarate (αKG) dioxygenase, or TauD, is a mononuclear non-heme iron hydroxylase that couples the oxidative decarboxylation of αKG to the decomposition of taurine, forming sulfite and aminoacetaldehyde. Prior studies revealed that taurine-free TauD catalyzes an O₂- and αKG-dependent self-hydroxylation reaction involving Tyr-73, yielding an Fe(III)-catecholate chromophore with a λ_max of 550 nm. Here, a chromophore (λ_max 720 nm) is described and shown to arise from O₂-dependent self-hydroxylation of TauD in the absence of αKG, but requiring the product succinate. A similar chromophore rapidly develops with the alternative oxidant H₂O₂. Resonance Raman spectra indicate that the ≈700-nm chromophore also arises from an Fe(III)-catecholate species, and site-directed mutagenesis studies again demonstrate Tyr-73 involvement. The ≈700-nm and 550-nm species are shown to interconvert by the addition or removal of bicarbonate, consistent with the αKG-derived CO₂ remaining tightly bound to the oxidized metal site as bicarbonate. The relevance of the metal-bound bicarbonate in TauD to reactions of other members of this enzyme family is discussed.

Footnotes

↵ ‡ Present address: Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS 39216.
↵ § To whom correspondence should be addressed: 6193 Biomedical and Physical Sciences, Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824-4320. E-mail: hausinge{at}msu.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

αKG,

α-ketoglutarate;

TauD,

taurine/α-ketoglutarate dioxygenase;

DAOCS,

deacetoxycephalosporin C synthase;

ACC,

1-aminocyclopropane-1-carboxylate