Interconversion of two oxidized forms of taurine/alpha-ketoglutarate dioxygenase, a non-heme iron hydroxylase: evidence for bicarbonate binding.
Taurinealpha-ketoglutarate (alphaKG) dioxygenase, or TauD, is a mononuclear non-heme iron hydroxylase that couples the oxidative decarboxylation of alphaKG to the decomposition of taurine, forming sulfite and aminoacetaldehyde. Prior studies revealed that taurine-free TauD catalyzes an O(2)- and alphaKG-dependent self-hydroxylation reaction involving Tyr-73, yielding an Fe(III)-catecholate chromophore with a lambda(max) of 550 nm. Here, a chromophore (lambda(max) 720 nm) is described and shown to arise from O(2)-dependent self-hydroxylation of TauD in the absence of alphaKG, but requiring the product succinate. A similar chromophore rapidly develops with the alternative oxidant H(2)O(2). Resonance Raman spectra indicate that the approximately 700-nm chromophore also arises from an Fe(III)-catecholate species, and site-directed mutagenesis studies again demonstrate Tyr-73 involvement. The approximately 700-nm and 550-nm species are shown to interconvert by the addition or removal of bicarbonate, consistent with the alphaKG-derived CO(2) 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.
Ryle
Matthew J
MJ
Department of Microbiology, Michigan State University, East Lansing, MI 48824-4320, USA.
Koehntop
Kevin D
KD
Liu
Aimin
A
Que
Lawrence
L
Hausinger
Robert P
RP
D001639:Bicarbonates / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D007656:Ketoglutaric Acids; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity; D013654:Taurine
D001639:Bicarbonates; D007656:Ketoglutaric Acids; D011994:Recombinant Proteins; D013654:Taurine; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C433064:taurine-alpha-ketoglutarate dioxygenase