Self-hydroxylation of taurine/alpha-ketoglutarate dioxygenase: evidence for more than one oxygen activation mechanism.

2-Aminoethanesulfonic acid (taurine)/alpha-ketoglutarate (alphaKG) dioxygenase (TauD) is a mononuclear non-heme iron enzyme that catalyzes the hydroxylation of taurine to generate sulfite and aminoacetaldehyde in the presence of O2, alphaKG, and Fe(II). Fe(II)TauD complexed with alphaKG or succinate, the decarboxylated product of alphaKG, reacts with O2 in the absence of prime substrate to generate 550- and 720-nm chromophores, respectively, that are interconvertible by the addition or removal of bound bicarbonate and have resonance Raman features characteristic of an Fe(III)-catecholate complex. Mutagenesis studies suggest that both reactions result in the self-hydroxylation of the active-site residue Tyr73, and liquid chromatography nano-spray mass spectrometry/mass spectrometry evidence corroborates this result for the succinate reaction. Furthermore, isotope-labeling resonance Raman studies demonstrate that the oxygen atom incorporated into the tyrosyl residue derives from H2 18O and 18O2 for the alphaKG and succinate reactions, respectively, suggesting distinct mechanistic pathways. Whereas the alphaKG-dependent hydroxylation likely proceeds via an Fe(IV) = O intermediate that is known to be generated during substrate hydroxylation, we propose Fe(III)-OOH (or Fe(V) = O) as the oxygenating species in the succinate-dependent reaction. These results demonstrate the two oxygenating mechanisms available to enzymes with a 2-His-1-carboxylate triad, depending on whether the electron source donates one or two electrons.