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

Abstract

Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme mononuclear Fe(II) oxygenase, liberates sulfite from taurine in a reaction that requires the oxidative decarboxylation of alpha-ketoglutarate (alphaKG). The lilac-colored alphaKG-Fe(II)TauD complex (lambda(max) = 530 nm; epsilon(530) = 140 M(-)(1) x cm(-)(1)) reacts with O(2) in the absence of added taurine to generate a transient yellow species (lambda(max) = 408 nm, minimum of 1,600 M(-)(1) x cm(-)(1)), with apparent first-order rate constants for formation and decay of approximately 0.25 s(-)(1) and approximately 0.5 min(-)(1), that transforms to yield a greenish brown chromophore (lambda(max) = 550 nm, 700 M(-)(1) x 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, (18)O labeling studies reveal that the added oxygen atom derives from solvent rather than from O(2). The transient yellow species, identified as a tyrosyl radical on the basis of EPR studies, is formed after alphaKG 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.

Oxidative dealkylation DNA repair mediated by the mononuclear non-heme iron AlkB proteins

“…The hydroxylation of the Trp side chain of AlkB, identified to be Trp 178 , suggests formation of an oxidative intermediate in AlkB [45]. This behavior is consistent with other members of the non-heme Fe II /αKG-dependent family [48][49][50]. The mechanism for the reactions for these enzymes, including AlkB, is proposed to use an Fe IV =O intermediate as the reactive oxidant.…”

Incorporation of oxygen into the succinate co‐product of iron(II) and 2‐oxoglutarate dependent oxygenases from bacteria, plants and humans

“…Results with some eukaryotic 2OG dependent hydroxylases, such as thymine 7-hydroxylase (T7H) [11], mammalian type I prolyl 4-hydroxylase [12], enzymes of flavonoid biosynthesis [13] and human prolyl and asparaginyl hydroxylases involved in hypoxic sensing, PHD1 [14] and FIH [15], show that >90% incorporation of oxygen from dioxygen occurs on hydroxylation of their substrates (in the case of some reactions catalysed by the flavonoid oxygenases exchange may occur after initial hydroxylation by a non-oxidative process [13]). This contrasts with results for eukaryotic lysyl hydroxylase where only approximately 10% of 18 O was reported to be incorporated into the peptide product [16] (data reviewed in [8]). In the case of taurine dioxygenase and 2,4-dichlorophenoxyacetate oxygenase where inactivating self-hydroxy-lation of the protein occurs, it was found that no oxygen from dioxygen was incorporated into the modified protein when the reaction was carried out under 18 O 2 gas.…”

“…The incorporation of oxygen from dioxygen into the alcohol product during hydroxylation reactions by this class of enzymes is well documented [8]. Further, incubations of prokaryotic 2OG oxygenases under an 18 O 2 atmosphere have demonstrated that, during hydroxylation reactions, a less than stoichiometric incorporation of oxygen into the hydroxyl group of the product can occur in some cases (e.g., hydroxylation of some substrates catalysed by clavaminic acid synthase and deacetoxy/deacetyl cephalosporin C synthase) [9,10]. This is thought to be due to solvent exchange of one of the reactive ironoxygen intermediates, although the identity of the particular intermediate(/s) that undergo exchange has not been defined.…”

“…The enzymes thymine-7-hydroxylase and c-butyrobetaine hydroxylase have been reported to incorporate >95% and 68% 18 O from 18 O 2 into succinate, respectively [11,19]. These data were obtained by mass spectrometric analysis of the succinate co-product derivatised with bis(trimethylsilyl)trifluoroacetamide to give the bis-trimethylsilyl ester.…”

“…These data were obtained by mass spectrometric analysis of the succinate co-product derivatised with bis(trimethylsilyl)trifluoroacetamide to give the bis-trimethylsilyl ester. In the case of the enzyme deacetoxy/deacetyl cephalosporin C synthase, greater than 90% 18 O incorporation into succinate from 18 O 2 was observed in experiments analysing the shift in 13 C resonance of succinate by NMR spectroscopy [20].…”