Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme Fe(II) oxygenase, catalyses the conversion of taurine (2-aminoethanesulfonate) to sulfite and aminoacetaldehyde concurrent with the conversion of alpha-ketoglutarate (alphaKG) to succinate and CO(2). The enzyme allows Escherichia coli to use taurine, widely available in the environment, as an alternative sulfur source. Here we describe the X-ray crystal structure of TauD complexed to Fe(II) and both substrates, alphaKG and taurine. The tertiary structure and fold of TauD are similar to those observed in other enzymes from the broad family of Fe(II)/alphaKG-dependent oxygenases, with closest structural similarity to clavaminate synthase. Using the TauD coordinates, a model was determined for the closely related enzyme 2,4-dichlorophenoxyacetate/alphaKG dioxygenase (TfdA), supporting predictions derived from site-directed mutagenesis and other studies of that biodegradative protein. The TauD structure and TfdA model define the metal ligands and the positions of nearby aromatic residues that undergo post-translational modifications involving self-hydroxylation reactions. The substrate binding residues of TauD were identified and those of TfdA predicted. These results, along with sequence alignment information, reveal how TauD selects a tetrahedral substrate anion in preference to the planar carboxylate selected by TfdA, providing insight into the mechanism of enzyme catalysis.

Taurine/alpha-ketoglutarate dioxygenase (TauD), a member of the broad class of non-heme Fe(II) oxygenases, converts taurine (2-aminoethanesulfonate) to sulfite and aminoacetaldehyde while decomposing alpha-ketoglutarate (alphaKG) to form succinate and CO(2). Under anaerobic conditions, the addition of alphaKG to Fe(II)TauD results in the formation of a broad absorption centered at 530 nm. On the basis of studies of other members of the alphaKG-dependent dioxygenase superfamily, we attribute this spectrum to metal chelation by the substrate C-1 carboxylate and C-2 carbonyl groups. Subsequent addition of taurine perturbs the spectrum to yield a 28% greater intensity, an absorption maximum at 520 nm, and distinct shoulders at 480 and 570 nm. This spectral change is specific to taurine and does not occur when 2-aminoethylphosphonate or N-phenyltaurine is added. Titration studies demonstrate that each TauD subunit binds a single molecule of Fe(II), alphaKG, and taurine. In addition, these studies indicate that the affinity of TauD for alphaKG is enhanced by the presence of taurine. alpha-Ketoadipate, the other alpha-keto acid previously shown to support TauD activity, and alpha-ketocaproate lead to the formation of weak 520 nm-like spectra with Fe(II)TauD in the presence of taurine; however, corresponding spectra at 530 nm are not observed in the absence of taurine. Pyruvate and alpha-ketoisovalerate fail to elicit absorption bands in this region of the spectrum, even in the presence of taurine. Stopped-flow UV-visible spectroscopy reveals that the 530 and 520 nm spectra associated with alphaKG-Fe(II)TauD and taurine-alphaKG-Fe(II)TauD are formed at catalytically competent rates ( approximately 40 s(-)(1)). The rate of chromophore formation was independent of substrate or enzyme concentration, suggesting that alphaKG binds to Fe(II)TauD prior to the formation of a chromophoric species. Significantly, the taurine-alphaKG-Fe(II)TauD state, but not the alphaKG-Fe(II)TauD species, reacts rapidly with oxygen (42 +/- 9 s(-)(1)). Using the data described herein, we develop a preliminary kinetic model for TauD catalysis.

The reaction of substrate-bound taurine/alpha-ketoglutarate dioxygenase with O2 has been studied using cryogenic continuous-flow spectroscopy. Transient absorption spectra acquired at -38 degrees C show an exponential decay of a 318-nm chromophore with an apparent rate of 1.3 s-1. The observed optical changes and their kinetics are consistent with the profile of an Fe(IV) species detected recently by Mössbauer spectroscopy (Price et al., Biochemistry 2003, 42, 7497-7508). Resonance Raman measurement upon excitation at 363.7 nm reveal at least two oxygen isotope-sensitive vibrations at 821/787 cm-1 and 583/555 cm-1 for 16O and 18O derivatives, respectively. An additional mode is likely to be obscured by an ethylene glycol vibration at 865 cm-1 and/or 1089 cm-1. The 821 cm-1 vibration is assigned to the stretching mode of Fe(IV)=O species on the basis of its frequency and isotopic shift amplitude. The 583 cm-1 band is likely to originate from an Fe-O2 precursor of the Fe(IV)=O species, although its structural details are unclear at present.

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.

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The present work defines one MgATP signal transduction pathway in the nitrogenase iron (Fe) protein. Deletion of an amino acid (Leu 127) by site-directed mutagenesis in the protein chain between Asp 125, located in the ATP binding site, and Cys 132, a ligand to the [4Fe-4S] cluster, resulted in protein conformational changes resembling the MgATP-bound state in the absence of any bound nucleotides. Specifically, 1H nuclear magnetic resonance, electron paramagnetic resonance, and circular dichroism spectroscopic properties, along with Fe chelation assays, suggested that deletion of Leu 127 in the Fe protein resulted in changes in the electronic properties of the [4Fe-4S] cluster similar to those normally observed upon MgATP binding to the wild-type Fe protein. Deletion of Leu 127 of the Fe protein lowered the redox potential of of the [4FE-4S] cluster by 112 mV compared to the wild-typeFe protein (-412mV compared to -294 mV). A nearly identical lowering of the redox potential by 120 mV occurs in the wild-type Fe protein upon binding MgATP (-294 mV compared to 420 mV). The L127delta Fe protein did not contain bound nucleotides which could account for the observed conformational changes. The present results support a model in which the protein chain from ASP 125 to Cys 132 acts as one pathway for MgATP signal transduction and suggests a mechanism for this transduction to the [4Fe-4S] cluster. The L127delta Fe protein was found to still bind 2 MgATP or 2 MgADP molecules/Fe protein. Unlike the wild-type Fe protein, the L127delta Fe protein bound 2 ADP molecules/Fe protein in the absence of Mg2+. Finally, the L127delta protein was found to bind to the MoFe protein, although the complex did not catalyze MgATP hydrolysis or substrate reduction. In concurrence with previous models, homologies between the Asp 125 to Cys 132 transduction pathway in Fe protein and the switch II region of the broad class of GTPase signal transduction proteins (G-proteins) are discussed.

The biological reduction of dinitrogen catalyzed by nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. The present work examines the role of a strictly conserved aspartic acid residue of nitrogenase iron protein (Fe protein) in coupling MgATP hydrolysis to electron transfer and substrate reduction. The aspartic acid residue at position 129 in the Azotobacter vinelandii Fe protein has been suggested to participate in nucleotide interactions from its location in the X-ray structure near several amino acids previously identified to participate in nucleotide binding and protein conformational changes. The function of this amino acid was probed by changing aspartic acid to glutamic acid (D129E) and asparagine (D129N) by site-directed mutagenesis. The D129N Fe protein proved to be unstable and could not be purified. Characterization of the purified D129E Fe protein revealed a central role for Asp 129 in the nucleotide-induced protein conformational changes in the Fe protein and possibly in the mechanism of MgATP hydrolysis. Data from EPR, circular dichroism spectroscopy, and Fe2+ chelation rates and the chemical shifts of isotropically shifted protons in the 1H NMR spectra implicate Asp 129 in the nucleotide-induced conformational changes in the Fe protein, which are reflected in changes in the environment of the [4Fe-4S] cluster. The D129E Fe protein was found to bind both MgATP and MgADP with high affinity. The Kd determined for MgADP binding (Kd = 131 microM) was comparable to that found for wild-type Fe protein (128 microM). The affinity for MgATP binding was 1.6 times tighter than that for wild-type Fe protein (370 compared to 580 microM). The midpoint reduction potential of the [4Fe-4S] cluster was similar to that determined for the wild-type Fe protein (-290 mV for wild-type Fe protein and -300 mV for D129E Fe protein). Upon the addition of MgATP or MgADP, the midpoint potentials for wild-type and D129E Fe proteins shifted to -430 and -440 mV, respectively. The D129E Fe protein was also found to bind to the molybdenum-iron protein (MoFe protein) with normal affinity, although it could not support electron transfer to the MoFe protein or MoFe protein-stimulated MgATP hydrolysis.