Steady-state and transient kinetic analyses of taurine/alpha-ketoglutarate dioxygenase: effects of oxygen concentration, alternative sulfonates, and active-site variants on the FeIV-oxo intermediate.

Taurine/alpha-ketoglutarate (alphaKG) dioxygenase (TauD), an archetype alphaKG-dependent hydroxylase, is a non-heme mononuclear Fe(II) enzyme that couples the oxidative decarboxylation of alphaKG with the conversion of taurine to aminoacetaldehyde and sulfite. The crystal structure of taurine-alphaKG-Fe(II)TauD is known, and spectroscopic studies have kinetically defined the early steps in catalysis and identified a high-spin Fe(IV)-oxo reaction intermediate. The present analysis extends our understanding of TauD catalysis by investigating the steady-state and transient kinetics of wild-type and variant forms of the enzyme with taurine and alternative sulfonates. TauD proteins substituted at residues surrounding the active site were shown to fold properly based on their abilities to form a diagnostic chromophore associated with the anaerobic Fe(II)-alphaKG chelate complex and to generate a tyrosyl radical upon subsequent reaction with oxygen. Steady-state studies of mutant proteins confirmed the importance of His 70 and Arg 270 in binding the sulfonate moiety of taurine and indicated the participation of Asn 95 in recognizing the substrate amine group. The N97A and S158A variants are likely to undergo an increase in hydrophobicity and expansion of the substrate-binding pocket, thus accounting for their decreased K(m) toward pentanesulfonic acid compared to wild-type TauD. Stopped-flow UV-visible spectroscopic examination of the reaction of oxygen with taurine-alphaKG-Fe(II)TauD confirmed a minimal three-step sequence of reactions attributed to Fe(IV)-oxo formation (k(1)), bleaching to the Fe(II) state upon substrate hydroxylation (k(2)), rebinding of excess substrates (k(3)), and indicated that none of the steps exhibit detectable solvent k(H)/k(D) isotope effects. This demonstrates that no protons are involved in the rate-determining step of Fe(IV)-oxo formation, in contrast to heme iron oxygenases. The Fe(IV)-oxo species is likely to be utilized in conversion of the alternative substrates pentanesulfonic acid and 3-N-morpholinopropanesulfonic acid; however, this spectroscopic intermediate was not detected because of the decreased k(1)/k(2) ratio. With taurine, k(1) was shown to depend on the oxygen concentration allowing calculation of a second-order rate constant of 1.58 x 10(5) M(-)(1) s(-)(1) for this irreversible reaction. Stopped-flow analyses of TauD variants provided several insights into how the protein environment influences the rates of Fe(IV)-oxo formation and decay. The Fe(IV)-oxo species was not detected in the N95D or N95A variants because of a reduced k(1)/k(2) ratio, likely related to a decreased substrate-dependent conversion of the six-coordinate to five-coordinate metal site.

Direct detection of oxygen intermediates in the non-heme Fe enzyme taurine/alpha-ketoglutarate dioxygenase.

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.