Steady-State and Transient Kinetic Analyses of Taurine/α-Ketoglutarate Dioxygenase:  Effects of Oxygen Concentration, Alternative Sulfonates, and Active-Site Variants on the FeIV-oxo Intermediate

• Published in: Biochemistry (Easton) 2005-03, Vol.44 (10), p.3845-3855
• Main Authors: Grzyska, Piotr K, Ryle, Matthew J, Monterosso, Greta R, Liu, Jian, Ballou, David P, Hausinger, Robert P
• Format: Article
• Language: English
• Subjects: Alkanesulfonates - chemistry; Alkanesulfonates - metabolism; Binding Sites - genetics; Catalysis; Electron Transport Complex IV - chemistry; Electron Transport Complex IV - metabolism; Escherichia coli Proteins - antagonists & inhibitors; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Ferrous Compounds - chemistry; Iron - chemistry; Ketoglutaric Acids - chemistry; Kinetics; Mixed Function Oxygenases - antagonists & inhibitors; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Mutagenesis, Site-Directed; Oxygen - chemistry; Oxygen - metabolism; Spectrophotometry, Ultraviolet; Substrate Specificity - genetics; Taurine - chemistry; Taurine - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi048746n

Taurine/α-ketoglutarate (αKG) dioxygenase (TauD), an archetype αKG-dependent hydroxylase, is a non-heme mononuclear FeII enzyme that couples the oxidative decarboxylation of αKG with the conversion of taurine to aminoacetaldehyde and sulfite. The crystal structure of taurine-αKG-FeIITauD is known, and spectroscopic studies have kinetically defined the early steps in catalysis and identified a high-spin FeIV-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 FeII-αKG 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-αKG-FeIITauD confirmed a minimal three-step sequence of reactions attributed to FeIV-oxo formation (k 1), bleaching to the FeII 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 FeIV-oxo formation, in contrast to heme iron oxygenases. The FeIV-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 × 105 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 FeIV-oxo formation and decay. The FeIV-oxo species was not detected in t