Kinetic and spectroscopic investigation of CoII, NiII, and N-oxalylglycine inhibition of the FeII/α-ketoglutarate dioxygenase, TauD

CoII, NiII, and N-oxalylglycine (NOG) are well-known inhibitors of FeII/α-ketoglutarate (αKG)-dependent hydroxylases, but few studies describe their kinetics and no spectroscopic investigations have been reported. Using taurine/αKG dioxygenase (TauD) as a paradigm for this enzyme family, time-dependent inhibition assays showed that CoII and NiII follow slow-binding inhibition kinetics. Whereas NiII-substituted TauD was non-chromophoric, spectroscopic studies of the CoII-substituted enzyme revealed a six-coordinate site (protein alone or with αKG) that became five-coordinate upon taurine addition. The CoII spectrum was not perturbed by a series of anions or oxidants, suggesting the CoII is inaccessible and could be used to stabilize the protein. NOG competed weakly (Ki ∼ 290 μM) with αKG for binding to TauD, with the increased electron density of NOG yielding electronic transitions for NOG-FeII-TauD and taurine-NOG-FeII-TauD at 380 nm (ε380 90-105 M−1 cm−1). The spectra of the NOG-bound TauD species did not change significantly upon oxygen exposure, arguing against the formation of an oxygen-bound state mimicking an early intermediate in catalysis.