Co-II, Ni-II, and N-oxalylglycine (NOG) are well-known inhibitors of Fe-II/alpha-ketoglutarate (alpha KG)-dependent hydroxylases, but few studies describe their kinetics and no spectroscopic investigations have been reported. Using taurine/alpha KG dioxygenase (TauD) as a paradigm for this enzyme family, time-dependent inhibition assays showed that Co-II and Ni-II follow slow-binding inhibition kinetics. Whereas Ni-II-substituted TauD was non-chromophoric, spectroscopic studies of the Co-II-substituted enzyme revealed a six-coordinate site (protein alone or with alpha KG) that became five-coordinate upon taurine addition. The Co-II spectrum was not perturbed by a series of anions or oxidants, suggesting the Co-II is inaccessible and could be used to stabilize the protein. NOG competed weakly (K-i similar to 290 mu M) with alpha KG for binding to TauD, with the increased electron density of NOG yielding electronic transitions for NOG-Fe-II-TauD and taurine-NOG-Fe-II-TauD and taurine-NOG-Fe-II-TauD at 380 nm (epsilon(380) 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. (c) 2005 Elsevier Inc. All rights reserved.