Kinetic and spectroscopic investigation of Co II, Ni II, and N-oxalylglycine inhibition of the Fe II/α-ketoglutarate dioxygenase, TauD

Abstract

Co II, Ni II, and N-oxalylglycine (NOG) are well-known inhibitors of Fe II/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 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 αKG) 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 ∼ 290 μM) with αKG 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 at 380 nm ( ε 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.