Metal ligand substitution and evidence for quinone formation in taurine/α-ketoglutarate dioxygenase

The three metal-binding ligands of the archetype FeII/α-ketoglutarate (αKG)-dependent hydroxylase, taurine/αKG dioxygenase (TauD), were systematically mutated to examine the effects of various ligand substitutions on enzyme activity and metallocenter properties. His99, coplanar with αKG and FeII, is unalterable in terms of maintaining an active enzyme. Asp101 can be substituted only by a longer carboxylate, with the D101E variant exhibiting 22% the kcat and threefold the Km of wild-type enzyme. His255, located opposite the O2-binding site, is less critical for activity and can be substituted by Gln or even the negatively charged Glu (81% and 33% active, respectively). Transient kinetic studies of the three highly active mutant proteins reveal putative FeIV-oxo intermediates as reported in wild-type enzyme, but with distinct kinetics. Supplementation of the buffer with formate enhances activity of the D101A variant, consistent with partial chemical rescue of the missing metal ligand. Upon binding FeII, anaerobic samples of wild-type TauD and the three highly active variants generate a weak green chromophore resembling a catecholate–FeIII species. Evidence is presented that the quinone oxidation state of dihydroxyphenylalanine, formed by aberrant self-hydroxylation of a protein side chain of TauD during aerobic bacterial growth, reacts with FeII to form this species. The spectra associated with FeII–TauD and CoII–TauD in the presence of αKG and taurine were examined for all variants to gain additional insights into perturbations affecting the metallocenter. These studies present the first systematic mutational analysis of metallocenter ligands in an FeII/αKG-dependent hydroxylase.