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

The three metal-binding ligands of the archetype Fe(II)/alpha-ketoglutarate (alphaKG)-dependent hydroxylase, taurine/alphaKG dioxygenase (TauD), were systematically mutated to examine the effects of various ligand substitutions on enzyme activity and metallocenter properties. His99, coplanar with alphaKG and Fe(II), 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 k(cat) and threefold the K(m) of wild-type enzyme. His255, located opposite the O(2)-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 Fe(IV)-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 Fe(II), anaerobic samples of wild-type TauD and the three highly active variants generate a weak green chromophore resembling a catecholate-Fe(III) 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 Fe(II) to form this species. The spectra associated with Fe(II)-TauD and Co(II)-TauD in the presence of alphaKG 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 Fe(II)/alphaKG-dependent hydroxylase.