ABSTRACT
The Fe(II)- and alpha-ketoglutarate (alphaKG)-dependent dioxygenases use mononuclear nonheme iron centers to effect hydroxylation of their substrates and decarboxylation of their cosubstrate, alphaKG, to CO(2) and succinate. Our recent dissection of the mechanism of taurine:alphaKG dioxygenase (TauD), a member of this enzyme family, revealed that two transient complexes accumulate during catalysis in the presence of saturating substrates. The first complex contains the long-postulated C-H-cleaving Fe(IV)-oxo intermediate, J, and the second is an enzyme.product(s) complex. Here, we demonstrate the accumulation of two transient complexes in the reaction of a prolyl-4-hydroxylase (P4H), a functional homologue of human alphaKG-dependent dioxygenases with essential roles in collagen biosynthesis and oxygen sensing. The kinetic and spectroscopic properties of these two P4H complexes suggest that they are homologues of the TauD intermediates. Most notably, the first exhibits optical absorption and Mössbauer spectra similar to those of J and, like J, a large substrate deuterium kinetic isotope on its decay. The close correspondence of the accumulating states in the P4H and TauD reactions supports the hypothesis of a conserved mechanism for substrate hydroxylation by enzymes in this family.
Subject(s)
Carbon/analysis , Hydrogen/analysis , Iron/analysis , Phycodnaviridae/enzymology , Procollagen-Proline Dioxygenase/analysis , Procollagen-Proline Dioxygenase/chemistry , Absorption , Amino Acid Sequence , Humans , Ketoglutaric Acids/metabolism , Kinetics , Mixed Function Oxygenases/metabolism , Molecular Sequence Data , Peptides/chemistry , Spectroscopy, Mossbauer , Substrate Specificity , TitrimetryABSTRACT
The Fe(II)- and alpha-ketoglutarate-dependent dioxygenases catalyze hydroxylation reactions of considerable biomedical and environmental significance. Recently, the first oxidized iron intermediate in the reaction of a member of this family, taurine:alpha-ketoglutarate dioxygenase (TauD), was detected and shown to be a high-spin Fe(IV) complex. In this study we have used X-ray absorption spectroscopy to demonstrate the presence of a short (1.62 A) interaction between the iron and one of its ligands in the Fe(IV) intermediate but not in the Fe(II) starting complex. The detection of this interaction strongly corroborates the hypothesis that the intermediate contains an Fe=O structural motif.
