Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 103, Issue 40, Pages 14738-14743Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0604005103
Keywords
Fe(IV)-oxo intermediates; hydroxylation; nonheme iron enzymes; oxygen activation
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Funding
- NIGMS NIH HHS [R01 GM069657, GM 69657] Funding Source: Medline
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The Fe(II)- and alpha-ketoglutarate (alpha KG)-dependent dioxygenases use mononuclear nonheme iron centers to effect hydroxylation of their substrates and decarboxylation of their cosubstrate, alpha KG, to CO2 and succinate. Our recent dissection of the mechanism of taurine:aKG 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 alpha KG-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 Mossbauer 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.
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