| Autor: |
Chaplin VD; Department of Chemistry , University of Massachusetts at Amherst , Amherst , Massachusetts 01003 , United States., Hangasky JA; Department of Chemistry , University of Massachusetts at Amherst , Amherst , Massachusetts 01003 , United States., Huang HT; Department of Chemistry , University of Massachusetts at Amherst , Amherst , Massachusetts 01003 , United States., Duan R; Department of Chemistry , University of Massachusetts at Amherst , Amherst , Massachusetts 01003 , United States., Maroney MJ; Department of Chemistry , University of Massachusetts at Amherst , Amherst , Massachusetts 01003 , United States., Knapp MJ; Department of Chemistry , University of Massachusetts at Amherst , Amherst , Massachusetts 01003 , United States. |
| Abstrakt: |
α-Ketoglutarate (αKG) dependent oxygenases comprise a large superfamily of enzymes that activate O 2 for varied reactions. While most of these enzymes contain a nonheme Fe bound by a His 2 (Asp/Glu) facial triad, a small number of αKG-dependent halogenases require only the two His ligands to bind Fe and activate O 2 . The enzyme "factor inhibiting HIF" (FIH) contains a His 2 Asp facial triad and selectively hydroxylates polypeptides; however, removal of the Asp ligand in the Asp201→Gly variant leads to a highly active enzyme, seemingly without a complete facial triad. Herein, we report on the formation of an Fe-Cl cofactor structure for the Asp201→Gly FIH variant using X-ray absorption spectroscopy (XAS), which provides insight into the structure of the His 2 Cl facial triad found in halogenases. The Asp201→Gly variant supports anion dependent peptide hydroxylation, demonstrating the requirement for a complete His 2 X facial triad to support O 2 reactivity. Our results indicated that exogenous ligand binding to form a complete His 2 X facial triad was essential for O 2 activation and provides a structural model for the His 2 Cl-bound nonheme Fe found in halogenases. |
