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Evidence That Two Enzyme-derived Histidine Ligands Are Sufficient for Iron Binding and Catalysis by Factor Inhibiting HIF (FIH)

DOI: 10.1074/jbc.M804999200 DOI Help
PMID: 18611856 PMID Help

Authors: K. S. Hewitson (Department of Chemistry, University of Oxford) , S. Holmes (Department of Chemistry, University of Oxford) , D. Ehrismann (Department of Chemistry, University of Oxford) , A. Hardy (Department of Chemistry, University of Oxford) , Rasheduzzaman Chowdhury (Department of Chemistry, University of Oxford) , C. Schofield (Department of Chemistry, University of Oxford) , Michael Mcdonough (Department of Chemistry, University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 283 (38) , PAGES 25971-25978

State: Published (Approved)
Published: September 2008

Abstract: A 2-His-1-carboxylate triad of iron binding residues is present in many non-heme iron oxygenases including the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases. Three variants (D201A, D201E, and D201G) of the iron binding Asp-201 residue of an asparaginyl hydroxylase, factor inhibiting HIF (FIH), were made and analyzed. FIH-D201A and FIH-D201E did not catalyze asparaginyl hydroxylation, but in the presence of a reducing agent, they displayed enhanced 2OG turnover when compared with wild-type FIH. Turnover of 2OG by FIH-D201A was significantly stimulated by the addition of HIF-1?786–826 peptide. Like FIH-D201A and D201E, the D201G variant enhanced 2OG turnover but rather unexpectedly catalyzed asparaginyl hydroxylation. Crystal structures of the FIH-D201A and D201G variants in complex with Fe(II)/Zn(II), 2OG, and HIF-1?786–826/788–806 implied that only two FIH-based residues (His-199 and His-279) are required for metal binding. The results indicate that variation of 2OG-dependent dioxygenase iron-ligating residues as a means of functional assignment should be treated with caution. The results are of mechanistic interest in the light of recent biochemical and structural analyses of non-heme iron and 2OG-dependent halogenases that are similar to the FIH-D201A/G variants in that they use only two His-residues to ligate iron.

Journal Keywords: Crystallography; X-Ray; Dioxygenases; Heme; Histidine; Humans; Iron; Ketoglutaric; Ligands; Metals; Mixed; Models; Chemical; Models; Molecular; Protein; Repressor Proteins

Subject Areas: Chemistry, Biology and Bio-materials


Instruments: I04-Macromolecular Crystallography