title: Evidence that two enzyme-derived histidine ligands are sufficient for iron binding and catalysis by factor inhibiting HIF (FIH).
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-1alpha(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-1alpha(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.
authors.affiliation: Chemistry Research Laboratory, The Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, United Kingdom.
authors.forename: Kirsty S
authors.initials: KS
authors.lastname: Hewitson
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Holmes
authors.forename: Dominic
authors.initials: D
authors.lastname: Ehrismann
authors.forename: Adam P
authors.initials: AP
authors.lastname: Hardy
authors.forename: Rasheduzzaman
authors.initials: R
authors.lastname: Chowdhury
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
authors.forename: Michael A
authors.initials: MA
authors.lastname: McDonough
mesh_terms: D002384:Catalysis; D018360:Crystallography, X-Ray / Q000379:methods; D049308:Dioxygenases / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D007501:Iron / Q000737:chemistry*; D007656:Ketoglutaric Acids / Q000737:chemistry; D008024:Ligands; D008670:Metals / Q000737:chemistry; D006899:Mixed Function Oxygenases; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D007656:Ketoglutaric Acids; D008024:Ligands; D008670:Metals; D012097:Repressor Proteins; D006418:Heme; D006639:Histidine; D007501:Iron; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C439790:HIF1AN protein, human