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-1α786–826 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-1α786–826/788–806 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