Human oxygenase variants employing a single protein Fe(II) ligand are catalytically active

Aspartate/asparagine-β-hydroxylase (AspH) is a human 2-oxoglutarate (2OG) and Fe(II) oxygenase that catalyzes C3 hydroxylations of aspartate/asparagine residues of epidermal growth factor-like domains (EGFDs). Unusually, AspH employs two histidine residues to chelate Fe(II) rather than the typical triad of two histidine and one glutamate/aspartate residue. We report kinetic, inhibition, and crystallographic studies concerning human AspH variants in which either of its Fe(II) binding histidine residues are substituted for alanine. Both the H725A and, in particular, the H679A AspH variant retain substantial catalytic activity. Crystal structures clearly reveal metal-ligation by only a single protein histidine ligand. The results have implications for the functional assignment of 2OG oxygenases and for the design of non-protein biomimetic catalysts.