Abstract
The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change.
Highlights
Iron is a common cofactor in enzymes that employ molecular oxygen as an oxidant
In related enzymes that catalyze halogenation reactions, instead of hydroxylations, the carboxylatecontaining residue is replaced with Ala, providing space for a halide ion to bind to the iron
The 2-His–1-carboxylate motif is common to many aketoglutarate-dependent iron(II) dioxygenases
Summary
Iron is a common cofactor in enzymes that employ molecular oxygen as an oxidant In addition to those iron-dependent enzymes that rely on heme, there is a class of non-heme, mononuclear iron(II) enzymes that catalyze the hydroxylation of aliphatic C–H bonds, dihydroxylation of arene double bonds, epoxidation of C–C double bonds, heterocyclic ring formation, and oxidative aromatic ring opening. Two histidine (His) and either one aspartic acid (Asp) or one glutamic acid (Glu) residue bind the iron at the vertices of one triangular face of the octahedral metal center, forming a 2-His–1carboxylate triad of facial ligands (Figure 1A) This arrangement leaves three coordination sites on the iron open for the substrate, oxygen, and other co-substrates. One atom of oxygen is incorporated into the product, and the other into succinate
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