Direct coordination of pterin to FeII enables neurotransmitter biosynthesis in the pterin-dependent hydroxylases

Abstract

Significance The mononuclear pterin-dependent nonheme iron enzymes catalyze the rate-limiting step in neurotransmitter biosynthesis and are essential in maintaining proper brain function. These enzymes utilize molecular oxygen, a redox active pterin cofactor, and a ferrous active site to generate an FeIV-oxo intermediate that catalyzes substrate oxidation. This study demonstrates that the pterin cofactor directly coordinates to the iron center before oxygen activation and also coordinates to a kinetically generated peroxy-FeII intermediate that is transiently observed in FeIV-oxo formation. The direct coordination of the pterin cofactor to the iron center enables facile electron transfer to promote rapid oxygen reduction that facilitates the biological function of this family of enzymes and thus defines a unified oxygen activation mechanism for the cofactor-dependent nonheme iron enzymes. The pterin-dependent nonheme iron enzymes hydroxylate aromatic amino acids to perform the biosynthesis of neurotransmitters to maintain proper brain function. These enzymes activate oxygen using a pterin cofactor and an aromatic amino acid substrate bound to the FeII active site to form a highly reactive FeIV = O species that initiates substrate oxidation. In this study, using tryptophan hydroxylase, we have kinetically generated a pre-FeIV = O intermediate and characterized its structure as a FeII-peroxy-pterin species using absorption, Mössbauer, resonance Raman, and nuclear resonance vibrational spectroscopies. From parallel characterization of the pterin cofactor and tryptophan substrate–bound ternary FeII active site before the O2 reaction (including magnetic circular dichroism spectroscopy), these studies both experimentally define the mechanism of FeIV = O formation and demonstrate that the carbonyl functional group on the pterin is directly coordinated to the FeII site in both the ternary complex and the peroxo intermediate. Reaction coordinate calculations predict a 14 kcal/mol reduction in the oxygen activation barrier due to the direct binding of the pterin carbonyl to the FeII site, as this interaction provides an orbital pathway for efficient electron transfer from the pterin cofactor to the iron center. This direct coordination of the pterin cofactor enables the biological function of the pterin-dependent hydroxylases and demonstrates a unified mechanism for oxygen activation by the cofactor-dependent nonheme iron enzymes.