Substrate Analogs Implicate a Free Radical Pathway in Tyrosine Hydroxylase Catalysis

Heme-dependent tyrosine hydroxylases (TyrH) are critical enzymes in catecholamine biosynthesis, yet the role of the substrate's <i>α</i>-amino group in their monooxygenation mechanism has been unclear. Using 3-(4-hydroxyphenyl)propionic acid (HPPA), an l-tyrosine analog lacking the <i>α</i>-amino group, we observed a distinct dimerization pathway that competes with the expected hydroxylation reaction. Several lines of evidence confirm that this process originates from a radical intermediate. First, the formation of this (HPPA)₂ dimer is selectively inhibited by a free radical scavenger. Second, ¹⁸O-labeling experiments show phenolic oxygen scrambling, indicating a disruption of substrate aromaticity during catalysis. Finally, EPR spectroscopy using nitrosobenzene as a substrate analog revealed a substrate-based free radical. This mechanistic divergence clarifies the role of the <i>α</i>-amino group. Its absence in HPPA creates a kinetic bottleneck for the final O atom transfer step, allowing a fraction of the substrate radical to form the off-pathway dimer. Thus, the native substrate's <i>α</i>-amino group acts as a crucial kinetic modulator, ensuring the rapid and efficient commitment of the substrate radical to productive hydroxylation. These results collectively establish a peroxidase-like free radical pathway for TyrH and reveal the nonessential yet significant role the amino group plays in controlling reaction outcomes.