Spectroscopy and kinetics of wild-type and mutant tyrosine hydroxylase: Mechanistic insight into O₂ activation

Tyrosine hydroxylase (TH) is a pterin-dependent nonheme iron enzyme that catalyzes the hydroxylation of L-tyr to L-DOPA in the rate-limiting step of catecholamine neurotransmitter biosynthesis. We have previously shown that the Fe^II site in phenylalanine hydroxylase (PAH) converts from six-coordinate (6C) to five-coordinate (5C) only when both substrate + cofactor are bound. However, steady-state kinetics indicate that TH has a different co-substrate binding sequence (pterin + O₂ + L-tyr) than PAH (L-phe + pterin + O₂). Using X-ray absorption spectroscopy (XAS), and variable-temperature-variable-field magnetic circular dichroism (VTVH MCD) spectroscopy, we have investigated the geometric and electronic structure of the wild-type (WT) TH and two mutants, S395A and E332A, and their interactions with substrates. All three forms of TH undergo 6C → 5C conversion with tyr + pterin, consistent with the general mechanistic strategy established for O ₂-activating nonheme iron enzymes. We have also applied single-turnover kinetic experiments with spectroscopic data to evaluate the mechanism of the O₂ and pterin reactions in TH. When the Fe ^II site is 6C, the two-electron reduction of O₂ to peroxide by Fe^II and pterin is favored over individual one-electron reactions, demonstrating that both a 5C Fe^II and a redox-active pterin are required for coupled O₂ reaction. When the Fe^II is 5C, the O₂ reaction is accelerated by at least 2 orders of magnitude. Comparison of the kinetics of WT TH, which produces Fe^IV=O + 4a-OH-pterin, and E332A TH, which does not, shows that the E332 residue plays an important role in directing the protonation of the bridged Fe ^II-OO-pterin intermediate in WT to productively form Fe ^IV=O, which is responsible for hydroxylating L-tyr to L-DOPA.