Spectroscopy and Kinetics of Wild-Type and Mutant Tyrosine Hydroxylase: Mechanistic Insight into O2 Activation

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Abstract
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 FeII 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 O2-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 FeII site is 6C, the two-electron reduction of O2 to peroxide by FeII and pterin is favored over individual one-electron reactions, demonstrating that both a 5C FeII and a redox-active pterin are required for coupled O2 reaction. When the FeII is 5C, the O2 reaction is accelerated by at least 2 orders of magnitude. Comparison of the kinetics of WT TH, which produces FeIV═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 FeII−OO−pterin intermediate in WT to productively form FeIV═O, which is responsible for hydroxylating l-tyr to l-DOPA.

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