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 + O-2 + L-tyr) than PAH (L-phe + pterin + O-2). 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 O-2 and pterin reactions in TH. When the Fe-II site is 6C, the two-electron reduction Of O-2 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 O-2 reaction. When the Fe-II is 5C, the O-2 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.