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

Marina S. Chow, Bekir E. Eser, Samuel A. Wilson, Keith O. Hodgson, Britt Hedman, Paul F. Fitzpatrick, Edward I. Solomon

Research output: Contribution to journalArticle

40 Scopus citations

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 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 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 Fe II-OO-pterin intermediate in WT to productively form Fe IV=O, which is responsible for hydroxylating L-tyr to L-DOPA.

Original languageEnglish (US)
Pages (from-to)7685-7698
Number of pages14
JournalJournal of the American Chemical Society
Volume131
Issue number22
DOIs
StatePublished - Jun 10 2009

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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