title: Spectroscopy and kinetics of wild-type and mutant tyrosine hydroxylase: mechanistic insight into O2 activation.
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 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.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: Marina S
authors.initials: MS
authors.lastname: Chow
authors.forename: Bekir E
authors.initials: BE
authors.lastname: Eser
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Wilson
authors.forename: Keith O
authors.initials: KO
authors.lastname: Hodgson
authors.forename: Britt
authors.initials: B
authors.lastname: Hedman
authors.forename: Paul F
authors.initials: PF
authors.lastname: Fitzpatrick
authors.forename: Edward I
authors.initials: EI
authors.lastname: Solomon
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D058085:Iron Compounds / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D011622:Pterins / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013052:Spectrometry, X-Ray Emission; D019265:Spectroscopy, Near-Infrared; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D058085:Iron Compounds; D011622:Pterins; D014443:Tyrosine; D014446:Tyrosine 3-Monooxygenase; D010100:Oxygen