TY - JOUR
T1 - Spectroscopy and kinetics of wild-type and mutant tyrosine hydroxylase
T2 - Mechanistic insight into O2 activation
AU - Chow, Marina S.
AU - Eser, Bekir E.
AU - Wilson, Samuel A.
AU - Hodgson, Keith O.
AU - Hedman, Britt
AU - Fitzpatrick, Paul F.
AU - Solomon, Edward I.
PY - 2009/6/10
Y1 - 2009/6/10
N2 - 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.
AB - 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.
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U2 - 10.1021/ja810080c
DO - 10.1021/ja810080c
M3 - Article
C2 - 19489646
AN - SCOPUS:67650510925
VL - 131
SP - 7685
EP - 7698
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
SN - 0002-7863
IS - 22
ER -