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HYSCORE Analysis of the Effects of Substrates on Coordination of Water to the Active Site Iron in Tyrosine Hydroxylase

Author:
McCracken, John, Eser, Bekir E., Mannikko, Donald, Krzyaniak, Matthew D., Fitzpatrick, Paul F.
Source:
Biochemistry 2015 v.54 no.24 pp. 3759-3771
ISSN:
1520-4995
Subject:
L-dopa, active sites, biosynthesis, catalytic activity, central nervous system, electron paramagnetic resonance spectroscopy, glutamic acid, histidine, hydroxylation, iron, ligands, neurotransmitters, nitric oxide, oxygen, tyrosine, tyrosine 3-monooxygenase
Abstract:
Tyrosine hydroxylase is a mononuclear non-heme iron monooxygenase found in the central nervous system that catalyzes the hydroxylation of tyrosine to yield l-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. Catalysis requires the binding of tyrosine, a tetrahydropterin, and O₂ at an active site that consists of a ferrous ion coordinated facially by the side chains of two histidines and a glutamate. We used nitric oxide as a surrogate for O₂ to poise the active site iron in an S = ³/₂ {FeNO}⁷ form that is amenable to electron paramagnetic resonance (EPR) spectroscopy. The pulsed EPR method of hyperfine sublevel correlation (HYSCORE) spectroscopy was then used to probe the ligands at the remaining labile coordination sites on iron. For the complex formed by the addition of tyrosine and nitric oxide, TyrH/NO/Tyr, orientation-selective HYSCORE studies provided evidence of the coordination of one H₂O molecule characterized by proton isotropic hyperfine couplings (Aᵢₛₒ = 0.0 ± 0.3 MHz) and dipolar couplings (T = 4.4 and 4.5 ± 0.2 MHz). These data show complex HYSCORE cross peak contours that required the addition of a third coupled proton, characterized by an Aᵢₛₒ of 2.0 MHz and a T of 3.8 MHz, to the analysis. This proton hyperfine coupling differed from those measured previously for H₂O bound to {FeNO}⁷ model complexes and was assigned to a hydroxide ligand. For the complex formed by the addition of tyrosine, 6-methyltetrahydropterin, and NO, TyrH/NO/Tyr/6-MPH₄, the HYSCORE cross peaks attributed to H₂O and OH– for the TyrH/NO/Tyr complex were replaced by a cross peak due to a single proton characterized by an Aᵢₛₒ of 0.0 MHz and a dipolar coupling (T = 3.8 MHz). This interaction was assigned to the N₅ proton of the reduced pterin.
Agid:
5320318