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

John McCracken, Bekir E. Eser, Donald Mannikko, Matthew D. Krzyaniak, Paul F Fitzpatrick

Research output: Contribution to journalArticle

5 Scopus citations

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 O<inf>2</inf> 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 O<inf>2</inf> to poise the active site iron in an S = 3/2 {FeNO}<sup>7</sup> 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 H<inf>2</inf>O molecule characterized by proton isotropic hyperfine couplings (A<inf>iso</inf> = 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 A<inf>iso</inf> of 2.0 MHz and a T of 3.8 MHz, to the analysis. This proton hyperfine coupling differed from those measured previously for H<inf>2</inf>O bound to {FeNO}<sup>7</sup> 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-MPH<inf>4</inf>, the HYSCORE cross peaks attributed to H<inf>2</inf>O and OH<sup>-</sup> for the TyrH/NO/Tyr complex were replaced by a cross peak due to a single proton characterized by an A<inf>iso</inf> of 0.0 MHz and a dipolar coupling (T = 3.8 MHz). This interaction was assigned to the N<inf>5</inf> proton of the reduced pterin.

Original languageEnglish (US)
Pages (from-to)3759-3771
Number of pages13
JournalBiochemistry
Volume54
Issue number24
DOIs
StatePublished - Jun 23 2015

ASJC Scopus subject areas

  • Biochemistry

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