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

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 (A(iso) = 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(iso) 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 A(iso) of 0.0 MHz and a dipolar coupling (T = 3.8 MHz). This interaction was assigned to the N₅ proton of the reduced pterin.

Structure of 3-mercaptopropionic acid dioxygenase with a substrate analog reveals bidentate substrate binding at the iron center

“…The peaks in the (−, +) quadrant represent strongly coupled 14 N nuclei; these peaks arise from nuclei where the hyperfine couplings are much greater than the 14 N Larmor frequency at 175 mT, $0.54 MHz. These peaks are characteristic of what is observed for directly coordinated 14 N from histidine ligands (53,57). The (+, +) quadrant contains peak from weakly coupled nuclei, namely those where the 14 N Larmor frequency is greater than the hyperfine coupling.…”

“…where T is the anisotropic contribution of the hyperfine interaction. The tensor was considered purely dipolar due to the distance of the coupled 1 H, which is consistent with other HYSCORE analyses of {FeNO} 7 centers (52)(53)(54). The dipolar contribution, T, was calculated using the geometry-optimized DFT structure of 3MPA-bound Av3MDO treated with NO and spin-projection factors that take into account the electronic structure of the {FeNO} 7 center (52,54).…”

“…The magnitude of coupling for each magnetic nucleus is highly dependent on the distance separating it from the paramagnetic center and its position relative to the magnetic axis. Similar HYSCORE experiments have been performed on a variety of nonheme enzymes to provide structural details about the (substrate/NO)-bound active site geometry (12,(51)(52)(53)(54)(55)(56). Moreover, relative to Mössbauer spectroscopy, HYSCORE is much more sensitive to subtle structural perturbations in the outer coordination sphere of the Fe-site, allowing for a more robust validation of the optimized (3MPA/NO)-bound Av3MDO structure.…”

“…In the case of an axial hyperfine interaction, only two angles are necessary. As with other {FeNO} 7 centers, the principal axis of the zero field splitting (zfs) and the molecular z axis are nearly coincident (52,53). As a result, these Euler rotations can be approximated using polar angles u and ɵ,…”

Spectroscopic analyses of 2-oxoglutarate-dependent oxygenases: TauD as a case study

“…Cross-peaks arising from the coupled protons of the histidyl imidazole side chains and bound water molecules are resolved in the (+,+) quadrant. The protons of bound water ligands are further characterized by strong dipolar couplings and easily distinguished by HYSCORE spectroscopy [54, 55]. The cross-peaks due to these protons are circled in red on the HYSCORE spectrum shown (Fig.…”