Direct Spectroscopic Evidence for a High-Spin Fe(IV) Intermediate in Tyrosine Hydroxylase

Abstract

Tyrosine hydroxylase (TyrH 1), the key enzyme in the biosynthesis of catecholamine neurotransmitters, is one of three members of the aromatic amino acid hydroxylase enzyme family. 2,3 The enzyme is found in the brain and adrenal gland where it catalyses the conversion of L-tyrosine to L-DOPA. The other members of the family are phenylalanine hydroxylase, which catabolizes excess phenylalanine to tyrosine, and tryptophan hydroxylase, which catalyzes the rate limiting step in the biosynthesis of the neurotransmitter serotonin. All three enzymes have a mononuclear non-heme iron, coordinated by the common His 2-Glu facial triad motif, 4,5 and use a tetrahydropterin to activate dioxygen for hydroxylation of the aromatic side chains of their corresponding amino acid substrates. 2,3 In the proposed mechanism 6-8 (Scheme 1), oxygen reacts with ferrous iron and tetrahydropterin to produce a Fe(IV)O (ferryl) hydroxylating intermediate and 4a-hydroxypterin (4a-HOPH 3). Then, through an electrophilic aromatic substitution, the ferryl species reacts with the aromatic side chain of the tyrosine substrate (Tyr) to form the product dihydroxyphenylalanine (DOPA). To date there has been no direct evidence for this ferryl species. Here, we report the detection of an Fe(IV) intermediate, which is likely to be the proposed ferryl species, in the TyrH reaction by the use of rapid reaction methods. The anaerobic TyrH•Fe(II) •6-MePH 4 •Tyr complex 9 was reacted with oxygen and quenched by rapid-freeze at time points from 20 ms to 390 ms. 10 Figure 1 (left panel) shows representative Mössbauer spectra of the samples from such a time course. The spectrum of the reactant complex reveals the presence of two broad lines with parameters typical of high-spin Fe(II). The asymmetry suggests the presence of at least two distinct Fe(II) complexes. A new line at ~0.9 mm/s is observed in the spectra of samples in which the reactant complex was exposed to oxygen for either 20 ms or 100 ms, but it is not detected in the spectrum of a sample reacted for 390 ms. Thus, this peak is associated with a reaction intermediate which exhibits a quadrupole doublet in a weak external magnetic field. The low-energy line of this quadrupole doublet overlaps with the low-energy line of the Fe(II). The features of the intermediate are similar to those observed for Fe(IV) intermediates in other mononuclear non-heme enzymes. 11,12

Versatility of biological non-heme Fe(II) centers in oxygen activation reactions

“…Heterolytic cleavage of the O-O bond would yield a 4a-hydroxy pterin and an Fe(IV)=O species prepared for hydroxylation chemistry. Recently, the Fe(IV)=O species has been directly detected and trapped 75 . The mechanism of hydroxylation remains under investigation.…”

Mono- and binuclear non-heme iron chemistry from a theoretical perspective

“…The S = 2 Fe IV =O intermediate was trapped in several pterin-dependent hydroxylases (i.e., tyrosine, [194] phenylalanine, [195] or tryptophan [196,197] hydroxylases). These enzymes hydroxylate the aromatic substrates by the electrophilic substitution mechanism that proceeds formally through 2e − transfer from the oxo group to Fe IV and direct OAT as depicted in Figure 8.…”

Protein Dynamics Control the Progression and Efficiency of the Catalytic Reaction Cycle of the Escherichia coli DNA-Repair Enzyme AlkB

“…1A). Subsequent binding of O 2 to the Fe(II) cofactor triggers relatively rapid decarboxylation of 2OG to generate carbon dioxide (CO 2 ), succinate (Suc), and a reactive oxyferryl (Fe(IV)ϭO) intermediate (16,(27)(28)(29) that slowly hydroxylates the nucleobase substrate (21,22). This long lived oxyferryl intermediate must remain tightly sequestered in the active site to avoid reaction with oxidizable compounds in the buffer (solvent-quench-ing), which results in uncoupled 2OG turnover (12,13,30) and release of toxic reactive oxygen species.…”