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

Just add water: Structurally, cyanobacterial aldehyde decarbonylases are members of the non‐heme diiron oxygenase family of enzymes. However, the enzyme catalyzes the hydrolysis of aliphatic aldehydes to alkanes and formate (see scheme), in an oxygen‐independent reaction. This unusual and chemically difficult reaction most likely involves free radical intermediates.

Cyanobacterial aldehyde decarbonylase (cAD) is, structurally, a member of the di-iron carboxylate family of oxygenases. We previously reported that cAD from Prochlorococcus marinus catalyzes the unusual hydrolysis of aldehydes to produce alkanes and formate in a reaction that requires an external reducing system but does not require oxygen (Das et al., 2011, Angew. Chem. 50, 7148–7152). Here we demonstrate that cADs from divergent cyanobacterial classes, including the enzyme from N. puntiformes that was reported to be oxygen dependent, catalyze aldehyde decarbonylation at a much faster rate under anaerobic conditions, and that the oxygen in formate derives from water. The very low activity (< 1 turn-over/h) of cAD appears to result from inhibition by the ferredoxin reducing system used in the assay and the low solubility of the substrate. Replacing ferredoxin with the electron mediator phenazine methosulfate allowed the enzyme to function with various chemical reductants, with NADH giving the highest activity. NADH is not consumed during turn-over, in accord with the proposed catalytic role for the reducing system in the reaction. With octadecanal, a burst phase of product formation, kprod = 3.4 ± 0.5 min−1 is observed indicating that chemistry is not rate-determining under the conditions of the assay. With the more soluble substrate, heptanal, kcat = 0.17 ± 0.01 min−1 and no burst phase is observed, suggesting that a chemical step is limiting in the reaction of this substrate.

Tyrosine Hydroxylase (TH) is a pterin-dependent non-heme 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 Fe II site in Phenylalanine Hydroxylase (PAH) converts from 6C to 5C only when both substrate + cofactor are bound. However, steady-state kinetics indicate that TH has a different cosubstrate binding sequence (pterin + O 2 + L-tyr) than PAH (L-phe + pterin + O 2 ). 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 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 non-heme iron enzymes. We have also applied single-turnover kinetic experiments with spectroscopic data to evaluate the mechanism of the O 2 and pterin reactions in TH. When the Fe II site is 6C, the two-electron reduction of O 2 to peroxide by Fe II and pterin is favored over individual one-electron reactions, demonstrating that both a 5C Fe II and a redox-active pterin are required for coupled O 2 reaction. When the Fe II is 5C, the O 2 reaction is accelerated by at least 2 orders of magnitude. Comparison of the kinetics of WT TH, which produces Fe IV =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.