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.

Bacteria and yeast utilize different strategies for sulfur incorporation in the biosynthesis of the thiamin thiazole. Bacteria use thiocarboxylated proteins. In contrast, Saccharomyces cerevisiae thiazole synthase (THI4p) uses an active site cysteine as the sulfide source and is inactivated after a single turnover. Here, we demonstrate that the Thi4 ortholog from Methanococcus jannaschii uses exogenous sulfide and is catalytic. Structural and biochemical studies on this enzyme elucidate the mechanistic details of the sulfide transfer reactions.

The triblock poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymers, Pluronics (L64, P65, and P123), form liquid crystalline (LC) mesophases with transition metal nitrate salts (TMS), [M(H2O)n](NO3)2, in the presence and absence of free water in the media. In this assembly process, M-OH2 plays an important role as observed in a TMS:CnEOm (CnEOm is oligo(ethylene oxide) nonionic surfactants) system. The structure of the LC mesophases and interactions of the metal ion-nitrate ion and metal ion-Pluronic were investigated using microscopy (POM), diffraction (XRD), and spectroscopy (FTIR and micro-Raman) techniques. The TMS:L64 system requires a shear force for mesophase ordering to be observed using X-ray diffraction. However, TMS:P65 and TMS:P123 form well structured LC mesophases. Depending on the salt/Pluronic mole ratio, hexagonal LC mesophases are observed in the TMS:P65 systems and cubic and tetragonal LC mesophases in the TMS:P123 systems. The LC mesophase in the water/salt/Pluronic system is sensitive to the concentration of free (H2O) and coordinated water (M-OH2) molecules and demonstrates structural changes. As the free water is evaporated from the H2O:TMS:Pluronic LC mesophase (ternary mixture), the nitrate ion remains free in the media. However, complete evaporation of the free water molecules enforces the coordination of the nitrate ion to the metal ion in all TMS:Pluronic systems.

Tryptophan hydroxylase (TrpH) uses a non-heme mononuclear iron center to catalyze the tetrahydropterin-dependent hydroxylation of tryptophan to 5-hydroxytryptophan. The reactions of the TrpH·Fe(II), TrpH·Fe(II)·tryptophan, TrpH·Fe(II)·6MePH 4 ·tryptophan, and TrpH·Fe(II) ·6MePH 4 ·phenylalanine complexes with O 2 were monitored by stopped-flow absorbance spectroscopy and rapid quench methods. The second-order rate constant for the oxidation of TrpH·Fe(II) has a value of 104 M −1 s −1 irrespective of the presence of tryptophan. Stopped-flow absorbance analyses of the reaction of the TrpH·Fe(II)·6MePH 4 ·tryptophan complex with oxygen are consistent with the initial step being reversible binding of oxygen, followed by the formation with a rate constant of 65 s −1 of an intermediate I that has maximal absorbance at 420 nm. The rate constant for decay of I, 4.4 s −1 , matches that for formation of the 4a-hydroxypterin product monitored at 248 nm. Chemical-quench analyses show that 5-hydroxytryptophan forms with a rate constant of 1.3 s −1 , and that overall turnover is limited by a subsequent slow step, presumably product release, with a rate constant of 0.2 s −1 . All of the data with tryptophan as substrate can be described by a five-step mechanism. In contrast, with phenylalanine as substrate, the reaction can be described by three steps: a second-order reaction with oxygen to form I, decay of I as tyrosine forms, and slow product release.Tryptophan hydroxylase (TrpH)1 catalyzes the formation of 5-hydroxytryptophan (5-HOtrp) from tryptophan, the first and rate-limiting step in the biosynthesis of melatonin and serotonin (1,2). The enzyme belongs to the family of aromatic amino acid hydroxylases that also includes phenylalanine hydroxylase (PheH) and tyrosine hydroxylase (TyrH) (3). These three enzymes catalyze the hydroxylation of their corresponding substrates utilizing a tetrahydropterin and molecular oxygen (Scheme 1) (4-6). While the physiological reactions of PheH, TyrH, and TrpH are all aromatic hydroxylations, these enzymes will also catalyze benzylic and aliphatic hydroxylation (7-9). The eukaryotic forms of each enzyme are * Address correspondence to: Paul F. Fitzpatrick, Department of Biochemistry, MC 7760, University of Texas Health Science Center at San Antonio, San Antonio, fitzpatrick@biochem.uthscsa.edu,. † This work was supported by NIH grant R01 GM047291 and Welch Foundation Grant A1245 to PFF and NIH grant F31 GM077092 to JAP. 1 Abbreviations: TyrH, tyrosine hydroxylase; PheH, phenylalanine hydroxylase; TrpH, tryptophan hydroxylase; TauD, taurine:α-ketoglutarate dioxygenase; 6MePH 4 , 6-methyltetrahydropterin; 4a-HO-6MePH 3 , 4a-hydroxypterin; 5-HO-trp, 5-hydroxytryptophan. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 September 7. (16,17). Our present understanding of this mechanism has come primarily from spectroscopy of enzyme-substrate or enzyme-inhibitor complexes and steady-state kinetics. The changes in the ligands to the iron as substr...

Thiamin diphosphate is an essential cofactor in all forms of life and plays a key role in amino acid and carbohydrate metabolism. Its biosynthesis involves separate syntheses of the pyrimidine and thiazole moieties, which are then coupled to form thiamin monophosphate. A final phosphorylation produces the active form of the cofactor. In most bacteria, six gene products are required for biosynthesis of the thiamin thiazole. In yeast and fungi only one gene product, Thi4, is required for thiazole biosynthesis. Methanococcus jannaschii expresses a putative Thi4 ortholog that was previously reported to be a ribulose 1, 5-bisphosphate synthase [Finn, M. W. and Tabita, F. R. (2004) J. Bacteriol. 186, 6360–6366]. Our structural studies show that the Thi4 orthologs from M. jannaschii and Methanococcus igneus are structurally similar to Thi4 from Saccharomyces cerevisiae. In addition, all active site residues are conserved except for a key cysteine residue, which in S. cerevisiae is the source of the thiazole sulfur atom. Our recent biochemical studies showed that the archael Thi4 orthologs use nicotinamide adenine dinucleotide, glycine and free sulfide to form the thiamin thiazole in an iron-dependent reaction [Eser, B., Zhang, X., Chanani, P. K., Ealick, S.E., and Begley, T.P. (2015) submitted]. Here we report X-ray crystal structures of Thi4 from M. jannaschii complexed with ADP-ribulose, the C205S variant of Thi4 from S. cerevisiae with a bound glycine imine intermediate, and Thi4 from M. igneus with bound glycine imine intermediate and iron. These studies reveal the structural basis for the iron-dependent mechanism of sulfur transfer in archael and yeast thiazole synthases.