Extracts of the plant Echinacea purpurea are widely used for medicinal purposes. Effective quality control of these extracts requires rapid methods to determine their chemical composition. A new method for analysis of caffeic acid derivatives and alkamides from Echinacea extracts has been developed. With this method, isomeric isobutylamides and 2-methylbutylamides can be distinguished, a capability that previously published methods have lacked. Quantitative analyses carried out with this method on E. purpurea extracts that have been stored for 18 months indicate that they contain caftaric acid, cichoric acid, and undeca-2Z,4E-diene-8,10-diynoic acid isobutylamide at concentrations of 0.7, 0.71 and 2.0mg/mL, respectively.

Haemophilus influenzae is a host adapted human mucosal pathogen involved in a variety of acute and chronic respiratory tract infections, including chronic obstructive pulmonary disease and asthma, all of which rely on its ability to efficiently establish continuing interactions with the host. Here we report the characterization of a novel molybdenum enzyme, TorZ/MtsZ that supports interactions of H. influenzae with host cells during growth in oxygen-limited environments. Strains lacking TorZ/MtsZ showed a reduced ability to survive in contact with epithelial cells as shown by immunofluorescence microscopy and adherence/invasion assays. This included a reduction in the ability of the strain to invade human epithelial cells, a trait that could be linked to the persistence of H. influenzae. The observation that in a murine model of H. influenzae infection, strains lacking TorZ/MtsZ were almost undetectable after 72 h of infection, while ∼3.6 × 103 CFU/mL of the wild type strain were measured under the same conditions is consistent with this view. To understand how TorZ/MtsZ mediates this effect we purified and characterized the enzyme, and were able to show that it is an S- and N-oxide reductase with a stereospecificity for S-sulfoxides. The enzyme converts two physiologically relevant sulfoxides, biotin sulfoxide and methionine sulfoxide (MetSO), with the kinetic parameters suggesting that MetSO is the natural substrate of this enzyme. TorZ/MtsZ was unable to repair sulfoxides in oxidized Calmodulin, suggesting that a role in cell metabolism/energy generation and not protein repair is the key function of this enzyme. Phylogenetic analyses showed that H. influenzae TorZ/MtsZ is only distantly related to the Escherichia coli TorZ TMAO reductase, but instead is a representative of a new, previously uncharacterized clade of molybdenum enzyme that is widely distributed within the Pasteurellaceae family of pathogenic bacteria. It is likely that MtsZ/TorZ has a similar role in supporting host/pathogen interactions in other members of the Pasteurellaceae, which includes both human and animal pathogens.

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Molybdenum enzymes contain at least one pyranopterin dithiolate (molybdopterin, MPT) moiety that coordinates Mo through two dithiolate (dithiolene) sulfur atoms. For sulfite oxidase (SO), hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of magnetic nuclei (I ≠ 0) near the Mo(V) (d1) center have been measured using high-resolution pulsed electron paramagnetic resonance (EPR) methods and interpreted with the help of the density functional theory (DFT) calculations. These have provided important insights about the active site structure and the reaction mechanism of the enzyme. However, it has not been possible to use EPR to probe the dithiolene sulfurs directly since naturally abundant 32S has no nuclear spin (I = 0). Here we describe direct incorporation of 33S (I = 3/2), the only stable magnetic sulfur isotope, into MPT using controlled in vitro synthesis with purified proteins. The electron spin echo envelope modulation (ESEEM) spectra from 33S-labeled MPT in this catalytically active SO variant are dominated by the ‘inter-doublet’ transition arising from the strong nuclear quadrupole interaction, as also occurs for the 33S-labeled exchangeable equatorial sulfite ligand [Klein, E. L., et al., Inorg. Chem. 2012, 51, 1408 – 1418]. The estimated experimental hfi and nqi parameters for 33S (aiso = 3 MHz and e2Qq/h = 25 MHz) are in good agreement with those predicted by DFT. In addition, the DFT calculations show that the two 33S atoms are indistinguishable by EPR and reveal a strong intermixing between their out-of-plane pz orbitals and the dxy orbital of Mo(V).

Several point mutations in the gene of human sulfite oxidase (hSO) result in isolated sulfite oxidase deficiency, an inherited metabolic disorder. Three conserved residues (H304, R309, K322) are hydrogen bonded to the phosphate group of the molybdenum cofactor, and the R309H and K322R mutations are responsible for isolated sulfite oxidase deficiency. The kinetic effects of the K322R mutation have been previously reported (Rajapakshe et al. 2012, Chem. Biodiversity 9, 1621-1634); here we investigate several mutants of H304 and R309 by steady-state kinetics, laser flash photolysis studies of intramolecular electron transfer (IET), and spectroelectrochemistry. An unexpected result is that all of the mutants show decreased rates of IET but increased steady-state rates of catalysis. However, in all cases the rate of IET is greater than the overall turnover rate, showing that IET is not the rate determining step for any of the mutations.

Human sulfite oxidase (hSO), an essential molybdoheme enzyme, catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic cycle includes two, one-electron intramolecular electron transfers (IET) between the molybdenum (Mo) and the heme domains. Rapid IET rates are ascribed to conformational changes that bring the two domains into close proximity to one another. Previous studies of hSO have focused on the roles of conserved residues near the Mo active site and on the tether that links the two domains. Here four aromatic surface residues on the heme domain (phenylalanine 57 (F57), phenylalanine 79 (F79), tyrosine 83 (Y83), and histidine 90 (H90)) have been mutated, and their involvement in IET rates, the heme midpoint potential, and the catalytic activity of hSO have been investigated using laser flash photolysis, spectroelectrochemistry, and steady-state kinetics, respectively. The results indicate that the size and hydrophobicity of F57 play an important role in modulating the heme potential and that F57 also affects the IET rates. The data also suggest that important interactions of H90 with a heme propionate group destabilize the Fe(III) state of the heme. The positive charge on H90 at pH ≤ 7.0 may decrease the electrostatic interaction between the Mo and heme domains, thereby decreasing the IET rates of wt hSO at low pH. Lastly, mutations of F79 and Y83, which are located on the surface of the heme domain, but not in direct contact with the heme or the propionate groups, have little effect on either IET or the heme potential.

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