EPR spectroscopy and electrospray ionization mass spectrometry reveal distinctive features of the iron site in leukocyte 12-lipoxygenase.

The procedure for the expression and purification of recombinant porcine leukocyte 12-lipoxygenase using Escherichia coli [K.M. Richards, L.J. Marnett, Biochemistry 36 (1997) 6692-6699] was updated to make it possible to produce enough protein for physical measurements. Electrospray ionization tandem mass spectrometry confirmed the amino acid sequence. The redox properties of the cofactor iron site were examined by EPR spectroscopy at 25K following treatment with a variety of fatty acid hydroperoxides. Combination of the enzyme in a stoichiometric ratio with the hydroperoxides led to a g4.3 signal in EPR spectra instead of the g6 signal characteristic of similarly treated soybean lipoxygenase-1. Native 12-lipoxygenase was also subjected to electrospray ionization mass spectrometry. There was evidence for loss of the mass of an iron atom from the protein as the pH was lowered from 5 to 4. Native ions in these samples indicated that iron was lost without the protein completely unfolding.

Benzyl Alcohol-Mediated Versatile Method to Fabricate Nonstoichiometric Metal Oxide Nanostructures.

Nanostructured metal oxides with cationic or anionic deficiency find applications in a wide range of technological areas including the energy sector and environment. However, a facile route to prepare such materials in bulk with acceptable reproducibility is still lacking; many synthesis techniques are still only bench-top and cannot be easily scaled-up. Here, we report that the benzyl alcohol (BA)-mediated method is capable of producing a host of nanostructured metal oxides (MOx, where M = Ti, Zn, Ce, Sn, In, Ga, or Fe) with inherent nonstoichiometry. It employs multifunctional BA as a solvent, a reducing agent, and a structure-directing agent. Depending on the oxidation states of metal, elemental or nonstoichiometric oxide forms are obtained. Augmented photoelectrochemical oxidation of water under visible light by some of these nonstoichiometric oxides highlights the versatility of the BA-mediated synthesis protocol.

Synthesis of glucaminium-based ionic liquids and their application in the removal of boron from water.

A novel class of ionic liquids (ILs), exhibiting high selectivity towards boron species as well as the ability to phase separate from water, were synthesized from N-methyl-D-glucamine. The complexation of boric acid/borate with the ILs was confirmed using (11)B NMR.

Theoretical and experimental studies of the isomeric protonation in solution for a prototype aliphatic ring containing two nitrogens.

Theoretical calculations were carried out for studying the tautomeric protonation of N-methylpiperazine as a prototype six-member aliphatic ring containing a secondary and a tertiary nitrogen atom. The protonation was investigated in three solvents, water, acetonitrile, and dichloromethane. Calculations were performed up to the B3LYP/aug-cc-pvtz and QCISD(T)/CBS levels by applying the IEF-PCM polarizable continuum dielectric solvent model. Relative solvation free energies also were calculated upon explicit solvent models by utilizing the free-energy perturbation theory as implemented in Monte Carlo simulations. The relative free energy for the N-methylpiperazine tautomer protonated at the secondary (NMps) rather than at the tertiary (NMpt) nitrogen was calculated at a ratio of 47/53 in infinitely dilute aqueous solution. The ratio further decreased in lower-polarity solvents. In contrast, NMR experiments suggested that the protonation takes place primarily at the secondary nitrogen in 0.37 molar aqueous solution with NMps/NMpt = 80/20. The NMps tautomer was exclusive in dichloromethane at the same concentration. The discrepancy between theory and experiment was resolved by considering association equilibria in parallel with the protonation for the solute. As a result, the theoretically predicted tautomer ratios were obtained in close agreement with the experimental values. The NMps tautomer could form a preferable dimeric structure, where one or two chloride anion(s) was/were in hydrogen bonds with protons of the associating monomers. The calculations suggest that the proton relocation may take place by solvent assistance in water or along an intramolecular proton jump in the twist-boat conformation. The predicted activation free energy was about 10 kcal/mol on the basis of variable-temperature NMR experiments in DCM.

Secondary alkyl hydroperoxides as inhibitors and alternate substrates for lipoxygenase.

Lipoxygenase plays a central role in polyunsaturated fatty acid metabolism, inaugurating the biosynthesis of eicosanoids in animals and phytooxylipins in plants. Redox cycling of the non-heme iron cofactor represents a critical element of the catalytic mechanism. Paradoxically, the isolated enzyme contains Fe(II), but the catalytically active form contains Fe(III), and the natural oxidant for the iron is the hydroperoxide product of the catalyzed reaction. Controlling the redox state of lipoxygenase iron with small molecules, inhibitors or activators, could be a means to modulate the activity of the enzyme. The effects of secondary alkyl hydroperoxides and the corresponding alcohols on soybean lipoxygenase-1 reaction rates were investigated and found to be very different. Secondary alcohols were noncompetitive or linear mixed inhibitors with inhibition constants in the millimolar concentration range, with more hydrophobic compounds producing lower values. Secondary alkyl hydroperoxides were inhibitors of lipoxygenase-1 primarily at high substrate concentration. They were more effective inhibitors than the alcohols, with dissociation constants in the micromolar concentration range. The hydroperoxides bearing longer alkyl substituents were the more effective inhibitors. Oxidation of the iron in lipoxygenase-1 by 2-hydroperoxyalkanes was evident in electron paramagnetic resonance (EPR) measurements, but the enzyme was neither activated nor was it inactivated. Instead there was evidence for an entirely different reaction catalyzed by the enzyme, a homolytic dehydration of the hydroperoxide to produce the corresponding carbonyl compound.

Evaluation of chiral discrimination of highly negatively charged enantiomers by quaternary ammonium beta-cyclodextrin using 1H NMR.

The positively charged quaternary ammonium cyclodextrin, QA-beta-CD, was previously used as a chiral selector to achieve baseline resolution of two of the dianionic enantiomers of disodium 3-(p-isothiocyanatophenoxy)-3-(p-isothiocyanatophenyl)propane-1,2-disulfate by capillary electrophoresis. The basis of the chiral discrimination between QA-beta-CD and the enantiomers was investigated by (1)H NMR spectroscopy. COSY and NOESY spectra were used to infer the role that molecular interactions and the stereocenters have upon association of QA-beta-CD with the enantiomers. A parallel two-step complexation model is used to rationalize the NMR and the chiral discrimination observed during separation of the enantiomers.

Interaction between non-heme iron of lipoxygenases and cumene hydroperoxide: basis for enzyme activation, inactivation, and inhibition.

Lipoxygenase catalysis depends in a critical fashion on the redox properties of a unique mononuclear non-heme iron cofactor. The isolated enzyme contains predominantly, if not exclusively, iron(II), but the catalytically active form of the enzyme has iron(III). The activating oxidation of the iron takes place in a reaction with the hydroperoxide product of the catalyzed reaction. In a second peroxide-dependent process, lipoxygenases are also inactivated. To examine the redox activation/inactivation dichotomy in lipoxygenase chemistry, the interaction between lipoxygenase-1 (and -3) and cumene hydroperoxide was investigated. Cumene hydroperoxide was a reversible inhibitor of the reaction catalyzed by lipoxygenase-1 under standard assay conditions at high substrate concentrations. Reconciliation of the data with the currently held kinetic mechanism requires simultaneous binding of substrate and peroxide. The enzyme also was both oxidized and largely inactivated in a reaction with the peroxide in the absence of substrate. The consequences of this reaction for the enzyme included the hydroxylation at C beta of two amino acid side chains in the vicinity of the cofactor, Trp and Leu. The modifications were identified by mass spectrometry and X-ray crystallography. The peroxide-induced oxidation of iron was also accompanied by a subtle rearrangement in the coordination sphere of the non-heme iron atom. Since the enzyme retains catalytic activity, albeit diminished, after treatment with cumene hydroperoxide, the structure of the iron site may reflect the catalytically relevant form of the cofactor.

An addition to the oxoacid family: H2B12(OH)12.

The acid H(2)B(12)(OH)(12) can be isolated as a crystalline solid by protonation of the hydroxylated borane anion, B(12)(OH)(12)(2)(-). This acidic compound has low solubility in water, conducts protons in the solid state, and has thermal stability to a temperature of 400 degrees C. The conductivity mechanism is a Grotthuss mechanism with a low activation enthalpy (9-13 kcal/mol). This new acid represents an addition to the class of oxoacids, of which sulfuric and phosphoric acid are the most prominent examples.