Effects of nitrous oxide on mitochondrial and cell respiration and growth in Distichlis spicata suspension cultures

The reversible inhibition of respiratory activity could provide a novel approach to the preservation of traditionally hard to store plant germplasm such as clonal materials and recalcitrant seed. The gaseous anesthetic nitrous oxide caused a reversible, dose-dependent, partial inhibition of dioxygen utilization in mitochondrial particles isolated from cell suspension cultures of the salt-tolerant marsh grass Distichlis spicata, with maximal inhibition of 33% after 30 minutes exposure to an atmosphere of 80% nitrous oxide plus 20% oxygen. Respiration of whole cells required longer time to be affected by the anesthetic, and was reversibly inhibited an average of 19% when measured using a differential respirometer. Exposure to 80% nitrous oxide plus 20% oxygen for up to 10 days caused no measurable effect on cell growth.Abbreviations PCV packed cell volume - EDTA sodium ethylenediaminetetraacetic acid - BSA bovine serum albumin - MOPS 3(N-morpholino) propanesulfonic acid - TMPD N,N,N',N'-tetramethyl-p-phenylene diamine - STP standard temperature and pressure     

Chemiluminescence of lucigenin by electrogenerated superoxide ions in aqueous solutions

The chemiluminescence reaction of lucigenin (Luc²⁺?2NO₃^?, N,N′‐dimethyl‐9,9′‐biacridinium dinitrate) at gold electrodes in dioxygen‐saturated alkaline aqueous solutions (pH 10) was investigated in detail by the use of electrochemical emission spectroscopy. We noted that both O₂ and Luc²⁺ are reduced on a gold electrode in aqueous solution of pH 10 in almost the same potential region. From this fact, we expected chemiluminescence based on a radical–radical coupling reaction of superoxide ion (O₂·^?) and one‐electron reduced form of Luc²⁺ (Luc·⁺, a radical cation). Chemiluminescence was actually observed in the potential range where O₂ and Luc²⁺ were simultaneously reduced at the electrodes. The effects were examined upon addition of enzymes, i.e. superoxide dismutase (SOD) and catalase, into the solution and the substitution of heavy water (D₂O) for light water (H₂O) as a solvent on the chemiluminescence. In the presence of native and active SOD, chemiluminescence was completely absent. On the other hand, chemiluminescence was observed, unchanged in the presence of either denatured and inert SOD or catalase. In addition, the amount of chemiluminescence in D₂O solution was about three times greater than that in H₂O solution. These results, together with cyclic voltammetric results, suggest that O₂·^? participates directly in the chemiluminescence but H₂O₂ does not, and the chemiluminescence results from the coupling reaction between O₂·^? and Luc^·+ under the present experimental conditions. These chemically unstable species, O₂·^? and Luc·⁺, are produced during the simultaneous electroreduction of O₂ and Luc²⁺. The coupling reaction between those radical species would lead to the formation of a dioxetane‐type intermediate and, finally, to chemiluminescence. The chemiluminescence reaction mechanism is discussed. Copyright © 2002 John Wiley & Sons, Ltd.     

The influence of dioxygen on luminol chemiluminescence

Assays of peroxy compounds are commonly performed after chromatographic separation of analysed mixtures. In high‐performance liquid chromatography (HPLC), solvent reservoirs are sparged by helium or inline vacuum‐degassed in order to control the compressibility of the solvents for efficient pumping. In this study, we investigated the influence of degassing the reaction solution on the light output of the hemin‐catalyzed luminol oxidation by various oxidants. We found that, when t‐butyl hydroperoxide, hydrogen peroxide, n‐butyl hydroperoxide, iodosobenzene and iodobenzene diacetate were used as oxidants, the luminol chemiluminescence was lowered by 50–70% compared with an equilibrated and degassed solution. The opposite effect was observed when dibenzoyl peroxide and 3‐chloroperoxybenzoic acid were used as oxidants, as the chemiluminescence increased by approximately 20–30%. The reduced chemiluminescence was explained based on the known role of dioxygen in luminol chemiluminescence. The enhancement of chemiluminescence was rationalized by suggesting an alternative mechanism of luminol oxidation valid for peroxyacids and diacyl peroxides in which the reaction of a peroxyacid anion with the diazaquinone led to light emission with a higher quantum yield than the usual path, which is suppressed by the removal of dioxygen from the reaction solution. Copyright © 2009 John Wiley & Sons, Ltd.     

Molecular Dynamics Simulation of Dioxygen Pathways through Mini Singlet Oxygen Generator (miniSOG), a Genetically Encoded Marker and Killer Protein

In this work, molecular dynamics (MD) simulations of the permeation of proteins by small gases of biological significance have been extended from gas carrier, sensor, and enzymatic proteins to genetically encoded tags and killer proteins. To this end, miniSOG was taken as an example of current high interest, using a biased form of MD, called random‐acceleration MD. Various egress gates and binding pockets for dioxygen, as an indistinguishable mimic of singlet dioxygen, were found on both above and below the isoalloxazine plane of the flavin mononucleotide cofactor in miniSOG. Of such gates and binding pockets, those lying within two opposite cones, coaxial with a line normal to the isoalloxazine plane, and with the vertex at the center of such a plane are those most visited by the escaping gas molecule. Out of residues most capable of quenching ¹O₂, Y30, lying near the base of one such a cone, and H85, near the base of the opposite cone, are held to be most responsible for the reduced quantum yield of ¹O₂ with folded miniSOG with respect to free flavin mononucleotide in solution.     

Lysine as a heme iron ligand: A property common to three truncated hemoglobins from Chlamydomonas reinhardtii

Background

The nuclear genome of Chlamydomonas reinhardtii encodes a dozen hemoglobins of the truncated lineage. Four of these, named THB1–4, contain a single ~130-residue globin unit. THB1, which is cytoplasmic and capable of nitric oxide dioxygenation activity, uses a histidine and a lysine as axial ligands to the heme iron. In the present report, we compared THB2, THB3, and THB4 to THB1 to gain structural and functional insights into algal globins.

Ambiguity associated with use of singlet oxygen trapping agents in myeloperoxidase-catalyzed oxidations.

It is shown that hypochlorous acid preferentially oxidizes 2,5-dimethylfuran, histidine, β-carotene and 1,4-diazabicyclo[2.2.2]octane in the presence of hydrogen peroxide without intermediary formation of singlet-excited molecular oxygen. It is therefore highly unlikely that the protective action of these compounds towards myeloperoxidase-catalyzed chlorination reactions is due to singlet oxygen deactivation or removal, and putative evidence based upon these effects for singlet oxygen participation in bactericidal reactions of myeloperoxidase-containing leukocytes is equivocal.     

Mutations at Asp112 adjacent to the trinuclear Cu center in CueO as the proton donor in the four-electron reduction of dioxygen

Asp112 adjacent to the trinuclear Cu center of a multicopper oxidase, CueO was mutated for Glu, Ala and Asn. Mutations on Asp112 affected not only spectroscopic and magnetic properties derived from the trinuclear Cu center but also enzyme activities. The uncoordinated Asp112 was found to play multiple roles to promote the binding of dioxygen at the trinuclear Cu center and to accelerate the conversion of dioxygen to water molecules by facilitating the supply of H+ to the reaction intermediates.     

Molecular simulations reveal a new entry site in quercetin 2,3‐dioxygenase. A pathway for dioxygen?

Molecular dynamics simulations performed on quercetin 2,3-dioxygenase have shown the existence of a channel linking the bulk solvent and the cavity of the enzyme. Although much is known about the the oxygenolysis reaction catalyzed by this enzyme, the way dioxygen enters the active site has not been firmly established. The size, orientation and hydrophobic character of this channel suggests that it could provide an entrance for molecular dioxygen into the cavity. Free energy calculations show that such a process is likely to occur.     

Encapsulation of Trigonopsis variabilis D-amino acid oxidase and fast comparison of the operational stabilities of free and immobilized preparations of the enzyme

A one-step procedure of immobilizing soluble and aggregated preparations of D-amino acid oxidase from Trigonopsis variabilis (TvDAO) is reported where carrier-free enzyme was entrapped in semipermeable microcapsules produced from the polycation poly(methylene-co-guanidine) in combination with CaCl2 and the polyanions alginate and cellulose sulfate. The yield of immobilization, expressed as the fraction of original activity present in microcapsules, was approximately 52 +/- 5%. The effectiveness of the entrapped oxidase for O2-dependent conversion of D-methionine at 25 degrees C was 85 +/- 10% of the free enzyme preparation. Because continuous spectrophotometric assays are generally not well compatible with insoluble enzymes, we employed a dynamic method for the rapid in situ estimation of activity and relatedly, stability of free and encapsulated oxidases using on-line measurements of the concentration of dissolved O2. Integral and differential modes of data acquisition were utilized to examine cases of fast and slow inactivation of the enzyme, respectively. With a half-life of 60 h, encapsulated TvDAO was approximately 720-fold more stable than the free enzyme under conditions of bubble aeration at 25 degrees C. The soluble oxidase was stabilized by added FAD only at temperatures of 35 degrees C or greater.     

Syntheses, characterization, and reactivity of copper complexes with tridentate N-donor ligands

The reaction of dioxygen with the copper(I) complex of the tridentate ligand 1,1,4,7,7-pentamethyldiethylenetriamine (Me₅dien) has been investigated using low-temperature stopped-flow techniques. The formation of a bis(μ-oxo)copper(III) complex as a reactive intermediate could be detected spectroscopically at low temperatures and a quantitative kinetic analysis was performed for this system. Crystal structures of the copper(II) complexes [(Me-bpa)Cu(Cl)₂] (1), [{(Me-bpa)Cu(Cl)(ClO₄)}₂] (2), [{(MeL)Cu(Cl)(ClO₄)}₂] (3), and [(MeL)Cu(NCS)₂] (4) (Me-bpa = N-methyl-[bis(2-pyridyl)methyl]amine; MeL = N-methyl-[(2-pyridyl)ethyl(2-pyridyl)methyl]amine) are reported.