Time-course curves suggest that nitrate is first converted to nitrite and subsequently to ammonia.

<p>Samples from <i>E.coli</i> (red line with circles) and <i>L</i>. <i>plantarum</i> (blue line with triangles) cultures grown at 2% O₂ with 5 mM nitrate were collected at regular intervals and analyzed for nitrite (panel A) and ammonia (panel B). (C). Ratio between nitrite and ammonia concentrations measured at each time point. Black lines represent <i>E.coli</i> cultures containing no added nitrate.</p

Bacterial NO generation and correlation with acidity of the growth medium.

<p>(A) Chemiluminescence detection of NO emission after injection of 100 μM nitrite in the vessel containing <i>E.coli</i> grown at different nitrate and oxygen conditions in modified LMRS broth for 24h. (B) Comparison of NO emission at 2% O₂ as in panel A, but using LAB cultures. Diagonal patter and solid bars indicate respectively the values detected before and after bacteria were re-suspended in fresh media at pH = 6.5 (as described in text). (C) Quantification of the amount of NO detected after injection of 100 μM or 250 μM nitrite in fresh LMRS media at different pH obtained by acidification with concentrated L-lactic acid. All data represent mean ± SD in duplicates.</p

Bacterial species and strains used in this study.

<p>Corresponding final pH of culture media and lactic acid concentration after 24 h growth at 2% O₂ in LMRS are indicated.</p><p>^a pH of LMRS media after 24 h of bacterial growth. All are ± 0.1 (initial pH = 6.5)</p><p>^b Values are ± 1 mM and each sample was assayed in triplicates.</p><p>^c ND = non detectable</p><p>Bacterial species and strains used in this study.</p

The effect of nitrate and oxygen gradients on the generation of nitrite and ammonia in different LAB cultures.

<p>Nitrite (A) and ammonia (B) concentrations were measured in LMRS media after 24 h growth at 2% O₂ with supplementation of different nitrate concentrations (0 to 10 mM). Similarly in (C) and (D) nitrate was fixed at 5 mM and we measured nitrite and ammonia dependence on oxygen concentrations (0, 2, 4, 6, 10 and 21%). Each point represents the mean ± SD (n = 3).</p

Nitrate and oxygen effect on <i>E.coli</i> bacterial cultures growth and formation of nitrite and ammonia.

<p>(A) Growth curves for <i>E.coli</i> MG1655 grown in the absence (black closed symbols) or in the presence of 5 mM nitrate (red open symbols) at 37°C in LMRS broth at 21%, 2%, and 0% O₂ concentrations (respectively square, circle and diamond symbols). (B) Concentration of nitrite and ammonia (blue and red solid lines) in <i>E.coli</i> pellets after 24 h growth at different oxygen levels with 5 mM nitrate. (C) and (D) Respectively dependence on nitrate (at 0% O₂) and oxygen (at 5 mM nitrate) of nitrite and ammonia concentrations in the cell-free culture media after 24h growth. The ammonia content of LMRS alone is indicated by the dashed lines. Values are means ± SD (n = 3). The average SD resulted smaller than the symbols dimensions (0.04 OD) and it is not shown for clarity.</p

Schematic representation of the link between different pathways of nitrogen oxides reduction in the human gut and the fate of ammonia.

<p>Each colored box represents a distinct pathway: Bacterial respiratory denitrification to dinitrogen in red box: the dissimilatory nitrate reduction to ammonia (DNRA) in blue box and the non-enzymatic conversion of nitrite to NO in green box (this route become significant only at pH<5.5). The endogenous L-arginine/NO synthase pathway from epithelial cells of the intestinal mucosa lining is also noted.</p

Nitrite Reductase Activity of Nonsymbiotic Hemoglobins from <i>Arabidopsis thaliana</i>

Plant nonsymbiotic hemoglobins possess hexacoordinate heme geometry similar to that of the heme protein neuroglobin. We recently discovered that deoxygenated neuroglobin converts nitrite to nitric oxide (NO), an important signaling molecule involved in many processes in plants. We sought to determine whether Arabidopsis thaliana nonsymbiotic hemoglobins classes 1 and 2 (AHb1 and AHb2, respectively) might function as nitrite reductases. We found that the reaction of nitrite with deoxygenated AHb1 and AHb2 generates NO gas and iron–nitrosyl–hemoglobin species. The bimolecular rate constants for reduction of nitrite to NO are 19.8 ± 3.2 and 4.9 ± 0.2 M–1 s–1, respectively, at pH 7.4 and 25 °C. We determined the pH dependence of these bimolecular rate constants and found a linear correlation with the concentration of protons, indicating the requirement for one proton in the reaction. The release of free NO gas during the reaction under anoxic and hypoxic (2% oxygen) conditions was confirmed by chemiluminescence detection. These results demonstrate that deoxygenated AHb1 and AHb2 reduce nitrite to form NO via a mechanism analogous to that observed for hemoglobin, myoglobin, and neuroglobin. Our findings suggest that during severe hypoxia and in the anaerobic plant roots, especially in species submerged in water, nonsymbiotic hemoglobins provide a viable pathway for NO generation via nitrite reduction

Heterobimetallic (Ferrocenyl)indenyl Rhodium Complexes. Synthesis, Crystal Structure, and Oxidative Activation of [η⁵-(1-Ferrocenyl)indenyl]RhL₂ [L₂ = COD, NBD, (CO)₂]^‖

The binuclear [η5-(1-ferrocenyl)indenyl]Rh(NBD) (1), [η5-(1-ferrocenyl)indenyl]Rh(COD) (1a), and [η5-(1-ferrocenyl)indenyl]Rh(CO)2 (2) complexes have been synthesized (NBD = norbornadiene; COD = cycloocta-1,5-diene). The crystal structure determination showed that the iron and rhodium nuclei are disposed in a transoid configuration in 1 probably to avoid steric repulsions. On the contrary, in 2 the metals are in a cisoid configuration due to the presence of stabilizing π-hydrogen bonds between the CO's and the hydrogens of the unsubstituted cyclopentadienyl ring. The results of the chemical and electrochemical oxidation of 2 are in favor of the existence of an effective interaction between the two metals

Reductase Domain of <i>Drosophila melanogaster</i> Nitric-Oxide Synthase: Redox Transformations, Regulation, and Similarity to Mammalian Homologues^†

The nitric oxide synthase of Drosophila melanogaster (dNOS) participates in essential developmental and behavioral aspects of the fruit fly, but little is known about dNOS catalysis and regulation. To address this, we expressed a construct comprising the dNOS reductase domain and its adjacent calmodulin (CaM) binding site (dNOSr) and characterized the protein regarding its catalytic, kinetic, and regulatory properties. The Ca2+ concentration required for CaM binding to dNOSr was between that of the mammalian endothelial and neuronal NOS enzymes. CaM binding caused the cytochrome c reductase activity of dNOSr to increase 4 times and achieve an activity comparable to that of mammalian neuronal NOS. This change was associated with decreased shielding of the FMN cofactor from solvent and an increase in the rate of NADPH-dependent flavin reduction. Flavin reduction in dNOSr was relatively slow following the initial 2-electron reduction, suggesting a slow inter-flavin electron transfer, and no charge-transfer complex was observed between bound NADP+ and reduced FAD during the process. We conclude that dNOSr catalysis and regulation is most similar to the mammalian neuronal NOS reductase domain, although differences exist in their flavin reduction behaviors. The apparent conservation between the fruit fly and mammalian enzymes is consistent with dNOS operating in various signal cascades that involve NO