Sugar beet hemoglobins: reactions with nitric oxide and nitrite reveal differential roles for nitrogen metabolism.

In contrast with human hemoglobin (Hb) in red blood cells, plant Hbs do not transport oxygen, instead research points towards nitrogen metabolism. Using comprehensive and integrated biophysical methods we characterized three sugar beet Hbs: BvHb1.1, BvHb1.2 and BvHb2. Their affinities for oxygen, CO, and hexacoordination were determined. Their role in nitrogen metabolism was studied by assessing their ability to bind NO, to reduce nitrite (NiR, nitrite reductase), and to form nitrate (NOD, NO dioxygenase). Results show that BvHb1.2 has high NOD-like activity, in agreement with the high nitrate levels found in seeds where this protein is expressed. BvHb1.1, on the other side, is equally capable to bind NO as to form nitrate, its main role would be to protect chloroplasts from the deleterious effects of NO. Finally, the ubiquitous, reactive, and versatile BvHb2, able to adopt 'open and closed forms', would be part of metabolic pathways where the balance between oxygen and NO is essential. For all proteins, the NiR activity is relevant only when nitrite is present at high concentrations and both NO and oxygen are absent. The three proteins have distinct intrinsic capabilities to react with NO, oxygen and nitrite; however, it is their concentration which will determine the BvHbs' activity.

Discovery of Fungal Denitrification Inhibitors by Targeting Copper Nitrite Reductase from Fusarium oxysporum.

The efficient application of nitrogenous fertilizers is urgently required, as their excessive and inefficient use is causing substantial economic loss and environmental pollution. A significant amount of applied nitrogen in agricultural soils is lost as nitrous oxide (N2O) in the environment due to the microbial denitrification process. The widely distributed fungus Fusarium oxysporum is a major denitrifier in agricultural soils and its denitrification activity could be targeted to reduce nitrogen loss in the form of N2O from agricultural soils. Here, we report the discovery of first small molecule inhibitors of copper nitrite reductase (NirK) from F. oxysporum, which is a key enzyme in the fungal denitrification process. The inhibitors were discovered by a hierarchical in silico screening approach consisting of pharmacophore modeling and molecular docking. In vitro evaluation of F. oxysporum NirK activity revealed several pyrimidone and triazinone based compounds with potency in the low micromolar range. Some of these compounds suppressed the fungal denitrification in vivo as well. The compounds reported here could be used as starting points for the development of nitrogenous fertilizer supplements and coatings as a means to prevent nitrogen loss by targeting fungal denitrification.

The concept, reality and utility of single-site heterogeneous catalysts (SSHCs).

Very substantial advances have recently been made in the design and construction of solid catalysts and in elucidating both their mode of operation and the factors that determine their selectivity and longevity. This Perspective explains how and why such progress has been made. One important factor, the deployment of single-site heterogeneous and enzymatic catalysts, used either alone or in conjunction with other strategies, including metabolic engineering, enables a multitude of new products (for example, environmentally clean jet fuel) to be readily manufactured. In a practical sense SSHCs enable the advantages of homogeneous and to a lesser degree enzymatic catalysts to be united with those of heterogeneous ones. With the aid of the vastly increasing families of nanoporous solids, desired catalytically active sites may be engineered in atomic detail on their inner, accessible surfaces, thereby opening up new possibilities in synthetic organic chemistry - as in the smooth formation of C-C and C[double bond, length as m-dash]N bonds in a number of intermolecular reactions - as well as in photocatalysts and in fluidized catalytic cracking of hydrocarbons.

Selenite-reducing capacity of the copper-containing nitrite reductase of Rhizobium sullae.

Rhizobium sullae strain HCNT1 contains a nitric oxide-producing nitrite reductase of unknown function due to the absence of a complementary nitric oxide reductase. HCNT1 had the ability to grow on selenite concentrations as high as 50 mM, and during growth, selenite was reduced to the less toxic elemental selenium. An HCNT1 mutant lacking nitrite reductase grew poorly in the presence of 5 mM selenite, was unable to grow in the presence of 25 or 50 mM selenite and also showed no evidence of selenite reduction. A naturally occurring nitrite reductase-deficient R. sullae strain, CC1335, also showed little growth on the higher concentrations of selenite. Mobilization of a plasmid containing the HCNT1 gene encoding nitrite reductase into CC1335 increased its resistance to selenite. To confirm that this ability to grow in the presence of high concentrations of selenite correlated with nitrite reductase activity, a new nitrite reductase-containing strain was isolated from the same location where HCNT1 was isolated. This strain was also resistant to high concentrations of selenite. Inactivation of the gene encoding nitrite reductase in this strain increased selenite sensitivity. These data suggest that the nitrite reductase of R. sullae provides resistance to selenite and offers an explanation for the radically truncated denitrification found uniquely in this bacterium.

Generation of nitric oxide by a nitrite reductase activity of xanthine oxidase: a potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity.

Nitric oxide (NO) synthesis is well-known to result from the oxidation of L-arginine by a family of NO synthases (NOS). However, under hypoxic conditions this mechanism of NO synthesis may be impaired and NO is formed by a NOS independent mechanism. This study was designed to examine the reduction of nitrite to NO by xanthine oxidase (XO) under hypoxia, because the bacterial nitrate/nitrite reductases have structural similarity to XO. We found that both purified and tissue containing XO catalyze the reduction of nitrite to NO, as demonstrated using a chemiluminescent NO meter. This redox reaction requires NADH as an electron donor, and is oxygen independent. The inhibitory profiles suggest that reduction of nitrite takes place at the molybdenum center of XO whilst NADH is oxidized at the FAD center. Heparin binding of XO caused an increase in the catalysis of nitrite reduction. The XO-catalyzed generation of NO may be important in redistribution of blood flow to ischaemic tissue as a supplement to NOS, since both nitrite and NADH have been shown to be elevated in hypoxic tissue.

Functional dissection and site-directed mutagenesis of the structural gene for NAD(P)H-nitrite reductase in Neurospora crassa.

Neurospora crassa NAD(P)H-nitrite reductase, encoded by the nit-6 gene, is a soluble, alpha2-type homodimeric protein composed of 127-kDa polypeptide subunits. This multicenter oxidation-reduction enzyme utilizes either NADH or NADPH as electron donor and possesses as prosthetic groups two iron-sulfur (Fe4S4) clusters, two siroheme groups, and two FAD molecules. The native activity of the enzyme is the NAD(P)H-dependent reduction of nitrite to ammonia. In addition, N. crassa nitrite reductase displays several partial activities in vitro, including a siroheme-independent NAD(P)H-cytochrome c reductase activity and an FAD-independent dithionite-nitrite reductase activity. These partial activities are presumed to be manifestations of discrete functional domains within the protein. A full-length nit-6 cDNA was constructed and used in developing an expression system within E. coli capable of yielding high levels of NADPH-nitrite reductase activity. Maximal expression was obtained in nirB- E. coli cells grown anaerobically at 22 +/- 1 degrees C, in conjunction with co-expression of a plasmid-borne cysG gene (encoding the rate-limiting enzyme in siroheme synthesis) and co-transformation with plasmid pGroESL (encoding bacterial chaperonins GroES and GroEL). Dissection of gene segments encoding putative functional domains within the nit-6 gene was performed. Expression of a partial cDNA construct encoding the FAD-/NAD-binding domain yielded extracts with NADPH-cytochrome c reductase activity but no NADPH-nitrite reductase activity or dithionite-nitrite reductase activity. Expression of a cDNA construct encoding the (Fe4S4)-siroheme-binding domain resulted in extracts possessing dithionite-nitrite reductase activity but no NADPH-nitrite reductase or NADPH-cytochrome c reductase activity. Analysis of site-directed mutations altering amino acid residues Cys-331 within the FAD-/NAD-binding domain and Ser-755 within the (Fe4S4)-siroheme-binding domain of the nitrite reductase demonstrated that these residues were not essential for native or partial enzyme activity. Cys-757 within the (Fe4S4)-siroheme-binding domain was essential for native enzyme activity.