Codenitrification and denitrification are dual metabolic pathways through which dinitrogen evolves from nitrate in Streptomyces antibioticus

We screened actinomycete strains for dinitrogen (N(2))-producing activity and discovered that Streptomyces antibioticus B-546 evolves N(2) and some nitrous oxide (N(2)O) from nitrate (NO(3)(-)). Most of the N(2) that evolved from the heavy isotope ([(15)N]NO(3)(-)) was (15)N(14)N, indicating that this nitrogen species consists of two atoms, one arising from NO(3)(-) and the other from different sources. This phenomenon is similar to codenitrification in fungi. The strain also evolved less, but significant, amounts of (15)N(15)N from [(15)N]NO(3)(-) in addition to (15)N(15)NO with concomitant cell growth. Prior to the production of N(2) and N(2)O, NO(3)(-) was rapidly reduced to nitrite (NO(2)(-)) accompanied by distinct cell growth, showing that the actinomycete strain is a facultative anaerobe that depends on denitrification and nitrate respiration for anoxic growth. The cell-free activities of denitrifying enzymes could be reconstituted, supporting the notion that the (15)N(15)N and (15)N(15)NO species are produced by denitrification from NO(3)(-) via NO(2)(-). We therefore demonstrated a unique system in an actinomycete that produces gaseous nitrogen (N(2) and N(2)O) through both denitrification and codenitrification. The predominance of codenitrification over denitrification along with oxygen tolerance is the key feature of nitrate metabolism in this actinomycete.     

Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro.

NO (nitric oxide) production from sunflower plants (Helianthus annuus L.), detached spinach leaves (Spinacia oleracea L.), desalted spinach leaf extracts or commercial maize (Zea mays L.) leaf nitrate reductase (NR, EC 1.6.6.1) was continuously followed as NO emission into the gas phase by chemiluminescence detection, and its response to post-translational NR modulation was examined in vitro and in vivo. NR (purified or in crude extracts) in vitro produced NO at saturating NADH and nitrite concentrations at about 1% of its nitrate reduction capacity. The K(m) for nitrite was relatively high (100 microM) compared to nitrite concentrations in illuminated leaves (10 microM). NO production was competitively inhibited by physiological nitrate concentrations (K(i)=50 microM). Importantly, inactivation of NR in crude extracts by protein phosphorylation with MgATP in the presence of a protein phosphatase inhibitor also inhibited NO production. Nitrate-fertilized plants or leaves emitted NO into purified air. The NO emission was lower in the dark than in the light, but was generally only a small fraction of the total NR activity in the tissue (about 0.01-0.1%). In order to check for a modulation of NO production in vivo, NR was artificially activated by treatments such as anoxia, feeding uncouplers or AICAR (a cell permeant 5'-AMP analogue). Under all these conditions, leaves were accumulating nitrite to concentrations exceeding those in normal illuminated leaves up to 100-fold, and NO production was drastically increased especially in the dark. NO production by leaf extracts or intact leaves was unaffected by nitric oxide synthase inhibitors. It is concluded that in non-elicited leaves NO is produced in variable quantities by NR depending on the total NR activity, the NR activation state and the cytosolic nitrite and nitrate concentration.     

Endogenous ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase that is not regulated by nitric oxide in Dictyostelium discoideum

A 41,000 M_r cytosolic protein (p41) in Dictyostelium discoideum was shown to be modified by ADP-ribosylation that was not regulated by nitric oxide (NO). This endogenous ADP-riboxylation was optimal at conditions distinct from those optimal for the NO-stimulated ADP-ribosylation of p41. These two activities were also differentially sensitive to reducing agents and modified different amino acids. The addition of haemoglobin, which sequesters NO, and 3 the NO synthase inhibitors failed to block the endogenous ADP-ribosylation. P41 was purified to homogeneity. The N-terminal sequence of the purified protein was shown to be highly homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both endogenous and NO-stimulated activities ADP-ribosylated three isoforms of the protein, with pI values of 6.6., 6.8 and 7.0. In each case, the isoform with pI 6.8 was preferentially modified. Experiments using purified GAPDH indicate that both the endogenous and NO-stimulated ADP-ribosylation are self-catalysed modifications.     

Cytochrome c' from Paracoccus denitrificans: spectroscopic studies consistent with a role for the protein in nitric oxide metabolism

Cytochrome c' was purified from the denitrifying bacterium Paracoccus denitrificans and the interaction of the protein with nitric oxide was examined spectroscopically. Two distinct types of haem-nitrosyl electronic absorption spectrum were observed, which were dependent upon [NO]. When cytochrome c' was saturated with NO, alpha and beta bands were centred at 562 nm and 530 nm, whereas with sub-saturating concentrations of NO the alpha and beta bands were red-shifted to 578 nm and 542 nm respectively. Further spectroscopic analysis showed that purified cytochrome c', added to suspensions of P. denitrificans, is able to complex with the NO which is formed as a freely diffusible intermediate of denitrification. In the presence of added NO-3 or NO-2, 40-60% of Fe(II)-cytochrome c' forms a 6-coordinate haem-nitrosyl complex. In the absence of nitrogen oxyanions or NO whole denitrifying cells are able to remove the NO from a Fe(II)-cytochrome c'-NO complex. These findings support the hypothesis that the physiological function of this enigmatic cytochrome involves the reversible binding of nitric oxide.     

Nitric oxide biosynthesis by Leishmania amazonensis promastigotes containing a high percentage of metacyclic forms

Due to the diversity of its physiological and pathophysiological functions and general ubiquity, the study of nitric oxide (NO) has become of great interest. In this work, it was demonstrated that Leishmania amazonensis promastigotes produces NO, a free radical synthesized from l-arginine by nitric oxide synthase (NOS). A soluble NOS was purified from L. amazonensis promastigotes by affinity chromatography (2′, 5′-ADP-agarose) and on SDS-PAGE the enzyme migrates as a single protein band of 116.2 (±6) kDa. Furthermore, the presence of a constitutive NOS was detected through indirect immunofluorescence using anti-cNOS and in NADPH consumption assays. The present work show that NO production, detected as nitrite in culture supernatant, is prominent in promastigotes preparations with high number of metacyclic forms, suggesting an association with the differentiation and the infectivity of the parasite.     

The cytochrome cbb3 from Pseudomonas stutzeri displays nitric oxide reductase activity.

The cytochrome cbb3 is an isoenzyme in the family of cytochrome c oxidases. This protein purified from Pseudomonas stutzeri displays a cyanide-sensitive nitric oxide reductase activity (Vmax=100+/-9 mol NO x mol cbb3(-1) x min(-1) and Km=12+/-2.5 microm), which is lost upon denaturation. This enzyme is only partially reduced by ascorbate, and readily re-oxidized by NO under anaerobic conditions at a rate consistent with the turnover number for NO consumption. As shown by transient spectroscopy experiments and singular value decomposition (SVD) analysis, these results suggest that the cbb3-type cytochromes, sharing structural features with bacterial nitric oxide reductases, are the enzymes retaining the highest NO reductase activity within the heme-copper oxidase superfamily.     

Nitric Oxide Upregulates Proteasomal Protein Degradation in Neurons

Nitric oxide (NO) is involved in many neuronal functions such as neuromodulation and intracellular signaling. Recent studies have demonstrated that nitric oxide is involved in regulation of proteasomal protein degradation. However, its role in neuronal protein degradation still remains unclear. In our study, we investigated the influence of endogenous nitric oxide production in this process. We have shown that nitric oxide synthase blockade prevents decline of the Ub^G76V-GFP fluorescence (GFP-based proteasomal protein degradation reporter) in neuronal processes of the cultured hippocampal neurons. It suggests that nitric oxide may regulate ubiquitin-dependent proteasomal protein degradation in neurons. Also, we have confirmed that the NO synthesis blockade alone significantly impairs long-term potentiation, and demonstrated for the first time that simultaneous blockade of the NO and proteins synthesis leads to the long-term potentiation amplitude rescue to the control values. Obtained results suggest that nitric oxide is involved in the protein degradation in proteasomes in physiological conditions.     

Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca²⁺/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases—protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases—are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation. shear stress; nitric oxide; endothelial cells; protein kinases     

Purification and properties of ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase) from Streptomyces antibioticus.

Two forms of ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase) have been purified from Streptomyces antibioticus. The larger form has an M(r) of 88,000, while the M(r) of a smaller form is 47,000. Both synthetase forms are active in the formation of guanosine 5'-triphosphate, 3'-diphosphate in reaction mixtures containing methanol. Unlike the RelA protein from Escherichia coli, the synthetases from S. antibioticus do not use GDP efficiently as a substrate. Experiments using crude extracts of S. antibioticus mycelium and the 88,000-M(r) form of guanosine pentaphosphate synthetase strongly suggest that the 47,000-M(r) species is produced by proteolysis of the larger species. This conclusion is supported by the observation that antibody to either protein reacts with the other protein. Thus, the 88,000-M(r) species may be the catalytically relevant protein in vivo. Unlike the RelA protein, the 88,000-M(r) protein is not activated by ribosomes. Modest levels of guanosine pentaphosphate synthesis were observed in mycelial extracts derived from nine other actinomycetes.     

The nitric oxide reductase of Paracoccus denitrificans.

The nitric oxide (NO) reductase activity of the cytoplasmic membrane of Paracoccus denitrificans can be solubilized in dodecyl maltoside with good retention of activity. The solubilized enzyme lacks NADH-dependent activity, but can be assayed with isoascorbate plus 2,3,5,6-tetramethylphenylene-1,4-diamine as electron donor and with horse heart cytochrome c as mediator. Reduction of NO was measured with an amperomeric electrode. The solubilized enzyme could be separated from other electron-transport components, including the cytochrome bc1 complex and nitrite reductase, by several steps of chromatography. The purified enzyme had a specific activity of 11 mumols.min-1.mg of protein-1 and the Km(NO) was estimated as less than 10 microM. The enzyme formed N2O from NO with the expected stoichiometry. These observations support the view that NO reductase is a discrete enzyme that participates in the denitrification process. The enzyme contained both b- and c-type haems. The former was associated with a polypeptide of apparent molecular mass 37 kDa and the latter with a polypeptide of 18 kDa. Polypeptides of 29 and 45 kDa were also identified in the purified protein which showed variable behaviour on electrophoresis in polyacrylamide gels.     

Identification of nitric oxide as an endogenous activator of the AMP-activated protein kinase in vascular endothelial cells

In endothelial cells, the AMP-activated protein kinase (AMPK) is stimulated by sheer stress or growth factors that stimulate release of nitric oxide (NO). We hypothesized that NO might act as an endogenous activator of AMPK in endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) to NO donors caused an increase in phosphorylation of both Thr-172 of AMPK and Ser-1177 of endothelial nitric oxide synthase, a downstream enzyme of AMPK. NO-induced activation of AMPK was not affected by inhibition of LKB1, an AMPK kinase. In contrast, inhibition of calcium calmodulin-dependent protein kinase kinase abolished the effect of NO in HUVECs. NO-induced AMPK activation in HeLa S3 cells was abolished by either 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalon-1-one, a potent inhibitor for guanylyl cyclase, or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), an intracellular Ca(2+) chelator, indicating that NO-induced AMPK activation is guanylyl cyclase-mediated and calcium-dependent. Exposure of HUVECs or isolated mice aortas to either calcium ionophore A23187 or bradykinin significantly increased AMPK Thr-172 phosphorylation, which was abolished by N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase. Finally, A23187- or bradykinin-enhanced AMPK activation was significantly greater in aortas from wild type mice than those in the aortas of endothelial nitric oxide synthase knock-out mice. Taken together, we conclude that NO might act as an endogenous AMPK activator.     

Purification and cDNA cloning of nitric oxide reductase cytochrome P450nor (CYP55A4) from Trichosporon cutaneum.

Cytochrome P450nor is involved in fungal denitrification as nitric oxide (NO) reductase. Although the heme protein has been known to occur in restricted species of fungi that belong to ascomycotina, we have previously suggested that it would also occur in the yeast Trichosporon cutaneum, which is phylogenetically far from those P450nor-producing ascomycetous fungi. Here we isolated and characterized the heme protein from the basidiomycetous yeast T. cutaneum. P450nor of the yeast (TcP450nor) exhibited properties in terms of catalysis, absorption spectrum and molecular mass that are almost identical to those of its counterparts in ascomycetous fungi. We also isolated and sequenced its cDNA. The predicted primary structure of TcP450nor showed high sequence identities (around 65%) to those of other P450nors, indicating that they belong to the same family. TcP450nor protein cofractionated with cytochrome c oxidase by subcellular fractionation and its predicted primary structure contained an extension on its amino terminus that is characteristic of a mitochondrial-targeting signal, indicating that it is a mitochondrial protein like some of the isoforms of other fungi. On the other hand, TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells. The occurrence of P450nor across the subdivisions of eumycota suggests that P450nor and denitrification are distributed more universally among fungi than was previously thought.     

Purification and properties of insulin-activated nitric oxide synthase from human erythrocyte membranes

A membrane bound form of nitric oxide synthase of human erythrocytes that could be activated by insulin was purified to homogeneity by detergent solubilization of the purified membrane preparation of these cells. The purified enzyme (M(r) 230 KD) was found to be composed of one heavy chain (M(r) 135 KD) and one light chain (Mr 95 KD) held together by disulphide bond(s). Scatchard plot analysis of insulin binding to the purified enzyme showed the presence of 2 different populations of the binding sites and the activation were directly related to the hormone binding to the protein. Line weaver Burk plot of the purified enzyme showed that the stimulation of the enzymic activity by insulin was related to the decrease of K(m) with simultaneous increase of V(max). Treatment of the purified enzyme with anti insulin receptor antibody inhibited the activation of the enzyme and the binding of the hormone to the protein. Furthermore NO itself, at low concentration (<0.4 microM) activated the enzyme, but at higher concentration (>0.8 microM) had no effect on the activation. Incubation of the purified enzyme with insulin simultaneously stimulated the tyrosine kinase and nitric oxide synthase activities of the preparations, that could be inhibited by genistein (an inhibitor of tyrosine kinase). These results indicated that the insulin activated nitric oxide synthase could be the insulin receptor itself.     

Increased expression of arginase II in human diabetic corpus cavernosum: in diabetic-associated erectile dysfunction

Nitric oxide (NO) is the principal mediator of penile erection. NO is synthesized by nitric oxide synthase (NOS). It has been well documented that the major causative factor contributing to erectile dysfunction in diabetic patients is the reduction in the amount of NO synthesis in the corpora cavernosa of the penis resulting in alterations of normal penile homeostasis. Arginase is an enzyme that shares a common substrate with NOS, thus arginase may downregulate NO production by competing with NOS for this substrate, l-arginine. The purpose of the present study was to compare arginase gene expression, protein levels, and enzyme activity in diabetic human cavernosal tissue. When compared to normal human cavernosal tissue, diabetic corpus cavernosum from humans with erectile dysfunction had higher levels of arginase II protein, gene expression, and enzyme activity. In contrast, gene expression and protein levels of arginase I were not significantly different in diabetic cavernosal tissue when compared to control tissue. The reduced ability of diabetic tissue to convert l-arginine to l-citrulline via nitric oxide synthase was reversed by the selective inhibition of arginase by 2(S)-amino-6-boronohexanoic acid (ABH). These data suggest that the increased expression of arginase II in diabetic cavernosal tissue may contribute to the erectile dysfunction associated with this common disease process and may play a role in other manifestations of diabetic disease in which nitric oxide production is decreased.     

Formation of the N-N bond from nitric oxide by a membrane-bound cytochrome bc complex of nitrate-respiring (denitrifying) Pseudomonas stutzeri.

Nitric oxide (NO) reductase was solubilized by Triton X-100 from the membrane fraction of Pseudomonas stutzeri ZoBell and purified 100-fold to apparent electrophoretic homogeneity. The enzyme consisted of two polypeptides of Mr 38,000 and 17,000 associated with heme b and heme c, respectively. Absorption maxima of the reduced complex were at 420.5, 522.5, and 552.5 nm, with a shoulder at 560 nm. The electron paramagnetic resonance spectrum was characteristic of high- and low-spin ferric heme proteins; no signals typical for iron-sulfur proteins were found. Nitric oxide reductase stoichiometrically transformed NO to nitrous oxide in an ascorbate-phenazine methosulfate-dependent reaction with a specific activity of 11.8 mumols/min per mg of protein. The activity increased to 40 mumols upon the addition of soybean phospholipids, n-octyl-beta-D-glucopyranoside, or its thio derivative to the assay system. Apparent Km values for NO and phenazine methosulfate were 60 and 2 microM, respectively. The pH optimum of the reaction was at 4.8. Cytochrome co was purified from P. stutzeri to permit its distinction from NO reductase. Spectrophotometric binding assays and other criteria also differentiated NO reductase from the respiratory cytochrome bc1 complex.     

PIN inhibits nitric oxide and superoxide production from purified neuronal nitric oxide synthase

A protein inhibitor of neuronal nitric oxide synthase (nNOS) was identified and designated as PIN. PIN was reported to inhibit nNOS activity in cell lysates through disruption of enzyme dimerization. However, there has been lack of direct characterization of the effect of PIN on NO production from purified nNOS. Furthermore, nNOS also generates superoxide (.O(2)(-)) at low levels of L-arginine. It is unknown whether PIN affects .O(2)(-) generation from nNOS. Therefore, we performed direct measurements of the effects of PIN on NO and .O(2)(-) generation from purified nNOS using electron paramagnetic resonance spin trapping techniques. nNOS was isolated by affinity chromatography and a fusion protein CBP-PIN was used to probe the effect of PIN. While the tag CBP did not affect nNOS activity, CBP-PIN caused a dose-dependent inhibition on both NO and L-citrulline production. In the absence of L-arginine, strong .O(2)(-) generation was observed from nNOS, and this was blocked by CBP-PIN in a dose-dependent manner. With low-temperature polyacrylamide gel electrophoresis, neither CBP nor CBP-PIN was found to affect nNOS dimerization. Thus, these results suggested that PIN not only inhibits NO but also .O(2)(-) production from nNOS, and this is through a mechanism other than decomposition of nNOS dimers.     

Use of a chemiluminescence detector for quantitation of nitric oxide produced in assays of denitrifying enzymes

We have developed a closed-flow system that continuously sweeps away gases evolved in enzyme assay mixtures into a commercially available oxides of nitrogen (NOx) analyzer for the quantitation of any nitric oxide present in these gases. The system enabled us to study both the stoichiometry and the kinetics of NO production by a copper-containing nitrite reductase (EC 1.7.99.3) purified from Achromobacter cycloclastes IAM 1013. In addition to its much greater sensitivity in comparison with standard gas chromatographic (GC) techniques, the method offers the advantage that NO, a very reactive free radical, is immediately swept away and quantitated, obviating the necessity for periodic manipulations and disturbances of the reaction mixture characteristic of other GC quantitations. The characteristics of the system are discussed and its utility in studies of the kinetics and stoichiometry of NO production from nitrite is confirmed by comparison with results obtained using manometric and GC techniques.     

Nitric oxide-reductase homologue that contains a copper atom and has cytochrome c-oxidase activity from an aerobic phototrophic bacterium Roseobacter denitrificans

A cytochrome cb-type enzyme with cytochrome c-oxidase activity was purified from an aerobic phototrophic bacterium Roseobacter denitrificans. The enzyme was solubilized with sucrose monodecanoate from the membranes of R. denitrificans grown aerobically under light conditions, and purified to electrophoretic homogeneity. Absorption spectra of the purified enzyme showed peaks at 410 nm and 530 nm in the oxidized state, and peaks at 420, 522, and 551 nm and a shoulder at around 560 nm in the reduced state. The enzyme is composed of two subunits with apparent molecular weights on SDS-PAGE of 37,000 and 18,000, the latter positive to heme staining. The protein contains heme c, heme b, and copper in a 1:2:1 stoichiometry. The spectral properties indicated that the heme c and one heme b are in low-spin states, while the other heme b is in a high-spin state. The base sequences of the genes and the deduced amino acid sequences are similar to those of known NorB and NorC subunits of nitric oxide reductases from other bacterial species. The enzyme is similar to nitric oxide reductase, but differs in that it contains copper. Virtually no nitric oxide reductase activity was detected in the purified enzyme.