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Nitric oxide (NO) signaling controls various metabolic pathways in bacteria and higher eukaryotes. Cellular enzymes synthesize and detoxify NO; however, a mechanism that controls its cellular homeostasis has not been identified. Here, we found a nitrogen oxide cycle involving nitrate reductase (Nar) and the NO dioxygenase flavohemoglobin (Fhb), that facilitate inter-conversion of nitrate, nitrite, and NO in the actinobacterium Streptomyces coelicolor. This cycle regulates cellular NO levels, bacterial antibiotic production, and morphological differentiation. NO down-regulates Nar and up-regulates Fhb gene expression via the NO-dependent transcriptional factors DevSR and NsrR, respectively, which are involved in the auto-regulation mechanism of intracellular NO levels. Nitrite generated by the NO cycles induces gene expression in neighboring cells, indicating an additional role of the cycle as a producer of a transmittable inter-cellular communication molecule.

Achromobacter denitrificans YD35 is an NO2 (-)-tolerant bacterium that expresses the aconitase genes acnA3, acnA4, and acnB, of which acnA3 is essential for growth tolerance against 100 mm NO2 (-). Atmospheric oxygen inactivated AcnA3 at a rate of 1.6 × 10(-3) min(-1), which was 2.7- and 37-fold lower compared with AcnA4 and AcnB, respectively. Stoichiometric titration showed that the [4Fe-4S](2+) cluster of AcnA3 was more stable against oxidative inactivation by ferricyanide than that of AcnA4. Aconitase activity of AcnA3 persisted against high NO2 (-) levels that generate reactive nitrogen species with an inactivation rate constant of k = 7.8 × 10(-3) min(-1), which was 1.6- and 7.8-fold lower than those for AcnA4 and AcnB, respectively. When exposed to NO2 (-), the acnA3 mutant (AcnA3Tn) accumulated higher levels of cellular citrate compared with the other aconitase mutants, indicating that AcnA3 is a major producer of cellular aconitase activity. The extreme resistance of AcnA3 against oxidation and reactive nitrogen species apparently contributes to bacterial NO2 (-) tolerance. AcnA3Tn accumulated less cellular NADH and ATP compared with YD35 under our culture conditions. The accumulation of more NO by AcnA3Tn suggested that NADH-dependent enzymes detoxify NO for survival in a high NO2 (-) milieu. This novel aconitase is distributed in Alcaligenaceae bacteria, including pathogens and denitrifiers, and it appears to contribute to a novel NO2 (-) tolerance mechanism in this strain., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The actinomycete Streptomyces antibioticus was shown to produce nitrite (NO-(2)) and ammonium (NH+(4)]) when aerobically incubated in an organic nitrogen-rich medium. The production of NO-(2) was synchronized with rapid cell growth, whereas most NH+(4)] was produced after cell proliferation had ceased. Intracellular formation of nitric oxide (NO) was also observed during the incubation. The production of these inorganic nitrogen compounds along with cell growth was prevented by several enzyme inhibitors (of nitric oxide synthase or nitrate reductase) or glucose. Distinct, membrane-bound nitrate reductase was induced in the NO-(2)-producing cells. Tungstate (a potent inhibitor of this enzyme) prevented the NO-(2) production and cell growth, whereas it did not prevent the NO formation. These results revealed the occurrence of novel nitrogen metabolic pathway in S. antibioticus forming NO-(2) from organic nitrogen by which rapid cell growth is possible. NO synthase, NO dioxygenase (flavohemoglobin), and dissimilatory nitrate reductase are possible enzymes responsible for the NO-(2) formation.

Nitric oxide (NO) is a toxic reactive nitrogen species that induces microbial adaption mechanisms. Screening a genomic DNA library identified a new gene, ntpA, that conferred growth tolerance upon Aspergillus nidulans against exogenous NO. The gene encoded a cysteine-rich 23-amino-acid peptide that reacted with NO and S-nitrosoglutathione to generate an S-nitrosated peptide. Disrupting ntpA increased amounts of cellular S-nitrosothiol and NO susceptibility. Thioredoxin and its reductase denitrosated the S-nitrosated peptide, decreased cellular S-nitrosothiol and conferred tolerance against NO, indicating peptide-mediated catalytic NO removal. The peptide binds copper(I) in vitro but is dispensable for metal tolerance in vivo. NO but not metal ions induced production of the peptide and ntpA transcripts. We discovered that the thionein family of peptides has NO-related functions and propose that the new peptide be named NO-inducible nitrosothionein (iNT). The ubiquitous distribution of iNT-like polypeptides constitutes a potent NO-detoxifying mechanism that is conserved among various organisms.

We cloned and characterized the gene and cDNA of Aspergillus oryzae cytochrome P450nor (Anor). The Anor gene (nicA; CYP55A5) has a different gene structure from other P450nor genes in that it has an extra intron. There were not only two kinds of mRNA but also two sets of TATA-box and CCAAT-box, and it appears that this gene has two expression patterns, like CYP55A1 of Fusarium oxysporum. A reporter analysis using the uidA gene indicated that gene expression of CYP55A5 was induced under anaerobic conditions, like CYP55A1. When the CYP55A5 gene was overexpressed in A. oryzae, a large amount of active Anor were accumulated as intracellular protein. Anor employed both NADH and NADPH as electron donors for reducing nitric oxide to nitrous oxide. Anor measured the amount of NO generated from 3-(2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine (NOC5) with a spectrophotometer. The sensitivity was 10 nmol/ml.

A flavocytochrome protein was isolated from the actinomycete Streptomyces antibioticus. The purified protein contained protoheme and FAD, and its M(r) was estimated to be 52000. The absorption spectra in its resting oxidized, dithionite-reduced, carbon monoxide-bound, and oxygenated (O(2)-bound) forms were characteristic of those of flavohemoglobin (Fhb). The N-terminal amino acid sequence showed high identities to those of other Fhb's. Furthermore, the actinomycete flavocytochrome scavenged nitric oxide in the presence of NADH. These results demonstrated that the flavocytochrome is the first Fhb purified from actinomycetes. The actinomycete Fhb was produced in S. antibioticus cells in large amounts without any external nitric oxide (NO) stress, which is indicative of a physiological function of Fhb other than detoxification of NO.

Nitric oxide (NO) is a natural reactive nitrogen species (RNS) that alters proteins, DNA, and lipids and damages biological activities. Although microorganisms respond to and detoxify NO, the regulation of the cellular metabolic mechanisms that cause cells to tolerate RNS toxicity is not completely understood. We found that the proline and arginine auxotrophic proA5 and argB2 mutants of the fungus Aspergillus nidulans require more arginine and proline for normal growth under RNS stress that starves cells by accumulating fewer amino acids. Fungal transcriptomes indicated that RNS stress upregulates the expression of the biosynthetic genes required for global amino acids, including proline and arginine. A mutant of the gene disruptant, cpcA, which encodes the transcriptional regulation of the cross-pathway control of general amino acid synthesis, did not induce these genes, and cells accumulated fewer amino acids under RNS stress. These results indicated a novel function of CpcA in the cellular response to RNS stress, which is mediated through amino acid starvation and induces the transcription of genes for general amino acid synthesis. Since CpcA also controls organic acid biosynthesis, impaired intermediates of such biosynthesis might starve cells of amino acids. These findings revealed the importance of the mechanism regulating amino acid homeostasis for fungal responses to and survival under RNS stress. [ABSTRACT FROM AUTHOR]

The article discusses the filamentous fungi's metabolic mechanisms under hypoxic conditions. It presents the denitrification process as a mechanism used by various types of fungi or microorganisms to reduce nitrate for nitric oxide generation. A discussion regarding hypoxic response of the filamentous fungus Aspergillus nidulans is also presented.

The article reports on results of a study of the denitrification by the fungus Fusarium oxysporum which involves NADH-nitrate reductase. A description of the experimental set-up and measurement methods is presented. The study concluded that the fungus denitrified nitric oxide through NADH-Nar activity in addition to the ubiquinol-Nar mechanism.

Nitric oxide synthases (NOS) are flavoheme enzymes with important roles in biology. The reductase domain of neuronal NOS (nNOSr) contains a widely conserved acidic residue (Asp1393) that is thought to facilitate hydride transfer between NADPH and FAD. Previously we found that the D1393V and D1393N mutations lowered the NO synthesis activity and the rates of heme and flavin reduction in full-length nNOS. To examine the mechanisms for these results in greater detail, we incorporated D1393V and D1393N substitutions into nNOSr along with a truncated NADPH-FAD domain construct (FNR) and characterized the mutants. D1393V nNOSr had markedly lower (≤ 1000×) cytochrome c reductase, ferricyanide reductase, and NADPH oxidase activities than the wild type. D1393N nNOSr also had lower reductase activities (≤ 10×) but had greater NADPH oxidase activity than that of the wild type, as did its FNR fragment. Both mutants had an altered interaction between FAD and the nicotinamide ring of NADP+, slower flavin reduction by NADPH, altered FAD midpoint potentials, a normal CaM response, and, in one case (D1393N), faster flavin oxidation by O2 and a lack of FMN shielding in response to NADPH binding. The results suggest that the two mutants have compromised catalysis for two different reasons. In D1393V nNOSr, hydride transfer from NADPH to FAD is so slow that it compromises all downstream electron-transfer events. In D1393N nNOSr, the increased oxidation of reduced flavins by O2 and thermodynamic destabilization of the FAD semiquinone uncouples or limits electron transfer to an extent that it inhibits downstream catalysis. These effects are due in part to the mutations eliminating (D1393V) or altering (D1393N) the native side-chain hydrogen-bonding properties of Asp1393 as well as removing its negative charge. [ABSTRACT FROM AUTHOR]

The haem-distal pocket of nitric oxide reductase cytochrome P450 contains many Arg and Lys residues that are clustered to form a putative access channel for NADH. Asp88 is the sole negatively charged amino acid in this positive charge cluster, and thus it would be interesting to know its functional role. Here we found the intriguing phenomenon that mutation at this site of P450nor (D88A or D88V) considerably decreased the overall nitric oxide reductase activity without blocking the reducing half reaction in which the ferric enzyme–NO complex is reduced with NADH to yield a specific intermediate ( I). The results indicate that the catalytic turnover subsequent to the I formation was blocked by such mutation. This property of the mutants made it possible to perform kinetic analysis of the reduction step, which is impossible with the wild-type P450nor. These results are the first kinetic evidence for direct complex formation between P450nor and an electron donor (NADH or NADPH). The kinetic analysis also showed that the inhibition by chloride ions (Cl–) is competitive with respect to NAD(P)H, which highlights the importance of the binding site for Cl– (the anion hole) in the interaction with NAD(P)H. We also characterized another mutant (D393A) of P450nor. The results demonstrated that both Asp residues play important roles in the interaction with NADH, whereas the role of Asp88 is unique in that it must be essential for the release of NAD+ rather than binding to NADH. [ABSTRACT FROM AUTHOR]

Studies Glu71-Arg64 couple by researchers from Japan. Implication of the couple in the access channel for NADH in cytochrome P450nor; Presence of many charged amino acid residues on the putative access channel for NADH in the heme-distal pocket of P450nor; Significance on the characterization of the E71A mutant protein of P450nor.

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. [ABSTRACT FROM AUTHOR]

Nitric oxide reductase cytochrome P450nor catalyzes an unusual reaction, direct electron transfer from NAD(P)H to bound heme. Here, we succeeded in determining the crystal structure of P450nor in a complex with an NADH analogue, nicotinic acid adenine dinucleotide, which provides conclusive evidence for the mechanism of the unprecedented electron transfer. Comparison of the structure with those of dinucleotide-free forms revealed a global conformational change accompanied by intriguing local movements caused by the binding of the pyridine nucleotide. Arg64 and Arg174 fix the pyrophosphate moiety upon the dinucleotide binding. Stereo-selective hydride transfer from NADH to NO-bound heme was suggested from the structure, the nicotinic acid ring being fixed near the heme by the conserved Thr residue in the I-helix and the upward-shifted propionate side-chain of the heme. A proton channel near the NADH channel is formed upon the dinucleotide binding, which should direct continuous transfer of the hydride and proton. A salt-bridge network (Glu71-Arg64-Asp88) was shown to be crucial for a high catalytic turnover. [Copyright &y& Elsevier]