Identification of B. anthracis N(5)-carboxyaminoimidazole ribonucleotide mutase (PurE) active site binding compounds via fragment library screening.

The de novo purine biosynthesis pathway is an attractive target for antibacterial drug design, and PurE from this pathway has been identified to be crucial for Bacillus anthracis survival in serum. In this study we adopted a fragment-based hit discovery approach, using three screening methods-saturation transfer difference nucleus magnetic resonance (STD-NMR), water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR, and surface plasmon resonance (SPR), against B. anthracis PurE (BaPurE) to identify active site binding fragments by initially testing 352 compounds in a Zenobia fragment library. Competition STD NMR with the BaPurE product effectively eliminated non-active site binding hits from the primary hits, selecting active site binders only. Binding affinities (dissociation constant, KD) of these compounds varied between 234 and 301µM. Based on test results from the Zenobia compounds, we subsequently developed and applied a streamlined fragment screening strategy to screen a much larger library consisting of 3000 computationally pre-selected fragments. Thirteen final fragment hits were confirmed to exhibit binding affinities varying from 14µM to 700µM, which were categorized into five different basic scaffolds. All thirteen fragment hits have ligand efficiencies higher than 0.30. We demonstrated that at least two fragments from two different scaffolds exhibit inhibitory activity against the BaPurE enzyme.

Differences in the purification and solution properties of PurC gene products from Streptococcus pneumoniae and Bacillus anthracis.

4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide synthetase (PurC) is a key enzyme in the de novo purine biosynthetic pathway of bacteria and an ideal target pathway for the discovery of antimicrobials. Bacillus anthracis (Ba) and Streptococcus pneumoniae (Sp) are two of the bacteria shown to be severe detriments to public health. To be able to carry out the experimentation that leads to drug discovery, high yields of pure soluble recombinant protein must first be obtained. We studied two recombinant PurC proteins from B. anthracis and S. pneumoniae, using Escherichia coli as the host cells. These two proteins, with very similar amino acid sequences, exhibit very different solution properties, leading to a large difference in yields during protein purification under the same conditions. The yield for SpPurC (>50mG per gram of cells) is ten times greater than that for BaPurC (<5mG per gram of cells). The BaPurC samples in solution consisted of oligomers and dimers, with dimers as its functional form. Comparing the yields of dimers, SpPurC is 25 times greater than that for BaPurC (∼2mG per gram of cell). Our studies suggest that the difference in exposed hydrophobic surface area is responsible for the difference in yields under the same conditions.

Identification of Bacillus anthracis PurE inhibitors with antimicrobial activity.

N(5)-carboxy-amino-imidazole ribonucleotide (N(5)-CAIR) mutase (PurE), a bacterial enzyme in the de novo purine biosynthetic pathway, has been suggested to be a target for antimicrobial agent development. We have optimized a thermal shift method for high-throughput screening of compounds binding to Bacillus anthracis PurE. We used a low ionic strength buffer condition to accentuate the thermal shift stabilization induced by compound binding to Bacillus anthracis PurE. The compounds identified were then subjected to computational docking to the active site to further select compounds likely to be inhibitors. A UV-based enzymatic activity assay was then used to select inhibitory compounds. Minimum inhibitory concentration (MIC) values were subsequently obtained for the inhibitory compounds against Bacillus anthracis (ΔANR strain), Escherichia coli (BW25113 strain, wild-type and ΔTolC), Francisella tularensis, Staphylococcus aureus (both methicillin susceptible and methicillin-resistant strains) and Yersinia pestis. Several compounds exhibited excellent (0.05-0.15µg/mL) MIC values against Bacillus anthracis. A common core structure was identified for the compounds exhibiting low MIC values. The difference in concentrations for inhibition and MIC suggest that another enzyme(s) is also targeted by the compounds that we identified.

Structures of SAICAR synthetase (PurC) from Streptococcus pneumoniae with ADP, Mg2+, AIR and Asp.

Streptococcus pneumoniae is a multidrug-resistant pathogen that is a target of considerable interest for antibacterial drug development. One strategy for drug discovery is to inhibit an essential metabolic enzyme. The seventh step of the de novo purine-biosynthesis pathway converts carboxyaminoimidazoleribonucleotide (CAIR) and L-aspartic acid (Asp) to 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) in the presence of adenosine 5'-triphosphate (ATP) using the enzyme PurC. PurC has been shown to be conditionally essential for bacterial replication. Two crystal structures of this essential enzyme from Streptococcus pneumoniae (spPurC) in the presence of adenosine 5'-diphosphate (ADP), Mg(2+), aminoimidazoleribonucleotide (AIR) and/or Asp have been obtained. This is the first structural study of spPurC, as well as the first of PurC from any species with Asp in the active site. Based on these findings, two model structures are proposed for the active site with all of the essential ligands (ATP, Mg(2+), Asp and CAIR) present, and a relay mechanism for the formation of the product SAICAR is suggested.

Elucidation of the bicarbonate binding site and insights into the carboxylation mechanism of (N(5))-carboxyaminoimidazole ribonucleotide synthase (PurK) from Bacillus anthracis.

Structures of (N(5))-carboxyaminoimidazole ribonucleotide synthase (PurK) from Bacillus anthracis with various combinations of ATP, ADP, Mg(2+), bicarbonate and aminoimidazole ribonucleotide (AIR) in the active site are presented. The binding site of bicarbonate has only been speculated upon previously, but is shown here for the first time. The binding involves interactions with the conserved residues Arg272, His274 and Lys348. These structures provide insights into each ligand in the active site and allow a possible mechanism to be proposed for the reaction that converts bicarbonate and AIR, in the presence of ATP, to produce (N(5))-carboxyaminoimidazole ribonucleotide. The formation of a carboxyphosphate intermediate through ATP phosphoryl transfer is proposed, followed by carboxylation of AIR to give the product, facilitated by a cluster of conserved residues and an active-site water network.

Nanomole-scale protein solid-state NMR by breaking intrinsic 1HT1 boundaries.

We present an approach that accelerates protein solid-state NMR 5-20-fold using paramagnetic doping to condense data-collection time (to approximately 0.2 s per scan), overcoming a long-standing limitation on slow recycling owing to intrinsic (1)H T(1) longitudinal spin relaxation. Using low-power schemes under magic-angle spinning at 40 kHz, we obtained two-dimensional (13)C-(13)C and (13)C-(15)N solid-state NMR spectra for several to tens of nanomoles of beta-amyloid fibrils and ubiquitin in 1-2 d.

Crystal structure of the nonerythroid alpha-spectrin tetramerization site reveals differences between erythroid and nonerythroid spectrin tetramer formation.

We have solved the crystal structure of a segment of nonerythroid alpha-spectrin (alphaII) consisting of the first 147 residues to a resolution of 2.3 A. We find that the structure of this segment is generally similar to a corresponding segment from erythroid alpha-spectrin (alphaI) but exhibits unique differences with functional significance. Specific features include the following: (i) an irregular and frayed first helix (Helix C'); (ii) a helical conformation in the junction region connecting Helix C' with the first structural domain (D1); (iii) a long A(1)B(1) loop in D1; and (iv) specific inter-helix hydrogen bonds/salt bridges that stabilize D1. Our findings suggest that the hydrogen bond networks contribute to structural domain stability, and thus rigidity, in alphaII, and the lack of such hydrogen bond networks in alphaI leads to flexibility in alphaI. We have previously shown the junction region connecting Helix C' to D1 to be unstructured in alphaI (Park, S., Caffrey, M. S., Johnson, M. E., and Fung, L. W. (2003) J. Biol. Chem. 278, 21837-21844) and now find it to be helical in alphaII, an important difference for alpha-spectrin association with beta-spectrin in forming tetramers. Homology modeling and molecular dynamics simulation studies of the structure of the tetramerization site, a triple helical bundle of partial domain helices, show that mutations in alpha-spectrin will affect Helix C' structural flexibility and/or the junction region conformation and may alter the equilibrium between spectrin dimers and tetramers in cells. Mutations leading to reduced levels of functional tetramers in cells may potentially lead to abnormal neuronal functions.

Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization.

With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697-2145) of non-erythroid beta spectrin (ßII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with ßII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with ßII-C (IP(ßII-C) s). The proteins include a fragment (residues 38-284) of "THAP domain containing, apoptosis associated protein 3, isoform CRA g", "glioma tumor suppressor candidate region gene 2" (residues 1-478), a fragment (residues 74-442) of septin 8 isoform c, a fragment (residues 704-953) of "coatomer protein complex, subunit beta 1, a fragment (residues 146-614) of zinc-finger protein 251, and a fragment (residues 284-435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these ßII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IP(αII-N) s) [1] on spectrin tetramer formation. The results showed that 3 IP(ßII-C) s were able to bind ßII-C even in the presence of αII-N, and 4 IP(αII-N) s were able to bind αII-N in the presence of ßII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and ßII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.

Yeast two-hybrid and itc studies of alpha and beta spectrin interaction at the tetramerization site.

Yeast two-hybrid (Y2H) and isothermal titration calorimetry (ITC) methods were used to further study the mutational effect of non-erythroid alpha spectrin (αII) at position 22 in tetramer formation with beta spectrin (ßII). Four mutants, αII-V22D, V22F, V22M and V22W, were studied. For the Y2H system, we used plasmids pGBKT7, consisting of the cDNA of the first 359 residues at the N-terminal region of αII, and pGADT7, consisting of the cDNA of residues 1697-2145 at the C-terminal region of ßII. Strain AH109 yeast cells were used for colony growth assays and strain Y187 was used for ß-galactosidase activity assays. Y2H results showed that the C-terminal region of ßII interacts with the N-terminal region of αII, either the wild type, or those with V22F, V22M or V22W mutations. The V22D mutant did not interact with ßII. For ITC studies, we used recombinant proteins of the αII N-terminal fragment and of the erythroid beta spectrin (ßI) C-terminal fragment; results showed that the K(d) values for V22F were similar to those for the wild-type (about 7 nM), whereas the K(d) values were about 35 nM for V22M and about 90 nM for V22W. We were not able to detect any binding for V22D with ITC methods. This study clearly demonstrates that the single mutation at position 22 of αII, a region critical to the function of nonerythroid α spectrin, may lead to a reduced level of spectrin tetramers and abnormal spectrin-based membrane skeleton. These abnormalities could cause abnormal neural activities in cells.

Important residue (G46) in erythroid spectrin tetramer formation.

Spectrin tetramerization is important for the erythrocyte to maintain its unique shape, elasticity and deformability. We used recombinant model proteins to show the importance of one residue (G46) in the erythroid alpha-spectrin junction region that affects spectrin tetramer formation. The G46 residue in the erythroid spectrin N-terminal junction region is the only residue that differs from that in non-erythroid spectrin. The corresponding residue is R37. We believe that this difference may be, at least in part, responsible for the 15-fold difference in the equilibrium constants of erythroid and non-erythroid tetramer formation. In this study, we replaced the Gly residue with Ala, Arg or Glu residues in an erythroid alpha-spectrin model protein to give G46A, G46R or G46E, respectively. We found that their association affinities with a beta-spectrin model protein were quite different from each other. G46R exhibited a 10-fold increase and G46E exhibited a 16-fold decrease, whereas G46A showed little difference, when compared with the wild type. The thermal and urea denaturation experiments showed insignificant structural change in G46R. Thus, the differences in affinity were due to differences in local, specific interactions, rather than conformational differences in these variants. An intra-helical salt bridge in G46R may stabilize the partial domain single helix in alpha-spectrin, Helix C', to allow a more stable helical bundling in the alphabeta complex in spectrin tetramers. These results not only showed the importance of residue G46 in erythroid alpha-spectrin, but also provided insights toward the differences in association affinity between erythroid and non-erythroid spectrin to form spectrin tetramers.

Inhibition of calpain but not caspase activity by spectrin fragments.

Calpains and caspases are ubiquitous cysteine proteases that are associated with a variety of cellular pathways. Calpains are involved in processes such as long term potentiation, cell motility and apoptosis, and have been shown to cleave non-erythroid (brain) alpha- and beta-spectrin and erythroid beta-spectrin. The cleavage of erythroid alpha-spectrin by calpain has not been reported. Caspases play an important role in the initiation and execution of apoptosis, and have been shown to cleave non-erythroid but not erythroid spectrin. We have studied the effect of spectrin fragments on calpain and caspase activities. The erythroid and non-erythroid spectrin fragments used were from the N-terminal region of alpha-spectrin, and C-terminal region of beta-spectrin, both consisting of regions involved in spectrin tetramer formation. We observed that the all spectrin fragments exhibited a concentration-dependent inhibitory effect on calpain, but not caspase activity. It is clear that additional studies are warranted to determine the physiological significance of calpain inhibition by spectrin fragments. Our findings suggest that calpain activity is modulated by the presence of spectrin partial domains at the tetramerization site. It is not clear whether the inhibitory effect is substrate specific or is a general effect. Further studies of this inhibitory effect may lead to the identification and development of new therapeutic agents specifically for calpains, but not for caspases. Proteins/peptides with a coiled coil helical conformation should be studied for potential inhibitory effects on calpain activity.

Structural approaches to pathway-specific antimicrobial agents.

This perspective provides an overview of the evolution of antibiotic discovery from a largely phenotypic-based effort, through an intensive structure-based design focus, to a more holistic approach today. The current focus on antibiotic development incorporates assay and discovery conditions that replicate the host environment as much as feasible. They also incorporate several strategies, including target identification and validation within the whole cell environment, a variety of target deconvolution methods, and continued refinement of structure-based design approaches.

Nonactive-Site Mutations in S. aureus FabI That Induce Triclosan Resistance.

The wide use of the antimicrobial agent/biocide, triclosan, promotes triclosan-resistant bacterial strains, including Staphylococcus aureus, as well as leads to accumulation in the aquatic and terrestrial environments. Knowledge of the molecular actions of triclosan on S. aureus is needed to understand the consequence of triclosan resistance and environmental accumulation of triclosan on S. aureus resistant strains, as well as to develop biphenyl ether analogs as antibiotic candidates. Triclosan inhibits an essential enzyme in the fatty acid biosynthetic pathway, the reduced nicotinamide adenine dinucleotide (NADH)/reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enoyl-acyl carrier protein (enoyl-ACP) reductase, or FabI. In this study, we used error-prone polymerase chain reaction (epPCR) to generate mutations in the S. aureus FabI enzyme. Instead of using an elaborate FabI enzyme activity assay that involves ACP-linked substrates to determine whether triclosan inhibits the enzyme activities of individual FabI mutants, we used an efficient and economical assay that we developed, based on thermal shift principles, to screen for triclosan binding to FabI mutants in cells. We identified four active-site mutations. More interestingly, we also identified nine triclosan-resistant mutations distant from the active site (G113V, Y123H, S166N, N220I, G227C, A230T, V241I, F252I, and H253P) but located in disparate positions in the monomer-monomer and dimer-dimer interface regions in S. aureus FabI. We suggest that these sites may serve as potential allosteric sites for designing potential therapeutic inhibitors that offer advantages in selectivity since allosteric sites are less evolutionarily conserved.

Glutamate racemase dimerization inhibits dynamic conformational flexibility and reduces catalytic rates.

Glutamate racemase (RacE) is a bacterial enzyme that converts l-glutamate to d-glutamate, an essential precursor for peptidoglycan synthesis. In prior work, we have shown that both isoforms cocrystallize with d-glutamate as dimers, and the enzyme is in a closed conformation with limited access to the active site [May, M., et al. (2007) J. Mol. Biol. 371, 1219-1237]. The active site of RacE2 is especially restricted. We utilize several computational and experimental approaches to understand the overall conformational dynamics involved during catalysis when the ligand enters and the product exits the active site. Our steered molecular dynamics simulations and normal-mode analysis results indicate that the monomeric form of the enzyme is more flexible than the native dimeric form. These results suggest that the monomeric enzyme might be more active than the dimeric form. We thus generated site-specific mutations that disrupt dimerization and find that the mutants exhibit significantly higher catalytic rates in the d-Glu to l-Glu reaction direction than the native enzyme. Low-resolution models restored from solution X-ray scattering studies correlate well with the first six normal modes of the dimeric form of the enzyme, obtained from NMA. Thus, along with the local active site residues, global domain motions appear to be implicated in the catalytically relevant structural dynamics of this enzyme and suggest that increased flexibility may accelerate catalysis. This is a novel observation that residues distant from the catalytic site restrain catalytic activity through formation of the dimer structure.

Association studies of erythroid alpha-spectrin at the tetramerization site.

The functional roles of residues 21-43 and 55-59 in the alpha-spectrin N-terminal region in forming tetramers were determined by the introduction of mutations at each of these positions. We measured association affinities for tetramer formation (K(d)), which can be used to predict clinical severity, of these mutants. A total of nine residues critical for association with beta-spectrin were found. The mutations of six of these residues have already been known to cause hereditary elliptocytosis or hereditary pyropoikilocytosis. Clinical symptoms associated with three mutations of residues 23, 57 and 58 have not yet been reported. We suggest that these mutations may also introduce abnormalities to erythrocytes.

Conformational change of erythroid alpha-spectrin at the tetramerization site upon binding beta-spectrin.

We previously determined the solution structures of the first 156 residues of human erythroid alpha-spectrin (SpalphaI-1-156, or simply Spalpha). Spalpha consists of the tetramerization site of alpha-spectrin and associates with a model beta-spectrin protein (Spbeta) with an affinity similar to that of native alpha- and beta-spectrin. Upon alphabeta-complex formation, our previous results indicate that there is an increase in helicity in the complex, suggesting conformational change in either Spalpha or Spbeta or in both. We have now used isothermal titration calorimetry, circular dichroism, static and dynamic light scattering, and solution NMR methods to investigate properties of the complex as well as the conformation of Spalpha in the complex. The results reveal a highly asymmetric complex, with a Perrin shape parameter of 1.23, which could correspond to a prolate ellipsoid with a major axis of about five and a minor axis of about one. We identified 12 residues, five prior to and seven following the partial domain helix in Spalpha that moved freely relative to the structural domain in the absence of Spbeta but when in the complex moved with a mobility similar to that of the structural domain. Thus, it appears that the association with Spbeta induced an unstructured-to-helical conformational transition in these residues to produce a rigid and asymmetric complex. Our findings may provide insight toward understanding different association affinities of alphabeta-spectrin at the tetramerization site for erythroid and non-erythroid spectrin and a possible mechanism to understand some of the clinical mutations, such as L49F of alpha-spectrin, which occur outside the functional partial domain region.

Brain proteins interacting with the tetramerization region of non-erythroid alpha spectrin.

The N-terminal region of non-erythroid alpha spectrin (Sp alpha II) is responsible for interacting with its binding partner, beta spectrin, to form functional spectrin tetramers. We used a yeast-two-hybrid system, with an N-terminal segment of alpha spectrin representing the functional tetramerization site, as a bait to screen human brain c-DNA library for proteins that interact with the alpha spectrin segment. In addition to several beta spectrin isoforms, we identified 14 proteins that interact with Sp alpha II. Seven of the 14 were matched to 6 known proteins: Duo protein, Lysyl-tRNA synthetase, TBP associated factor 1, two isoforms (b and c) of a protein kinase A interacting protein and Zinc finger protein 333 (2 different segments). Four of the 6 proteins are located primarily in the nucleus, suggesting that spectrin plays important roles in nuclear functions. The remaining 7 proteins were unknown to the protein data base. Structural predictions show that many of the 14 proteins consist of a large portion of unstructured regions, suggesting that many of these proteins fold into a rather flexible conformation. It is interesting to note that all but 3 of the 14 proteins are predicted to consist of one to four coiled coils (amphiphilic helices). A mutation in Sp alpha II, V22D, which interferes with the coiled coil bundling of Sp alpha II with beta spectrin, also affects Sp alpha II interaction with Duo protein, TBP associated factor 1 and Lysyl-tRNA synthetase, suggesting that they may compete with beta spectrin for interaction with Sp alpha II. Future structural and functional studies of these proteins to provide interaction mechanisms will no doubt lead to a better understanding of brain physiology and pathophysiology.

An Efficient and Economical Assay to Screen for Triclosan Binding to FabI.

Triclosan is an effective inhibitor for enoyl acyl carrier protein reductase (ENR) in fatty acid biosynthesis. Triclosan-resistant mutants of ENR have emerged. Thus, it is important to detect these triclosan-resistant mutations in ENR. Generally, enzyme activity assays on the mutants are used to determine the effect of triclosan on ENR activity. Since the substrates are linked to acyl carrier protein (ACP), the assays are challenging due to the need to prepare the ACP and link it to the substrates. Non-ACP-linked (coenzyme A [CoA]-linked) substrates can be used in some ENR, but not in all. Consequently, screening for triclosan-resistant mutants is also challenging. We have developed a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the ENR active site, and thus it can be used for screening for triclosan-resistant mutants. Staphylococcus aureus FabI enzyme and its mutants were used to demonstrate the binding ability of triclosan with NADP(+) to FabI. The direct correlation between the binding ability and enzyme activity was demonstrated with Francisella tularensis FabI. This method may also be applied to select effective triclosan analogues that inhibit ENR activity.

Mutational effects at the tetramerization site of nonerythroid alpha spectrin.

Spectrin, a prominent cytoskeletal protein, exerts its fundamental role in cellular function by forming a sub-membrane filamentous network. An essential aspect of spectrin network formation is the tetramerization of spectrin alphabeta heterodimers. We used laboratory methods, the yeast two-hybrid system and random mutagenesis, to investigate, for the first time, effects of amino acid mutations on tetramerization of nonerythroid (brain) spectrin (fodrin). Based on high sequence homology with erythroid spectrin, we assume the putative tetramerization region of nonerythroid alpha-spectrin at the N-terminal region. We introduced mutations in the region consisting of residues 1-45 and studied mutational effects on spectrin alphabeta association to form tetramers. We detected single, double, and triple mutations involving 24 residues in this region. These amino acid mutations of nonerythroid alpha-spectrin exhibit full, partial, or no effect on the association with nonerythroid beta-spectrin. Single amino acid mutations in the region of residues 1-9 (D2Y, G5V, V6D, and V8M) did not affect the association. However, seven single mutations (I15F, I15N, R18G, V22D, R25P, Y26N, and R28P) affected the alphabeta association. These mutations were clustered in the region predicted by sequence alignment to be crucial in nonerythroid alpha-spectrin for tetramerization, a region that spanned residues 12-36, corresponding to the partial domain Helix C' (residues 21-45) in erythroid alpha-spectrin. In addition, two other mutations, one upstream and one downstream of this region at positions 10 (E10D) and 37 (R37P), also affected the alphabeta association. Our results implied nonerythroid alpha-spectrin partial domain helix may be longer than Helix C' (residues 21-45 and a total of 25 residues) in erythroid alpha-spectrin and spanned at least residues 10-37. It is interesting to note that seven out of these nine single mutations (I15F, I15N, R18G, V22D, R25P, Y26N, R37P) were at the a, d, e or g heptad positions based on sequence alignment with erythroid alpha-spectrin. Four of the mutated residues (I15, R18, V22, R25) are conserved in both erythroid and nonerythroid spectrin. These positions were previously identified as hot spots in erythroid alpha-spectrin that lead to severe hematological symptoms. This study clearly demonstrated that single mutation in a region predicted to be critical functionally in nonerythroid alpha-spectrin indeed leads to functional abnormalities and may lead to neurological disorders.

Dinitrosyliron complexes are the most abundant nitric oxide-derived cellular adduct: biological parameters of assembly and disappearance.

It is well established that nitric oxide ((•)NO) reacts with cellular iron and thiols to form dinitrosyliron complexes (DNIC). Little is known, however, regarding their formation and biological fate. Our quantitative measurements reveal that cellular concentrations of DNIC are proportionally the largest of all (•)NO-derived adducts (900 pmol/mg protein, or 45-90 µM). Using murine macrophages (RAW 264.7), we measured the amounts, and kinetics, of DNIC assembly and disappearance from endogenous and exogenous sources of (•)NO in relation to iron and O(2) concentration. Amounts of DNIC were equal to or greater than measured amounts of chelatable iron and depended on the dose and duration of (•)NO exposure. DNIC formation paralleled the upregulation of iNOS and occurred at low physiologic (•)NO concentrations (50-500 nM). Decreasing the O(2) concentration reduced the rate of enzymatic (•)NO synthesis without affecting the amount of DNIC formed. Temporal measurements revealed that DNIC disappeared in an oxygen-independent manner (t(1/2)=80 min) and remained detectable long after the (•)NO source was removed (>24 h). These results demonstrate that DNIC will be formed under all cellular settings of (•)NO production and that the contribution of DNIC to the multitude of observed effects of (•)NO must always be considered.