The hemerythrin-like diiron protein from Mycobacterium kansasii is a nitric oxide peroxidase.

The hemerythrin-like protein from Mycobacterium kansasii (Mka HLP) is a member of a distinct class of oxo-bridged diiron proteins that are found only in mycobacterial species that cause respiratory disorders in humans. Because it had been shown to exhibit weak catalase activity and a change in absorbance on exposure to nitric oxide (NO), the reactivity of Mka HLP toward NO was examined under a variety of conditions. Under anaerobic conditions, we found that NO was converted to nitrite (NO2-) via an intermediate, which absorbed light at 520 nm. Under aerobic conditions NO was converted to nitrate (NO3-). In each of these two cases, the maximum amount of nitrite or nitrate formed was at best stoichiometric with the concentration of Mka HLP. When incubated with NO and H2O2, we observed NO peroxidase activity yielding nitrite and water as reaction products. Steady-state kinetic analysis of NO consumption during this reaction yielded a Km for NO of 0.44 µM and a kcat/Km of 2.3 × 105 M-1s-1. This high affinity for NO is consistent with a physiological role for Mka HLP in deterring nitrosative stress. This is the first example of a peroxidase that uses an oxo-bridged diiron center and a rare example of a peroxidase utilizing NO as an electron donor and cosubstrate. This activity provides a mechanism by which the infectious Mycobacterium may combat against the cocktail of NO and superoxide (O2•-) generated by macrophages to defend against bacteria, as well as to produce NO2- to adapt to hypoxic conditions.

Ordered Motions in the Nitric-Oxide Dioxygenase Mechanism of Flavohemoglobin and Assorted Globins with Tightly Coupled Reductases.

Nitric-oxide dioxygenases (NODs) activate and combine O2 with NO to form nitrate. A variety of oxygen-binding hemoglobins with associated partner reductases or electron donors function as enzymatic NODs. Kinetic and structural investigations of the archetypal two-domain microbial flavohemoglobin-NOD have illuminated an allosteric mechanism that employs selective tunnels for O2 and NO, gates for NO and nitrate, transient O2 association with ferric heme, and an O2 and NO-triggered, ferric heme spin crossover-driven, motion-controlled, and dipole-regulated electron-transfer switch. The proposed mechanism facilitates radical-radical coupling of ferric-superoxide with NO to form nitrate while preventing suicidal ferrous-NO formation. Diverse globins display the structural and functional motifs necessary for a similar allosteric NOD mechanism. In silico docking simulations reveal monomeric erythrocyte hemoglobin alpha-chain and beta-chain intrinsically matched and tightly coupled with NADH-cytochrome b5 oxidoreductase and NADPH-cytochrome P450 oxidoreductase, respectively, forming membrane-bound flavohemoglobin-like mammalian NODs. The neuroprotective neuroglobin manifests a potential NOD role in a close-fitting ternary complex with membrane-bound NADH-cytochrome b5 oxidoreductase and cytochrome b5. Cytoglobin interfaces weakly with cytochrome b5 for O2 and NO-regulated electron-transfer and coupled NOD activity. The mechanistic model also provides insight into the evolution of O2 binding cooperativity in hemoglobin and a basis for the discovery of allosteric NOD inhibitors.

Allostery in the nitric oxide dioxygenase mechanism of flavohemoglobin.

The substrates O2 and NO cooperatively activate the NO dioxygenase function of Escherichia coli flavohemoglobin. Steady-state and transient kinetic measurements support a structure-based mechanistic model in which O2 and NO movements and conserved amino acids at the E11, G8, E2, E7, B10, and F7 positions within the globin domain control activation. In the cooperative and allosteric mechanism, O2 migrates to the catalytic heme site via a long hydrophobic tunnel and displaces LeuE11 away from the ferric iron, which forces open a short tunnel to the catalytic site gated by the ValG8/IleE15 pair and LeuE11. NO permeates this tunnel and leverages upon the gating side chains triggering the CD loop to furl, which moves the E and F-helices and switches an electron transfer gate formed by LysF7, GlnE7, and water. This allows FADH2 to reduce the ferric iron, which forms the stable ferric-superoxide-TyrB10/GlnE7 complex. This complex reacts with internalized NO with a bimolecular rate constant of 1010 M-1 s-1 forming nitrate, which migrates to the CD loop and unfurls the spring-like structure. To restart the cycle, LeuE11 toggles back to the ferric iron. Actuating electron transfer with O2 and NO movements averts irreversible NO poisoning and reductive inactivation of the enzyme. Together, structure snapshots and kinetic constants provide glimpses of intermediate conformational states, time scales for motion, and associated energies.

Plant hemoglobins: a journey from unicellular green algae to vascular plants.

Globins (Glbs) are widely distributed in archaea, bacteria and eukaryotes. They can be classified into proteins with 2/2 or 3/3 α-helical folding around the heme cavity. Both types of Glbs occur in green algae, bryophytes and vascular plants. The Glbs of angiosperms have been more intensively studied, and several protein structures have been solved. They can be hexacoordinate or pentacoordinate, depending on whether a histidine is coordinating or not at the sixth position of the iron atom. The 3/3 Glbs of class 1 and the 2/2 Glbs (also called class 3 in plants) are present in all angiosperms, whereas the 3/3 Glbs of class 2 have been only found in early angiosperms and eudicots. The three Glb classes are expected to play different roles. Class 1 Glbs are involved in hypoxia responses and modulate NO concentration, which may explain their roles in plant morphogenesis, hormone signaling, cell fate determination, nutrient deficiency, nitrogen metabolism and plant-microorganism symbioses. Symbiotic Glbs derive from class 1 or class 2 Glbs and transport O2 in nodules. The physiological roles of class 2 and class 3 Glbs are poorly defined but could involve O2 and NO transport and/or metabolism, respectively. More research is warranted on these intriguing proteins to determine their non-redundant functions.

Starved Escherichia coli preserve reducing power under nitric oxide stress.

Nitric oxide (NO) detoxification enzymes, such as NO dioxygenase (NOD) and NO reductase (NOR), are important to the virulence of numerous bacteria. Pathogens use these defense systems to ward off immune-generated NO, and they do so in environments that contain additional stressors, such as reactive oxygen species, nutrient deprivation, and acid stress. NOD and NOR both use reducing equivalents to metabolically deactivate NO, which suggests that nutrient deprivation could negatively impact their functionality. To explore the relationship between NO detoxification and nutrient deprivation, we examined the ability of Escherichia coli to detoxify NO under different levels of carbon source availability in aerobic cultures. We observed failure of NO detoxification under both carbon source limitation and starvation, and those failures could have arisen from inabilities to synthesize Hmp (NOD of E. coli) and/or supply it with sufficient NADH (preferred electron donor). We found that when limited quantities of carbon source were provided, NO detoxification failed due to insufficient NADH, whereas starvation prevented Hmp synthesis, which enabled cells to maintain their NADH levels. This maintenance of NADH levels under starvation was confirmed to be dependent on the absence of Hmp. Intriguingly, these data show that under NO stress, carbon-starved E. coli are better positioned with regard to reducing power to cope with other stresses than cells that had consumed an exhaustible amount of carbon.

Type I flavohemoglobin of mycobacterium smegmatis is a functional nitric oxide dioxygenase.

Two flavohemoglobins, type I and type II, displaying distinct structural features and cofactor binding sites coexist in Mycobacterium smegmatis; however, none of these flavohemeproteins are characterized so far. We have cloned and expressed type I flavohemoglobin (FHb1) of Mycobacterium smegmatis, encoded by MSMEG_1336, and characterized its spectral and functional properties. FHb1 exists as a monomer and displays spectral and functional characteristics similar to HMP of E. coli. Specific NO dioxygenase (NOD) activity of FHb1 was estimated to be 63.5 nmol heme(-1) sec(-1) , which was nearly eightfold higher than the HbN of M. tuberculosis and matched closely to the HMP of E. coli on the basis of cellular heme content. FHb1 preferred NADH for the NO dioxygenation and exhibited rapid reduction of flavin adenine dinucleotide and heme iron using NADH as electron donor. Level of FHb1 transcript increased significantly in M. smegmatis in the presence of acidified nitrite, and a nitric oxide-responsive transcriptional regulator of Rrf2 family exists together with the FHb1 under the same operon. These results suggested that FHb1 of M. smegmatis is a functional NOD and may be involved in the stress management of its host toward nitric oxide and nitrosative stress.

Insight into metabolic sensors of nitrosative stress protection in Phytophthora infestans.

Phytophthora infestans, a representative of phytopathogenic oomycetes, have been proven to cope with redundant sources of internal and host-derived reactive nitrogen species (RNS). To gain insight into its nitrosative stress resistance mechanisms, metabolic sensors activated in response to nitrosative challenge during both in vitro growth and colonization of the host plant were investigated. The conducted analyses of gene expression, protein accumulation, and enzyme activity reveal for the first time that P. infestans (avirulent MP946 and virulent MP977 toward potato cv. Sarpo Mira) withstands nitrosative challenge and has an efficient system of RNS elimination. The obtained data indicate that the system protecting P. infestans against nitric oxide (NO) involved the expression of the nitric oxide dioxygenase (Pi-NOD1) gene belonging to the globin family. The maintenance of RNS homeostasis was also supported by an elevated S-nitrosoglutathione reductase activity and upregulation of peroxiredoxin 2 at the transcript and protein levels; however, the virulence pattern determined the expression abundance. Based on the experiments, it can be concluded that P. infestans possesses a multifarious system of metabolic sensors controlling RNS balance via detoxification, allowing the oomycete to exist in different micro-environments flexibly.

Modulating Nitric Oxide Dioxygenase and Nitrite Reductase of Cytoglobin through Point Mutations.

Cytoglobin is a hexacoordinate hemoglobin with physiological roles that are not clearly understood. Previously proposed physiological functions include nitric oxide regulation, oxygen sensing, or/and protection against oxidative stress under hypoxic/ischemic conditions. Like many globins, cytoglobin rapidly consumes nitric oxide under normoxic conditions. Under hypoxia, cytoglobin generates nitric oxide, which is strongly modulated by the oxidation state of the cysteines. This gives a plausible role for this biochemistry in controlling nitric oxide homeostasis. Mutations to control specific properties of hemoglobin and myoglobin, including nitric oxide binding/scavenging and the nitrite reductase activity of various globins, have been reported. We have mapped these key mutations onto cytoglobin, which represents the E7 distal ligand, B2/E9 disulfide, and B10 heme pocket residues, and examined the nitric oxide binding, nitric oxide dioxygenase activity, and nitrite reductase activity. The Leu46Trp mutation decreases the nitric oxide dioxygenase activity > 10,000-fold over wild type, an effect 1000 times greater than similar mutations with other globins. By understanding how particular mutations can affect specific reactivities, these mutations may be used to target specific cytoglobin activities in cell or animal models to help understand the precise role(s) of cytoglobin under physiological and pathophysiological conditions.

Interdomain electron transfer in flavohaemoglobin from Candida norvegensis with antibiotic azole compounds.

Flavohaemoglobins (FlavoHbs) function as nitric oxide dioxygenases, oxidizing nitric oxide with nitrite and shuttling electrons from NAD(P)H via FAD and O2 . Here, using pulse radiolysis, we investigate intramolecular electron transfer between FAD and haem b in FlavoHbs. We found that reduction of FlavoHb with hydrated electrons proceeded via two phases: an initial fast phase and a second slower process. Absorbance measured at 600 nm revealed fast flavin reduction followed by a slower decrease corresponding to reoxidation of FAD. The slower process was partially lost in FlavoHbs lacking FAD. These results suggest that the slower phase is attributable to intramolecular electron transfer from FAD to the haem iron. The rate constant in the absence of azole compound (3.3 × 103 s-1 ) was accelerated ~ 10-fold (2.7 × 104 s-1 ) by the binding of econazole, reflecting a conformational change in the open-to-closed transition.

Development of a microtiter plate-based analysis method of nitric oxide dioxygenase activity.

Nitric oxide (NO) functions in cell protection or cell death, depending on its concentration. Therefore, regulation of the intracellular concentrations of NO by its degradation systems is important for cellular functions. One of the NO degrading enzymes, flavohemoglobin (FHb), which has NO dioxygenase (NOD) activity, is a promising target for antibiotics, based on the finding that FHb-deficient pathogens exhibited reduced host toxicity. Here, we developed a high-throughput method to measure the NOD activity. Our newly developed method could contribute to the screening of potential antibiotics with NOD inhibitory activity.

A Nitric Oxide-Responsive Transcriptional Regulator NsrR Cooperates With Lrp and CRP to Tightly Control the hmpA Gene in Vibrio vulnificus.

Nitric oxide (NO) is an important antimicrobial effector produced by the host innate immune system to counteract invading pathogens. To survive and establish a successful infection, a fulminating human pathogen Vibrio vulnificus expresses the hmpA gene encoding an NO dioxygenase in an NO-responsive manner. In this study, we identified an Rrf2-family transcriptional regulator NsrR that is predicted to contain the Fe-S cluster coordinated by three cysteine residues. Transcriptome analysis showed that NsrR controls the expression of multiple genes potentially involved in nitrosative stress responses. Particularly, NsrR acts as a strong repressor of hmpA transcription and relieves the repression of hmpA upon exposure to NO. Notably, nsrR and hmpA are transcribed divergently, and their promoter regions overlap with each other. Molecular biological analyses revealed that NsrR directly binds to this overlapping promoter region, which is alleviated by loss of the Fe-S cluster, leading to the subsequent derepression of hmpA under nitrosative stress. We further found that a leucine-responsive regulatory protein (Lrp) negatively regulates hmpA in an NsrR-dependent manner by directly binding to the promoter region, presumably resulting in a DNA conformation change to support the repression by NsrR. Meanwhile, a cyclic AMP receptor protein (CRP) positively regulates hmpA probably through repression of nsrR and lrp by directly binding to each promoter region in a sequential cascade. Altogether, this collaborative regulation of NsrR along with Lrp and CRP enables an elaborate control of hmpA transcription, contributing to survival under host-derived nitrosative stress and thereby the pathogenesis of V. vulnificus.

Quantifying Nitric Oxide Flux Distributions.

Nitric oxide (NO) is a radical that is used as an attack molecule by immune cells. NO can interact and damage a range of biomolecules, and the biological outcome for bacteria assaulted with NO will be governed by how the radical distributes within their biochemical reaction networks. Measurement of those NO fluxes is complicated by the low abundance and transience of many of its reaction products. To overcome this challenge, we use computational modeling to translate measurements of several biochemical species (e.g., NO, O2, NO2-) into NO flux distributions. In this chapter, we provide a detailed protocol, which includes experimental measurements and computational modeling, to estimate the NO flux distribution in an Escherichia coli culture. Those fluxes will have uncertainty associated with them and we also discuss how further experiments and modeling can be employed for flux refinement.

Transcriptomic Identification and Biochemical Characterization of HmpA, a Nitric Oxide Dioxygenase, Essential for Pathogenesis of Vibrio vulnificus.

Nitric oxide (NO) and its derivatives are important effectors of host innate immunity, disrupting cellular function of infecting pathogens. Transcriptome analysis of Vibrio vulnificus, an opportunistic human pathogen, identified a set of genes induced upon exposure to NO. Among them, VvhmpA (V. vulnificus hmpA), encoding a multidomain NO dioxygenase, was the most greatly induced upon exposure to NO and was thus further characterized. Absorption spectra demonstrated that VvHmpA is a heme protein in which the heme iron can exist in either reduced, NO-bound, or oxidized state. Biochemical studies revealed that VvHmpA is a flavohemoglobin containing equimolar amounts of heme and FAD as cofactors. The K M and k cat values of VvHmpA for NO at 37°C, the temperature encountered by V. vulnificus in the host, were greater than those at 30°C, indicating that VvHmpA detoxifies high levels of NO effectively during infection. Compared with the wild type, the VvhmpA mutant exhibited a lower NO-decomposition activity and impaired growth in the presence of NO in vitro. Also, the cytotoxicity and survival of the VvhmpA mutant infecting the NO-producing murine macrophage cells were lower than those of the wild type. Furthermore, the mouse lethality of the VvhmpA mutant was reduced compared to that of the parental wild type. The combined results revealed that VvHmpA is a potent virulence factor that is induced upon exposure to NO and important for the survival and pathogenesis of V. vulnificus during infection.

Lysine as a heme iron ligand: A property common to three truncated hemoglobins from Chlamydomonas reinhardtii.

BACKGROUND: The nuclear genome of Chlamydomonas reinhardtii encodes a dozen hemoglobins of the truncated lineage. Four of these, named THB1-4, contain a single ~130-residue globin unit. THB1, which is cytoplasmic and capable of nitric oxide dioxygenation activity, uses a histidine and a lysine as axial ligands to the heme iron. In the present report, we compared THB2, THB3, and THB4 to THB1 to gain structural and functional insights into algal globins. METHODS: We inspected properties of the globin domains prepared by recombinant means through site-directed mutagenesis, electronic absorption, CD, and NMR spectroscopies, and X-ray crystallography. RESULTS: Recombinant THB3, which lacks the proximal histidine but has a distal histidine, binds heme weakly. NMR data demonstrate that the recombinant domains of THB2 and THB4 coordinate the ferrous heme iron with the proximal histidine and a lysine from the distal helix. An X-ray structure of ferric THB4 confirms lysine coordination. THB1, THB2, and THB4 have reduction potentials between -65 and -100 mV, are capable of nitric oxide dioxygenation, are reduced at different rates by the diaphorase domain of C. reinhardtii nitrate reductase, and show different response to peroxide treatment. CONCLUSIONS: Three single-domain C. reinhardtii hemoglobins use lysine as a distal heme ligand in both Fe(III) and Fe(II) oxidation states. This common feature is likely related to enzymatic activity in the management of reactive oxygen species. GENERAL SIGNIFICANCE: Primary structure analysis of hemoglobins has limited power in the prediction of heme ligation. Experimental determination reveals variations in this essential property across the superfamily.

An integrated network analysis identifies how ArcAB enables metabolic oscillations in the nitric oxide detoxification network of Escherichia coli.

The virulences of many pathogens depend on their abilities to detoxify the immune antimicrobial nitric oxide (NO•). The functions of bacterial NO• detoxification machinery depend on oxygen (O2 ), with O2 inhibiting some enzymes, whereas others use it as a substrate. Previously, Escherichia coli NO• detoxification was found to be highly attenuated under microaerobic conditions and metabolic oscillations were observed. The oscillations in [NO•] and [O2 ] were found to result from the inhibitory action of NO• on aerobic respiration, the catalytic inactivation of NO• by Hmp (an NO• dioxygenase), and an imbalanced competition for O2 between Hmp and cytochrome terminal oxidase activity. Here the authors investigated the role of the ArcAB two component system (TCS) in microaerobic NO• detoxification. The authors observed that wild-type, ΔarcA, and ΔarcB had comparable initial NO• clearance times; however, the mutant cultures failed to exhibit [NO•] and [O2 ] oscillations. Using an approach that employed experimentation and computational modeling, the authors found that the loss of oscillations in ΔarcA was due to insufficient induction of cytochrome bd-I expression. Collectively, these results establish ArcAB as a TCS that influences NO• detoxification in E. coli within the physiologically-relevant microaerobic regime.

Characterization of the Heme Pocket Structure and Ligand Binding Kinetics of Non-symbiotic Hemoglobins from the Model Legume Lotus japonicus.

Plant hemoglobins (Hbs) are found in nodules of legumes and actinorhizal plants but also in non-symbiotic organs of monocots and dicots. Non-symbiotic Hbs (nsHbs) have been classified into two phylogenetic groups. Class 1 nsHbs show an extremely high O2 affinity and are induced by hypoxia and nitric oxide (NO), whereas class 2 nsHbs have moderate O2 affinity and are induced by cold and cytokinins. The functions of nsHbs are still unclear, but some of them rely on the capacity of hemes to bind diatomic ligands and catalyze the NO dioxygenase (NOD) reaction (oxyferrous Hb + NO → ferric Hb + nitrate). Moreover, NO may nitrosylate Cys residues of proteins. It is therefore important to determine the ligand binding properties of the hemes and the role of Cys residues. Here, we have addressed these issues with the two class 1 nsHbs (LjGlb1-1 and LjGlb1-2) and the single class 2 nsHb (LjGlb2) of Lotus japonicus, which is a model legume used to facilitate the transfer of genetic and biochemical information into crops. We have employed carbon monoxide (CO) as a model ligand and resonance Raman, laser flash photolysis, and stopped-flow spectroscopies to unveil major differences in the heme environments and ligand binding kinetics of the three proteins, which suggest non-redundant functions. In the deoxyferrous state, LjGlb1-1 is partially hexacoordinate, whereas LjGlb1-2 shows complete hexacoordination (behaving like class 2 nsHbs) and LjGlb2 is mostly pentacoordinate (unlike other class 2 nsHbs). LjGlb1-1 binds CO very strongly by stabilizing it through hydrogen bonding, but LjGlb1-2 and LjGlb2 show lower CO stabilization. The changes in CO stabilization would explain the different affinities of the three proteins for gaseous ligands. These affinities are determined by the dissociation rates and follow the order LjGlb1-1 > LjGlb1-2 > LjGlb2. Mutations LjGlb1-1 C78S and LjGlb1-2 C79S caused important alterations in protein dynamics and stability, indicating a structural role of those Cys residues, whereas mutation LjGlb1-1 C8S had a smaller effect. The three proteins and their mutant derivatives exhibited similarly high rates of NO consumption, which were due to NOD activity of the hemes and not to nitrosylation of Cys residues.

Covalent attachment of the heme to Synechococcus hemoglobin alters its reactivity toward nitric oxide.

The cyanobacterium Synechococcus sp. PCC 7002 produces a monomeric hemoglobin (GlbN) implicated in the detoxification of reactive nitrogen and oxygen species. GlbN contains a b heme, which can be modified under certain reducing conditions. The modified protein (GlbN-A) has one heme-histidine C-N linkage similar to the C-S linkage of cytochrome c. No clear functional role has been assigned to this modification. Here, optical absorbance and NMR spectroscopies were used to compare the reactivity of GlbN and GlbN-A toward nitric oxide (NO). Both forms of the protein are capable of NO dioxygenase activity and both undergo heme bleaching after multiple NO challenges. GlbN and GlbN-A bind NO in the ferric state and form diamagnetic complexes (FeIII-NO) that resist reductive nitrosylation to the paramagnetic FeII-NO forms. Dithionite reduction of FeIII-NO GlbN and GlbN-A, however, resulted in distinct outcomes. Whereas GlbN-A rapidly formed the expected FeII-NO complex, NO binding to FeII GlbN caused immediate heme loss and, remarkably, was followed by slow heme rebinding and HNO (nitrosyl hydride) production. Additionally, combining FeIII GlbN, 15N-labeled nitrite, and excess dithionite resulted in the formation of FeII-H15NO GlbN. Dithionite-mediated HNO production was also observed for the related GlbN from Synechocystis sp. PCC 6803. Although ferrous GlbN-A appeared capable of trapping preformed HNO, the histidine-heme post-translational modification extinguished the NO reduction chemistry associated with GlbN. Overall, the results suggest a role for the covalent modification in FeII GlbNs: protection from NO-mediated heme loss and prevention of HNO formation.

Structure of Chlamydomonas reinhardtii THB1, a group 1 truncated hemoglobin with a rare histidine-lysine heme ligation.

THB1 is one of several group 1 truncated hemoglobins (TrHb1s) encoded in the genome of the unicellular green alga Chlamydomonas reinhardtii. THB1 expression is under the control of NIT2, the master regulator of nitrate assimilation, which also controls the expression of the only nitrate reductase in the cell, NIT1. In vitro and physiological evidence suggests that THB1 converts the nitric oxide generated by NIT1 into nitrate. To aid in the elucidation of the function and mechanism of THB1, the structure of the protein was solved in the ferric state. THB1 resembles other TrHb1s, but also exhibits distinct features associated with the coordination of the heme iron by a histidine (proximal) and a lysine (distal). The new structure illustrates the versatility of the TrHb1 fold, suggests factors that stabilize the axial ligation of a lysine, and highlights the difficulty of predicting the identity of the distal ligand, if any, in this group of proteins.

Synergistic effects between catalase inhibitors and modulators of nitric oxide metabolism on tumor cell apoptosis.

Inhibitors of catalase (such as ascorbate, methyldopa, salicylic acid and neutralizing antibodies) synergize with modulators of nitric oxide (NO) metabolism (such as arginine, arginase inhibitor, NO synthase-inducing interferons and NO dioxygenase inhibitors) in the singlet oxygen-mediated inactivation of tumor cell protective catalase. This is followed by reactive oxygen species (ROS)-dependent apoptosis induction. TGF-beta, NADPH oxidase-1, NO synthase, dual oxidase-1 and caspase-9 are characterized as essential catalysts in this process. The FAS receptor and caspase-8 are required for amplification of ROS signaling triggered by individual compounds, but are dispensable when the synergistic effect is established. Our findings explain the antitumor effects of catalase inhibitors and of compounds that target NO metabolism, as well as their synergy. These data may have an impact on epidemiological studies related to secondary plant compounds and open new perspectives for the establishment of novel antitumor drugs and for the improvement of established chemotherapeutics.

Characterization of denitrifying activity by the alphaproteobacterium, Sphingomonas wittichii RW1.

Sphingomonas wittichii RW1 has no reported denitrifying activity yet encodes nitrite and nitric oxide reductases. The aims of this study were to determine conditions under which S. wittichii RW1 consumes nitrite (NO(-) 2) and produces nitrous oxide (N2O), examine expression of putative genes for N-oxide metabolism, and determine the functionality of chromosomal (ch) and plasmid (p) encoded quinol-dependent nitric oxide reductases (NorZ). Batch cultures of wildtype (WT) and a norZ ch mutant of S. wittichii RW1 consumed NO(-) 2 and produced N2O during stationary phase. The norZ ch mutant produced N2O, although at significantly lower levels (c.a. 66-87%) relative to the WT. Rates of N2O production were 2-3 times higher in cultures initiated at low relative to atmospheric O2 per unit biomass, although rates of NO(-) 2 consumption were elevated in cultures initiated with atmospheric O2 and 1 mM NaNO2. Levels of mRNA encoding nitrite reductase (nirK), plasmid-encoded nitric oxide dioxygenase (hmp p) and plasmid-encoded nitric oxide reductase (norZ p) were significantly higher in the norZ ch mutant over a growth curve relative to WT. The presence of NO(-) 2 further increased levels of nirK and hmp p mRNA in both the WT and norZ ch mutant; levels of norZ p mRNA compensated for the loss of norZ ch expression in the norZ ch mutant. Together, the results suggest that S. wittichii RW1 denitrifies NO(-) 2 to N2O and expresses gene products predicted to detoxify N-oxides. So far, only S. wittichii strains within four closely related taxa have been observed to encode both nirK and norZ genes, indicating a species-specific lateral gene transfer that may be relevant to the niche preference of S. wittichii.