The identification of a periplasmic nitrate reductase in Paracoccus denitrificans

Abstract A nitrate reductase activity has been identified in periplasmic extracts of Paracoccus denitrificans . The enzyme is relatively insensitive to azide and does not reduce chlorate, features which distinguish it from the well-characterised membrane-associated nitrate reductase. The specific activity of the enzyme was higher in intact cells grown with butyrate rather than succinate as the sole source of carbon.     

Identification of an assimilatory nitrate reductase in mutants of Paracoccus denitrificans GB17 deficient in nitrate respiration

A Paracoccus denitrificans strain (M6Ω) unable to use nitrate as a terminal electron acceptor was constructed by insertional inactivation of the periplasmic and membrane-bound nitrate reductases. The mutant strain was able to grow aerobically with nitrate as the sole nitrogen source. It also grew anaerobically with nitrate as sole nitrogen source when nitrous oxide was provided as a respiratory electron acceptor. These growth characteristics are attributed to the presence of a third, assimilatory nitrate reductase. Nitrate reductase activity was detectable in intact cells and soluble fractions using nonphysiological electron donors. The enzyme activity was not detectable when ammonium was included in the growth medium. The results provide an unequivocal demonstration that P. denitrificans can express an assimilatory nitrate reductase in addition to the well-characterised periplasmic and membrane-bound nitrate reductases. Received: 12 August 1996 / Accepted: 29 October 1996     

Properties of the periplasmic nitrate reductases from Paracoccus pantotrophus and Escherichia coli after growth in tungsten-supplemented media

Paracoccus pantotrophus grown anaerobically under denitrifying conditions expressed similar levels of the periplasmic nitrate reductase (NAP) when cultured in molybdate- or tungstate-containing media. A native PAGE gel stained for nitrate reductase activity revealed that only NapA from molybdate-grown cells displayed readily detectable nitrate reductase activity. Further kinetic analysis showed that the periplasmic fraction from cells grown on molybdate (3 microM) reduced nitrate at a rate of V(max)=3.41+/-0.16 micromol [NO(3)(-)] min(-1) mg(-1) with an affinity for nitrate of K(m)=0.24+/-0.05 mM and was heat-stable up to 50 degrees C. In contrast, the periplasmic fraction obtained from cells cultured in media supplemented with tungstate (100 microM) reduced nitrate at a much slower rate, with much lower affinity (V(max)=0.05+/-0.002 micromol [NO(3)(-)] min(-1) mg(-1) and K(m)=3.91+/-0.45 mM) and was labile during prolonged incubation at >20 degrees C. Nitrate-dependent growth of Escherichia coli strains expressing only nitrate reductase A was inhibited by sub-mM concentrations of tungstate in the medium. In contrast, a strain expressing only NAP was only partially inhibited by 10 mM tungstate. However, none of the above experimental approaches revealed evidence that tungsten could replace molybdenum at the active site of E. coli NapA. The combined data show that tungsten can function at the active site of some, but not all, molybdoenzymes from mesophilic bacteria.     

Cold adaptation of the mononuclear molybdoenzyme periplasmic nitrate reductase from the Antarctic bacterium Shewanella gelidimarina

The reduction of nitrate to nitrite is catalysed in bacteria by periplasmic nitrate reductase (Nap) which describes a system of variable protein subunits encoded by the nap operon. Nitrate reduction occurs in the NapA subunit, which contains a bis-molybdopterin guanine dinucleotide (Mo–MGD) cofactor and one [4Fe–4S] iron–sulfur cluster. The activity of periplasmic nitrate reductase (Nap) isolated as native protein from the cold-adapted (psychrophilic) Antarctic bacterium Shewanella gelidimarina (Nap_Sgel) and middle-temperature adapted (mesophilic) Shewanella putrefaciens (Nap_Sput) was examined at varied temperature. Irreversible deactivation of Nap_Sgel and Nap_Sput occurred at 54.5 and 65 °C, respectively. When Nap_Sgel was preincubated at 21–70 °C for 30 min, the room-temperature nitrate reductase activity was maximal and invariant between 21 and 54 °C, which suggested that Nap_Sgel was poised for optimal catalysis at modest temperatures and, unlike Nap_Sput, did not benefit from thermally-induced refolding. At 20 °C, Nap_Sgel reduced selenate at 16% of the rate of nitrate reduction. Nap_Sput did not reduce selenate. Sequence alignment showed 46 amino acid residue substitutions in Nap_Sgel that were conserved in NapA from mesophilic Shewanella, Rhodobacter and Escherichia species and could be associated with the Nap_Sgel cold-adapted phenotype. Protein homology modeling of Nap_Sgel using a mesophilic template with 66% amino acid identity showed the majority of substitutions occurred at the protein surface distal to the Mo–MGD cofactor. Two mesophilic ↔ psychrophilic substitutions (Asn ↔ His, Val ↔ Trp) occurred in a region close to the surface of the NapA substrate funnel resulting in potential interdomain π–π and/or cation–π interactions. Three mesophilic ↔ psychrophilic substitutions occurred within 4.5 Å of the Mo–MGD cofactor (Phe ↔ Met, Ala ↔ Ser, Ser ↔ Thr) resulting in local regions that varied in hydrophobicity and hydrogen bonding networks. These results contribute to the understanding of thermal protein adaptation in a redox-active mononuclear molybdenum enzyme and have implications in optimizing the design of low-temperature environmental biosensors.     

Effect of ammonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium species

BACKGROUND AND AIMS: Most Vaccinium species have strict soil requirements for optimal growth, requiring low pH, high iron availability and nitrogen primarily in the ammonium form. These soils are limited and are often located near wetlands. Vaccinium arboreum is a wild species adapted to a wide range of soils, including high pH, low iron, and nitrate-containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake compared with the cultivated Vaccinium species. METHODS: Nitrate, ammonium and iron uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in two Vaccinium species grown hydroponically in either nitrate or ammonia, with or without iron. The species studied were the wild V. arboreum and the cultivated V. corymbosum interspecific hybrid, which exhibits the strict soil requirements of most Vaccinium species. RESULTS: Ammonium uptake was significantly greater than nitrate uptake in both species, while nitrate uptake was greater in the wild species, V. arboreum, compared with the cultivated species, V. corymbosum. The increased nitrate uptake in V. arboreum was correlated with increased root NR activity compared with V. corymbosum. The lower nitrate uptake in V. corymbosum was reflected in decreased plant dry weight in this species compared with V. arboreum. Root FCR activity increased significantly in V. corymbosum grown under iron-deficient conditions, compared with the same species grown under iron-sufficient conditions or with V. arboreum grown under either iron condition. CONCLUSIONS: V. arboreum appears to be more efficient in acquiring nitrate compared with V. corymbosum, possibly due to increased NR activity and this may partially explain the wider soil adaptation of V. arboreum.     

EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

Nitrate reductases are enzymes that catalyze the conversion of nitrate to nitrite. We report here electron paramagnetic resonance (EPR) studies in the periplasmic nitrate reductase isolated from the sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774. This protein, belonging to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes, comprises a single 80-kDa subunit and contains a Mo bis(molybdopterin guanosine dinucleotide) cofactor and a [4Fe–4S] cluster. EPR-monitored redox titrations, carried out with and without nitrate in the potential range from 200 to −500 mV, and EPR studies of the enzyme, in both catalytic and inhibited conditions, reveal distinct types of Mo(V) EPR-active species, which indicates that the Mo site presents high coordination flexibility. These studies show that nitrate modulates the redox properties of the Mo active site, but not those of the [4Fe–4S] center. The possible structures and the role in catalysis of the distinct Mo(V) species detected by EPR are discussed.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Induction of nitrate reductase and membrane cytochromes in wild type and chlorate-resistant Paracoccus denitrificans

An experimental system has been devised for induction of nitrate reductase in suspensions of wild type Paracoccus denitrificans incubated with limited aeration in the presence of azide, nitrate or nitrite. Azide promoted maximum synthesis of enzyme, accompanied by formation of excess b-type cytochrome; the level of enzyme attained with nitrate was less and c-type cytochrome predominated in the membrane. The nitrate reductase was solubilized with deoxycholate from membranes of azide-induced cells and was identified as a major polypeptide M _r =150,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Mutants strains lacking nitrate reductase activity were isolated on the basis of resistance to chlorate and mutant M-1 was examined in detail. When incubated in the cell suspension system M-1 formed a membrane protein M _r =150,000 similar to that attributed to nitrate reductase in the wild type. Maximum formation of the protein by M-1 occurred without inducer and it was accompanied by synthesis of excess b-type cytochrome. The observations with wild type and M-1 indicate that nitrate reductase protein and b-type cytochrome are coregulated and that the active enzyme has a role in regulating its own synthesis.Non-standard Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - DOC sodlum deoxycholate     

Effect of glutamine on the induction of nitrate reductase

Nitrate reductase (NR. EC 1.6.6.1/2) is a substrate inducible enzyme that could be repressed by its end product glutamine or amino acids. To test this hypothesis, 6-day-old maize seedlings ( Zea mays cv. W64A × W182E) were grown hydroponically in a 1/10 strength Hougland's salt solution modified to contain no nitrogen. Previous experiments had established that after a 24-h induction with NO₃⁻ (5 mM KNO₃⁻) the level of NR activity and protein had reached a constant level. In the present experiments when glutamine (5 mM) was included together with NO₃⁻, there was a significant reduction in NR activity (34% of the control values). NR protein and NR mRNA accumulation in the root. In the shoot, on the other hand, glutamine additions had little or no effect on the levels of either NR activity (81% of control) or NR protein. Inhibition of glutamine synthetase by methionine sulfoximine (MSX) resulted in reduced levels of glutamine in both root and shoot tissues. Contrary in our prediction, however, it had no effect on NR activity and mRNA content in roots. In the shoot, on the other hand, there was a marked reduction of NR activity (34% of the control value) and NR protein, but no apparent effect on NR mRNA. When detached shoots were treated with MSX and other inhibitors of glutamine synthetase (tabtoxinine-β-lactam or phosphinothricin) the induction of NR activity by NO₃⁻ was also inhibited. Glutamine additions 15 or 50 mM to detached shoots had essentially no effect on the induction of NR activity (90% of control). These results demonstrate that the influence of glutamine and MSX on the induction of NR in maize root and shoot tissues, respectively, is very different.     

A method for the determination of nitrate reductase

A procedure for the assay of nitrate reductase based on Szekely's diaminodiphenylsulphone method of nitrate determination (Szekely, E. (1967) Talanta 14, 941–950) is described. The method is simple and sensitive, allowing determination of 1 μg of nitrate in a volume of 1 ml or less. It is particularly suited to the determination of nitrate reductase.     

Kinetics of substrate inhibition of periplasmic nitrate reductase

Periplasmic nitrate reductase catalyzes the reduction of nitrate into nitrite using a mononuclear molybdenum cofactor that has nearly the same structure in all enzymes of the DMSO reductase family. In previous electrochemical investigations, we found that the enzyme exists in several inactive states, some of which may have been previously isolated and mistaken for catalytic intermediates. In particular, the enzyme slowly and reversibly inactivates when exposed to high concentrations of nitrate. Here, we study the kinetics of substrate inhibition and its dependence on electrode potential and substrate concentration to learn about the properties of the active and inactive forms of the enzyme. We conclude that the substrate-inhibited enzyme never significantly accumulates in the EPR-active Mo(+ V) state. This conclusion is relevant to spectroscopic investigations where attempts are made to trap a Mo(+ V) catalytic intermediate using high concentrations of nitrate.     

Novel growth characteristics and high rates of nitrate reduction of an Escherichia coli strain, LCB2048, that expresses only a periplasmic nitrate reductase

Escherichia coli strain LCB2048 is a double mutant defective in the synthesis of the two membrane-associated nitrate reductases A and Z. This strain can grow anaerobically on a non-fermentable carbon source, glycerol, in the presence of nitrate even in media supplemented with high concentrations of tungstate. This growth was totally dependent upon a highly active, periplasmic nitrate reductase (Nap). Due to the presence of a previously unreported narL mutation, synthesis of the periplasmic nitrate reductase by this strain was induced during anaerobic growth by nitrate. We have also demonstrated that methyl viologen is an ineffective electron donor to Nap: its use leads to an underestimation of the contribution of Nap activity to the rate of nitrate reduction in vivo.     

Distribution of nitrate reductase activity in Vicia faba: Effect of nitrate and plant genotype

The short term effect of NO₃⁻ (12 mM) on nitrate reductase (NR. EC 1.6.6.1) activity has been studied in the roots, nodules and leaves of different genotypes of Vicia faba L. at the end of vegetative growth. Root and leaf NR activity responded positively to NO₃⁻ while nodule activity, where detected, proved to he strongly inhibited. The withdraw of this NO₃⁻ from the solution consistently reduced activity in the roots and leaves but surprising, promoted a significant increase in nodule activity, which matched or surpassed that of control plants On the other hand, nodules developed in the presence of 8 mM NO₃⁻ expressed an on average 141% higher level of NR activity than did controls. This effect was observed even in nodules with negligible control activity. In any case, a naturally occurring mutant (VF17) lacking root and nodule NR activity is described. The results indicate that in V. faba. the effects of NO₃⁻ and plant genotype on NR activity depended on plant organ and time of NO₃⁻ application, hut the distribution of NO₃⁻ reduction through the plain was mainly dependent on plant genotype, and to a lesser extent on NO: supply and plant age.     

The activation state of nitrate reductase is not always correlated with total nitrate reductase activity in leaves

The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRA_max) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRA_max and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRA_max and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRA_max and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRA_max was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRA_max in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NR_max by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRA_max. Received: 24 March 1999 / Accepted: 31 May 1999     

Phytochrome regulation of nitrate reductase in wheat

In excised wheat leaves, the activity of nitrate reductase was enhanced by a brief pulse of red light and this increase was reversed by far-red light irradiation. Even under continuous far-red light, nitrate reductase activity increased by 258% after 18 h. When leaves were kept in distilled water during exposure to red light and then transferred to potassium nitrate, there was no difference in endogenous nitrate concentration. The nitrate reductase activity was the same whether leaves were floated in potassium nitrate or in distilled water during irradiation. Partial to complete inhibition of enzyme activity was observed when leaves were incubated in actinomycin-D and cycloheximide respectively, following 4 h of red light irradiation.In vitro irradiation of extract had no significant effect on nitrate reductase activity     

Assignment of haem ligands and detection of electronic absorption bands of molybdenum in the di-haem periplasmic nitrate reductase of Paracoccus pantotrophus.

The periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a soluble two-subunit enzyme (NapAB) that binds two c-type haems, a [4Fe-4S] cluster and a bis-molybdopterin guanine dinucleotide cofactor that catalyses the reduction of nitrate to nitrite. In the present work the NapAB complex has been studied by magneto-optical spectroscopy to probe co-ordination of both the NapB haems and the NapA active site Mo. The absorption spectrum of the NapAB complex is dominated by features from the NapB c-type cytochromes. Using a combination of electron paramagnetic resonance spectroscopy and magnetic circular dichroism it was demonstrated that both haems are low-spin with bis-histidine axial ligation. In addition, a window between 600 and 800 nm was identified in which weak absorption features that may arise from Mo could be detected. The low-temperature MCD spectrum shows oppositely signed bands in this region (peak 648 nm, trough 714 nm) which have been assigned to S-to-Mo(V) charge transfer transitions.     

Respiratory Rate as a Regulatory Factor in the Biosynthesis of the Denitrification Pathway of the Bacterium Paracoccus denitrificans

The decrease in the electron flow of the aerobic respiratory chain of the bacterium Paracoccus denitrificans, owing to either the drop in the saturation of terminal oxidases by oxygen or to the inhibition of the rate of respiration by azide or nitrite, resulted in the synthesis of dissimilatory nitrate reductase and nitrite reductase. The dependence of the resulting activities of the two enzymes (after a three-hour adaptation) on the initial value of the parameter V_max/k_La (oxidase activity of the volume unit of the culture divided by the volumetric oxygen transfer coefficient) or on the concentrations of the inhibitors had a similar form, characterized by the appearance of a maximum. The increasing parts of the obtained curves reflect the synthesis of enzymes, probably initiated by the increase in the intracellular degree of reduction, the subsequent drop being evidently in connection with the lack of metabolic energy for biosynthesis. The possible mechanisms of the effect of nitrogenous terminal acceptors (NO^-₃ and NO^-₂) on the formation of the denitrification pathway are discussed.     

Critical evaluation of thein situ nitrate reductase assay

Anin situ method, derived from anin vivo method, was used to determine nitrate reductase activity (NRA) in:i) excised barley and corn shoots and excised soybean leaves during a N-depletion experiment and; ii) roots and shoots of N-depleted barley and corn seedlings during induction of nitrate, reductase (NR). Nitrate reduction, calculated from thesein situ RNA measurements, was compared with estimates of each organ's nitrate reduction in light aerobic conditions from NO ₃ ⁻ consumption and a¹⁵N model (Gojonet al., 1986b). Thein situ RNA of roots strongly underestimated their¹⁵NO ₃ ⁻ reduction. In contrast, in barley and corn shoots and in the first trifoliolate leaves from 26-day-old, soybean, thein situ NRA assay gave a fair approximation of the true NO ₃ ⁻ reduction rate (relative differences ranging from −14 to +32%). In young soybean leaves (from 20-day-old plants), however, thein situ NRA strongly underestimated the actual NO ₃ ⁻ reduction. The physiological significance of thein situ NRA assay in shoots and roots, and its value for field studies are discussed from these results.     

Circadian rhythmicity of nitrate reductase activity in barley leaves

Nitrate reductase (EC 1.6.6.1) activity showed circadian rhythmicity in the first leaf of 8–11 days old barley ( Hordeum vulgare L. cv. Herta) plants. Circadian rhythms were found using both the in vitro and in vivo method for testing the enzyme activity. When the light intensity was reduced from 65 to 20 W m⁻², the amplitude was smaller and the oscillations were damped sooner. In continuous darkness nitrate reductase activity decreased in a two step process. Three different light qualities were tested which all gave the same results.