Radiation inactivation analysis of assimilatory NADH:nitrate reductase. Apparent functional sizes of partial activities associated with intact and proteolytically modified enzyme

Recently we demonstrated that target sizes for the partial activities of nitrate reductase were considerably smaller than the 100-kDa subunit which corresponded to the target size of the full (physiologic) activity NADH:nitrate reductase. These results suggested that the partial activities resided on functionally independent domains and that radiation inactivation may be due to localized rather than extensive damage to protein structure. The present study extends these observations and addresses several associated questions. Monophasic plots were observed over a wide range of radiation doses, suggesting a single activity component in each case. No apparent differences were observed over a 10-fold range of concentration for each substrate, suggesting that the observed slopes were not due to marked changes in Km values. Apparent target sizes estimated for partial activities associated with native enzyme and with limited proteolysis products of native enzyme suggested that the functional size obtained by radiation inactivation analysis is independent of the size of the polypeptide chain. The presence of free radical scavengers during irradiation reduced the apparent target size of both the physiologic and partial activities by an amount ranging from 24 to 43%, suggesting that a free radical mechanism is at least partially responsible for the inactivation. Immunoblot analysis of nitrate reductase irradiated in the presence of free radical scavengers revealed formation of distinct bands at 90, 75, and 40 kDa with increasing doses of irradiation rather than complete destruction of the polypeptide chain.     

Physical studies on assimilatory nitrate reductase from Chlorella vulgaris.

Assimilatory nitrate reductase (EC 1.6.6.1 NADH:nitrate oxidoreductase) from Chlorella vulgaris purified by affinity chromatography was found to be homogeneous as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel and by analytical ultracentrifugal techniques. The molecular weight of the intact enzyme and that of the enzyme dissociated in 6 M GuHCl, determined by sedimentation equilibrium studies, were 280,000 +/- 10,000 and 90,000 +/- 5,000, respectively. Comparable values were obtained using the S20,w value and the D20,w values in Svedberg's equation. The D20,w values were determined by laser light-scattering measurements. Active enzyme centrifugation showed that the monomer is an active species. A quantitative re-evaluation of the prosthetic groups present (FAD, heme, and molybdenum) was also made and was consistent with the conclusion that the active monomer contains three subunits as previously deduced by Solomonson et al. ((1975) J. Biol. Chem. 250, 4120). Electron micrographs showed images which corresponded to three subunits, supporting the data obtained by hydrodynamic studies. The enzyme is not cigar-shaped, as previously surmised, but has a roughly globular structure.     

The role of the essential sulfhydryl group in assimilatory NADH: nitrate reductase of Chlorella

Incubation of the complex metalloflavoprotein, assimilatory nitrate reductase with N-ethylmaleimide, or a spin-labeled analog, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl, resulted in a time-dependent inactivation of NADH:nitrate reductase and NADH: cytochrome-c reductase activity with no effect on reduced methyl viologen:nitrate reductase activity. Inactivation of the enzyme, which could be prevented by incubation in the presence of NADH, was achieved following modification of a single sulfhydryl group determined from [3H]N-ethylmaleimide incorporation and quantitation of the EPR spectrum of the spin-labeled enzyme. Sulfhydryl group modification precluded reduction of the enzyme by NADH and NAD+ binding. The EPR spectrum of the spin-labeled enzyme revealed the presence of a single species with the nitroxide retaining substantial motional freedom. Cleavage of the spin-labeled enzyme using corn-inactivating protease and separation into its flavin and molybdenum/heme domains followed by EPR spectroscopy revealed the modified sulfhydryl group to be associated with the latter fragment suggesting a close interaction of these domains in the region of the nucleotide-binding site.     

Reversible inactivation by NADH and ADP on Chlorella fusca nitrate reductase

The active form of $\text{Chlorella fusca}$ nitrate reductase can be reversibly converted into its inactive form by reduction with NADH in the presence of ADP. Under the experimental conditions used, no inactivation occurs when nitrate is simultaneously present or when the nucleotides act isolately, the inactivating effect being maximal at a concentration of ADP (0.3 mM) equimolecular with that of NADH. The inactive enzyme thus attained can be completely reactivated by reoxidation with ferricyanide. The redox state of the pyridine nucleotide and the phosphorylation degree of the adenine nucleotide are critical for the inactivation process to ensue, since neither NAD⁺ nor AMP or ATP do exert any effect. ADP is also a powerful, although rather unspecific, protector against thermal inactivation of the NADH-diaphorase moiety of the NADH-nitrate reductase complex.     

Reversible inactivation of nitrate reductase in Chlorella vulgaris in vivo

Summary The NADH-nitrate oxidoreductase of Chlorella vulgaris has an inactive form which has previously been shown to be a cyanide complex of the reduced enzyme. This inactive enzyme can be reactivated by treatment with ferricyanide in vitro. In the present study, the activation state of the enzyme was determined after different prior in vivo programs involving environmental variations. Oxygen, nitrate, light and CO₂ all affect the in vivo inactivation of the enzyme in an interdependent manner. In general, the inactivation is stimulated by O₂ and inhibited by nitrate and CO₂. Light may stimulate or inhibit, depending on conditions. Thus, the effects of CO₂ and nitrate (inhibition of reversible inactivation) are clearly manifested only in the light. In contrast, light stimulates the inactivation in the presence of oxygen and the absence of CO₂ and nitrate. Since the inactivation of the enzyme requires HCN and NADH, and it is improbable that O₂ stimulates NADH formation, it is reasonable to conclude that HCN is formed as the result of an oxidation reaction (which is stimulated by light). The formation of HCN is probably stimulated by Mn²⁺, since the formation of reversibly-inactivated enzyme is impaired in Mn²⁺-deficient cells. The prevention of enzyme inactivation by nitrate in vivo is in keeping with previous in vitro results showing that nitrate prevents inactivation by maintaining the enzyme in the oxidized form. A stimulation of nitrate uptake by CO₂ and light could account for the effect of CO₂ (prevention of inactivation) which is seen mainly in the presence of nitrate and light. Ammonia added in the presence of nitrate has the same effect on the enzyme as removing nitrate (promotion of reversible inactivation). Ammonia added in the absence of nitrate has little extra effect. It is therefore likely that ammonia acts by preventing nitrate uptake. The uncoupler, carbonylcyanide-m-chloro-phenylhydrazone, causes enzyme inactivation because it acts as a good HCN precursor, particularly in the light. Nitrite, arsenate and dinitrophenol cause an enzyme inactivation which can not be reversed by ferricyanide in crude extracts. This suggests that there are at least two different ways in which the enzyme can be inactivated rather rapidly in vivo.     

Regulation of the Neurospora crassa assimilatory nitrate reductase.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase from Neurospora crassa was purified and found to be stimulated by certain amino acids, citrate, and ethylenediaminetetraacetic acid (EDTA). Stimulation by citrate and the amino acids was dependent upon the prior removal of EDTA from the enzyme preparations, since low quantities of EDTA resulted in maximal stimulation. Removal of EDTA from enzyme preparations by dialysis against Chelex-containing buffer resulted in a loss of nitrate reductase activity. Addition of alanine, arginine, glycine, glutamine, glutamate, histidine, tryptophan, and citrate restored and stimulated nitrate reductase activity from 29- to 46-fold. The amino acids tested altered the Km of NADPH-nitrate reductase for NADPH but did not significantly change that for nitrate. The Km of nitrate reductase for NADPH increased with increasing concentrations of histidine but decreased with increasing concentrations of glutamine. Amino acid modulation of NADPH-nitrate reductase activity is discussed in relation to the conservation of energy (NADPH) by Neurospora when nitrate is the nitrogen source.     

Activation of nitrate reductase from rice seedlings by NADH

Rice leaf nitrate reductase was specifically activated by preincubation both at 0° and 25°, with low concentrations of NADH. The nucleotide acted as a positive effector of the enzyme after a time lag of 20 min. NADPH, FMNH₂ and NAD were without any effect.     

Regulation of assimilatory nitrate reductase formation in Klebsiella aerogenes W70.

Klebsiella aerogenes W70 could grow aerobically with nitrate or nitrite as the sole nitrogen source. The assimilatory nitrate reductase and nitrite reductase responsible for this ability required the presence of either nitrate or nitrite as an inducer, and both enzymes were repressed by ammonia. The repression by ammonia, which required the NTR (nitrogen regulatory) system (A. Macaluso, E. A. Best, and R. A. Bender, J. Bacteriol. 172:7249-7255, 1990), did not act solely at the level of inducer exclusion, since strains in which the expression of assimilatory nitrate reductase and nitrite reductase was was independent of the inducer were also susceptible to repression by ammonia. Insertion mutations in two distinct genes, neither of which affected the NTR system, resulted in the loss of both assimilatory nitrate reductase and nitrite reductase. One of these mutants reverted to the wild type, but the other yielded pseudorevertants at high frequency that were independent of inducer but still responded to ammonia repression.     

Quaternary structure and composition of squash NADH:nitrate reductase

NADH:nitrate reductase (EC 1.6.6.1) was isolated from squash cotyledons (Cucurbita maxima L.) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on Bio-Gel A-1.5m. These preparations gave a single protein staining band (Mr = 115,000) on sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is homogeneous. The native Mr of nitrate reductase was found to be 230,000, with a minor form of Mr = 420,000 also occurring. These results indicate that the native nitrate reductase is a homodimer of Mr = 115,000 subunits. Acidic amino acids predominate over basic amino acids, as shown both by the amino acid composition of the enzyme and an isoelectric point for nitrate reductase of 5.7. The homogeneous nitrate reductase had a UV/visible spectrum typical of a b-type cytochrome. The enzyme was found to contain one each of flavin (as FAD), heme iron, molybdenum, and Mo-pterin/Mr = 115,000 subunit. A model is proposed for squash nitrate reductase in which two Mr = 115,000 subunits are joined to made the native enzyme. Each subunit contains 1 eq of FAD, cytochrome b, and molybdenum/Mo-pterin.     

Tuning a nitrate reductase for function. The first spectropotentiometric characterization of a bacterial assimilatory nitrate reductase reveals novel redox properties

Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. In the present study the ferredoxin-dependent nitrate reductase NarB of the cyanobacterium Synechococcus sp. PCC 7942 was analyzed by spectropotentiometry and protein film voltammetry. Metal and acid-labile sulfide analysis revealed nearest integer values of 4:4:1 (iron/sulfur/molybdenum)/molecule of NarB. Analysis of dithionite-reduced enzyme by low temperature EPR revealed at 10 K the presence of a signal that is characteristic of a [4Fe-4S](1+) cluster. EPR-monitored potentiometric titration of NarB revealed that this cluster titrated as an n = 1 Nernstian component with a midpoint redox potential (E(m)) of -190 mV. EPR spectra collected at 60 K revealed a Mo(V) signal termed "very high g" with g(av) = 2.0047 in air-oxidized enzyme that accounted for only 10-20% of the total molybdenum. This signal disappeared upon reduction with dithionite, and a new "high g" species (g(av) = 1.9897) was observed. In potentiometric titrations the high g Mo(V) signal developed over the potential range of -100 to -350 mV (E(m) Mo(6+/5+) = -150 mV), and when fully developed, it accounted for 1 mol of Mo(V)/mol of enzyme. Protein film voltammetry of NarB revealed that activity is turned on at potentials below -200 mV, where the cofactors are predominantly [4Fe-4S](1+) and Mo(5+). The data suggests that during the catalytic cycle nitrate will bind to the Mo(5+) state of NarB in which the enzyme is minimally two-electron-reduced. Comparison of the spectral properties of NarB with those of the membrane-bound and periplasmic respiratory nitrate reductases reveals that it is closely related to the periplasmic enzyme, but the potential of the molybdenum center of NarB is tuned to operate at lower potentials, consistent with the coupling of NarB to low potential ferredoxins in the cell cytoplasm.     

Reduction of ferric citrate catalyzed by NADH:nitrate reductase

We show that NADH:nitrate reductase from squash cotyledons can catalyze the reduction of ferric citrate. When nitrate reductase was purified to homogeneity using a two-step affinity chromatography procedure, an NADH:Fe(III)-citrate reductase activity copurified with it and had identical electrophoretic mobility to it. The iron reductase activity was optimum near pH 6.3, had an apparent Km for Fe(III)-citrate of 0.02 mM, and was inhibited by monospecific anti-nitrate reductase rabbit sera. Differential inhibition of the enzyme's activities indicated iron and nitrate were reduced at different sites. In addition to its role in nitrogen assimilation, nitrate reductase catalyzes ferric citrate reduction and could have a role in iron assimilation.     

Stoichiometry of electron uptake and oxidation-reduction midpoint potentials of NADH:nitrate reductase.

Upon detergent or hypo-osmotic lysis of CHO-cell postnuclear supernatants or isolated lysosomes at pH 4.8, the lysosomal enzymes beta-hexosaminidase, beta-galactosidase, alpha-fucosidase and cathepsin C were readily pelleted, whereas the exogenous marker, long-term-internalized horseradish peroxidase, was not. Salt or pH elevation greatly decreased lysosomal-enzyme pelletability. The results suggest that, under native conditions, lysosomal hydrolases may be aggregated. Aggregation could promote enzyme retention within the organelle.     

Assimilatory nitrate reductase from Chlorella. Effect of ionic strength and pH on catalytic activity

Initial velocity studies of Chlorella nitrate reductase showed that increased ionic strength stimulated NADH:nitrate reductase activity by increasing both Vmax and Km for nitrate. Examination of the effect of ionic strength on the various partial activities of nitrate reductase revealed that while NADH:ferricyanide and reduced methyl viologen:nitrate reductase activities were unaffected by ionic strength, NADH:cytochrome c and reduced flavin:nitrate reductase activities were inhibited and stimulated by increased ionic strength, respectively. Comparison of the rates for the partial activities indicated electron transfer from heme to molybdenum to be the rate-limiting step in enzyme turnover. The pH optimum for NADH:nitrate reductase activity was found to be 7.9 while values for the partial activities ranged from 5.5 to 8.1. Phosphate was found to stimulate both NADH:nitrate and reduced methyl viologen:nitrate reductase activities indicating the molybdenum center as the site of interaction.     

Electrochemical and kinetic analysis of electron-transfer reactions of Chlorella nitrate reductase.

Assimilatory nitrate reductase (NR) from Chlorella is homotetrameric, each subunit containing FAD, heme, and Mo-pterin in a 1:1:1 stoichiometry. Measurements of NR activity and steady-state reduction of the heme component under conditions of NADH limitation or competitive inhibition by nitrite suggested intramolecular electron transfer between heme and Mo-pterin was a rate-limiting step and provided evidence that heme is an obligate intermediate in the transfer of electrons between FAD and Mo-pterin. In addition to the physiological substrates NADH and nitrate, various redox mediators undergo reactions with one or more of the prosthetic groups. These reactions are coupled by NR to NADH oxidation or nitrate reduction. To test whether intramolecular redox reactions of NR were rate-determining, rate constants for redox reactions between NR and several chemically diverse mediators were measured by cyclic voltammetry in the presence of NADH or nitrate. Reduction of ferrocenecarboxylic acid, dichlorophenolindophenol, and cytochrome c by NADH-reduced NR was coupled to reoxidation at a glassy carbon electrode (ferrocene and dichlorophenolindophenol) or at a bis(4-pyridyl) disulfide modified gold electrode (cytochrome c), yielding rate constants of 10.5 x 10(6), 1.7 x 10(6), and 2.7 x 10(6) M-1 s-1, respectively, at pH 7. Kinetics were consistent with a second-order reaction, implying that intramolecular heme reduction by NADH and endogenous FAD was not limiting. In contrast, reduction of methyl viologen and diquat at a glassy carbon electrode, coupled to oxidation by NR and nitrate, yielded similar kinetics for the two dyes. In both cases, second-order kinetics were not obeyed, and reoxidation of dye-reduced Mo-pterin of NR by nitrate became limiting at low scan rates.(ABSTRACT TRUNCATED AT 250 WORDS)