Hydroxylamine reductase enzymes from maize scutellum and their relationship to nitrite reductase

Three enzymes contribute to the total hydroxylamine reductase activity of corn (Zea mays L.) scutellum extracts. Two of these resemble enzymes previously prepared from leaves, while the third, which accounts for a major part of the activity, appears to have no counterpart in leaf tissue. One of the hydroxylamine reductases found only in small amounts is associated with nitrite reductase and is induced, together with nitrite reductase, by nitrite. The other two enzymes are noninducible by nitrite and can be totally separated from nitrite reductase, which subsequently remains capable of catalyzing the reduction of nitrite to ammonia. Possible causes of the decline of hydroxylamine reductase activity during the induction of nitrite reductase are discussed.     

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.     

Starch Gel Electrophoresis of Plant NADH-Nitrate Reductase and Nitrite Reductase

Isozymes of both nitrate reductase (NR) and nitrite reductase(NiR) have been found in plant tissues, mainly after partialpurification. We have used starch gel electrophoresis to examineboth NR and NiR in crude extracts. Only one NR and one NiR enzymewere found in wheat tissues and no difference in mobilitiescould be detected between root and leaf enzymes. It was confirmedthat some tissues of corn have two NiR isozymes.     

Activation of Nitrite Reductase in a Cell-Free System from the Denitrifier Alcaligenes sp. by Freeze-Thawing

Nitrite reductase (NiR) activity of the cell-free extract orthe soluble fraction prepared from cells of Alcaligenes sp.NC1B 11015 grown anaerobically in the presence of nitrate wasexamined by measuring the rate of nitrite disappearance withdithionitemethyl viologen (MV) as an electron donor. Freezingat — 20?C and subsequent thawing of the fraction resultedin 5-40 times increase of the specific activity of NiR. Fromthe experiments on the effect of freezing conditions on theactivation, the phase change of solvent water due to freezingis considered to play an important role in the activation. Thisactivation occurred with the preparation in the exponentialgrowth phase, but not that in the stationary growth phase. Clearly,the low-molecular-weight (< 12,000) component which was obtainedfrom the soluble fraction through a collodion bag participatedin the activation. The activated enzyme proved to be the dissimilatory NiR, becauseNO production from nitrite, one of the typical characteristicsof the dissimilatory NiR, was also activated when assayed withascorbate-tetramethyl-p-phenylene diamine (TMPD) as an electrondonor. Nevertheless, the reaction products of nitrite reductionwere identified as hydroxylamine and ammonia with dithionite-MV.The possible pathway of nitrite reduction with this electrondonor is discussed. (Received May 26, 1983; Accepted February 2, 1984)     

Some properties of ferredoxin-nitrite reductase from green shoots of bean and an immunological comparison with nitrite reductase from roots and etiolated shoots

Ferredoxin-nitrite reductase (EC 1.7.7.1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, has been isolated from green shoots of bean (Phaseolus angularis). The isolated enzyme (GR-NiR), having a molecular mass of 68 000, showed 1.4 times higher ferredoxin-dependent activity than methyl viologen-linked activity. The enzyme was homogeneous by polyacrylamide gel electrophoresis (PAGE). In the oxidized form, the enzyme had absorption maxima at 275, 393 (Soret band), 535 and 571 (α band) nm, indicating that siroheme is involved in the catalysis of nitrite reduction. The absorbance ratios, A₃₉₃ : A₂₇₅ and A₅₇₁ : A₃₉₃ were 0.26 and 0.32, respectively. Antibody against the isolated enzyme was raised in rabbits. Analysis of the antiserum by immunodiffusion and immunoelectrophoresis suggested that it was a specific antiserum against GR-NiR. Using the antiserum, immunodiffusion and immunoprecipitation procedures were employed to compare the immunological similarity of NiR from green shoots, etiolated shoots and roots of bean. These tests revealed that the three forms of assimilatory NiR have antigenic determinants in common.     

Characterization of a heme c nitrite reductase from a non-ammonifying microorganism, Desulfovibrio vulgaris Hildenborough

A cytochrome c nitrite reductase (NiR) was purified for the first time from a microorganism not capable of growing on nitrate, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. It was isolated from the membranes as a large heterooligomeric complex of 760 kDa, containing two cytochrome c subunits of 56 and 18 kDa. This complex has nitrite and sulfite reductase activities of 685 micromol NH(4)(+)/min/mg and 1.0 micromol H(2)/min/mg. The enzyme was studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies. The overall redox behavior was determined through a visible redox titration. The data were analyzed with a set of four redox transitions, with an E(0)' of +160 mV (12% of total absorption), -5 mV (38% of total absorption), -110 mV (38% of total absorption) and -210 mV (12% of total absorption) at pH 7.6. The EPR spectra of oxidized and partially reduced NiR show a complex pattern, indicative of multiple heme-heme magnetic interactions. It was found that D. vulgaris Hildenborough is not capable of using nitrite as a terminal electron acceptor. These results indicate that in this organism the NiR is not involved in the dissimilative reduction of nitrite, as is the case with the other similar enzymes isolated so far. The possible role of this enzyme in the detoxification of nitrite and/or in the reduction of sulfite is discussed.     

A random-sequential mechanism for nitrite binding and active site reduction in copper-containing nitrite reductase

The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a random-sequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO(-)(2)] reduction of the type-2 site precedes nitrite binding, at high [NO(-)(2)] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an approximately 50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism.     

Nitrite reduction in barley-root plastids: Dependence on NADPH coupled with glucose-6-phosphate and 6-phosphogluconate dehydrogenases,and possible involvement of an electron carrier and a diaphorase

Plastids from roots of barley (Hordeum vulgare L.) seedlings were isolated by discontinuous Percoll-gradient centrifugation. Coinciding with the peak of nitrite reductase (NiR; EC 1.7.7.1, a marker enzyme for plastids) in the gradients was a peak of a glucose-6-phosphate (Glc6P) and NADP⁺-linked nitrite-reductase system. High activities of phosphohexose isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 2.7.5.1) as well as glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) were also present in the isolated plastids. Thus, the plastids contained an overall electron-transport system from NADPH coupled with Glc6PDH and 6PGDH to nitrite, from which ammonium is formed stoichiometrically. However, NADPH alone did not serve as an electron donor for nitrite reduction, although NADPH with Glc6P added was effective. Benzyl and methyl viologens were enzymatically reduced by plastid extract in the presence of Glc6P+ NADP⁺. When the plastids were incubated with dithionite, nitrite reduction took place, and ammonium was formed stoichiometrically. The results indicate that both an electron carrier and a diaphorase having ferredoxin-NADP⁺ reductase activity are involved in the electron-transport system of root plastids from NADPH, coupled with Glc6PDH and 6PGDH, to nitrite.Abbreviations Cyt cytochrome - Glc6P glucose-6-phosphate - Glc6PDH glucose-6-phosphate dehydrogenase - MVH reduced methyl viologen - NiR nitrite reductase - 6PG 6-phosphogluconate - 6PGDH 6-phosphogluconate dehydrogenase     

Purification and properties of nitrite reductase from roots of pea (Pisum sativum cv. Meteor)

Nitrite reductase (EC 1.6.6.4) prepared from pea roots was found to be immunologically indistinguishable from pea leaf nitrite reductase. Comparisons of the pea root enzyme with nitrite reductase from leaf sources showed a close similarity in inhibition properties, light absorption spectrum, and electron paramagnetic resonance signals. The resemblances indicate that the root nitrite reductase is a sirohaem enzyme and that it functions in the same manner as the leaf enzyme in spite of the difference in reductant supply implicit in its location in a non-photosynthetic tissue.Abbreviations DEAE diethylaminoethyl - EPR electron paramagnetic resonance - NIR nitrite reductase - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Reactions of spinach nitrite reductase with its substrate, nitrite, and a putative intermediate, hydroxylamine

Plant nitrite reductase (NiR) catalyzes the reduction of nitrite (NO(2)(-)) to ammonia, using reduced ferredoxin as the electron donor. NiR contains a [4Fe-4S] cluster and an Fe-siroheme, which is the nitrite binding site. In the enzyme's as-isolated form ([4Fe-4S](2+)/Fe(3+)), resonance Raman spectroscopy indicated that the siroheme is in the high-spin ferric hexacoordinated state with a weak sixth axial ligand. Kinetic and spectroscopic experiments showed that the reaction of NiR with NO(2)(-) results in an unexpectedly EPR-silent complex formed in a single step with a rate constant of 0.45 +/- 0.01 s(-)(1). This binding rate is slow compared to that expected from the NiR turnover rates reported in the literature, suggesting that binding of NO(2)(-) to the as-isolated form of NiR is not the predominant type of substrate binding during enzyme turnover. Resonance Raman spectroscopic characterization of this complex indicated that (i) the siroheme iron is low-spin hexacoordinated ferric, (ii) the ligand coordination is unusually heterogeneous, and (iii) the ligand is not nitric oxide, most likely NO(2)(-). The reaction of oxidized NiR with hydroxylamine (NH(2)OH), a putative intermediate, results in a ferrous siroheme-NO complex that is spectroscopically identical to the one observed during NiR turnover. Resonance Raman and absorption spectroscopy data show that the reaction of oxidized NiR ([4Fe-4S](2+)/Fe(3+)) with hydroxylamine is binding-limited, while the NH(2)OH conversion to nitric oxide is much faster.     

Studies on nitrite reductase in barley

Summary Nitrite reductase from barley seedlings was purified 50–60 fold by ammonium sulphate precipitation and gel filtration. No differences were established in the characteristics of nitrite reductases isolated in this way from either leaf or root tissues. The root enzyme accepted electrons from reduced methyl viologen, ferredoxin, or an unidentified endogenous cofactor. Enzyme activity in both tissues was markedly increased by growth on nitrate. This activity was not associated with sulphite reductase activity. Microbial contamination could not account for the presence of nitrite reductase activity in roots. Nitrite reductase assayed in vitro with reduced methyl viologen as the electron donor was inhibited by 2,4-dinitrophenol but not by arsenate.Abbreviations DNP 2,4-dinitrophenol - DEAE diethyl amino ethyl     

微生物亚硝酸盐还原酶的研究进展

亚硝酸盐还原酶(Nitrite reductase,简称NiR,EC1.7.2.1)是催化亚硝酸盐(Nitrite,简称NIT)还原的一类酶,可降解NIT为NO或NH3,是自然界氮循环过程的关键酶。本文详细阐述亚硝酸盐还原酶的分类、结构特点、催化机制以及现阶段的应用领域,为深入研究亚硝酸还原酶提供参考。     

Effect of light/dark cycles on expression of nitrate assimilatory genes in maize shoots and roots

The level of nitrate reductase (NR) and nitrite reductase (NiR) varied in both shoot and root tissue from nitrate-fed Zea mays L. grown under a 16-hour light/8-hour dark regime over a 10-day period postgermination, with peak activity occurring in days 5 to 6. To study the effect of different light regimes on NR and NiR enzyme activity and mRNA levels, 6-day-old plants were grown in the presence of continuous KNO₃ (10 millimolar). Both shoot NRA and mRNA varied considerably, peaking 4 to 8 hours into the light period. Upon transferring plants to continuous light, the amplitude of the peaks increased, and the peaks moved closer together. In continuous darkness, no NR mRNA or NR enzyme activity could be detected by 8 hours and 12 hours, respectively. In either a light/dark or continuous light regime, root NRA and mRNA did not vary substantially. However, when plants were placed in continuous darkness, both declined steadily in the roots, although some remained after 48 hours. Although there was no obvious cycling of NiR enzyme activity in shoot tissue, changes in mRNA mimicked those seen for NR mRNA. The expression of NR and NiR genes is affected by the light regime adopted, but light does not have a direct effect on the expression of these genes.     

Immunological studies of ferredoxin-nitrite reductases and ferredoxin-glutamate synthases from photosynthetic organisms

Polyclonal antiserum specific for ferredoxin-nitrite reductase (EC 1.7.7.1) from the green alga Chlamydomonas reinhardii recognized the nitrite reductase from other green algae, but did not cross-react with the corresponding enzyme from different cyanobacteria or higher plant leaves. An analogous situation was also found for ferredoxin-glutamate synthase (EC 1.4.7.1), using its specific antiserum. Besides, the antibodies raised against C. reinhardii ferredoxin-glutamate synthase were able to inactivate the ferredoxin-dependent activity of nitrite reductase from green algae.These results suggest that there exist similar domains in ferredoxin-nitrite reductases and ferredoxin-glutamate synthases from green algae. In addition, both types of enzymes share common antigenic determinants, probably located at the ferredoxin-binding domain. In spite of their physicochemical resemblances, no apparent antigenic correlation exists between the corresponding enzymes from green algae and those from higher plant leaves or cyanobacteria.Abbreviations Fd ferredoxin - GOGAT glutamate synthase - MV⁺ reduced methyl viologen (radical cation) - NiR nitrite reductase - PMSF phenylmethylsulphonyl fluoride - SDS sodium dodecyl sulfate     

The purification and properties of nitrite reductase from higher plants, and its dependence on ferredoxin

1. NADPH-dependent nitrite reductase from the leaves of higher plants was purified at least 70-fold and separated into two enzyme fractions. The first enzyme, a diaphorase with ferredoxin-NADP-reductase activity, is required only to transfer electrons from NADPH to a suitable electron acceptor, which then donates electrons to nitrite reductase proper. 2. Purified nitrite reductase accepted electrons from ferredoxin (the natural donor) or from reduced dyes. Ferredoxin was reduced by illuminated chloroplasts or dithionite, or by NADPH when diaphorase was present. The purified enzyme did not accept electrons directly from NADPH. 3. Ferredoxins purified from maize, spinach or Clostridium were interchangeable in the nitrite-reductase system. 4. Nitrite reductase had K(m) 0.15mm for nitrite. The pH optimum varied with plant and method of assay. The preparation had low sulphite-reductase activity. Ammonia was the product of nitrite reduction. 5. For some plants, the assay of crude preparations with NADPH was limited by diaphorase and the addition of diaphorase gave a better estimate of nitrite-reductase activity. A simple method of assay is described that uses dithionite with benzyl viologen as electron donor.     

Effects of freezing on purified nitrite reductase from a denitrifier, Alcaligenes sp. NCIB 11015

The effects of freezing on Alcaligenes sp. nitrite reductase [nitric-oxide: ferricytochrome c oxidoreductase, EC 1.7.2.1] dissolved in sodium phosphate (pH 7.2) were investigated. The nitrite reductase was gradually activated with time in the frozen state, resulting in an increase in its activity of 2.5-4.5 times. The final freezing temperature influenced the enzyme activation, maximal activation being observed at around -20 degrees C. All the enzymatic activities that the nitrite reductase is known to catalyze were enhanced by freeze-thawing. The activation was followed by neither association-dissociation nor any gross conformational change of the enzyme molecule, but was accompanied by an increase in the fluorescence intensity of 2-p-toluidinonaphthalene-6-sulfonate used as a hydrophobic probe. The results are consistent with the hypothesis that the activation of the NiR is due to a limited conformational change of the enzyme molecule, particularly in the hydrophobic region. The mechanism of the activation of NiR by freeze-thawing is discussed, in comparison with the mechanisms of inactivation by freeze-thawing of many enzymes reported by previous workers.     

Mutagenesis of nitrite reductase from Pseudomonas aeruginosa: tyrosine-10 in the c heme domain is not involved in catalysis

In Pseudomonas aeruginosa, conversion of nitrite to NO in dissimilatory denitrification is catalyzed by the enzyme nitrite reductase (NiR), a homodimer containing a covalently bound c heme and a d₁ heme per subunit. We report the purification and characterization of the first single mutant of P. aeruginosa cd₁ NiR in which Tyr¹⁰ has been replaced by Phe; this amino acid was chosen as a possibly important residue in the catalytic mechanism of this enzyme based on the proposal (Fülöp, V., Moir, J.W.B., Ferguson, S.J. and Hajdu, J. (1995) Cell 81, 369–377) that the topologically homologous Tyr²⁵ plays a crucial role in controlling the activity of the cd₁ NiR from Thiosphaera pantotropha. Our results show that in P. aeruginosa NiR substitution of Tyr¹⁰ with Phe has no effect on the activity, optical spectroscopy and electron transfer kinetics of the enzyme, indicating that distal coordination of the Fe³⁺ of the d₁ heme is provided by different side-chains in different species.     

Measurement of nitrite reductase in leaf tissue of Vigna mungo

The enzyme nitrite reductase (EC 1.6.6.4) is generally assayed in terms of disappearance of nitrite from the assay medium. We describe a technique which allowed estimation of the enzyme level in leaf tissues of Vigna mungo (L). Hepper in terms of the release of the product (NH₃) of the enzyme reaction. The technique is offered as an alternative, possibly more convenient method for assay of nitrite reductase in plant tissue in vivo.