The Conversion of Nitrite to Nitrogen Oxide(s) by the Constitutive NAD(P)H-Nitrate Reductase Enzyme from Soybean

Abstract

A two-step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase INAD(P)H-NR, pH 6.5; EC 1.6.6.21 activity from the nitric oxide and nitrogen dioxide (NO(X)) evolution activity extracted from soybean (Glycine max [L.] Merr.) leaflets.Both of these activities were eluted with NADPH from Blue Sepharose columns loaded with extracts from either wild-type or LNR-5 and LNR-6 (lack constitutive NADH-NR [pH 6.51) mutant soybean plants regardless of nutrient growth conditions. Fast protein liquid chromatographyanion exchange (Mono Q column) chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO(X) formation. The Blue Sepharose-purified soybean enzyme has a pH optimum of 6.75, an apparent K. for nitrite of 0.49 millimolar, and an apparent K. for NADPH and NADH of 7.2 and 7.4 micromolar, respectively, for the NO(X) evolution activity. In addition to NAD(P)H, reduced flavin mononucleotide (FMNH2) and reduced methyl viologen (MV) can serve as electron donors for NO(X) evolution activity. The NADPH-, FMNH2-, and reduced MV-NO(,) evolution activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymercuribenzoate, whereas, the FMNH2 and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrite to NO(X). NADPH eluted both NR and NO(X) evolution activities from Blue Sepharose columns loaded with extracts of either nitrate-or zero N-grown winged bean (Psophocarpus tetragonolobus [L.]), whereas NADH did not elute either type of activity. Winged bean appears to contain only one type of NR enzyme that is similar to the constitutive NAD(P)H-NR (pH 6.5) enzyme of soybean.It has been shown that the predominant compound evolved from soybean leaves during the purged in vivo NR3 assay is nitric oxide (NO) with trace amounts of nitrous oxide (N20) and 'Supported by an American Soybean Association research grant, project number 84953.

Emission of nitrous oxide (N2O) from transgenic tobacco expressing antisense NiR mRNA

“…The difference in the N 2 O emission rates of soybean and clone 271 tobacco may be due to differences in the experimental systems used or differences in the source of N 2 O in the plants. Dean and Harper (1988) (c) Clone 271 or wild-type plants grown on 15 NO 2 ± -containing medium for 2 weeks were transferred to 14 NO 3 ± -containing medium and grown for 2 weeks. The NO 2 ± levels in these plants were determined by capillary electrophoresis (Kawamura et al, 1996).…”

Proteomics Approach to Identify Dehydration Responsive Nuclear Proteins from Chickpea (Cicer arietinum L.)

“…Recently, it has been shown that tobacco cells transformed with an antisense construct of 14-3-3 no longer accumulate ROS (48) suggesting 14-3-3 protein as a very important component in this signaling pathway. NR (CaN-710) is known to catalyze a one electron transfer from NADPH to nitrite to produce nitric oxide (NO) (49,50), a potential signaling intermediate (51). Dehydration stress causes increased NR protein turnover and accelerated mRNA turnover (30,52) and 14-3-3 proteins are instrumental in bringing about this degradation.…”

Nitrite–dependent nitric oxide production pathway: implications for involvement of active nitrogen species in photoinhibitionin vivo

“…Although there was controversy over the NOx evolution pathway from NO 3 via NO (Mulvaney & Hageman 1984), evidence that NO and N 2 O were produced from NO 3 during nitrate reduction was provided by gas chromatography, mass spectrometry and 15 N tracer techniques (Dean & Harper 1986). Because the c 1 NAD(P)H:NR (EC 1.6.6.2) is unique to the Phaseoleae tribe of the family Leguminosae (Dean & Harper 1986), the evolution of NOx through the NR-dependent pathway was thought to be limited to legume species among higher plants (Dean & Harper 1988).…”