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Exposure of Arabidopsis leaves to nitrogen dioxide (NO2) results in nitration of specific chloroplast proteins. To determine whether NO2 itself and/or nitrite derived from NO2 can nitrate proteins, Arabidopsis thylakoid membranes were isolated and treated with NO2-bubbled or potassium nitrite (KNO2) buffer, followed by protein extraction, electrophoresis, and immunoblotting using an anti-3-nitrotyrosine (NT) antibody. NO2 concentrations in the NO2-bubbled buffer were calculated by numerically solving NO2 dissociation kinetic equations. The two buffers were adjusted to have identical nitrite concentrations. Both treatments yielded an NT-immunopositive band that LC/MS identified as PSBO1. The difference in the band intensity between the 2 treatments was designated nitration by NO2. Both NO2 and nitrite mediated nitration of proteins, and the nitration ability per unit NO2 concentration was ~100-fold greater than that of nitrite.
We previously reported that exposure of Arabidopsis leaves to nitrogen dioxide (NO2) resulted in almost exclusive nitration of PsbO and PsbP, extrinsic proteins of photosystem II (PSII), and highly selective nitration of non-PSII proteins such as peroxiredoxin IIE (PRXIIE).1 We also showed using 15N-labeled NO2 that the nitro group of proteins nitrated by exposure to NO2 is derived from exogenously supplied NO2.2
However, the mechanism underlying NO2-mediated protein nitration is unclear. For example, whether exogenous NO2 per se or nitrite derived from NO2 mediates NO2-mediated protein nitration is unknown. NO2-mediated protein nitration may be induced directly by exogenously applied NO2 molecules because NO2 is a potent nitrating agent in vitro3 and a primary intermediate of protein nitration in vivo in mammals and humans.4,5 However, foliar uptake of NO2 occurs through reactive absorption, in which NO2 is absorbed by reacting with apoplastic ascorbate, the primary product of which, nitrite, enters cells.6 Nitrite is converted into NO2 by a variety of peroxidases in the presence of hydrogen peroxide, which then nitrates proteins in mammals, humans, and plants7-11 (see below). Oxyhemoglobin also oxidizes nitrite to NO2, resulting in nitration of proteins.12
To identify the nitrogen species involved in NO2-mediated protein nitration, the following 2 buffers were used: a buffer through which NO2 gas was bubbled (NO2-bubbled buffer) and the same buffer containing only potassium nitrite (KNO2) (KNO2 buffer). In the NO2-bubbled buffer, a portion of the dissolved NO2 undergoes disproportionation (as shown in reaction 1) to form nitrate and nitrite; thus, this buffer contains 2 nitrating agents, NO2 and NO2−. Nitration following incubation in the NO2-bubbled buffer is attributable to nitration by NO2 or by NO2−, compared to nitration by NO2- alone in KNO2 buffer. The concentration of the latter can be determined using the capillary ion analyzer (CIA) method, and that of the former can be calculated mathematically as described below. Therefore, in the presence of identical NO2− concentrations in the 2 buffers, nitration by NO2 can be calculated by subtraction of nitration due to incubation in the latter buffer from that by incubation in the former buffer. In this study, nitration by NO2 and nitration by NO2− were quantified and compared.
According to Huie (1994),13 references therein the kinetic equations of the NO2 dissociation shown in reaction 1 are as follows:
where k1, k2, and k3 are the rate constants 4.5 × 108 L mol−1 s−1, 6.4 × 103 s−1, and 103 s−1, respectively,13 references therein In reaction 1, there is a linear relationship between the initial concentration of NO2 and the concentrations of NO2− and NO3−. The NO2− and NO3− concentrations in the buffer were determined by the CIA method (Code CIA; Millipore Corp., Milford, MA, USA) as described previously.14 Using these values, the differential equation was solved numerically, and the concentration of NO2 in the buffer was calculated as described in the (Fig. S1). Alternatively, KNO2 buffer containing only KNO2 at the same concentration as that in the NO2-bubbled buffer was prepared, and used for treatment with nitrite only.
As a plant material, thylakoid membranes isolated from Arabidopsis leaves were used because PSBO1 has been reported to be the sole nitratable protein of ~30 kDa in the thylakoid membrane fraction from Arabidopsis leaves following exposure to NO2.1
Seeds of Arabidopsis thaliana (L.) Heynh. accession Columbia (Arabidopsis) were sown in vermiculite and perlite (1:1, v/v) in plastic containers and incubated in a growth chamber (model ER-20-A; Nippon Medical & Chemical Instruments, Osaka, Japan) at 22.0 ± 0.3°C and a relative humidity of 70 ± 4% under continuous fluorescent light (70 µmol photons/m2/s). The plants were grown for 4 weeks with irrigation every 4 d in a half-strength solution of inorganic salts of Murashige and Skoog medium,15 as described previously.1 Thylakoid membranes were isolated from Arabidopsis leaves according to Suorsa et al.16 NO2 gas (5% in N2 gas at a flow rate of 0.1 L/min) was bubbled through a suspension buffer containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH (pH 7.0), 15 mM NaCl2, 5 mM MgCl2 and 400 mM sucrose for 0 to 60 s in a NOx chamber, as described previously.1 CIA analysis of the resulting NO2-bubbled buffer indicated the presence of 0–4.35 mM NO2−. The NO2 concentrations (0–36.8 μM NO2) in the buffer were calculated by solving differential eqs. 1 to 3 numerically, as described above (see also Supplemental Information). Suspension buffer containing KNO2 at 0–4.35 mM was also prepared.
Thylakoid membranes suspended in NO2-bubbled buffer or KNO2 buffer were incubated for 60 min at 25°C under illumination (100 µmol photons per m2 per s) in the presence of 1000 U mL−1 catalase and 40 U mL−1 SOD to prevent photoinhibition.17 After incubation, thylakoid membranes were collected by centrifugation at 2,500 g for 5 min at 4°C, and resuspended in 50 mM potassium phosphate buffer (pH 7.5) containing 0.5% (v/v) Triton-X100. After centrifugation at 15,000 g for 10 min at 4°C, the supernatant was collected. The protein content of the supernatant was determined using the Bio-Rad DC Protein Assay Kit 2 (Bio-Rad, CA, USA), with BSA as a standard. Then the supernatant was added to sodium dodecyl sulfate (SDS) sample buffer consisting of 2% SDS, 50 mM Tris-HCl (pH 6.8), 10% glycerol, and a trace of bromophenol blue. Protein solution containing 10 µg protein was loaded onto 12% (w/v) polyacrylamide-SDS slab gels, and electrophoresed for 1 h at 20 mA. Then sample proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, MA, USA) using an electroblotter (Atto, Tokyo, Japan), and subjected to immunoblot analysis using a polyclonal antibody against NT (Upstate Biotechnology, NY, USA) diluted 1:1000 in phosphate-buffered saline containing Tween (Tween-PBS). After three washes with Tween-PBS, the membranes were incubated for 1 h with goat anti-rabbit peroxidase-conjugated secondary antibody (Vector Labs, CA, USA) diluted 1:2000 in Tween-PBS. After three washes with Tween-PBS, immunoreactive bands were detected using enhanced Western Blot Chemiluminescence Reagent Plus (NEN Life Science Products, MA, USA) and visualized using a VersaDoc Imager (Bio-Rad).
On an immunoblot gel of protein extracted from thylakoid membranes following incubation in NO2-bubbled buffer containing 36.8 µM NO2 and 4.35 mM NO2− in light, a distinct NT-positive band of ~32.5 kDa was detected (Fig. 1A). A similar NT-immunopositive band was observed at around 32.5 kDa on an immunoblot of protein extracted from thylakoid membranes following incubation in KNO2 buffer containing 4.35 mM NO2− in light (Fig. 1A). In contrast, proteins extracted from thylakoid membranes following incubation with suspension buffer containing 4.35 mM KNO3 did not show NT-immunopositive bands (data not shown), indicating that nitrate did not induce protein nitration.
The SYPRO Ruby-stained protein band corresponding to the NT-immunopositive band was excised and subjected to tryptic in-gel digestion according to Shevchenko et al.18 The peptide samples were subjected to liquid chromatography electrospray ionization mass spectrometry (MS)/MS analysis using an LCQ Advantage ion-trap mass spectrometer (Thermo Fisher Scientific, CA, USA). Proteins were identified using the Mascot search engine against the NCBInr database (Arabidopsis thaliana) and the TurboSEQUEST software (Thermo Fisher Scientific).
LC/MS analysis followed by a Mascot search identified this band as PSBO1. This result is in agreement with a report that PSBO1 is the sole nitrated protein in the insoluble (thylakoidal membranous) fraction of chloroplast proteins isolated and purified from leaves of Arabidopsis following exposure to NO2 in light.1 Furthermore, the NT-positive band on an immunoblot gel of protein extracted from thylakoid membranes following incubation in KNO2 buffer in light (see above, Fig. 1A) was also identified as PSBO1 by LC/MS analysis.
Taken together, these findings suggest that PSBO1 in isolated thylakoid membranes was specifically nitrated by incubation with the NO2-bubbled and KNO2 buffers. Nitration in the former is attributable to NO2 and nitrite, while that in the latter is attributable to nitrite. Therefore, both NO2 and nitrite are involved in protein nitration following exposure to NO2.
The two nitrating agents exhibited different nitration abilities. The intensity of the PSBPO1 immunoblot band was analyzed using the PDQuest software ver. 7.0 (Bio-Rad) as described previously.1 The intensity of the NT-immunopositive PSBPO1 band increased with increasing NO2 and NO2− concentrations in a dose-dependent manner (Fig. 1B). The fold-change in nitration of PSBPO1 following incubation with NO2 buffer was calculated using the following equation and designated ‘FCPSBO1 by NO2 buffer’: FCPSBO1 by NO2 buffer = (intensity of the NT-immunopositive band of PSBPO1 after incubation with NO2-bubbled buffer) / (intensity of the same band before incubation with NO2-bubbled buffer). Similarly, the fold-change in intensity of the NT-immunopositive band of PSBPO1 following incubation with KNO2 buffer was calculated and designated ‘FCPSBO1 by nitrite.’ The difference between FCPSBO1 by NO2 buffer and FCPSBO1 by nitrite was designated ‘FCPSBO1 by NO2’ [ = (FCPSBO1 by NO2 buffer) – (FCPSBO1 by nitrite)], which corresponds to the fold-change in the intensity of the NT-immunopositive band of PSBPO1 due to NO2 alone. The FCPSBO1 by NO2-bubbled buffer was higher than the FCPSBO1 by nitrite in the presence of an identical NO2− concentration (Fig. 1 C). As a result, the value of FCPSBO1 by NO2 was positive irrespective of NO2 concentration (Fig. 1C).
The FCPSBO1 by NO2 per unit concentration of NO2, which reflects the ability of NO2 to nitrate protein, was calculated to be 0.2/μM NO2. Similarly, the ability of NO2− to nitrate protein was calculated to be 0.9/mM NO2−. Therefore, NO2 possesses a ~100-fold greater ability to nitrate proteins than NO2−.
Furthermore, the mechanism of nitration by NO2 differs from that by nitrite. In mammals and humans, a variety of peroxidases such as myeloperoxidase, eosinophil peroxidase,7,9 heme peroxidase,8 and pathogen-inducible peroxidases10 catalyze the oxidation of nitrite to NO2 radical to nitrate proteins in the presence of H2O2. In addition, plant horseradish peroxidase reportedly converts nitrite into NO2, which mediates the nitration of phytophenolics, in the presence of hydrogen peroxide.11 In addition, thylakoid membranes harbor membrane-bound peroxidases such as ascorbate peroxidases.19 Light-induced generation of hydrogen peroxide in isolated thylakoids has also been reported.20 Thus, H2O2 produced by thylakoid and thylakoid membrane-bound peroxidases is likely involved in nitrite-mediated nitration of PSBO1 in isolated thylakoid membranes.
Nitration by peroxidase/H2O2/nitrite is reported to proceed as follows: Nitrite is oxidized to NO2 (at a rate of 3.2 × 106 M−1s−1),5 tyrosine residues are oxidized to tyrosyl radicals (at a rate of 7.7 × 105 M−1s−1),21 and the 2 radicals combine at a diffusion-controlled rate (3 × 109 M−1s−1)5 to form NT. The overall rate of this nitration is limited by the rate of conversion of tyrosine residues into tyrosyl radicals.
In contrast, tyrosine nitration by NO2 is reported to proceed as follows: One molecule of NO2 oxidizes a target tyrosine residue to a tyrosyl radical at a rate of 3.2 × 105 M−1s−1,22 and the tyrosyl radical combines with a second molecule of NO2 to form NT at a rate of 3.2 × 109 M−1s−1.5,21 The overall rate of nitration by NO2 is limited by the rate of conversion of tyrosine residue to tyrosyl radical by NO2.5,23
However, these 2 mechanisms result in similar overall rates (3.2–7.7 × 105 M−1s−1) of nitration by NO2 and nitrite. Therefore, these mechanisms cannot explain our finding of a 2 orders-of-magnitude difference in the nitration abilities of NO2 and nitrite unless the following are assumed: (i) the rate of nitration of PSBO1 by the putative ascorbate peroxidase/H2O2/nitrite system proceeds at a rate considerably lower than 3.2 × 105 M−1s−1 and/or (ii) the rate of oxidation of PSBO1 tyrosine residues is considerably greater than that of oxidation by NO2. For example, they may be oxidized by more rapid reactions, such as photo-oxidation, which has a half-life on the order of nanoseconds (Takahashi et al., unpublished results). Further studies are required to clarify this point.
It should be noted, however, that the incubation buffers used in this study contained scavengers of H2O2 (such as catalase and SOD) to prevent photoinhibition,16 as described above. H2O2 is required for peroxidase-mediated conversion of nitrite into NO2, and thus the presence of scavengers of H2O2 in the incubation buffer might have inhibited protein nitration by nitrite, possibly leading to underestimation of the nitration ability of nitrite. However, the presence of these scavengers of H2O2 in incubation buffers is unlikely to be the cause of the observed ~100-fold difference in nitration abilities between NO2 and nitrite because withdrawal of both scavengers from the incubation buffer increases nitration by a factor of only ≤2 (Takahashi et al., unpublished results).
This discussion can be extended to the mechanism of protein nitration by exposure of the intact cells of leaves to NO2 gas. When leaves are exposed to NO2, nitrite derived from NO2 is one of the major nitrogen species that enters cells because foliar uptake of NO2 is governed by the reactive absorption mechanism,6 which involves diffusion of NO2 through cell walls and simultaneous reaction of NO2 with apoplastic ascorbate to form nitrite. Thus, nitrite/peroxidase must play a major role in NO2-mediated protein nitration in intact cells of leaves. However, given that NO2 has twice the nitration ability of nitrite, and that its hydrophobicity facilitates permeation of the cell membrane,24 it is possible that at least some NO2 molecules diffuse freely, before reacting with apoplastic ascorbate, to enter cells and nitrate proteins. The time available for free diffusion of NO2 is estimated based on the half-life (τ) of the reaction between NO2 and ascorbate, as given by eq. 5.25,26
where k and c are the reaction constant and ascorbate concentration in cell-wall water, respectively. Assuming c and k to be 1 mM and 3.5 × 107 M−1s−1, respectively,25,27 τ is calculated23,26 to be 19.8 μs. The distance (d) to which NO2 can diffuse during its half-life is estimated by the Einstein-Smoluchowski equation (eq. 6):
Substituting τ for t, and assuming the diffusion constant D of NO2 to be 1.4 × 10−9 (m2/s),25 d is calculated to be 0.2 μm. Thus, NO2 can diffuse through 0.2 µm of cell-wall water before reacting with ascorbate. Primary cell walls are 0.1 to 10 μm thick.28 Thus, depending on the cell-wall thickness, NO2 molecules can enter cells by diffusion. Taken together, our findings suggest that NO2 and nitrite are involved in the NO2-mediated nitration of cellular proteins in intact cells of leaves.
No potential conflicts of interest were disclosed.
We thank Professor Junichi Mano, Yamaguchi University, for his interest in this work and invaluable discussions during the course of this study.
This work was supported by grants from the Nippon Life Insurance Foundation (to MT) and the Nissan Science Foundation (to MT), a Grant-in-Aid for Creative Scientific Research from the Japan Science and Technology Agency (no. 13GS0023 to HM), and a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no.15710149 to MT).