Red-light effects sensitized by methylene blue on nitrate reductase from spinach (Spinacia Oleracea L.) leaves

Red-Light Effects Sensitized by Methylene Blue on Nitrate Reductase from Spinach (Spinacia oleracea L.) Leaves S. G. Mauriño, M. A. Vargas, C. Echevarría, P. J. Aparicio, and J. M. Maldonado Departamento de Fisiologia Vegetal, Faeultad de Biologia, Universidad de Sevilla, 41012 Sevilla, Spain, and Departamento de Fisiologia Vegetal, Faeultad de Ciencias, Universidad de Cordoba, 14005 Cordoba, Spain Z. Naturforsch. 39c, 1079-1084 (1984); received July 27, 1984 Methylene Blue, Nitrate Reductase, Photosensitization, Singlet Oxygen, Spinacia Nitrate reductase from spinach (Spinacia oleracea L.) leaves, which had been inactivated in vitro by incubation with NADH and cyanide, was fully reactivated in minutes when irradiated in anaerobic conditions with red light in the presence of methylene blue. Both the rate and the extent of reactivation increased with light intensity ( 6 to 100 W -m “ 2) and dye concentration (1 to 10 jiM). On the contrary, photoreactivation was completely abolished when NADH or ethylenediaminetetra-acetic acid were present during irradiation. We propose that methylene blue, when photo excited, exhibits a redox potential positive enough to reoxidise the CN~-reduced molybdenum complex settled in the inactive enzyme, thus causing its reactivation. On the other hand, prolonged irradiation of nitrate reductase, under air and in the presence of methylene blue, promoted an oxygen-dependent irreversible inactivation of the two partial activities of the enzyme. This inactivation was markedly enhanced in 77% deuterated water and greatly prevented by azide, which indicates that singlet oxygen is the species primarily involved in the photooxidative inactivation of the enzyme. 1. Introduction Nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) from spinach leaves catalyses the reduc tion of nitrate to nitrite with NADH as physio logical electron donor. Besides this overall reaction, the enzyme complex exhibits two partial activities which can be independently assayed: a) a NADHdehydrogenase activity, which reduces cytochrome c, ferricyanide or dichlorophenolindophenol; and b) the so-called terminal NR activity, which cata lyzes nitrate reduction with reduced flavins or viologens. The functionality of both partial activ ities is required for the reduction of nitrate with NADH. The enzyme molecule contains FAD, cyto chrome 6-557 and molybdenum as redox prosthetic groups. Whereas the flavin and, presumably, the heme components seem to be associated with the Abbreviations: NADH, nicotinamide adenine dinucleotide, reduced form; NR, nitrate reductase; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; ' 0 2, singlet oxygen; CN—NR, cyanide-inactivated nitrate reductase; MB, methylene blue; M V\ methyl viologen, reduced form; Tris, tris(hydroxymethyl)-aminomethane; EDTA, ethylene diaminetetra-acetic acid. Reprint requests to Prof. J. M. Maldonado. 0341-0382/84/1100-1079 $01.30/0 dehydrogenase function, molybdenum is clearly in volved in the catalytic reduction of nitrate [1 -3]. NR from algae can exist in two metabolically interconvertible forms, either active or inactive, in response to changes in nutritional and environ mental conditions [2, 4], Except for the spinach enzyme, the interconversion mechanism of NR has been scarcely studied in higher plants [1, 2]. Spinach NR is inactivated in vitro by CNor C2H2 when previously reduced with a low potential reductant such as NADH or S2C>4- [5, 6]. The inactivation affects the terminal NR but not to its dehydro genase activity [5-7]. The CN—NR can be re activated by ferricyanide [5, 7], peroxidase systems [8] or trivalent manganese ions generated by illu minated chloroplasts [7], Apparently, these treat ments reoxidise the CN“-reduced molybdenum complex, thus causing the release of the bound CNand the concomitant restoration of the enzyme activity [3, 5, 6]. Inactivation by NADH plus cyanide, and corresponding reactivation by ferri cyanide, have been also demonstrated on NR from rice [9] wheat [10], barley [11] and maize [12], It has been established that in vitro inactivated NR from Chlorella fusca [13], Chlamydomonas reinhardii [14], Neurospora crassa [15], spinach [5, 13, 16, 17], wheat [10] and maize [12], is likewise readily reactivated by exposure of the inactive enzyme to This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution-NoDerivs 3.0 Germany License. On 01.01.2015 it is planned to change the License Conditions (the removal of the Creative Commons License condition “no derivative works”). This is to allow reuse in the area of future scientific usage. Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Creative Commons Namensnennung-Keine Bearbeitung 3.0 Deutschland Lizenz. Zum 01.01.2015 ist eine Anpassung der Lizenzbedingungen (Entfall der Creative Commons Lizenzbedingung „Keine Bearbeitung“) beabsichtigt, um eine Nachnutzung auch im Rahmen zukünftiger wissenschaftlicher Nutzungsformen zu ermöglichen. 1080 S. G. Maurino et al. ■ Photosensitized Effects on Spinach Nitrate Reductase light in the presence of flavins. Further, light plus FAD also promoted efficiently the reactivation of an inactive form of NR isolated from Chlorella [5, 13], Chlamydomonas [14] and wheat leaf [10]. On the other hand, it has been reported [18], that prolonged irradiation of spinach NR with blue light in the presence of FMN, provokes an irreversible damage of the enzyme due more likely to the harmful effect of photogenerated '0 2. We report herein that photoreactivation of spinach C N -N R can also be sensitized by MB. Moreover, in the presence of oxygen, the dye photo sensitizes an irreversible inactivation of the two partial activities of NR complex where '0 2 seems to be involved. 2. Materials and Methods 2.1. Plant material and enzyme assays NR from leaves of field-grown spinach (Spinacia oleracea L.) was partially purified as previously described [16]. Th enzymatic assays for MV*-nitrate reductase and NADH-dehydrogenase activities were reported elsewhere [6]. One unit of MV’-nitrate reductase or NADH-dehydrogenase is the amount of enzyme which catalyzes the formation of 1 |imol N 0 2 or 1 (imol reduced cytochrome c per min, respectively. 2.2. Inactivation of NR To prepare CN -N R , active enzyme was in cubated for 10 min at 4 °C in 0.2 m Tris-HCl buffer (pH 7.5), 0.6 m M NADH and 0.2 m M KCN. In order to remove the excess of NADH and CN_, the preparation was filtered through a Sephadex G-25 column equilibrated with 0.1 m K-phosphate buffer (pH 7.5). The eluted inactive NR was used for experiments on photoreactivation. 2.3. Conditions of irradiation Unless otherwise indicated, NR, in 0.5 ml of 0.1 m K-phosphate buffer (pH 7.5), was irradiated in open-air glass tubes (5 cm high x 1 cm diameter) placed in small transparent methacrylate baths. Temperature was kept at 4°C by continuous circu lation around the tubes of iced water. Actinic redlight was provided by an ordinary slide projector (24 V, 250 W lamp) fitted with a Balzer interference filter DT 606 transmitting from 606 nm into the infrared. Red-light irradiance at sample position was 100 W • m-2 except in the experiment of Fig. 2 where different light irradiances were used, as indicated. Irradiances were measured with a Yellow-Spring 65 A radiometer. To establish anaerobic conditions, glucose (30 m M ), glucose oxidase (1.5 units) and catalase (1400 units) were added to the corresponding mixtures. Imme diately after, the tubes were fitted with rubber stoppers (Becton-Dickinson Vacutainer) and sub jected to three 1 min-cycles of evacuation and flush ing with N2. Aliquots for determination of en zymatic activities were taken out in this case with the aid of hypodermic needles. 2.4. Analytical methods Protein was determined according to the method of Lowry et al. [19], using bovine serum albumin as standard. Nitrite was measured by the diazo-coupling colorimetric assay of Snell and Snell [20]. Spectrophotometric enzyme assays were per formed in a Beckman DK-2A spectrophotometer, and colorimetric measurements in a Bausch & Lomb Spectronic 100 spectrophotometer. 3. Results 3.1. The two light-effects mediated by MB on spinach NR Results of Fig. 1 show that red-light irradiation, in the presence of MB, of C N -N R brought about two different effects on the terminal activity, depending on whether or not oxygen was present during irradiation. When irradiation was carried out under anaerobic conditions (Fig. 1 A), a rapid reactivation of MV'-nitrate reductase took place. Highest rates were attained at MB concentrations of 5 to 10 (iM , whereas at 1 |iM the rate decreased to about half. In the absence of sensitizer, photore activation could hardly proceed. Under air (Fig.IB), the MB-photosensitized reactivation was greatly impeded and, moreover, upon extended irradiation, the reactivated enzyme was progressively inactivated, especially with 10 (iM MB. In order to investigate separately both processes, photoreactivation was performed in anaerobic sys tems, whereas photoinactivation experiments were conducted under air and using fully active NR. S. G. Maurino et al. ■ Photosensitized Effects on Spinach Nitrate Reductase 1081 TIME OF IRRADIATION (min) Fig. 1. Antagonistic effects of red-light irradiation on spinach C N -N R in the presence of MB. Samples of C N -N R were irradiated with red light under anaerobic (A) or aerobic (B) conditions, either alone (o) or in the presence of 1 (iM (•), 5 (iM (a) and 10 |!M MB (a ). At the indicated times, 0.05 ml aliquots were taken out from the different mixtures to measure MV'-nitrate reductase activity. The specific activity of the enzyme preparation before inactivation with CNwas 32 mU • mg protein“ 1. 3.2. Reactivation of spinach NR photosensitized by MB Fig. 2 shows the time courses of photoreactiva tion of CN—NR when the enzyme, in the presence of 5 pM MB, was irradiated with red light of 6, 25 and 100 W • m~2. The rate of MB-photosensitized reactivation increased with irradiance. Even at an irradiance as low as 6 W • m~2, a remarkable re activation rate was observed. In the dark, however, the enzyme activity did not show any significant increase, at least within the period of time tested. It has been reported [21, 22] that photoexcited MB is easily reduced by NADH or EDTA. Inter estingly, the MB-mediated photoreactivation of spinach C N -N R was completely abolished when TIME OF IRRADIATION (min) Fig. 2. Effect of light intensity on the MB-sensitized photoreactivation of spinach C N -N R . Samples containing CN—NR and 5 |iM MB were made anaerobic and then either kept in the dark (o) or illuminated with red light at irradiances of 6 W • m - 2 (•), 25 W • m~ 2 (a) or 100 W • n r 2 (a). Other conditions as in Fig. 1. TIME OF IRRADIATION (min) Fig. 3. Oxygen-dependent MB-sensitized photoinactivation of the partial activities of spinach NR. Samples containing active NR and 0.1 mM MB were irradiated with red light, either under air (o, a) or under anaerobic conditions (•, a). At the indicated times, 0.05 ml aliquots were taken out from the different mixtures to measure MV"-nitrate reductase (o, •) and NADH-dehydrogenase (a, a ) activities. 1082 S. G. Mauriiio et al. • Photosensitized Effects on Spinach Nitrate Reductase TIME OF IRRADIATION (min) Fig. 4. Enhancement by deuterium oxide of the MBsensitized photoinactivation of the partial activities o f spinach NR. Samples containing active NR and 0.1 mM MB. in a medium without (o, a) and with (•, a) 77% D 20 , were irradiated under air with red light. Other experi mental conditions as in Fig. 3. MV'-niträte reductase (o, •); NADH-dehydrogenase (a, a). Table I. Protection by azide of the partial activities of spinach NR against MB-sensitized photoinactivation. Conditions MV'-nitrate reductase NADH-dehydrogenase U • mg protein1 Dark 0.49 2.25 Light 0.08 0.13 Light plus azide 0.25 0.90 Samples containing active NR and 0.1 mM MB were either kept in the dark or irradiated under air for 25 min with red light both in the absence and presence of 2 mM N aN 3. Activities were then assayed on aliquots from the mixtures. either EDTA or NADH at 5 mM concentration were included in the irradiation mixture (data not shown). 3.3. Photodynamic inactivation of the partial activities of spinach NR Fig. 3 shows that irradiation with red light of active NR, under air and in the presence of 0.1 mM MB, provoked a marked inactivation of the two partial activities of the enzyme. In the absence of oxygen, the activities were practically not altered. The absolute requirement of oxygen seems to in dicate that, in contrast to the photoreactivation process, photoinactivation is not promoted by ex cited MB per se but rather by some reactive chemical species produced by interaction of the excited photosensitizer with molecular oxygen. Many photosensitized biological reactions in volving molecular oxygen (photodynamic reactions) take place by the action of the highly reactive '0 2 generated by direct energy transfer from the excited triplet state of the sensitizer to the ground triplet state of molecular oxygen [23]. The specific quench ing effect of N j on ‘0 2 and the longer lifetime of *02 in D20 than in H20 are sensitive parameters successfully used to demonstrate the participation of '0 2 in photodynamic reactions [18, 23]. Table I shows that MB-photosensitized inactivation of the two partial activities of spinach NR was greatly prevented in the presence of 2 mM N aN3. On the other hand, photoinactivation rates substantially increased in a deuterated medium (77% D 20 ) as compared to regular water (Fig. 4). These results indicate that '0 2 is the major reactive oxygen species involved in the MB-sensitized photoinac tivation of spinach NR. 4. Discussion As demonstrated in this paper, spinach C N -N R is rapidly reactivated by red light in the presence of MB. In contrast to the flavin-mediated photoreac tivation of N. crassa NR [15], photoreactivation of the spinach enzyme sensitized by either flavins [16] or MB (this paper) did not require oxygen. More over, reactivation was faster and more complete under anaerobic conditions. The experimental evi dence presented above indicates that inhibition of photoreactivation by oxygen is due to the interac tion of molecular oxygen with the excited dye, which yields two different effects. Firstly, molecular oxygen readily quenches the lowest excited triplet state of MB [24], thus diminishing the actual concentration of excited MB molecules, which appears to be the chemical species primarily involved in the photo reactivation process. This interpretation is further sustained by the fact that either EDTA or NADH, which efficiently reduce photoexcited MB [21, 22], completely impeded the MB-sensitized reactivation. Secondly, some reactive species, more likely '0 2, generated from the interaction of molecular oxy gen with excited MB, brings about a subsequent inactivation of the enzyme. Indeed, the experiments S. G. Maurino et al. • Photosensitized Effects on Spinach Nitrate Reductase 1083 carried out with active NR using D20 and N J, suggest that ‘0 2 is the major reactive species in volved in the enzyme inactivation which takes place under air during extended light exposure. Incuba tion of photoinactivated NR with ferricyanide or dithioerythritol did not restore the original en zymatic activities (data not shown), indicating that, as in the FMN-mediated photoinactivation [18], NR might be irreversibly damaged. By means of redox titrations with ferricyanide it has been demonstrated [25] that the reactivation process of C N -N R from Ankistrodesmus braunii shows a “midpoint potential” of 4-0.23 V at pH 7.5. On the other hand, from thermodynamic and kinetic aspects of photoinduced electron transfer reactions [21, 26], it has been proposed [21] that the redox potential of MB shifts upon irradiation from — 0.23 V, in the ground state, to -I-1.6 V, in the excited triplet state. The redox potential of the photoexcited dye is therefore positive enough to reoxidise readily the CN“-reduced molybdenum complex and, hence, reactivate the enzyme. Similar light-induced changes in the redox potential of flavins [27] might be responsible for the reported flavin-mediated photoreactivation [5, 13, 15, 16]. In fact, assuming that the photochemical active form of flavins is also the triplet state [28] and that the energy of the triplet state above the ground-state electronic level is 2.07 eV [29], it can be easily cal culated [26] that the redox midpoint potential of flavins might shift from - 0.24 V, in the dark, to about + 1.83 V, in the light, namely their oxidizing power would be substantially increased upon photo excitation. As a comprehensive conclusion from results reported in this and previous related papers [5, 13, 16-18], in Fig. 5 are summarized the mechanisms proposed by ourselves for the sensitized light-effects on NR activity from spinach leaves: a) photore activation of the C N -N R , and b) irreversible photodynamic inactivation of its two partial activ ities. Both photoreactions can be triggered either by blue light plus flavins or red light plus MB. In the ground singlet state (S0), the sensitizer, either flavin or MB, by absorption of light becomes photoexcited to, eventually, its triplet state (3S). In this state, the sensitizer exhibits a redox potential positive enough to reoxidise the CN~-reduced molybdenum complex settled in the inactive nitrate reductase (CN -NR), thus bringing about the restoration PHOTODYNAMIC INAC TIVATIO N PHOTOREACTIVATION NR "V " NR-Or (Irreversib ly dam aged) CN-NR ' (Inactive) ^ NR S (Active) C N* o„ H2°2 S„ -------- - h\> Fig. 5. Proposed mechanisms for the light effects and the roles of oxygen on the activity of spinach NR sensitized by either flavins or methylene blue. S0: sensitizer in the ground singlet state; 3S: sensitizer in the excited triplet state; Sr: semiquinone form of the sensitizer; 30 2: m olec ular oxygen (ground triplet state); Ot 2: superoxide radical; N R —0 2: oxygenated nitrate reductase. of the enzyme activity. The initial rate of the photo reactivation process increases under anaerobic con ditions since ^ can be unenergized by ground triplet dioxygen (30 2) through energy transfer (see left half of the figure). Notwithstanding, sustained photoreactivation might show a definite require ment of 30 2, capable by itself of regenerating S0 from the semiquinone form of the sensitizer (S7), if the initial concentration of S0 were too low, which does not appear to be the case under the ordinary experimental conditions. On the other hand, under prolonged irradiation in air, the highly reactive '0 2, generated by energy transfer from 3S to 30 2, pro motes oxidation or oxygenation of some aminoacids of the NR molecule (N R -0 2) [27], thus causing the irreversible loss of its various activities. Acknowledgements We wish to thank Professor Losada and Drs. F. F. de la Rosa and M. A. de la Rosa for helpful discussions during the course of this work, and Miss C. Santos for typing the manuscript. S. G. M. also acknowledges the tenure of a fellowship from Ministerio de Education y Ciencia (Spain). 1084 S. G. Maurino et al. • Photosensitized Effects on Spinach Nitrate Reductase [1] L. Beevers and R. H. Hageman, The Biochemistry of Plants, Vol. 5 (ed. B. J. Miflin), pp. 115-168, Aca demic Press, New York 1980. [2] M. G. Guerrero, J. M. Vega, and M. Losada, Ann. Rev. Plant Physiol. 32,169-240 (1981). [3] E. J. Hewitt and B. A. Notton, Molybdenum and Molybdenum-containing Enzymes (ed. M. Coughlan), pp. 273-325, Pergamon Press, Oxford 1980. [4] B. Vennesland and M. G. Guerrero, Encyclopedia of Plant Physiology, New Ser. (eds. M. Gibbs and E. Latzko), pp. 425-444, Springer-Verlag, Berlin 1979. [5] P. J. Aparicio and J. M. Maldonado, Nitrogen As similation of Plants (eds. E. J. Hewitt and C. V. Cutting), pp. 207-215, Academic Press, London 1979. [6] J. M. Maldonado, M. A. Vargas, S. G. Maurino, and P. J. Aparicio, Biochim. Biophys. Acta 661, 112-119(1981). [7] J. M. Maldonado, B. A. Notton, and E. J. Hewitt, Planta 150,242-248 (1980). [8] J. M. Maldonado, B. A. Notton, and E. J. Hewitt, Planta 156,289-294(1982). [9] C. C. Leong and T. C. Shen, Experientia 35, 584-585 (1979). [10] A P. Aryan, R. G. Batt, and W. Wallace, Plant Physiol. 71,582-587 (1983). [11] D. A Somers, T. M. Kuo, A. Kleinhofs, R. L. Warner, and A. Oaks, Plant Physiol. 72,949-952 (1983). [12] C. Echevarria, S. G. Maurino, and J. M. Maldonado, Phytochemistry 23,2155-2158 (1984). [13] P. J. Aparicio, J. M. Roldan, and F. Calero, Biochem. Biophys. Res. Commun. 70,1071 - 1077 (1976). [14] M. P. Azuara and P. J. Aparicio, Plant Physiol. 71, 286-290 (1983). [15] J. M. Roldan and W. L. Butler, Photochem. Photobiol. 32,375-381 (1980). [16] S. G. Maurino, M. A. Vargas, P. J. Aparicio, and J. M. Maldonado, Physiol. Plant. 57 ,41 1-4 16 (1983). [17] J. M. Roldan, F. Calero, and P. J. Aparicio, Z. Pflanzenphysiol. 90,467-4 7 4 (1978). [18] M. A. Vargas, S. G. Maurino, J. M. Maldonado, and P. J. Aparicio, Photochem. Photobiol. 36, 223-228 (1982). [19] O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193,265-275 (1951). [20] F. D. Snell and C. T. Snell, Colorimetric Methods of Analysis, pp. 802-807, Van Nostrand, New York 1949. [21] M. Julliard and J. Le Petit, Photochem. Photobiol. 36,283-290 (1982). [22] G. Oster and N. Wotherspoon, J. Am. Chem. Soc. 79, 4836-4838 (1957). [23] J. D. Spikes, The Science of Photobiology (ed. K C. Smith), pp. 87-112, Plenum Press, New York 1977. [24] N. Hasty, P. B. Merkel, P. Radlik, and D. R. Kearns, Tetrahedron Lett. 1,49 -51 (1972). [25] M. A. de la Rosa, C. Gömez-Moreno, and J. M. Vega, Biochim. Biophys. Acta 6 6 2 ,77-8 5 (1981). [26] V. Balzani and F. Scandola, Photochemical Con version and Storage of Solar Energy (ed. J. S. Con nolly), pp. 9 7 - 125, Academic Press, New York 1981. [27] M. Losada, Bioelectrochem. Bioenerg. 6, 205-225 (1979). [28] P. S. Song, Flavins and Flavoproteins (ed. H. Kamin), pp. 37-51, Univ. Park Press, Baltimore 1971. [29] P. J. Heelis, Chem. Soc. Rev. 11, 15-39 (1982).