Mehler activity in nitrogen fixing cyanobacteria: A key role in oxygen consumption.

Transcription

1 Mehler activity in nitrogen fixing cyanobacteria: A key role in oxygen consumption. Allen J. Milligan Institute of Marine and Coastal Studies, Rutgers, The State University of New Jersey, New Brunswick, New Jersey Ilana Berman-Frank Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 52900, Israel Yoram Gerchman Department of Chemistry, Princeton University, Princeton, New Jersey G. Charles Dismukes Department of Chemistry, Princeton University, Princeton, New Jersey Paul G. Falkowski Institute of Marine and Coastal Studies and Department of Geology, Rutgers, The State University of New Jersey, New Brunswick, New Jersey Corresponding Author: Allen J Milligan Department of Zoology 3029 Cordley Hall Oregon State University Corvallis, OR Allen.Milligan@science.oregonstate.edu Cell: (541) Fax: (541) Running head: Mehler activity in diazotrophs Key Words: Trichodesmium, diazotroph, Mehler activity, PSI, Oxygen consumption. 1

2 Summary Many colonial diazotrophic cyanobacteria are capable of simultaneously evolving O 2 through oxygenic photosynthesis and fixing nitrogen via nitrogenase. As nitrogenase is irreversibly inactivated by O 2, accommodation of the two metabolic pathways has led to biochemical and/or structural adaptations that protect nitrogenase from O 2. In some species differentiated cells (heterocysts) are produced within the colony in which PSII is absent, while photosystem I (PSI) activity is maintained. In other species, cells temporally share a division of labor whereby individual cells in a colony fix nitrogen while others evolve oxygen. Using membrane inlet mass spectrometry (MIMS) in conjunction with tracer 18 O 2 and inhibitors of photosynthetic and respiratory electron transport, we examined the light dependence of O 2 consumption in Trichodesmium sp. IMS 101, a non-heterocyctous colonial cyanobacterium and Anabaena flos-aquae, a heterocystous species. Our results indicate that in both species, intracellular O 2 concentrations are maintained at low levels by the light-dependent reduction of the gas via the Mehler reaction. In nitrogen fixing Trichoidesmium colonies, Mehler activity can be fully three quarters of gross O 2 production. In purified heterocysts of Anabaena, light accelerates O 2 consumption by 3 fold. Our results suggest that a major role for PSI is to consume O 2 and through pseudocyclic electron transport supply ATP in both heterocystous and non-heterocystous diazotrophs. 2

3 Introduction Nitrogen fixation is inherently an anaerobic process, but in cyanobacteria, it occurs under aerobic conditions. Given that the interatomic distance between the two atoms of N in N 2 is 1.09 A, while that for the two atoms of O in O 2 is 1.11 A, both gases can freely diffuse across the plasmamembrane. Hence, to protect the iron-sulfur clusters in nitrogenase from becoming irreversibly oxidized (and thereby rendered catalytically inactive) by molecular oxygen, nitrogen-fixing cyanobacteria must consume O 2 at high rates to maintain low O 2 concentrations in the vicinity of the enzyme. In this paper we examine the metabolic pathway(s) responsible for the consumption of O 2 in cyanobacteria. In all oxygenic photoautotrophs, O 2 may be consumed via three pathways, namely oxygendependent respiration,, the oxygenase activity of RubisCO (photorespiration) and the photocatalyzed reduction of O 2 to H 2 O in photosystem I (i.e., the Mehler reaction; (Mehler, 1957)). In single celled nitrogen fixing cyanobacteria, dark respiration is engaged to consume intracellular O 2 at night, however it is not clear whether this process is also engaged during the photoperiod in colonial nitrogen fixing species (Berman-Frank et al., 2003). Although photorespiration may occur, in cyanobacteria this process is relatively inefficient at consuming O 2 because of the one or more CO 2 concentrating mechanisms that raises the CO 2 concentration in the vicinity of RubisCO thereby reducing the oxygenase activity of the enzyme (Shibata et al., 2002). However, most cyanobacteria contain a relatively high ratio of PSI to PSII reaction centers (Kawamura et al., 1979). It has generally been assumed that the primary role of PSI in cyanbacteria is to provide ATP via cyclic electron flow around the reaction center (Wolk et al., 1994), however, the photocatalyzed reduction of O 2 via the Mehler reaction, which also generates ATP, is potentially a major sink for both photosynthetically generated and ambient O 2 (Berman-Frank et al., 2001). Cyanobactera in the genus Trichodesmium do not form heterocysts, yet simultaneously fix N 2 and evolve O 2 (Ohki et al., 1991). Immunolocalization and biophysical studies have demonstrated that in these organisms, nitrogenase is compartmentalized in a fraction of the cells along a trichome, however, active photosynthetic components (such as PSI and PSII complexes, RubisCO, carboxysomes) are found 3

4 in all cells, even those harboring nitrogenase (Janson et al., 1994; Fredriksson and Bergman, 1997; Fredriksson et al., 1998; Berman-Frank et al., 2001). Protection against oxygen in Trichodesmium is a complex interaction comprising a spatial and temporal segregation of the photosynthetic, respiratory and nitrogen fixation processes (Chen et al., 1999; Berman-Frank et al., 2001). Kana (1992) demonstrated that Mehler activity may be a large fraction of gross O 2 production in field populations of Trichodesmium collected from the sub-tropical Atlantic, and it was subsequently hypothesized that this reaction pathway protected nitrogenase from inactivation by O 2. Although Anabaena flos-aquae fixes N 2 in heterocysts, high rates of O 2 consumption are required to maintain low O 2 concentrations within heterocysts. While dark respiration has been proposed to be the primary mechanism consuming O 2 in heterocysts, the role of light dependent O 2 consumption has not been investigated. Here we provide evidence that the Mehler reaction is a critically important O 2 consuming pathway in both non-heterocystic nitrogen fixing cyanobacteria and in heterocysts. Our results suggest that the coevolution of oxygenic photosynthesis and nitrogen fixation in colonial cyanobacteria led to the appropriation of PSI as an oxygen consuming reaction which protects nitrogenase, and supplies ATP through pseudocyclic electron flow rather than via cyclic electron flow. Results and Discussion We utilized membrane inlet mass spectrometry to monitor 16O2 and 18O2 consumption rates The effect of light and inhibitors on O 2 consumption in Trichodesmium is presented in Table 1. Light greatly stimulated O 2 consumption, but the rates of light-dependent O 2 consumption are similar in the presence of the RubisCO inhibitor glycoaldehyde (Miller and Canvin, 1989). This result suggests that oxygenase activity of RubisCO is not responsible for the large light-dependent O 2 consumption rates observed (Table 1). Inhibition of RubisCO nearly stopped O 2 evolution and enhanced O 2 consumption, consistent with a decrease in the consumption of reducing equivalents and an electron-sink negative feedback to PSI (Table 1). Moreover, the addition of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a specific inhibitor of 4

5 linear photosynthetic electron transport that prevents reduction of the plastiquinone pool by PSII, lowered the light-dependent O 2 consumption to rates similar to those obtained in the dark. These results suggest that respiratory electron transport resulting from the oxygen dependent oxidation of internal stores of reduced carbon is not strongly stimulated or repressed by light. Light dependent consumption rates were similar with and without the addition of both sodium azide and sodium cyanide, two compounds which inhibit terminal oxidase activity (data not shown), supporting the hypothesis that respiratory O 2 consumption is not significantly influenced by light. Based on these observations, we conclude that the enhancement of O 2 consumption in the light is primarily due to the Mehler activity of PSI. It would appear that the shared electron carriers of the respiratory and photosynthetic pathways allow electron flow to be shunted from cytochrome C oxidase to PSI, where they can be used to reduce O 2 (Fig. 1) (Scherer, 1990). The regulation of electron transfer from cyt c 553 to either PSI or cyt aa 3 is not well understood, but the half saturation constant (Km) for reduced cyt c 553 in each of these pathways is approximately equal, and greater electron transfer to PSI in the light is likely due to its >10 fold turnover rate (Scherer, 1990). Mehler activity is generally considered a process which can only consume photosynthetically derived O 2 and it cannot cause net consumption of O 2 because PSI activity relies on photosynthetically derived electrons (Kana, 1993). In cyanobacteria the arrangement of photosynthetic and respiratory electron transport chains allows electrons derived from NAD(P)H to feed into the photosynthetic electron transport chain and reduce PSI (Fig. 1). In Trichodesmium, where the light-dependence O 2 consumption is completely abolished upon addition of DCMU, it is clear that PSII activity is responsible for supplying reductant to PSI (Table 1). We are not able to determine whether abolishment of Mehler activity is due to direct blocking of PSII in N 2 fixing cells or whether DCMU blocks PSII activity in non-n2 fixing cells and interrupts the supply of translocated reductant. If reductant pools are small one would expect Mehler activity to be sensitive to DCMU addition even if NAD(P)H is supplying PSI with electrons. Hence, in principle, Mehler activity can result in a net consumption of O 2 in the nitrogen-fixing cells which have little or no PSII activity. 5

6 Mehler activity in Trichodesmium sp. depends both on time of day and on the nitrogen source. Both net O 2 production and variable fluorescence (Fv/Fm) in nitrogen fixing cultures of Trichodesmium sp. exhibited two maxima over the light period while nitrate-amended cultures showed a single peak at midday (Fig. 2 A, C). The depression of net O 2 production in N 2 -fixing cultures coincided with a peak in light-dependent O 2 consumption and a concomitant peak in N 2 fixation (Fig. 2A, B). In nitrate-amended cultures N 2 fixation was absent and there was low and constant light-dependent O 2 consumption (Fig. 2 D). The period of maximum N 2 fixation is coincident with a decline in the net production of O 2 and a rise in the consumption of oxygen via Mehler activity. Approximately 75% of the gross O 2 production in N 2 -fixing Trichodesmium is consumed by the Mehler reaction. This process is about three times the rate of that in a non-n 2 fixing strain of Synechococcus (~25% of gross O 2 production) exposed to high enough irradiances to cause light stress (Kana, 1992). While Mehler activity can potentially exceed gross O 2 production in Trichodesmium during mid-day when N 2 fixation is greatest, (Berman-Frank et al., 2001), we did not observe a negative O 2 production in this study (Fig 2 B) (Carpenter et al., 1990; Kana, 1993; Roenneberg and Carpenter, 1993). When cultures were grown on nitrate, Mehler activity was initially similar to that observed in N 2 fixing cultures, but quickly fell to low and constant rates through the rest of the photoperiod (Fig 2). Mehler activity was about 10% of gross photosynthetic O 2 production when nitrate was used as a nitrogen source, similar to rates observed in Synechococcus that is not exposed to light stress (Kana, 1992). Based on the results presented here and elsewhere, we propose a basic model that explains how Trichodesmium simultaneously fixes N 2 and CO 2 in the light. Trichodesmium relies on short term regulation of PSII and nitrogenase activities to separate these functions within a trichome (Berman-Frank et al., 2001). PSII activity is regulated on time scales of min and appears to involve the association/disassociation of the phycobilisome antenna from PSII (Kupper et al., 2004). Nitrogenase activity is also post-translationally regulated on similar time scales (Berman-Frank et al., 2001). While 6

7 PSII activity is repressed in N 2 fixing cells, PSI is engaged in Mehler reaction to lower intracellular O 2 concentrations. The Mehler reaction is maintained by the translocation of reductant. Purified heterocysts have increased O 2 consumption rates relative to whole filaments (Fay and Walsby, 1966; Smith et al., 1985). Hence, O 2 consumption rates be high in heterocysts. We examined whether the Mehler reaction might enhance oxygen consumption in purified heterocysts of Anabaena flos-aquae. Oxygen consumption rates were measured on purified heterocysts (Fay 1980) suspended in 400 mm sorbitol, 20 mm HEPES ph 7.5 with 4 mm glucose 1-6 phosphate supplied as a carbon source. In a series of six independent estimates of O 2 consumption, rates were enhanced 3 fold (dark rate = 0.4 ± 0.01 µmol O 2 mgchla -1 min -1 ; light rate 1.2 ± 0.03 µmol O 2 mgchla -1 min -1 ) in the presence of light. Although Brown and Webster (1953) reported light-enhanced O 2 consumption in whole cells of Anabaena sp., to our knowledge this is the first report of light-dependent O 2 consumption in purified heterocysts. The purity of heterocyst preparations was verified through four observations: (1) fluorescence of PSII was undetectable using FRR fluorometery (data not shown); (2) both 16 O 2 and 18 O 2 light induced consumption rates are indistinguishable, which expected if there is no production of 16 O 2 via water splitting; (3) lowtemperature fluorescence emission spectra of heterocysts lack peaks at 685 and 694 nm, which are indicative of core antenna complexes associated with PSII (Fig. 3); (4) a western blot of D1 clearly reveals the presence of the reaction center protein of PSII in whole cell extracts of both Trichodesmium and A. flos-aquaea but this protein was absent in heterocyst preparations (Fig. 4). The intracellular concentration O 2 is likely to be controlled by consumption rather than the permeability of the heterocyst wall. Calculation of the intracellular concentration of O 2 using a range of O 2 consumption rates and membrane permeabilities reveals that O 2 concentration is relatively independent of 7

8 membrane permeability over a large range, while increasing O 2 consumption rates gives a linear decrease in O 2 concentration (Subczynski et al., 1992) (Fig 6.). The dependence of nitrogen fixation on light and PSI activity has been well established. Indeed, Fay s (1970) action spectrum of nitrogen fixation in purified heterocysts of Anabaena cylindrica indicates primary involvement of PSI. Many observations of the dependence of N 2 fixation on PSI activity in heterocystous cyanobacteria have lead to the hypothesis that cyclic electron flow around PSI is necessary to provide the ATP required in for nitrogenase activity (LaRoche and Breitbarth, 2005). We suggest that Mehler activity of PSI provides ATP and the additional advantage of accelerating O 2 consumption in cyanobacteria. Mehler activity is thought to be a mechanism for energy dissipation under high light intensities or when carbon fixation is limited by supply of inorganic carbon (Asada, 2000). Since the product of O 2 reduction is superoxide and hydrogen peroxide through the activity of superoxide dismutase, it has been suggested that Mehler activity is a metabolic defect rather than an adaptive strategy (Patterson and Myers, 1973). The Mehler reaction, however, does not necessarily lead to peroxide production in cyanobacteria. In the cyanobacterium Synechocystis sp. strain PCC 6803, superoxide is reduced directly to water without a hydrogen peroxide intermediate (Helman et al., 2003). This single step reduction of superoxide to water is catalyzed by A-type flavoproteins, two of which (flv1, flv3) were identified as essential for this activity (Helman et al., 2003). Sequence analysis of cyanobacterial genomes reveals homologous genes to flv1 and flv3 are present in Trichodesmium erythraeum, with 62% and 67% sequence identity and Anabaena sp. PCC 7120 with 65% and 67% sequence identities, respectively. Conclusions It is well established that concomitant O 2 production and N 2 fixation in cyanobacteria is dependent on respiratory sinks of O 2. We show that the photosynthetically driven acceleration in O 2 consumption 8

9 (Mehler activity) is correlated to N 2 fixation in Trichodesmium and that purified heterocysts of Anabaena also have high Mehler activities. The reductant requirement for the observed O 2 consumption is likely to be derived through translocation and used to consume O 2 through the acceleration of electron flow through photooxidized PSI. We suggest that the evolution of a colonial habit was a necessary precursor to the simultaneous N 2 and O 2 evolution and that the short term regulation of these processes as occurs in Trichodesmium was proceeded by the evolution of cell differentiation as occurs in Anabaena. Acknowledgements John Waterbury graciously provided cultures of Trichodesmium sp. IMS 101. We thank Yael Helman and Aaron Kaplan for helpful discussions and Liti Haramaty for laboratory assistance. The D1 antibody was kindly provided by Autar K. Mattoo, USDA. XX anonymous reviewers provided comments on the manuscript. Funding was provided by the National Science Foundation Center for Bioinorganic Chemistry NSF/CEBIC (NSF# CHE ), and NSF OCE , Biocomplexity: The Evolution and Raditation of Eukaryotic Phytoplankton Taxa. 9

10 References Asada, K. (2000). The water-water cycle as alternative photon and electron sinks. Phil Trans R Soc Lon B 355: Berman-Frank, I., Lundgren, P., and PG, F. (2003). An anaerobic enzyme in an aerobic world: Nitrogen fixation and photosynthetic oxygen evolution in cyanobacteria. Res Microbiol 154: Berman-Frank, I., Lundgren, P., Chen, Y.-B., Kupper, H., Kolber, Z., Bergman, B., and Falkowski, P. (2001). Segregation of nitrogen fixation and oxygenic photosynthesis in the marine cyanobacterium Trichodesmium. Science 294: Brown, A.H. and Webster, G.C. (1953) The influence of light on the rate of respiration of the blue-green alga Anabaena. Am. J. Botany 40: Capone, D.G. (1993) Determination of nitrogenase activity in aquatic samples using the acetylene reduction procedure. In Handbook of Methods in Aquatic Microbial Ecology. Kemp, P. F., Sherr, B. F., Sherr, E. B. and Cole, J. J. (eds.) Boca Raton, USA: Lewis Press, pp Carpenter, E.J., Chang, J., Cottrell, M., Schubauer, J., Paerl, H.W., Bebout, B.M., and Capone, D.G. (1990). Reevaluation of nitrogenase oxygen-protective mechanisms in the planktonic marine cyanobacterium-trichodesmium. Mar Ecol Prog Ser 65: Chen, Y.B., Zehr, J.P., and Mellon, M. (1996). Growth and nitrogen fixation of the diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp IMS 101 in defined media: Evidence for a circadian rhythm. J Phycol 32: Chen, Y.B., Dominic, B., Zani, S., Mellon, M.T., and Zehr, J.P. (1999). Expression of photosynthesis genes in relation to nitrogen fixation in the diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp IMS 101. Plant Mol Biol 41: Fay, P Photostimulation of nitrogen fixation in Anabaena cylindrica. Biochim. Biophys. Acta 216: Fay, P Nitrogen fixation in heterocysts. In Recent advances in biological nitrogen fixation. Subba Rao N.S. (ed.). London, England: Edward Arnold, pp

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13 Wolk, C.P., Ernst, A., and Elhai, J. (1994) Heterocyst metabolism and development. In The molecular biology of cyanobacteria. Bryant E.D.E (ed.). Dordecht, Netherlands: Kluwer Academic Publishers, pp

14 Figure legends Figure 1. A flow diagram of photosynthetic and respiratory electron transport chains in cyanobacteria illustrating the shared electron carries of each pathway (Schmetterer, 1994). Figure 2. Time course of oxygen production and consumption during the photoperiod in Trichodesmium sp. IMS 101 grown with N 2 (A) and nitrate (C) as nitrogen sources. Time course of acetylene reduction and variable fluorescence in cultures grown with N 2 (B) and nitrate (D) as nitrogen sources. Error bars are ±1 standard deviation. Axenic cultures of Trichosdesmium sp. IMS 101 were grown in YBC II medium (Chen et al., 1996) under 12L:12D cycle with light provided by fluorescent bulbs (150 µmol quanta m -2 s - 1 ) T = 26 o C. To suppress N 2 fixation, nitrate was added at a concentration of 100 µm. Nitrogen fixation rates were estimated using the reduction of acetylene to ethylene (Capone 1993). Production and consumption of oxygen was followed using a membrane inlet system attached to a described in Tchernov et al. (2004). Samples of Trichodesmium (5 10 µg Chl a) were harvested by filtration on 5 µm pore-size PC membranes. Trichomes were resuspeded in 0.9 ml of culture medium equilibrated to atmospheric O 2 and placed in the membrane inlet chamber in the dark. The 18 O 2 spike (100 µl or about 15% of total O 2 ) was added prior to turning on the light source and incubations were run for 2 cycles of 5-6 min in light and 5-6 min in dark. Light was provided by a mercury arc lamp at a fluence rate of 200 µmol quanta m -2 s -1 controlled by neutral density filters. The photochemical quantum yield of PSII (F v /F m ) was determined using a custom built fast repetition rate fluorometer (Kolber and Falkowski, 1993). The initial dark adapted fluorescence (F 0 ) is measured when Q A is fully oxidized, maximal fluorescence (F m ) occurs when Q A is fully reduced. Photochemical quantum yield was calculated as F v /F m = (F m -F 0 )/F m.. 14

15 Figure 3. Low temperature (77 o K) fluorescence emission spectra of purified heterocysts. Excitation = 480 nm. PSII emission is nm. Heterocysts of A. flos-aquaea were purified following the methods of Fay (1980). Cells were harvested by centrifugation (5000 x g for 10 min) transferred to a 25ml septum vial, resuspended in 400 mm sorbitol with 20 mm HEPES buffer (ph 7.5) and 2 mg ml -1 lysozyme and sparged with N 2 gas for 5 min to remove O 2. The vegetative cells were digested at 37 o C for two hours while heterocysts remained intact. Following the incubation cells were concentrated to 2 ml by centrifugation, sonicated under flowing N 2 gas for 5 rounds of 15 s pulse, 15s rest cycles with a sonicator (Microson, Misonix Inc.) fitted with a microprobe tip. The resulting suspension was layered onto a N 2 sparged, Percoll step gradient (5ml 100%, 5ml 50% and 5ml 20% Percoll with 400mM sorbitol and 20mM HEPES ph = 7.5) and centrifuged at 5000 x g for 20min. The purified heterocysts appeared as a narrow brown upper layer easily identifiable with a long-wavelength UV light source as a weakly fluorescent band. Figure 4. A) Western blot of the PSII protein D1 (monomer 33 kda, dimer 66 kda). Lane 1) Purified heterocysts 2) N 2 grown and 3) NO 3 grown Anabaena flos-aquae. Samples were loaded on an equal protein basis, separated on 12% SDS PAGE denaturing gels, and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were probed with polyclonal antibodies for Photosystem II subunit D1 and detected using a horseradish peroxidase chemiluminescence system (SuperSignal, Pierce). Figure 5. Relative dependence of intracellular O 2 concentration on O 2 consumption rate (V) and membrane permeability (P m, note log scale). Calculated according to (Subczynski et al., 1992). Assuming a O 2 concentration of 218 µm and a seawater diffusivity of 2.32 x 10-5 cm 2 s -1 ( T = 26 o C, S = 30), starting V = 6 x mol O 2 cell -1 s -1 ; P m = 21 cm s

16 Table 1. Daily integrated O 2 production and consumption (µmol O 2 µg chla -1 ) in Trichodesmium. Values in parentheses indicate 1 standard error. Treatment Net Production (from 16 O 2 production rate) Light dependent O 2 consumption rate ( 18 O 2 light dark) Light 945 (±53) 2500 (±63) Dark (±47) --- DCMU addition (20 µm) -5 (±7) -25 (±26) Glycoaldehyde addition (2 mm) 254 (±21) 2991 (±40) 16

17 Figure 1 Milligan et al. 17

18 Figure 2 Milligan et al. 18

19 Figure 3. Milligan et al. 19

20 Figure 4. Milligan et al. 20

21 Figure 5. Milligan et al. 21