Mercury ions inhibit photosynthetic electron transport at multiple sites in the cyanobacterium Synechococcus 6301

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1 J. Biosci., Vol. 18, Number 3, September 1993, pp Printed in India. Mercury ions inhibit photosynthetic electron transport at multiple sites in the cyanobacterium Synechococcus 6301 S D S MURTHY and PRASANNA MOHANTY* School of Life Sciences, Jawaharlal Nehru University, New Delhi , India MS received 15 April 1992; revised 5 March 1993 Abstract. Inhibition of electron transport activities in the spheroplasts of Synechococcus 6301 by HgCl 2 is dependent on the concentration of mercury ions. The inhibition of whole chain electron transport activity occurs at low concentration of Hg 2+ (6 μμ). This inhibition occurs mostly due to interaction of Hg 2+ on plastocyanin. At an elevated concentration (24 μμ), mercury induces inhibition chiefly in photosystem II catalyzed electron transport. At this concentration it also alters both the absorption and emission characteristics of the phycocyanin. The photosystem I catalyzed electron transport was inhibited by 50% only at high concentrations (36 μμ) of HgCl 2. However, electron transport catalyzed by photosystems I and II from reduced duroquinone to methylviologen which involves intersystem electron transport is extremely sensitive to mercury (low concentration 6 9 μμ) like that of whole chain assay indicating that the observed inhibition in whole chain electron transport at low concentrations is mostly contributed by the damage involving other intersystem electron transport carrier(s) like plastocyanin. Thus mercury ions depending on the concentration affects the electron transport at multiple sites in the spheroplasts of Synechococcus. 1. Introduction Keywords. Electron transport; mercury; phycocyanin; sepheroplasts; Synechococcus Heavy metals are known to interfere with a variety of photosynthetic functions (Clijsters and Van Assche 1985; Mohanty and Mohanty 1988; Murthy and Mohanty 1991a, b). Among the many heavy metals that affect photosynthetic electron flow in plants mercury has been shown to interrupt the flow of electrons at multiple sites, such as oxidizing side of photosystem (PS) II (Honeycutt and Krogmann 1972; Samson and Popovic 1990), plastocyanin (Pcy) (Katoh and Takamiya 1964), the P700, the reaction centre of PSI (Golbeck et al 1977; Kojima et al 1987). In addition, it has been shown that mercury interferes with enzymes like NADPoxidoreductase (Honeycutt and Krogmann 1972). Recently, we have shown that mercury at low enough (3 μμ) concentration inhibits the energy transfer from phycocyanin (PC) Chl a in the intact cells of the cyanobacterium Spirulina platensis (Murthy et al 1989; Murthy and Mohanty 1991a,b). Unlike in Spirulina, the intact cells of Synechococcus 6301 when exposed to Hg 2+ ions for short duration (5 min) they remain resistant. Therefore, we have investigated the effect of this heavy metal ion on the photochemical activities of Synechococcus spheroplasts. Our results indicate that mercury also affects the electron transport at multiple *Corresponding author. Abbreviations used: PS, Photosystem; Pcy, plastocyanin; PC, phycocyanin; MV, methylviologen; DCPIP, 2,6-dichlorophenol indophenol; DQ, duroquinone; DCMU, 3(3,4-dichlorophenyl)-l, 1-dimethyl urea. 355

2 356 S D S Murthy and Prasanna Mohanty sites, in this cyanobacterium. The site of inhibition is dependent on the concentration of this ion used. 2. Materials and methods Synechococcus 6301 was grown axenically in BG-11 medium (Stanier et al 1971) at C under continuous illumination (~15Wm -2 ). Throughout the growth period the culture was agitated by the passage of filtered air. After five days of growth (late log phase) cells were harvested by centrifuging at 9,000 g for 5min, washed twice with 20 mm Tricine-KOH buffer (ph 7 5) that contained 400 mm sucrose, 10 mm KCl and 10 mm EDTA (disodium salt) and centrifuged as above. Spheroplasts were prepared by incubating the intact cells at 37 C in the presence of lysozyme (1 mg/ml) for 3 h according to Newman and Sherman (1978). Photochemical activities were assayed polarographically with a Clark-type oxygen electrode according to Murthy et al (1989). The three ml of reaction mixture used for the assay of whole chain electron transport contained suspension buffer, 0 5 mm methylviologen (MV) and 1 mm sodium azide. The three ml of reaction mixture used for assay of the PSII catalyzed electron transport assay contained suspension buffer and 0 5 mm 2, 6 dichlorophenol indophenol (DCPIP). The three ml of reaction mixture for reduced duroquinone (DQH 2 ) supported PSI catalyzed electron transport consisted of suspension buffer reduced DQ, 5 μμ 3(3, 4- dichlorophenyl)-l, 1-dimethyl urea (DCMU), 0 5 mm MV and 1 mm sodium szide. Reduced DQ was prepared according to Izawa and Pan (1978). The reaction mixture (3 ml) used for assaying the PSI catalyzed electron transport consisted suspension buffer, 0 1 mm DCPIP, 5 mm ascorbate, 1 mm azide, 5 μμ DCMU and 0 5 mm MV. In all the assays spheroplasts equivalent to 15 µg Chl was used. Spheroplasts were incubated with or without Hg 2+ for 5 min in the dark before measurements were carried out at 25 C under saturating illumination by white light (intensity ~ 480 Wm -2 ). The absorption spectra of a suspension of spheroplasts with or without Hg 2+ were recorded using Shimadzu UV-3000 double beam spectrophotometer operated in the split beam mode (Murthy et al 1989). Fluorescence emitted by spheroplasts was measured at room temperature with excitation at 545 nm in a Perkin Elmer (LS-5) spectrofluorometer (Murthy et al 1989; Murthy and Mohanty 1991 a). Fluorescence spectra were not corrected for the spectral sensitivity of the photomultiplier. Cells equivalent to 5 μg of Chl were used for all fluorometric assays. The concentration of Chl was determined by the method of MacKinney (1941). 3. Results The addition of a low concentration of Hg 2+ (6μΜ) to the spheroplasts of Synechococcus caused approximately 50% inhibition in whole chain electron transport (H 2 O MV) activity whereas in the presence of 18 μμ Hg 2+, the activity was inhibited by 70% (see table 1). At this latter concentration (18 μμ) ~36% inhibition of DCPIP supported evolution of O 2 by PSII was observed (table 1). Further rise in mercury concentration (24 μμ) caused approximately 50% inhibition in the PSII catalyzed electron transport (see table 1). To examine whether

3 Mercury induces alterations in photoelectron transport 357 Table 1. Effect of various concentrations of HgCl 2 on whole chain (H 2 O MV) PS II (H 2 O DCPIP) and PS I without including cyto b 6 f (DCPIPH 2 MV) with including cyt b 6 f. (DQH 2 MV) catalyzed electron transport activities of spheroplasts. Spheroplasts were incubated in the presence and absence of mercury for 5 min in dark. Other details were given in 2. the observed inhibition of Hill activity by Hg 2+ is linked to the Hg 2+ induced changes in the spectral properties of phycobilisomes, we measured the spectral characteristics of spheroplasts in the presence of mercury (24 μμ) where it inhibits PSII catalyzed electron transport activity by 50% (see figure 1). In the presence of low concentrations of Hg 2+ no appreciable change in the absorption by PC in spheroplasts was seen (data not shown). The increase in the concentration to 24 μμ partially reduced the absorption capacity by PC (see figure 1). At this same concentration, both partial changes in the absorption by PC and significant alterations in the PC-fluorescence intensity were detected (figure 2). In control spheroplasts upon excitation at 545 nm, an emission peak at 649 nm, emanating from PC was clearly observed in the spectrum (see figure 2). Incubation of spheroplasts with higher concentrations of mercury (24 μμ) (where it affects slightly the absorption of PC) caused 25% decrease in the fluorescence intensity and induced a blue shift of 2 nm towards the blue region of the spectrum. Unlike whole chain electron transport and PSII catalyzed electron transport, PSI catalyzed electron transport (DCPIPH 2 MV) was less sensitive to low concentrations of mercury (see table 1). At 6 and 18 μμ HgCl 2, only 8% and 20% inhibition in the PSI catalyzed electron transport activity was observed. After further increase in the concentration to 36 μμ, the PSI activity gets inhibited by 55%. To prove the exact site of inhibition, the effect of mercury was studied on the intersystem electron transport by measuring DQH 2 catalyzed PSI electron transport activity. DQH 2 is known to donate electrons at the plastoquinone level in the PSI catalyzed electron transport (Izawa and Pan 1978; Sane et al 1979). At low

4 358 S D S Murthy and Prasanna Mohanty Figures 1 and 2. (1) Effect of mercury ions on the absorption spectrum of spheroplasts. Spheroplasts were incubated with HgCl 2 (24 μμ) or without it for 5 min in dark, before measurements were made. Other details were given in 2. (2) Mercury (24 μm) induced alterations in phycocyanin fluorescence emission spectrum of spheroplasts at room temperature. Spheroplasts were incubated with or without mercury for 5 min in dark. Spheroplasts were excited at 545 nm to excite PBsome specifically. Slit width for both excitation and emission was 5 nm. Cells equivalent to 5 µg of Chl a were used for spectral measurements. concentration (9 μμ) mercury caused 34% inhibition in the DQH 2 supported PSI electron transport indicating that the most sensitive site lies among the intersystem electron transport carrier(s) (see table 1). 4. Discussion The results presented in this study clearly demonstrate that HgCl 2, depending on the concentration of heavy metal used, affects different segments of photosynthetic electron transport chain of Synechococcus 6301 spheroplasts. At 6μΜ of mercury the whole chain electron transport activity gets inhibited by 50% suggesting the Figure 3. Schematic diagram showing various sites of inhibition in the photosynthetic electron transport chain by mercury. The effective concentration for 50% inhibition is indicated.

5 Mercury induces alterations in photoelectron transport 359 presence of an inhibitor site between PSII and PSI (see table 1). The inhibition of whole chain electron transport appears to be associated with the inactivation of plastocyanin (Kotoh and Takamiya 1964; Honeycutt and Krogmann 1972) in the intersystem electron transport chain as has been reported earlier for chloroplasts. Further increase in the concentration to 24 μμ induced 50% inhibition in PSII catalyzed electron transport. To prove the exact site of mercury action in PSII, the effect of mercury on light harvesting pigment complex (PBsomes) has been studied (see figures 1 and 2). This elevated concentrations (24 μμ) mercury induced alterations in absorption and emission properties of PC is due to the structural changes that affect the pigment-protein interactions. Hence the observed inhibition in PSII catalyzed electron transport could be partially due to the alteration in the spectral properties of PC as has been reported earlier in the intact cells of Spirulina (Murthy et al 1989). At this concentration mercury induced inhibition in PSII activity could also be due to the presence of an inhibitory site near PSII catalyzed electron transport as has been suggested by Honeycutt and Krogmann (1972) in spinach chloroplasts and Samson and Popovic (1990) in green alga, Dunaliella tertiolecta. The observed inhibition in PSI catalyzed electron transport activity by mercury at 36 μμ could be either due to the alterations at the oxidizing side of PSI (at the level of Pcy) as has been reported earlier for spinach thylakoids by Honeycutt and Krogmann (1972) or due to the inhibitory effect of mercury on P700 as has been shown by Golbeck et al (1977). Thus the difference in the I 50 values of mercury (whole chain, 6μΜ; PSII, 24 μμ; PSI, 36 μμ) indicate that mercury at low concentration (6μΜ) may be able to induce maximal inhibition in whole chain electron transport by mostly affecting the intersystem electron transport carrier(s) with exerting marginal effect on PSII and PSI catalyzed electron transport activities. Measurements of intersystem catalyzed PSI electron transport activity (DQH 2 MV) indicated that the observed inhibition in whole chain electron transport activity at low concentrations is due to the presence of most sensitive site which lies among the intersystem electron transport carrier (s) (see table 1). Thus as shown in figure 3 mercury, depending on the concentration exerts multiple effects on photosynthetic electron transport in the spheroplasts of Synechococcus. At low concentrations (6μΜ) mercury inhibits the whole chain electron transport activity by affecting at the level of intersystem electron transport carrier level. The further increase in mercury concentration brings additional inhibition in electron transport activity due to its effect on partial electron transport activity catalyzed by either PSII or PSI. Acknowledgements Supported in part by ICAR-USDA FG-IN-679 and grant No. J 12017/70/82-ENI from Department of Environment, New Delhi. SDSM acknowledges the support of Senior Research Fellowship (project of PM) by the Council of Scientific and Industrial Research, New Delhi. References Clijsters Η and Van Assche F 1985 Inhibition of photosynthesis by heavy metals; Photosynth. Res

6 360 S D S Murthy and Prasanna Mohanty Golbeck J Η, Stephen L and San-Pietro A 1977 Isolation and characterization of sub-chloroplast particle enriched in iron-sulphur protein and Ρ 700; Arch. Biochem. Biophys Honeycutt R C and Krogmann D W 1972 Inhibition of chloroplast reactions with phenylmercuric acetate; Plant Physiol Izawa S and Pan R Ν 1978 Photosystem I electron transport and phosphorylation supported by electron donation to the plastoquinone region; Biochem. Biophys. Res. Commun Katoh S and Takämiya A 1964 Nature of Copper-protein binding in spinach plastocyanin; J. Biochem Kojima Υ, Hiyama Τ and Sakurai Η 1987 Effects of mercurials on iron-sulfur centres of photosystem I of Anacystis nidulans; in Progress in photosynthesis research (ed.) J Biggins (Amsterdam: Nijhoff/Junk Publishers) Vol. 2, pp MacKinney G 1941 Absorption of light by chlorophyll solutions; J. Biol. Chem Mohanty Ν and Mohanty Ρ 1988 Cation effects on primary processes of photosynthesis; in Advances in frontier areas of plant biochemistry (eds) R Singh and S Κ Sawhney (New Delhi: Printice Hall of India) pp 1 18 Murthy S D S and Mohanty Ρ 1991a Mercury induces alteration of energy transfer in phycobilisomes by selectively affecting the pigment protein, phycocyanin in the cyanobacterium Spirulina platensis; Plant Cell Physiol Murthy S D S and Mohanty Ρ 1991 b Inhibitory effects of heavy metal ions on bioenergetic processes of photosynthesis; in Trends in bioenergetics and biotechnological processes (eds) G S Singhal and Τ Rama Sarma (New Delhi: Today and Tomorrow Publishers) pp Murthy S D S, Sabat S C and Mohanty Ρ 1989 Mercury induced inhibition of photosystem II activity and changes in the emission of fluorescence from phycobilisome in intact cells of the cyanobacterium Spirulina platensis; Plant Cell Physiol Newman Ρ J and Sherman L A 1978 Isolation and characterization of photosystem I and II membrane particles from the blue green alga, Synechococcus cedrorum; Biochim. Biophys. Acta Samson G and Popovic R 1990 Inhibitory effects of mercury on photosystem II photochemistry in Dunaliella tertiolecta under in vivo conditions; J. Photochem. Photobiol. B Sane Ρ V, Johnningmeyer U and Trebst A 1979 The inhibition of photosynthetic electron flow by DCCP; an indication for proton channels; FEBS Lett Stanier R Y, Kunisawa R, Mandai Μ and Cohen-Bazire G 1971 Purification and properties of unicellular blue green algae (order chrococcales); Bacteriol. Rev