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Department of Plant Science, Macdonald College of McGill University, P. 0 . Box 4000 ... GEORGE M. CURRY ..... CODD, G. A., and W. D. P. STEWART. 1976.
The effect of light-dependent oxygen consumption on nitrogenase activity in Anabaena cylindrica DONALD L. SMITH' Department of Plant Science, Macdonald College of McGill University, P. 0 . Box 4000, Ste-Anne-de-Bellevue, P. Q., Canada H9X I CO

DAVIDG. PATRIQUIN Biology Department, Dalhousie University, Hal*,

N.S., Canada B3H 4J1

MARGARETA DIJAK Can. J. Bot. Downloaded from www.nrcresearchpress.com by Renmin University of China on 06/04/13 For personal use only.

Agriculture Canada, Plant Research Centre, Building 21, Central Experimental Farm, Ottawa, Ont., Canada KIA OC6 AND

GEORGEM. CURRY Biology Department, Acadia University, Wolfville, N.S., Canada BOP 1 x 0 Received September 30, 1985 SMITH,D. L., D. G. PATRIQUIN, and G. M. CURRY.1986. The effect of light-dependent oxygen consumption on nitrogenase activity in Anabaena cylindrica. Can. J . Bot. 64: 1843 - 1848. Light-dependent oxygen consumption (LDOC) was observed in isolated heterocysts and in intact and sonicated C0,-fixing Anabaena cylindrica cells. The rate of LDOC in heterocysts was about three times that of C0,-fixing cells. Photosynthetic oxygen production by A. cylindrica became light saturated at 0.3 to 0.5 mW cm-,. LDOC and nitrogenase activity (acetylene reduction) increased with light intensity up to 2.5 mW cm-, and incubation under air resulted in much larger relative acetylene reduction increases than incubation under N,. Carbonyl cyanide-m-chlorophenyl-hydrazone, 3-(3,4-dichloropheny1)-1,ldimethylurea, and cyanide did not affect the rate of LDOC in isolated heterocysts or cell-free preparations of C0,-fixing cells. However, all three substances induced LDOC in C0,-fixing cells. Heat treatment (100°C for 1 min) caused a doubling of LDOC. Depletion of reduced carbon reserves by dark incubation caused a similar decrease in LDOC and dark respiration. The higher rates of LDOC observed in heat-treated materials were removed by catalase, but not by superoxide dismutase. Catalase injection released half of the 0, consumed through LDOC by heated preparations. LDOC increased with temperature up to 85"C, and increased threefold with pH between pH 10 and 11.5. The possibility that LDOC may act to protect the nitrogenase of the heterocyst from oxygen inactivation is discussed. et G. M. CURRY.1986. The effect of light-dependent oxygen consumption on nitrogenase SMITH,D. L., D. G. PATRIQUIN activity in Anabaena cylindrica. Can. J . Bot. 64: 1843-1848. Une consommation d'oxygkne dependante de la lumikre (CODL) est observCe dans les hCtCrocystes isolCs ainsi que dans les cellules fixatrices de CO, intactes ou traitCes aux ultrasons, d'Anabaena cylindrica. Le taux de CODL dans les hCtCrocystes est environ trois fois plus ClevC que dans les cellules fixatrices de CO,. La production photosynthCtique d'oxygkne par A. cylindrica devient saturCe en lumikre i 0,3-0,5 mW cm-,. CODL et I'activitC de la nitrogCnase (rkduction de 1'acCtylkne) augmentent avec 1'intensitC lumineuse jusqu'i 2,5 mW cm-,. L'incubation sous l'air rCsulte en une augmentation de la rCduction d'acCtylkne beaucoup plus grande que l'incubation sous l'azote. Carbonyl cyanide-m-chlorophCny1-hydrazone, 3-(3,4-dichlorophCny1)-1,l-dimkthylureaet le cyanure n'affecte pas le taux de CODL dans les hCtCrocystes isolCs ou dans les prkparations de cellules (detmites par sonication) fixatrices de CO,. Cependant, les trois substances induisent CODL dans les cellules fixatrices de CO,. Un traitement i la chaleur (100°C durant 1 min) fait doubler CODL. L'Cpuisement des rkserves de carbone rCduit, suite i une incubation i l'obscuritC, cause une diminution similaire de CODL et de la respiration sombre. Les taux plus ClevCs de CODL observCs dans les Cchantillons traitCs par la chaleur sont effaces par la catalase, mais non par la superoxyde dismutase. L'injection de catalase entraine le dCgagement de la moitiC de l'oxygkne consommC via CODL par les prkparations chauffkes. CODL augmente avec la temperature jusqu'i 8SoC, et aux pH entre 10 et 1 1 3 , son augmentation est triple. La possibilitk que CODL puisse agir dans la protection de la nitrogCnase de llhCtCrocyste contre l'inactivation par l'oxygkne est discutCe. [Traduit par la revue]

Introduction Exposure of nitrogenase to 0, causes inactivation of the enzyme complex (9). Aerobic diazotrophes have evolved a number of mechanisms for the protection of nitrogenase from 02:mucilagenous sheaths (17), uncoupled respiration (4), conformational protection of the nitrogenase proteins (13), leghaemoglobin (2), and, among the cyanobacteria, temporal (29) and spatial (26) separation of photosynthesis and N, fixation. The best characterized of the cyanobacterial O2protection 'Author to whom reprint requests should be addressed Printed In Canada I lmprimi au Canada

mechanisms is the heterocyst. Heterocysts do not fix CO, (8) or carry on photolysis of water, but they house photosystem I of photosynthesis and the nitrogenase enzyme complex (26). In addition to not evolving 0 2 , these specialized cells may have mechanisms for scrubbing O2 that diffuses in from adjacent water-splitting cells. It has been demonstrated that heterocysts consume O2 through the combined action of the respiratory electron transport chain and an uptake hydrogenase (5, 10) utilizing H2 produced by nitrogenase reduction of protons. This activity is reported to stabilize N2 fixation by A. cylindrica, possibly through 0, protection of nitrogenase (5). Bradley and Carr (6, 7) reported that the oxygen consumption

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CAN. J. BOT. VOL. 64, 1986

of isolated hetereocysts was greatly enhanced by exposure to bright white light. They found that this light-driven 0, consumption (light-dependent oxygen consumption, LDOC) was not diminished by treatment with azide or by boiling. The light-driven reduction of O,, by pseudocyclic electron flow through photosystems I and 11, has been reported for several algae and for isolated chloroplasts of spinach (19, 22). This flow may be important in providing ATP to dark CO, fixation (12) or may play a role in poising the photosystems, during the first few seconds after illumination, for more conventional electron flow once dark CO, fixation reactions have begun (23). As heterocysts do not retain an operative photosystem I1 (26), photosystem I of these cells cannot receive electrons from water. However, it has been reported that NADPH, NADH, and Hz can donate electrons to photosystem I of isolated heterocysts (16), replacing photosystem 11. The LDOC of isolated heterocysts may represent the operation of the photosystem I portion of pseudocyclic electron transport, functioning with an alternate electron source. This research has attempted to assess the possible role of heterocyst LDOC in Oz protection of nitrogenase.

1:6

' 2.4 ' 3:2 L i g h t intensity (mW cm-')

FIG. 1. The effect of light intensity on acetylene reduction (nitrogenase activity) by intact, heterocystous filaments of A. cylindrica. Each line represents a different trial with a different batch culture. The vertical bars are st, with n = 6. Material was cultured on Nfree nutrient medium (0) or medium containing one-tenth of fullstrength NaNO, (0.17 M) (A). chl, chlorophyll.

+

Materials and methods Batch cultures of Anabaena cylindrica Lemm., strain B629 (Stan Culture Collection, University of Texas) were grown axenically in petri dishes on agar solidified medium (1). When heterocystous material was required, NaNO, was omitted. Two to 3 weeks of incubation at 25°C under 0.2 mW cm-, light produced dense cyanobacterial lawns. When material depleted in carbon reserves was required, cultures were incubated in darkness for 48 h. Heterocyst isolation was achieved by sonication, following the methods of Fay and Lang (11). Chlorophyll concentrations were determined according to Amon (3). For the acetylene reduction assay, cyanobacteria were suspended in 0.1 Mcarbonate buffer, pH 8.9. Ten millilitres of this suspension was added to each of the desired number of 50-mL flasks. Each flask was closed with a serum stopper, then 6 mL of the gas phase was removed and replaced with C,H,. Control flasks were vigorously flushed with N, immediately before being stoppered. During experiments, the flasks were shaken and maintained at 25°C in a shaker bath. At regular intervals, 1-mL gas samples were removed from each flask. These samples were injected into a Carle Basic 9500 gas chromatograph fitted with a 0.5-m Poropak T column. Changes in PO, were measured with a Clark type Beckman polarographic oxygen electrode. The experimental material was held in a water-jacketed 11-mL flask (+0.2"C). A port in the flask - water jacket assembly allowed for syringe injection of various substances while PO, levels were being monitored. The standard conditions for Po, measurement were 25"C, pH 7.9 (0.1 M phosphate buffer), and 2.5 mW cm-2 light. A blank, of buffer only, was subjected to the experimental protocol after each set of experimental measurements. This allowed determination of 0, consumption by the electrode itself. Light was provided by a bank of Sylvania Cool-White fluorescent tubes. The intensity of the light was varied by altering the number of illuminated tubes within the bank. Light intensity, at the site of the flask - water jacket assembly was measured with an 820A Gamma Scientific photometer. As tests for H,O, or 0, production, catalase (200 units) or superoxide dismutase (200 units), respectively, were injected into heated preparations of A. cylindrica while LDOC was being monitored. All enzymes, 3-(3,4-dichlorophenyl)-l , 1-dimethylurea (DCMU), carbonyl cyanide-m-chlorophenyl-hydrazone (CCCP), 2,6-dichlorophenol indophenol (DPIP), and rotenone were purchased from the Sigma Chemical Co. of St. Louis, Missouri. Superoxide dismutase was Type I; protease, Type VI; lysozyme, grade I from egg white; catalase, from bovine liver.

45 T i m e (min)

90

FIG. 2. The typical pattern of light-dependent oxygen consumption in sonicated, C02-fixing cells of A. cylindrica, as measured by Po, electrode. The bar along the X-axis indicates the light regime: white segments represent illumination periods, black segments represent darkness. The row of numbers above the X-axis gives the rates of 0, consumption, in microlitres of 0, consumed per milligram chlorophyll per hour, of segments of the curve above. A control flask, containing buffer only, showed 0, consumption under the same light-dark regime necessitating subtraction of 1.82 pL. mg chlorophyll-'. h-' from the rates given. The chlorophyll concentration in the sample illustrated here was 14.9 mg L-'. The chlorophyll concentration for sonicates was always beteen 5 and 15 mg L-', and for preparations of isolated heterocysts, it was always 0.5-2.0 mg L-'.

Results Increasing light intensity on A. cylindrica cultures from photosynthetically saturating levels (0.5 mW cm-,) to LDOC saturating levels (3 mW cm-,) resulted in increased nitrogenase activity (C2H2reduction) under air (Fig. 1) or N2 (data not shown). An increase from 0.5 to 2.5 mW cm-2 caused a 2.5- to 3.5-fold increase in the rate of C2H2 reduction by cultures under air and a 1.2- to 1.%fold increase under N,. The pattern of increase was the same for cultures fully dependent on N2

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SMITH ET AL.

TABLE1. Effects of light intensity and DCMU (2 X M) on 0, exchange by intact C02-fixing cells of A. cylindrica

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Trial No.

1:0

2:O

3:0

Light intensity (mW ~ m - ~ )

Light intensity (mW ~ r n - ~ )

Rate of 0, exchange (pL 02.mg chlorophyll-'. h-')

+ DCMU

- DCMU

TABLE2. The effects of pH and temperature on the rate of LDOC by isolated heterocysts and by sonicates of C02-fixing cells of A. cylindrica

FIG. 3. The effect of light intensity on LDOC by sonicates of A. cylindrica C02-fixing cells. Vertical bars represent f sf; n = 5 for sonicates and n = 9 for heterocysts.

Rate (pL O2 consumed. mg chlorophyll-'. h-') Heterocysts* Sonicatest pH

2.5 4.0 6.0 7.0 8.0 10.0 11.O

457 433 A

A

1780

Temperature ("C)

Time (minl

I

10 15 25 35 45 80

-

4 14 767 1058 -

B *Each value is the result of a single trial.

tValues shown are averages of three trials for pH and two for temperature, and are given si (standard error).

*

Time ( m i d

FIG. 4. The induction of LDOC in intact filaments of A. cylindrica M CCCP, and (b) M KCN. Chlorophyll concenby (a) 2 x tration was 11.2 mg L-' for (a) and 11.9 for (b). Arrows indicate the point at which the KCN or CCCP was added. Other symbols as in caption for Fig. 2.

fixation and those receiving some combined N (Fig. 1). LDOC was characterized with sonicates of C0,-fixing cells (Fig. 2), then verified in heterocysts. This procedure was adopted as large quantitites of heterocysts were difficult to obtain. The effects of light intensity (Table 1, Figs. 1 and 3), temperature, and pH (Table 2) were similar for LDOC by sonicates of C0,-fixing cells and LDOC by isolated heterocysts, indicating that the same activity was measured in both types of material. LDOC remained fairly constant over the pH range from 2.5 to 10, while it increased sharply between 10 and 11.5 (Table 2). LDOC showed a general increase with temperature from 15 to 80°C. It increased with increasing light intensity up to 2.5 mW cm-2 (Fig. 3). LDOC by sonicates and heterocysts was unaffected by KCN, NaN,, CCCP, rotenone, or DCMU (data not shown). Treatment with CCCP (Fig. 4A), KCN (Fig. 4B), or DCMU (Table 1) induced LDOC in C0,-fixing cells. Rates of LDOC

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TABLE3. The effect of 48 h of dark incubation on dark respiration and LDOC by preparations of the darkincubated cultures

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Initial rate*

Final rate* Decrease?

Dark respiration by intact filaments Trial 1 2 3 4 5 LDOC by heterocysts Trial 1 2 3 4 5 LDOC by sonicates of C02-fixing cells Trial 3 4 5 *Rates are in pL O x .mg chlorophyll-'. h-'. tPercent decrease = rate after dark incubation x loo rate at beginning of dark incubation

by C02-fixing cells treated with these compounds were comparable to those observed in sonicates. We were always able to observe LDOC without the addition of exogenous reductant. After 48 h of dark incubation, both dark respiration and LDOC were decreased by comparable amounts (Table 3). A broad-band action spectrum for LDOC showed much lower activity in the green spectral region than in the red and blue regions. M) inhibited LDOC by almost 30% (Table DPIP (5 x 4). This was not due to light absorption by the blue dye, as increasing the light intensity above 2.5 mW cm-2 did not increase the LDOC rate in DPIP-treated material. Injection of catalase into A. cylindrica preparations carrying on LDOC did not produce a burst of O2 unless the material had been heated (100°C for 1 min) before the testing (Table 5). The 0, burst observed in heat-treated materials was equivalent to about half the 0, consumed by LDOC operation up to the time of catalase injection (Table 4). The heat treatment also caused a doubling of the LDOC rates; addition of catalase halved the LDOC rate of heat-treated A. cylindrica preparations (Table 5). Superoxide dismutase had no effect on LDOC in heated or unheated A. cylindrica material.

Discussion Both nitrogenase activity and LDOC exhibited bright-light enhancement (Figs. 1 and 3). These enhancements reached light saturation in the same range, a range well above that of photosynthetic light saturation, measured by us at 0.3 to 0.4 mW cm-,. The bright-light stimulation of nitrogenase occurred in the presence of atmospheric 0, plus O2 derived from photosynthesis in adjacent C0,-fixing cells; it also occurred under nitrogen (in the presence of 0, only from adjacent C0,-fixing cells) but was much greater under air. These data suggest that

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TABLE 4. The effect of DPIP (5 X lo-' M) on LDOC (pL 0 2 .mg chlorophyll-'. h-') in sonicates of C02-fixing cells of A. cylindrica Light intensity 2.5 mW cm-2 Trial DPIP % inhibition No. 3.5 mW cm-2 - DPIP

+

TABLE5. The effect of heating and of catalase on LDOC by various A. cylindrica materials, as well as the release of 0, by catalase in

previously heated materials

Material*

Preheat rate?

Postheat ratel.

Sonicate Sonicate Heterocysts Sonicate Sonicate

132 110 396 102 117

320 273 883 Not heated Not heated

Amount of Catalase- Post0, consumed released catalase (&)$ 0 2 (pL)§ rate? 11.3 8.1 6.3 5.8 7.4

5.4 4.1 2.9 0.0 0.0

183 152 485 175 184

*The heterocysts were isolated and contained no CO1-fixing cell material; the sonicates were cell-free preparations of C0,-fixing cells. ?The rates are in pL 0, consumed. rnS chlorophyll-'. h-'. $The amounts of 0, consumed are in pL. 11 mL-' of experimental material in 20 min. $The amounts of 0, released are in pL. 11 mL-' of experimental material.

LDOC operates in heterocysts in situ and protects nitrogenase from O2 inactivation. Some response to LDOC under N, might be expected as heterocysts were still exposed to 0, diffusing from adjacent C0,-fixing cells through microplasmodesmatal pores between the two cell types. Under a N2 atmosphere the gradient of 0, concentration between the inside of the C02-fixing cells and the medium would have been much steeper than under air, and so a greater proportion of the photosynthetically produced 0, would diffuse into the surrounding medium, instead of into the heterocysts. However, a portion would still be expected to move toward the site of nitrogenase activity so that an O2 scrubbing mechanism would have some beneficial effect. Pienkos et al. (21) presented data indicating that the nitrogenase of cyanobacteria may be capable of conformational protection, and that it can recover activity very quickly upon removal of Oz. The high light intensity saturation of LDOC may result in significant levels of LDOC activity only under conditions where CO, is limiting. If this were the case, LDOC would not compete with C 0 2 fixation for reductant, a situation analogous to that of leaf NO; reduction in higher plants. It has recently been proposed (23) that pseudocyclic electron flow represents an alternative electron pathway within the photosystems allowing electrons to flow to 0, instead of CO, when C 0 2 is limiting and electron pressure on the photosystems is high (i.e., under high light intensities). This may also be the situation in heterocysts, except that electrons supplied to photosystem I are drawn from reduced carbon compounds instead of photosystem 11. Wang et al. (28) described an oxygen-scavenging system found in membranes isolated from sonicated C0,-fixing cells

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SMITH ET AL.

of Anabaena 7120 and Plectonema boryanum. This system appears to be like that of Honeycutt and Krogmann (14), and like that of the sonicates of C02-fixing cells that we studied. Wang et al. (28) found that their LDOC was heat stable and did not produce HzOz in unboiled material. They also reported that addition of this system to an 0,-sensitive Anabaena mutant resulted in increased nitrogenase activity, and that addition of DCMU to this mutant gave a similar (although smaller) increase. Unfortunately they gave no measure of the light intensities used in this experiments. We suggest that the system characterized by these authors is the LDOC of sonicates of C02-fixing cells only, as characterized in this paper, and induced by disruption of the normal photosystem I photosystem I1 relationship in intact membranes. Interestingly, Tozum and Gallon (27) have recently noted the apparent light-activated protection of the nitrogenase complex of the unicellular cyanobacterium Anacystis nidulans. This system involves the photosystems of photosynthesis and produces H202. As heterocysts contain no photosystem I1 (26), electrons must come from reduced carbon and not water. Electrons would be removed from some compound, e.g., NADH or NADPH (16), and used to reduce an oxidized P700 in photosystem I, they would then move to some acceptor molecule (ferredoxin, P430, or ferredoxin-reducing substance (FRS) ). This acceptor would make a two-electron transfer to 0, producing H z 0 2 . Catalase, if present, would convert two HzOz molecules to two HzO and one 0 , . LDOC would appear to represent the functioning of the photosystem I half of pseudocyclic electron flow. Evidence for this proposal is discussed below. Depletion of reduced carbon reserves by dark incubation caused equivalent reductions in LDOC and dark respiration (Table 5), suggesting a common pool of electrons for both activities. Compounds formed in the citrid acid or pentose phosphate cycles (NADPH, and NADH,) can provide electrons to photosystem I of heterocysts (16). It is known that light can drive such electrons from photosystem I to nitrogenase (16, 18). The association of LDOC systems with the photosynthetic apparatus (6, 15, 25) together with our action spectrum imply that chlorophyll is the photoreceptor in this activity. However, the light saturation of LDOC (about 3.5 mW cm-,) is much higher than that of cyanobacterial photosynthesis (normally about 0.3 mW cm-,), indicating that electron flow through the LDOC is limited by factors different from those that limit the photosynthetic pathway. DPIP inhibits LDOC because it promotes cyclic electron transport within photosystem I. The light-driven production of H 2 0 z detected in many cyanobacteria (25) may represent the functioning of an LDOC acting with photosystem I1 as an electron donor (i.e., pseudocyclic electron transport). The initial product of LDOC was HzOz (Table 5 and lack of superoxide dismutase effect). The LDOC observed in Anabaena variabilis yielded HzOz as its final product (25) while that of our A. cylindrica produced water (Table 3). Anabaena variabilis does not contain catalase (14), while A. cylindrica does (10). Radmer and Kok (22) suggested that ferredoxin directly reduced 0, in pseudocyclic electron flow. Bradley and Carr (6) suggested FRS for this role in LDOC. We found that removal of FRS, by the methods of Yocum and San Pietro (30), did cause a reduction in LDOC. The subsequent return of the FRS fraction to the material restored the rate of LDOC to pre-

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removal levels (data not shown). Heterocysts consume 0, in the light and dark via a Knallgas reaction involving Hz produced by nitrogenase, an uptake hydrogenase, and the respiratory electron transport chain (20). This activity stabilizes N, fixation by heterocysts and 0, protection of nitrogenase has been suggested (5, 10). However, when respiratory electron flow was disrupted by KCN, CCCP, and rotenone, LDOC was not (Fig. 4). Also the uptake hydrogenase is, like nitrogenase, inactivated by 0, (10) and inhibited by acetylene (24). LDOC was observed in sonicates of C02-fixing cells, where no protection from 0, was possible (e.g., Table 1). In addition, bright-light enhancement of nitrogenase activity, presumably because of LDOC, was measured in the presence of acetylene. It would seem that LDOC and hydrogenase 0, consumption are independent. In summary, the light-activated oxygen consumption by A. cylindrica heterocysts seems to be a mechanism for maintaining lowered Poz within the heterocysts, thereby protecting the nitrogenase enzyme from 0, inactivation. 1. ALLEN,M. M. 1968. Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4: 1-4. 2. APPLEBY, C. A. 1974. Leghemoglobin. In The biology of nitrogen fixation. Edited by A. Quispel. American Elsevier Publishing Co., Inc., New York. pp. 521 -554. 3. ARNON,D. I. 1949. Copper enzymes in isolated chloroplasts: polyphenoloxidase in Beta vulgaris. Plant Physiol. 24: 1- 15. 4. BIGGINS, D. R., and J. R. POSTGATE. 1971. Nitrogen fixation by extracts of Mycobacterium flavum 301: use of natural electron donors and oxygen sensitivity of cell-free preparations. Eur. J. Biochem. 19: 408 -415. 5. BOTHE,H., J. TENNIGKEIT, G. EISBRENNER, and M. G. YATES. 1977. The hydrogenase-nitrogenase relationship in the biuegreen alga Anabaena cylindrica. Planta, 133: 237-242. 6. BRADLEY, S., and N. G. CARR.1971. The absence of functional photosystem I1 in heterocysts of Anabaena cylindrica. J. Gen. Microbiol. 68: xiii -xiv. 7. BRADLEY, S., and N. G. CARR.1973. Aspects of development of blue-green algae. Symp. Soc. Gen. Microbiol. 23: 161- 188. 8. CODD,G. A., and W. D. P. STEWART. 1976. Polyhedral bodies and ribulose-1,s-diphosphate carboxylase of the blue-green alga Anabaena cylindrica. Planta, 103: 323 -326. 9. EADY, R. R., B. E. SMITH,K. A. COOK,and J. R. POSTGATE. 1972. Nitrogenase of Klebsiella pneumoniae: purification and properties of the component proteins. Biochem. J. 128: 655 -675. 10. EISBRENNER, G., E. DISTLER, L. FLOENER, and H. BOTHE. 1978. The occurrence of the hydrogenase in some blue-green algae. Arch. Microbiol. 118: 177- 184. 11. FAY,P., and N. J. LANG.1971. The heterocysts of blue-green algae. Ultrastructure and integrity after isolation. Proc. R. Soc. London B, 178: 185- 192. 12. FURBANK, R. T., M. R. BADGER, and C. B. OSMOND. 1982. Photosynthetic oxygen exchange in isolated cells and chloroplasts of C, plants. Plant Physiol. 70: 927-931. 13. HILL,S., W. DROZD, and J. R. POSTGATE. 1972. Environmental effects on the growth of nitrogen-fixing bacteria. J. Appl. Chem. Biotechnol. 22: 541 -558. 14. HONEYCUTT, R. C., and D. W. KROGMANN. 1970. A lightdependent oxygen-reducing system from Anabaena variabilis. Biochim. Biophys. Acta, 197: 267-275. 15. HONEYCUTT, R. C., and D. W. KROGMANN. 1972. Further studies in the oxygen-reducing system of Anabaena variabilis. Biochim. Biophys. Acta, 256: 467-476. 16. HOUGHINS, J. P., and G. HIND.1982. Electron donation to the photosynthetic and respiratory electron transfer chains in Anabaena heterocysts. plant ~ h ~ s i o(~uppl.) l. 69: 74

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17. JENSEN,H. L., E. J. PETERSEN, P. K. DE, and R. BHATTACHARYA. 1960. A new nitrogen-fixing bacterium: Derxia gummose nov. gen. nov. spec. Arch. Mikrobiol. 36: 182- 195. 18. LOCKAU, W., R. B. PETERSON, C. P. WOLK,and R. H. BURRIS. 1978. Modes of reduction of nitrogenase in heterocysts isolated from Anabaena species. Biochim. Biophys. Acta, 502: 298-308. and R. J. RADMER.1979. 19. MARSHO,T. V., P. W. BEHRENS, Photosynthetic oxygen reduction in isolated chloroplasts and cells of spinach. Plant Physiol. 64: 656-659. 20. PETERSON, R. B., and R. H. BURRIS.1978. H2 metabolism in isolated heterocysts of Anabaena 7120. Arch. Microbiol. 116: 125- 132. 21. PIENKOS, P, T., S. BODMER, and F. R. TABITA.1983. Oxygen inactivation and recovery of nitrogenase in cyanobacteria. J. Bacteriol. 153: 182- 190. 22. RADMER, R. J., and B. KOK. 1976. Photoreduction of 0, primes and replaces CO, assimilation. Plant Physiol. 58: 336 - 340. 23. SATOH,K. 1982. Mechanism of photoactivation of electron transport in Bryopsis chloroplasts. Plant Physiol. 70: 1413- 1426.

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24. SMITH,L. A., S. HILL,and M. G. YATES.1976. Inhibition by acetylene of conventional hydrogenase in nitrogen-fixing bacteria. Nature (London), 262: 209 -2 10. and J. MYERS.1973. The 25. STEVENS, S. E., C. 0 . P. PATTERSON, production of hydrogen peroxide by blue-green algae: a survey. J. Phycol. 9: 127- 130. 26. TEL-OR,E., and W. D. P. STEWART.1977. Photosynthetic components and activities of nitrogen-fixing heterocysts of Anabaena cylindrica. Proc. R. Soc. London B, 198: 61 -86. , D. R., and J. R. GALLON.1978. The effects of 27. T o z u ~ S. methyl viologen on Gloeocapsa sp. LB795 and their relationship to inhibition of acetylene reduction (nitrogen fixation) by oxygen. J. Gen. Microbiol. 111: 313-326. 28. WANC,Y., J. HE, and S. LI. 1982. The oxygen-scavenging system protecting nitrogenase from oxygen in blue-green algal cells. Acta Bot. Sin. 23: 231 -240. 29. WYATT,J. T., and J. K. G. SILVEY.1969. Nitrogen fixation by Gloeocapsa. Science (Washington, D.C.), 165: 908 -909. 30. YOCUM,C. F., and A. SANPIETRO.1970. Ferredoxin-reducing substance (FRS) from spinach. 11. Separation and assay. Arch. Biochem. Biophys. 140: 152 - 157.