Photosynthetic Production of Hydrogen Peroxide by Anacystis ... - NCBI

Plant Physiol. (1973) 51, 104-109

Photosynthetic Production of Hydrogen Peroxide Anacystis nidulans'

by

Received for publication July 18, 1972

C. 0. PAT PATTERSON2 AND JACK MYERS Department of Zoology, University of Texas, Austiii, Texas 78712 tained 1 nmole of scopoletin and 80 units of peroxidase in a total volume of 3.3 ml. Cuvette temperature did not rise signifiA sensitive assay based upon fluorescence of scopoletin al- cantly above room temperature (24-26 C). We confirmed the lowed continuous recording of H20, production in illuminated previously reported requirement for peroxidase and the 1:1 intact cells of Anacytis nidulans. Onset of illumination was fol- stoichiometry for scopoletin-H20,2 in our apparatus using H202 lowed by a 5 to 10 second lag, a burst of very rapid production solutions standardized against KMnO,. Oxygen exchange was continuing for up to 5 minutes, and finally a slow and contin- measured by a Beckman oxygen macroelectrode in a cuvette of uing steady rate of H202 production. Size of the H202 burst was about 1.5 ml at a sensitivity of about 0.02 ,l 02/ml (32). decreased by 3-(3,4-dichlorophenyl)-1,l-dimethvlurea, by low Anacystis nidulans Richter (strain Tx 20 of our laboratory) 02, and by certain Calvin cycle intermediates; it was increased was grown in medium C (24) in a continuous culture apparatus by high light intensity, CO2 depletion, Calvin cycle inhibitors at 25 C or 30 C, aerated with 1% CO2 in air. Although no (as iodoacetamide), cold shock, carbonyl cyanide ,n-chloro- detailed study of culture conditions was made, it was noted that phenylhydrazone, and certain organic acids as glycolate). The cells grown under comparatively low light intensity (four 40-w H202 burst was explained by the following hypothesis: a low tubular tungsten lamps at 40 cm, cell doubling time about 24 potential reductant is produced more rapidly than it can be hr) consistently gave greater initial bursts and higher steady used in the normal pathway to C02 reduction and, instead, re- rates of H202 production than cells grown under higher inacts with oxygen. H20. production is regarded as a metabolic tensity light at higher growth rates. Cells grown in this way defect observable in Anacystis most dramatically during the were routinely used. For experiments, harvested cell suspentransition from a very low rate of oxidative dark nmetabolism sions (about 3 ,1l cells/ml) were centrifuged at 3300g for 10 to a high rate of photosynthetic metabolism. min and resuspended in a medium C-m containing only the major salts (1 mMMgSO,, 5 mm NaNO., 2 mm KNO2, 6 mM K2HPO,; pH 7.3). This stock cell suspension (about 1 1-l cells/ ml) was held at 26 C, illuminated with two 20-w warm white fluorescent lamps, and aerated with 1 % C02 in air until use. Scopoletin and pCMB3 (sodium salt) were purchased from Mann Research Laboratories. Scopoletin solutions were preThe classical experiments of Mehler and coworkers (28-30) pared in 1 or 2 liter quantities (2 mg/ 1) and stored frozen undemonstrated the production of H202 by isolated chloroplasts. til use. Peroxidase (type II, horseradish) and iodoacetamide In subcellular preparations the role of molecular oxygen as a were from Sigma Chemical Company. Carbonyl cyanide inHill oxidant and production of H202 has been well established, chlorophenylhydrazone was a gift of Dr. F. P. Healey. Diquat even in the absence of autoxidizable additives (3, 7, 15, 17, (ethylene viologen) was obtained as the dibromide salt from 20). However, it has remained uncertain whether reduction Ortho Division of California Chemical Company. of oxygen to H202 is a normal event or an artifact induced by Cell concentrations were determined by centrifuging the damage to the photosynthetic apparatus during isolation. Very cell suspension for 1 hr at 2200g in centrifuge tubes with calismall amounts of H202 production have been reported in flash- brated capillary bottoms. Chlorophyll a was estimated in 80% illuminated cells of Chlorella (l1) and in Anacystis nidulatns acetone extracts (absorption coefficient = 82.04 cm2/ mg at 663 (38). We now report the direct, continuous measurement of nm). photosynthetic production of H.O by intact cells of Anacystis nidulans. RESULTS Hydrogen peroxide production, as described herein for MATERIALS AND METHODS Anacystis niduilans, is not a universal phenomenon in algae nor Modification of the scopoletin fluorescence assay (. 34) al- in blue-green algae. By the same methods we sought but did lowed continuous recording of production of H202 by illumi- not find evidence of H202 production by Anabaetia cylindrica, nated algal cells. Scopoletin is oxidized by H202 in the presence Agmtienellum quadrutplicatumti, and Chlorella pyrenoidosa (Emof peroxidase so that decrease in scopoletin fluorescence is a erson strain), either in freshly harvested or in C02-starved cells. direct measure of amount of H202. The assay apparatus is Subsequent to the main body of our work, further exploration described in Figure 1. The routine assay reaction mixture con- has revealed H202 production in some other blue-green algae, although not always with the same pattern seen in Anacystis. Time Course. Production of H202 by A nacystis after onset 'This study was supported by Grant GM 11300 from the NaABSTRACT

tional Institutes of Health. 2Present address: Division of Biological Sciences, Indiana University, Bloomington, Ind. 47401.

:'Abbreviations: CCCP: carbonyl cyanide-m-chlorophenylhydrazone; pCMB: p-chloromercuribenzoate. 104

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PEROXIDE PRODUCTION BY ANACYSTIS

51, 1973 cz;

2FF2

LIZIFO-

F1

F;-

FIG. 1. Apparatus for H202 assay. Fluorescence of scopoletin in cuvette C was excited by 365 nm provided by a 100-w mercury lamp; UV, was via a Corning 5860 plus interference filter, F1. Scopoletin fluorescence at 410 to 470 nm was measured by the IP21 photomultiplier; PM was of an Aminco photometer via a compound filter, F2, containing 1 cm of 1.0 M CUSO4, and Wratten filters 2A, 2B(2), and 47B. Photosynthesis of algal cells in the cuvette was driven at 620 or 675 nm by a Kodak projector; IL, was via an 8 cm water cell and blocked interference filters, F3.

105

(up to 20) sec in darkness. The character of the time course was not dependent on wavelength of illumination. Illumination Effects. While the length of the lag period and the size of the steady rate varied with light intensity, the most dramatic effect of intensity was on the size of the burst (Fig. 2). Figure 3 shows burst size and rate of oxygen evolution versus light intensity as determined in parallel experiments on the same cell suspension. The burst disappeared at low intensity; illumination at 200 to 500 /tw/cm2, 620 nm, gave no burst but supported a low steady rate. Still lower intensities (< 200 juw/cm', 620 nm) gave no measurable H202 production at all. Light saturation of the burst size required intensi-

0-

0

E

0

~~~~~~~46

l 0

lo

20

TIME, minutes

FIG. 2. Time course of H202 production in Anacystis. The reaction cuvette (Fig. 1) contained 2 ml of 50 mm phosphate buffer equilibrated with 1% C02, 100 Id of scopoletin stock solution (2 mg/l in water), 1 ml of cell suspension in medium C-m containing 1 Al cells and equilibrated with 1% C02 in air, and 50 Al of a peroxidase solution (4 mg/ml in water). In all experiments, the complete reaction mixture was equilibrated in the cuvette in the dark for 5 min before onset of illumination. The reaction mix was aerated with 1% C02 in air throughout each experiment. Illumination was at 620 nm, and the intensity shown on each curve is in ,Awatts/cm2. The immediate upward displacements at the beginnings ( a ) and equal downward displacements at the ends ( T) of illumination are artifacts resulting from incomplete protection of the photomultiplier against 620 nm. Estimation of burst size is shown for one intensity.

of illumination typically followed a complex time course (Fig. 2) consisting of three stages: an initial lag of several (510) sec, a burst or very rapid production continuing for up to 5 min, and finally a slower and continuing steady rate of production. Return to darkness during the steady rate dropped H202 production rate to zero within the instrumental time response (about 2 sec). If illumination ended during the burst stage, H202 production was observed to continue for several

4000 6000 )0 LIGHT INTENSITY, fLwatts/cm2

FIG. 3. Light intensity curves for steady-state oxygen evolution (A) and for the H202 burst (B) in Anacystis. Harvested cells were centrifuged, resuspended in medium C-m at 0.6 Al cells/ml, and aerated with 1% C02 in air at 25 C under very low tungsten illumination until use. Chlorophyll concentration was 6.6 ug/gul cells. Illumination was provided by a Kodak projector using Baird Atomic interference filters at 620 nm (15 nm half band width) or 675 (30 nm half band width) together with infrared blocking filter 6143 and a 8 cm H20 cell. Intensity was varied by screens and measured by a calibrated thermopile (A) or a silicon cell calibrated against the thermopile (B). Compensating intensity for 620 nm was estimated to be 30 uwatts/cm2. A: The curves for 620 and 675 nm were normalized only on the intensity scale by adjustment factor 4.04. B: The 620-nm curve was drawn for best fit by inspection; the 675nm curve was obtained by doubling intensity values for the 620-nm curve and drawing a new curve for the points thus generated. Reasonable fit is thereby attained with experimental points for 675 nm. The curve fitting procedure was selected to make evident that for equal photosynthetic effectiveness 620 nm is about two times as effective as 675 nm in producing the peroxide burst.

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PATTERSON AND MYERS

ties much higher than those necessary to saturate photosynthetic oxygen evolution. Figure 3 also shows that for equal photosynthetic effectiveness, 620 nm is about twice as effective as 675 nm in producing the H22O burst. The burst could be entirely eliminated by slow (rather than abrupt) increase from zero to high light intensity or by illumination by long flashes (6, 10 sec, 1500 /w/cm2, 620 nm) 10 sec). However, neither of separated by dark periods these regimens eliminated the final steady rate. The entire time

()

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z^

20

u

, 4C

0

z

C C

10-7

0

10-6

5X10-7

5X]10)6

CCCP, Molar FIG. 4. Effect of CCCP. The H202 burst (A) was measured at 3.3 mw/cm2 of 620 nm, and reaction conditions are the same as described for Figure 2. Small aliquots of CCCP in ethanol were added to the reaction cuvette just prior to addition of cell suspension, 5 min before illumination. Rates of dark respiration (0) and light-saturated oxygen evolution at 7.7 mw/cm2 of 620 nm (0) were measured by oxygen electrode. Small aliquots of CCCP in ethanol were added to the cell suspension in the electrode cuvette 5 min before measurement. CONCENTRATION OF

A

v

25

.

a)

LU

0.12 co Ul)

cc

6

7

8

9

pH FIG. 5. Effect of pH on the H202 burst. Stock suspension of cells was prepared in medium C-m aerated with 1% C02 in air. One-ml aliquots of cell suspension were added to 2 ml of buffer also equilibrated with 1% C02 in air and previously adjusted by KHCO3 or HCI to give the desired final pH. Buffers used were 50 mM phosphate (0), medium C-m (0), 0.1 M

KHCO3-Na2CO3

Warburg buffer No. 9 (A) and No. 11 (A). Illumination was at 1500 Aw/cm2 of 620 nm and other reaction conditions were as for Figure 2.

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course (lag, burst, steady production) could be observed repeatedly from a single cell suspension if sufficient dark time (5-10 min) was allowed between light doses of sufficient intensity and duration. CONDITIONS INFLUENCING THE BURST

Decrease of Burst Size. DCMU cut burst size in half at 0.01 pLM and abolished all H202 production at 0.1 ptm. Similarly all H202 production was abolished by 0.5 mM-o-phenanthroline. These results are taken as evidence that the photosynthetic apparatus is the source of reductant for H2O2 production. H202 production was also abolished by decreased 02 concentration obtained by aeration with 1% CO2 in N2. Increase of Burst Size. Production of H,02 was markedly increased after 5-min incubation with pCMB or iodoacetamide, two Calvin cycle inhibitors (2, 14). At 1 mm, pCMB doubled burst size and increased the final steady rate 3- to 4-fold. lodoacetamide at 1 mm increased burst size 10-fold and increased the steady rate 20-fold. H202 production was also increased by CO2 depletion. After aeration with alkali-scrubbed air, burst was increased 3- to 4-fold; steady rate was increased 4- to 5-fold. Effects of cold shock were studied with cell suspensions held at 4 C for 45 min in darkness and rewarmed to 25 C before assay. This cold shock tripled the burst size and doubled the final steady rate. Similar cold treatment is known to decrease photosynthetic rates and cause pteridine excretion in Anacystis (12). The uncoupler CCCP (18) gave increasing burst size with increasing concentration (3-30 pM) as indicated in Figure 4. At 30 -M, burst size was doubled, but photosynthetic 02 evolution was decreased to about half its control rate. Both H202 burst and rate of 02 evolution declined at higher concentrations of CCCP. We note the contrast between inhibition by the uncoupler CCCP and by blocking of electron flow with DCMU. Effects of the autoxidizable electron acceptor, diquat (40), were of a different kind. Production of H202 occurred in darkness at a measureable rate which decayed slowly with time. At the onset of illumination the rate increased abruptly with no detectable lag, reached a very high maximum rate in about 1 min, and continued without decrease until return to darkness. By introduction of a catalase-ethanol trap (28), we also made conventional measurements of 02 uptake mediated by diquat. At saturating concentration (15 mM) and light intensity, the rate of 02 uptake was somewhat greater than the rate of 02 production in unpoisoned cells. The burst was sensitive to pH and to the buffer system (Fig. 5). The H202 burst was absent or very small below pH 7.0 where growth of Anacystis is inhibited (24). The H202 burst is increased by higher pH values during the brief (5 min) dark incubation preceding illumination. Our curve for burst size versus pH in 50 mm phosphate buffer is remarkably similar to the curve of Honeycutt and Krogmann (20), describing 02 reduction by a subcellular preparation of Anabaena variabilis. Effects of Cell History. Effects of cell history were examined for cells grown under illumination varied from our standard conditions. Although effects of light intensity during growth were not explored in detail, it was noted that cells grown at higher light intensities and higher specific growth rate gave lowered burst size. A particular condition examined was that of red light illumination which gives a high phycocyaninchlorophyll ratio and an increased ratio of system 2-system 1 activity as viewed by enhancement (22, 23). Such cells grown under BCJ red (>660 nm) lamps gave greatly increased burst

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02/mg chl min) or about 15% of the average rate during the same portion of the induction period (15-75 sec). Such calculations, and indeed all of our measurements based on extracellular H202, necessarily underestimate rates of intracellular H202 production. Anacystis has a strong catalase activity evident equally in whole cells and broken cell preparations (27,

33).

10 02

0

E c

0.5~~~ ~ ~ U)

a,a:/

0

0

2000

4000

/I watts /cm2 FIG. 6. The H202 burst versus light intensity for two types of cells. Cells grown under clear tungsten lamps (0) and cells grown under red BCJ lamps (I) were adjusted to equal concentrations of 1.0,X cells/ml, and the burst size was determined under 620 nm and reaction conditions cited in Figure 2. Cells grown under clear tungsten lamps (our standard cell material) contained 5.9 ,ug chlorophyll a and 74 ,ug phycocyanin per ,ul cells. Cells grown under BCJ lamps contained 2.6 ,sg chlorophyll a and 85 Ag phycocyanin per ul. Phycocyanin was estimated (26) after cell disruption by a French press.

size observed at lower intensity of 620 nm actinic illumination (Fig. 6). Unfortunately, the culturing illumination was effectively low and gave only slow growth with doubling time of about 96 hr. Hence, it is not clear whether the increased burst size is attributable to the preceding very low growth rate or to the increased system 2-system 1 activity. Effects of Metabolic Intermediates. Recalling effects of various intermediates in decreasing induction lags in 02 production or CO2 uptake by chloroplasts (4, 5, 8, 39), we examined effects of intermediates added at the beginning of the 5-min dark incubation period before assay. Four of the five Calvin cycle intermediates tested depressed H20, production (Table IA). We also examined effects of five carboxylic acids (Table IB). Here the effects were more complex and more dependent on concentration. The most dramatic effects were seen in the 3- to 4-fold increase in burst size with added succinate and malate. None of the additives examined gave measurable H202 production in darkness. DISCUSSION In Anacystis the transient high rate of H202 formation (the burst) can be a significant fraction of the total electron flow. At 7 mw of 620 nm illumination a typically measured rate of H,02 production was 0.4 ,umole H20,/mg chl-min during the 1st min of the burst. This represents about 5% of the steady light-saturated rate of electron transport (3.7 ptmoles

It is instructive to consider the metabolic peculiarity of Anacystis. As with several other blue-green algae, it will assimilate a number of exogenous substrates into cellular material in light but not in darkness (19, 36). A proposed explanation of its obligate autotrophy hinges partly on lack of ability to generate ATP oxidatively (6, 36). As compared to other blue-green algae, Anacystis is unusual in its very low rate of dark oxidative metabolism, especially when grown at low light intensities (25). From these considerations alone it might be expected that Anacystis must have a special metabolic problem in adjustment of ATP level and pool sizes of intermediates following transition from darkness to high light intensity. Almost all of our data are consistent with the following simple hypothesis as an explanation of the H202 burst in Anacystis (Fig. 2). A low potential reductant is produced more rapidly than it can be used in the normal pathway to CO, reduction and instead, reacts with 02. The preceding initial lag represents time required to deplete pool sizes or otherwise establish limitations on rate of electron acceptance by the Calvin cycle. The burst ends when such limitations are removed. In support of this hypothesis, we note especially the following. (a) The burst requires sudden transition from darkness to high light intensity; it can be abolished by gradual transition or by repeated flashes if each is shorter than the lag. (b) Partial inhibition of electron transport (DCMU) decreases the burst, while partial inhibition of phosphorylation (CCCP) or of the Calvin cycle (low CO2, pCMB, iodoacetamide) increases the burst. (c) Cold shock, which increases the burst, has been shown to inhibit phosphorylation far more than electron transport (21). (d) Exogenous Calvin cycle intermediates Table I. Effects of Metabolic Intermediates on H202 Production Intermediates

Burst

% control

mm

A. Ribulose-5-P

Fructose-1, 6-diP'

Ribose-5-P1 DihydroxyacetoneP 3-Phosphoglycerate B. Glycolate Succinate

L-Malate DL-Isocitrate

Acetate'

0.3 0.9 0.35 0.8

Steady Rate

70 58 77 39 12 63 21 83

1.7 0.12 0.6 0.26 0.88 0.6 3 30 45 6

100 98 150 220 300

15

400

6 15 0.84 1.4 15 30

270 480 24

51

16 135 56

50 50 50 28 0 100 10 77 80 100 100 100 110 100 100 50 50 38 30 100 50

1 Control experiments indicated the compound did not interfere with the scopoletin assay.

108

PATTERSON AND MYERS

which decrease induction effects in chloroplasts also decrease the burst. (e) More complete diversion of electron flow to oxygen by diquat abolishes the lag, showing that there is no lag in electron transport. We note from the light intensity curves (Fig. 3) several characteristics which support or are consistent with the above hypothesis. Below a threshold light intensity the burst disappears. Here photochemical generation of reductant is slow, and the necessary adjustment in pool sizes is small enough that there is no accumulation of excess reductant. At high light intensities the burst size is still increasing when the steady rate of photosynthesis is saturated. We suppose that in these cells the rate-limiting step in photosynthesis is located somewhere in the Calvin cycle, at least during the first several minutes of illumination. The lower effectiveness of 675 nm (versus 620 nm) is attributed to its relatively higher contribution to cyclic photophosphorylation and a more rapid increase in available ATP as one of the pools requiring adjustment. We have no evidence on the site of H202 production. However, none of the electron transfer intermediates between the photoreactions has been reported capable of reducing 02 to H202. Hence, we tentatively assign H202 production to a reductant near the terminus of system 1. Production of H202 by autoxidation of ferredoxin has been reported (37), but H2O, production also has been reported in membrane preparations of Anabaena, presumably washed free of ferredoxin (20). The cytochrome reducing substance has been shown to be readily oxidized by 02 but without evidence for concurrent H502 production (13). The final steady production of H202, though possibly related to the burst in terms of site of H202 formation, appears to be an independent phenomenon partially overlapping in time. A succession of flashes or gradual transition from darkness to high light, which eliminate the burst, have no effect on the final steady production. The final steady rate, as measured, never represented more than 0.5% of the light-saturated electron throughput rate as estimated from rate of oxygen evolution. Hence, we regard it as a small defect or leak. We have considered the notion that H202 might play some metabolic role, e.g. biosynthesis of glycolate (35), but find no supporting evidence pertinent to blue-green algae. Effects of some added intermediates were explored but without intent of providing detailed analysis of metabolic pathways. With whole cells interpretation of such effects is clouded by questions of intracellular availability. In fact we were surprised to find effects at all and by such an array of compounds. Depression of the burst by ribose-5-P, ribulose-5-P, fructose-1 , 6-diP, and dihydroxyacetone-P is consistent with observations on shortening of induction effects in chloroplasts (4, 5, 8, 39). We suppose that these additives are effective by maintaining pool sizes of Calvin cycle intermediates closer to their light levels. Activity of glycolate, succinate, and malate in increasing the H202 burst suggests that these compounds may be supplying reductant (e.g., reduced pyridine nucleotide) to the cells via succinate dehydrogenase, malate dehydrogenase (36), and glycolate oxidase (16). This would cause still greater excess of reductant at onset of illumination. Such activity suggests the operation of the tricarboxylic acid cycle or the glyoxylate shunt. However, on the basis of labeling experiments in which Anacystis cells were fed '4C-acetate, previous investigators (19, 36) have concluded that the tricarboxylic acid cycle in this organism is blocked at a-ketoglutarate and that no glyoxylate shunt is present to bypass the block. Our experiments indicate that the light metabolism in Anacystis is somehow disturbed by substrate levels of exogenous organic acids; our observations may

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be related to those of Miller et al. (31) who observed light stimulation of glycolate uptake and oxidation in two bluegreen species. Our findings contribute to but do not resolve a three-part anomaly surrounding Anacystis and H202: (a) a high sensitivity of single-cell isolates to exogenous H202 (27, 38); (b) a strong catalase-like activity observable in whole cells and not increased by cell disruption (33); and (c) a photochemical production of H202 by whole cells observable outside the cells. We have treated a relatively small production of peroxide observed under special conditions in Anacystis. This observation and our explanation for it are not novel (9, 10), save in the sense that we have considered events occurring in intact, unpoisoned cells. We have been led to the conclusion that the peroxide production results from a metabolic defect understandable from other known metabolic characteristics of Anacystis. In a broader context we are led to great respect for the elegance of metabolic machinery of other algae. It is common practice to grow algae in the laboratory at a relatively low effective light intensity and then to study the cells at light saturation and a metabolic rate perhaps 10-fold higher than they had ever previously experienced. Detailed attention has been given relatively minor aberrant events observable during the induction period of photosynthesis. We wonder if there occurs anywhere other than in photosynthetic cells the up to 100-fold increase in over-all metabolic rate which attends transition from darkness to high light intensity. LITERATURE CITED

hydrogen

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D. I., M. LoSADA, F. R. WHATLEY, H. Y. TSUjIMOTo, D. 0. HALL, AN'D A. A. HORTON. 1961. Photosynthetic phosphorylation and molecular oxygen. Proc. Nat. Acad. Sci. U.S.A. 47: 1314-1334. 4. BALDRY, C. W., D. A. WALKER, AND C. BUCKE. 1966. Calvin-cycle inter-

3.

mediates in relation to induction phenomena in photosynthetic carbon

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KOUCHIKOVSKY, Y. 1963. Induction photosynthetique des chloroplastes

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