pending fresh chloroplasts in 10 ms PMA-0.4 M sucrose, and then washed twice with 20 ... appears to be insensitive to PMA up to 500 ItM. The closed and open ...
Plant Physiol. (1972) 49, 376-380
Inhibition of Chloroplast Reactions with Phenylmercuric Acetate1'2 Received for publication July 30, 1971
RIcHARD C. HONEYCUTT3 AND DAVID W. KROGMANN
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907 ABSTRACT
Phenyhnercuric acetate is a selective inhibitor of the photosynthetic activities of isolated spinach (Spinacia oleracea) chloroplasts. At 5 /M concentration of phenylmercuric acetate, photophosphorylation is inhibited. At 33 4M phenylmercuric acetate, ferredoxin is inactivated. Ferredoxin-NADP oxidoreductase is 50% inhibited at 100 4M phenylmercuric acetate. Photosystem II reactions are 50% inhibited at 150 pM phenylmercuric acetate and very much higher cooncentrations-500 MM-are needed to approach complete inhibition. Phenylmercuric acetate inhibition of photosystem II appears to be selective, blocking a site between the 3-(3,,4-dichlorophenyl)-1,1dimethyl urea sensitive site and the site inactivated by high concentrations of tris buffer.
Mercurials have long been known to inhibit photosynthetic electron transport. Siegenthaler and Packer (13) reported that phenylmercuric acetate inhibits both Hill activity and photophosphorylation. Davenport and Hill (7) determined that PMA4 inhibited methemoglobin reduction supported by ferredoxin. Hiyama et al. (9) found that PMA inhibits the dark reduction of cytochrome C5M,4 in whole cells of Chlamydomonas reinhardi and inhibits either ferredoxin or ferredoxin-NADP oxidoreductase. The work presented in this paper shows that 33 tsM PMA inhibits ferredoxin specifically, thereby blocking the photoreduction of NADP. The diaphorase activity associated with ferredoxin-NADP oxidoreductase in inhibited at higher concentrations of PMA than inhibit the ferredoxin-dependent reactions of photosystem I. Recently many workers have directed their attention to the dark reactions of photosystem II associated with oxidation of water and oxygen evolution. From these studies several different sites of inhibition of photosystem II have been uncovered. For example, photosystem II is inhibited by heat (10), UV light (17). and high concentrations of tris buffer (18). Epel and 'Publication No. 4513 of the Purdue Agriculture Experiment Station. 'This work was supported by National Science Foundation Grant GB-27466. 'Present address: Radiation Biology Laboratory, Smithsonian Institution, 12441 Parklawn Drive, Rockville, Md. 20852. 'Abbreviations: PMA: phenylmercuric acetate; TCIP: 2,3', 6trichlorophenol indophenol; TCIPH2: reduced TCIP; DCMU, 3(3 .4-dichlorophenyl)-1,1-dimethyl urea; DPC: diphenyl carbazide; Fd: ferredoxin. 376
Levine (8) have found mutants of C. reinhardi which have lost partial function in photosystem II. These blocks in photosystem II occur on the oxidizing side of the photoact, and many of the manifestations of this photosystem can be restored with the appropriate electron donor system. This paper presents evidence that PMA inhibits photosystem II at a site between the tris lesion and the photoact. Alternate oxidation sites for artificial donors such as hydroxylamine, hydroquinone, and diphenyl carbazide have been discerned through the use of PMA to study the complex series of reactions associated with the evolution of oxygen. MATERIALS AND METHODS The method for the spectrophotometric measurement of the photoreduction of NADP has been described previously (15). A typical 3-ml reaction mixture contained the following components in ,umoles: sodium phosphate buffer, pH 7.8, 50: NADP, 0.5; ADP, 1: MgCl2, 10; and a saturating amount of spinach ferredoxin. If TCIP-ascorbate was to be used as a donor, 5 nmoles of DCMU, 20 ,umoles of sodium ascorbate, and 0.3 ,umole of TCIP were added. For metmyoglobin reduction NADP was replaced by 0.4 ,umole of metmyoglobin, and the light-induced absorbance change at 580 nm was measured. When ferricyanide or TCIP was used as acceptor. NADP and ferredoxin were replaced by 1.0 1lmole of potassium ferricyanide or 0.1 1Lmole of TCIP, and the light-induced change at 410 or 620 nm respectively was followed. The luminous flux for such experiments was 5 x 10° erg cm 2 sec1. Methyl viologen reduction was measured by taking advantage of its autoxidizability. Oxygen uptake was measured using a Yellow Springs Instrument Company oxygen electrode. A representative 3-ml reaction mixture usually contained the following in amoles: Tricine buffer, pH 7.8, 45: methyl viologen, 2: sodium azide, 1; and NaCl, 6. When TCIP was the electron donor. 5 nmoles of DCMU, 30 nmoles of TCIP, and 15 /Lmoles of sodium ascorbate were used. The light intensity used for methyl viologen reduction was the same as noted above. Photophosphorylation was assayed according to the procedure of Krogmann and Olivero (12). The diaphorase activity of ferredoxin-NADP oxidoreductase was measured with a modification of the method of Avron and Jagendorf (2). A 3-ml reaction mixture containing 50 nmoles of TCIP, 50 ,umoles of sodium phosphate buffer, pH 7.0, and 0.04 ml of partially purified spinach flavoprotein (approximately 3 mg protein/ml) was placed in a cuvette and the absorbance measured at 620 nm. The reaction was initiated by adding 0.3 ,umole NADPH. The rate of decrease in absorbance at 620 nm is a measure of diaphorase activity. Mn2+ photooxidation was measured according to Ben-Hayyim and Avron (4). A standard reaction mixture contained exactly one-half of the amounts of components described by
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PMA INHIBITION OF PHOTOSYNTHESIS
the above authors except that 3 ,tmoles of methyl viologen were used, and the final reaction volume was 3 ml. The reaction mixture was placed in a 50-ml Erlenmeyer flask and illuminated in a Warburg bath for 3 min with red light at an intensity of 8 X 10' ergs cm-2 sec'. Aliquots were then taken for Mn3+ determination. Chloroplasts from Spinacia oleracea were prepared by the procedure of Avron et al. (3). Tris treated chloroplasts were prepared according to the procedure of Yamashita and Butler (17). The chloroplasts were incubated in 0.4 M tris buffer, pH 7.8, for 30 min, then washed twice with 0.4 M sucrose-50 mM NaCl solution. PMA-treated chloroplasts were prepared by suspending fresh chloroplasts in 10 ms PMA-0.4 M sucrose, and then washed twice with 20 ml of 0.4 M sucrose-50 mm NaCI solution. Chemicals. PMA and myoglobin were purchased from Sigma Chemical Company. Metmyoglobin was prepared from myoglobin using the procedure of Smith (14). DPC was purchased from Aldrich Chemical Company, and hydroxylamine was obtained from Matheson, Coleman and Bell. Ferredoxin was prepared by the method of Boger et al. (5). Chlorophyll concentration was estimated by the method of Arnon (1).
RESULTS While studying various manifestations of Hill activity in spinach chloroplasts, a variety of effects of PMA were observed. Figure 1 is representative of such experiments. The top curve shows the effects of PMA on the reduction of methyl viologen using TCIPH2 as an electron donor. Photosystem I appears to be insensitive to PMA up to 500 ItM. The closed and open triangles show the effect of PMA on photosystem II reactions, i.e., ferricyanide reduction and TCIP reduction respectively. There is a gradual inhibition of photosystem II with incomplete inhibition above 500 ftM PMA. The open circles and closed circles respectively represent ferredoxin re-
Table I. PMA Inihibition of Soluble Ferredoxin Methods for NADP reduction have been described previously. The same amount of chloroplasts as in Figure 1 were added. PMA was added to a sample of ferredoxin to a final concentration of 33 Mm. Ferredoxin (200 yg) was used for each experiment. Dialysis of control and PMA-treated ferredoxin was carried out for 18 hr in 600 ml of 0.005 M tris, pH 8.0, in separate containers. Additions to Fresh Chloroplasts
Rate of C-
Experiment 1 200Mug Fd 200,ugFd + PMA 400,ugFd + PMA Experiment 2 200 ,g Fd before dialysis 200 jug Fd after dialysis (200 Mg Fd + PMA) before dialysis (200,Mg Fd + PMA) after dialysis (200,g Fd + PMA) after dialysis + 200 Mg excess Fd
control
100 0 71 100 100 0 0 100
quiring NADP and metmyoglobin photoreduction using electrons trom water. PMA appears to inhibit these activities at
much lower concentrations (50 p.M). Identical results were obtained when TCIPH2 was used to replace water as the electron donor in the NADP reducing reaction. Pyocyanin-mediated cyclic photophosphorylation (closed rectangles) is inhibited at much lower PMA concentrations than inhibit photosynthetic electron transport. We found that noncyclic photophosphorylation is inhibited by PMA at the same concentration that inhibits cyclic photophosphorylation. Although the data are not shown, methyl viologen reduction using electrons from water behaves toward PMA as does TCIP photoreduction. Thus PMA inhibits photosynthetic electron transport in at least 2 sites, a sensitive site in the ferredoxin-dependent reactions and a less sensitive site in photosystem II. We attempted to determine whether the flavoprotein ferredoxin-NADP oxidoreductase or ferredoxin itself was being inhibited by low PMA. Hiyama et al. (9) have suggested either protein might be the site of PMA inhibition. Table I shows that PMA specifically inhibits the ability of exogenous fero 0 redoxin to support NADP photoreduction using electrons from TCIPH2. Adding excess ferredoxin overcomes the PMA effect. 0 Experiment 2 shows that PMA does irreversible damage to ferredoxin. Dialysis of PMA-treated ferredoxin does not relieve the inhibitory effect of PMA. Keister and San Pietro (11) have shown that the mercurial p-chloromercury phenyl sulfonic acid 0 50 100 200 300 bleached the visible spectrum of ferredoxin. We have found 400 500 [PM A] fJmolar that PMA causes a similar bleaching of ferredoxin. The effect of PMA on the diaphorase activity of isolated FIG. 1. Differential inhibition of photosynthetic electron transferredoxin-NADP oxidoreductase is represented in Figure 2. port by PMA. The reaction conditions for all measurements are given in "Materials and Methods." Numbers within brackets below PMA inhibits soluble or bound diaphorase at much higher indicate the control (100%) rate in terms of Mumoles electrons per concentrations than those needed to inhibit ferredoxin. At 33 mg chl per hour. The open blocks represent methyl viologen re- /tM PMA, where NADP photoreduction is inhibited 90%, the duction by electrons from TCIP-ascorbate [170]. The closed blocks chloroplast diaphrase activity is inhibited only 30%. One must represent pyocyanine-mediated cyclic photophosphorylation [360 conclude that the inhibition of NADP reduction caused by 33 Mmoles ATP/mg chl*hr = 100%]. The open triangles represent utM PMA is due to inhibition of ferredoxin. TCIP photoreduction by electrons from water [320], while the Figure 1 presented evidence that high concentrations of closed triangles represent ferricyanide photoreduction by electrons PMA inhibited the photoreduction of TCIP or ferricvanide from water [77]. The open circles represent photoreduction of NADP by electrons from water [315], while the closed circles rep- using water as an electron donor. An attempt was made to reresent photoreduction of metmyoglobin by electrons from water late PMA inhibition of photosystem II to tris inhibition and [50]. Chloroplasts representing 40 ug of chlorophyll were used in other known lesions occurring in this part of the electron transthis experiment. port sequence. Figure 3 presents evidence to show that PMA
378
3iONEYCUTT AND KROGMANN
is Mn2+ photooxidation. Tris-treated chloroplasts which have photosystem II activity supported by DPC behave toward PMA as do the control chloroplasts. Table II indicates that PMA inhibition of photosystem II is of a specific nature. Both PMA and tris inhibit the flow of electrons from water to methyl viologen. DPC and hydroxylamine reverse the tris inhibition in a DCMU-sensitive fashion. However, hydroxylamine but not DPC reverses the PMA block. The reversal by hydroxylamine is DCMU sensitive, indicating PMA inhibits on the
100
80 -2
0
60~ 55 0 cr
Plant Physiol. Vol. 49, 1972
40C
20~ I
50
c
I
55
0
1000
500
100
[PMA]
0-)
Lmolar
=
-a
V) -_ cr -
FIG. 2. Inhibition of Fd: NADP oxidoreductase by PMA. The procedure for assay of diaphorase is outlined in "Materials and Methods." The open circles represent the assay of the soluble diaphorase which had been prepared by the method of Boger et al. (5) The closed circles show diaphorase bound to chloroplast. Chloroplasts representing 40 ,ug of chlorophyll were used in these assays.
ECP.I a)
inhibits
dark reaction associated with photosystem II. The shows the effect of varying light intensity on TCIP photoreduction in the absence of PMA. The bottom curve shows the same experiment with PMA present (165 tIM). The inhibitory effect of PMA appears to be on a dark reaction since saturating light intensities do not overcome the inhibition. Figure 4 presents some observations of PMA effects on photosystem II. PMA at low concentrations stimulates the photooxidation of Mn2+ by whole chloroplasts. At higher concentrations, PMA inhibits this same reaction. As the concentration of PMA approaches 150 [Mm TCIP photoreduction by photosystem II is inhibited in the same concentration range as top
a curve
Light Intensity (erg cm-2 sec-1 x 10-5
FIG. 3. Inhibition of TCIP reduction by PMA. The rates of reaction are expressed as ,umoles electrons transferred to indophenol dye per mg chlorophyll per hour. The reaction conditions are described in the "Materials and Methods." The open circles represent the photoreduction of TCIP in the absence of PMA. The closed circles represent the photoreduction of TCIP in the presence of 165 AM PMA. Chloroplasts containing 40 ,ug of chlorophyll were added just prior to illumination.
DPC TC
IP
0
50) cc
50
100
150
350
[PMA] Smolar FIG. 4. Mn2+ oxidation and TCIP reduction as a function of PMA concentration. The procedure for assay of Mn'+ photooxidation and TCIP reduction have been described in "Materials and Methods". Chloroplasts representing 50 j.g of chlorophyll and 50 j,g of catalase were added to the 3-ml mixture. DPC (1.6 MAmoles) was employed where indicated. The open circles represent the reaction DPC to TCIPH2 in tris-treated chloroplasts. The closed circles represent the effect of PMA on the reaction water to TCIPH2 in control chloroplasts. Maximum rates of electron transport were 75 Omoles electrons per mg chlorophyll per hour for Mn'+ photooxidation (open squares), 187 ,umoles electrons per mg chlorophyll per hour for TCIP reduction from water, and 95 ,umoles electrons per mg chlorophyll per hour for TCIP reduction from DPC. All of these reactions were DCMU sensitive.
Plant Physiol. Vol.
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1972
379
PMA INHIBITION OF PHOTOSYNTHESIS
oxidizing side of photosystem II. These data also show that the PMA block is closer to the photoact than is the tris inhibition site. PMA pretreatment of chloroplasts followed by washing with sucrose-sodium chloride solution does not relieve the inhibition of photosystem II and causes a gradual loss in the ability of the chloroplasts to use hydroxylamine as an electron donor. A ratio of 1 jtmole of PMA to 300 jtg of chlorophyll is optimal for inhibition of oxygen evolution when attempting to substitute another donor for water. Table III presents the effect of donor systems on tris- and PMA-treated chloroplasts. DPC reverses inhibition of methyl viologen reduction caused by tris. This reversal is DCMU sensitive. DPC will not reverse the PMA inhibitory effect. Semicarbazide is a poor substitute for DPC in tris-washed chloroplasts. Hydroxylamine now acts like DPC when used for reversal of inhibition of PMA-pretreated plastid. There appears to be a time-dependent PMA inhibition phenomenon associated with hydroxylamine oxidation resulting in a much poorer reversal of PMA inhibition by hydroxylamine. Benzidine and MnCl2 are poor donors to both tris- and PMA-treated chloroplasts. The reduced aromatic donors such as hydroquinone or phenylenediamine appear to reverse both types of inhibition in a DCMU-sensitive fashion. Ascorbate alone is a poor donor to either tris- or PMA-treated chloroplasts. DISCUSSION Siegenthaler and Packer (13) reported that 10 ,uM PMA inhibits photophosphorylation as well as NADP and 2,6'-dichlorophenol indophenol reduction. Our findings indicate that while 10 /uM PMA inhibits photophosphorylation, it takes much higher concentrations of PMA (50 /uM) to inhibit NADP reduction. Even higher concentrations (200 ,uM) are required to inhibit photosystem II-mediated TCIP reduction. In 1960 Davenport and Hill (7) showed that 150 juM PMA gave 50% inhibition of methemoglobin photoreduction supported by ferredoxin. We find that metmyoglobin reduction is inhibited 50% at lower concentrations of PMA (25 [kM). The discrepancies between our data and Davenport and Hill's data probably arise from the use of different amounts of ferredoxin. Tagawa and Arnon (16) have shown that mercurials react stoichiometrically with pure spinach ferredoxin. We have further established that PMA inhibits at least three sites of photosynthetic electron transport. PMA at 33 [kM inhibits ferredoxin specifically and thereby inhibits NADP photoreduction. PMA at 33 /AM also inhibits metmyoglobin photoreduction, a reaction which requires only ferredoxin and not
Table III. Effect of Donior Systems Oii Methyl Viologeni Redulctiont by Tris- anzd PMA-treated Chloroplasts The method and reaction conditions for measuring methyl viologen reduction are described in "Materials and Methods". Chloroplasts representing 350,ug of chlorophyll were added to the reaction mixture prior to illumination. The ascorbate concentration was 0.2 mm where indicated. The PMA-chlorophyll ratio was 250 for this experiment. Preincubation time for both tris- and PMA-treated chloroplasts was 30 min. A control rate using water as a donor with fresh chloroplast was 100 Mmoles electrons/mg chl- hr. Rate of AMethyl V-iologen Reduction
PMA chloroplasts Tris chloroplasts Donor
Concn
I
+
Amnoles e-,'rng cll-hr
tmM
Water
Semicarbazide Diphenyl carbazide Hydroxylamine Benzidine Manganous chloride Hydroquinone-ascorbate Phenylenediamineascorbate Benzidine-ascorbate Ascorbate
3 0.5
14
3 0
11
-
30 0.06
23 18
9 9
11
0.1
10
0
0.3
41
20
23 38 105 200 20 36 110
0.03
93
50
138
0
0.06
26 12
9
76 15
18
0.2
10 10 15
29 0
23
the flavoprotein (6). Inhibition of diaphorase activity occurs at higher concentrations of PMA. Thus ferredoxin-NADP oxidoreductase is eliminated as a point of inhibition when using 33 ktM PMA to inhibit NADP photoreduction. Hiyama et al. (9) established that either ferredoxin or ferredoxin-NADP oxidoreductase of C. reinhardi could be inhibited by 100 ,uM PMA. We have confirmed their observations for spinach chloroplasts by measuring the effect of 100 /tM PMA on cytochrome c, turnover using dual wavelength spectrophotometry. The third site of inhibition by PMA is on the oxidizing side of photosystem II and occurs around 200 pLM. PMA inhibits TCIP and ferricyanide photoreduction and also inhibits Mn25 photooxidation. Yamashita and Butler (17) have established that high concentrations of tris buffer inhibit on the oxidizing Table II. Reversal of PMA anid Tris nl1hibitioni Methyl viologen reduction is described in "Materials and Meth- side of photosystem II and that various donors will re-estabods". The concentration of tris was 0.6 M. The concentration of lish electron flow by donating electrons past the tris block at PMA was 500 M. The control rate of electron transport was 89 a point closer to the photoact (see scheme below). Our data ,umoles e- 'mg chl hr. The reaction conditions were the same as indicate that PMA blocks photosystem II at a point between those described in "Materials and Methods.' Chloroplasts con- the tris block and the photoact. DPC will reverse the tris block taining 156 ug of chlorophyll were added for each experiment. but not PMA inhibition. Thus DPC must be oxidized at a Where indicated, DCMU was present at a concentration of 5 Am. point between the two inhibitory sites. Hydroquinone-ascorbate, phenylene diamine-ascorbate, and hydroxylamine (under the proper conditions) will reverse both PMA and tris inhibiRate of 02 Uptake tion. It appears that hydroquinone-ascorbate and other related Donor PMIA inhibition Tris inhibition donors are oxidized at a site closer to the photoact than are donors such as DPC or semicarbazide. It also appears that Mn2+ N o DCNMU N o DCMIU I DCMIU DCMIU donates electrons at a site further from the photoact than the tris or PMA block since tris or PMA inhibit Mn2+ photooxidajvncles e,/lmg cil- hr tion. The scheme (Fig. 5) represents our interpretation of the 21 20 30 12 H20 above data. PMA inhibition suggests the sequential relations of 32 42 107 159 NH20H two inhibitor sites and three donor sites on the oxidizing side of 32 0 126 18 DPC photosystem II.
380
HONEYCUTT AND KROGMANN HQ/Asc or
DPC NH20H
Mn
Mn ~Eb>2~~
TRIS PMA
I C yt % 54 ZZQ-kCyt.C.."P 700iQ
~FRS
Z
DCMU
-
Fd
FpP
NADP
PMA
FIG. 5. Inhibition of photosynthetic electron transport. Tris buffer prevents the use of electrons from manganous ions (Mn) and this block is circumvented by diphenyl carbazide (DPC). High concentrations of phenyl mercuric acetate (PMA) will block electron flow from both Mn and DPC but can be bypassed by using either hydroquinone and ascorbate (HQ/Asc) or hydroxylamine (NH20H) as electron donors to the photoact (Z -- Q). All of these reactions are inhibited by DCMU. Low concentrations of PMA block the activity of ferredoxin (Fd) and the ferredoxin-NADP oxidoreductase (Fp) which prevents the flow of electrons from cytochrome c574 (Cyt. c5zi4) and other carriers in the intermediary dark electron transport chain through P7o0, photosystem I and the ferredoxin reducing substance (FRS) to NADP. LITERATURE CITED 1. AiN\oN. P.I. 1949. Copper enzy-mes in isolated chloroplasts. PolyphenolAxidlase in Beta vulgaris. Plant Physiol. 24: 1-15. 2. AvRoN 'M.A.ND A. T. JAGEN DORF. 1956. A TPNH diaphor-ase from chloroplasts. Arch. Biochem. Biophys. 65: 475-490. 3. Avsrox-. MT.. A. T. JAGENDORF. AN-D M. EVANS. 1957. Photosynthetic phosphorIlasilon in a partially purifiecl system. Biochim. Biophys. Acta 26: 262269. 4. BEN--HAYYIMI. B. AND M. AvRON. 1970. MIn2+ as electron donor in isolated chloroplasts. Biochim. Biophys. Acta 205: 86-94. 5. BO6GER, P.. C. C. BLACK AND A. SAN PIETRO. 1966. Photosynthetic reactions with pyridine nucleotide analogs. Arch. Biochem. Biophys. 115: 35-43.
Plant Physiol. Vol. 49, 1972
6. DAVENPORT, H. E. 1963. The pathway of metmyoglobin and NADP reduction by illuminated chloroplasts. In: A. T. Jagendorf and B. Kok, eds., Photosynthetic Mechanisms in Green Plants. NAS-NRC publication 1145. pp. 278-283. 7. DAVEN-PORT, H. E. AND R. HILL. 1960. A protein from leaves catalyzing the reduction of haem-protein compounds by illuminated chloroplasts. Biochem. J. 74: 493-501. 8. EPEL, B. L. AND R. P. LEVINE. 1971. Mutant strains of Chlamydomonas reinhardi wvith lesions on the oxidizing side of photosystem II. Bicchim. Biophys. Acta 226: 154-160. 9. HIYAMA, T., M. NISHIMLRA, AND B. CHANCE. 1970. Energy and electron transfer systems of Chlamydomosias reinhardi. Plant Physiol. 46: 163-168. 10. KATOH, S. AND A. SAN PIETRO. 1968. Ascorbate supported NADP plhotoreduction by heatedl Euiglena chloroplasts. Arch. Biochem. Biophys. 122: 144-152. 11. KEISTER, D. AND A. SAN- PIETRO. 1963. The photoreduction of cytochrome c by chlsoloplasts. Arch. Biochem. Biophys. 103: 45-53. 12. KROGMANIN-. D. W. AN-D E. OLIVERO. 1962. The specificity of plastoquinone as a cofactor for photophosphorylation. J. Biol. Chem. 237: 3292-3295. 13. SIEGEN-THALER. P. AN-D L. PACKER. 1965. Light dependent volume changes and reactions in cliloroplasts. Action of alkenylsuccinic acid and phenylmercturie acetate and possible relation to mechanisms of stomatal control. Plant Plivsiol. 40: 785-791. 14. SNIITH. L. 1955. Cytochlromes a, al, a2, a3. Isi: S. P. Colosviek and N. 0. Kaplan, eds., 'Methods in Enzymology, V'ol. II. Academllic Press Inc., Newt York. pp. 732-740. 15. SuSOR, W. A. AND D. W. KROGNIANIN. 1966. Triphosphopyridine nucleoticle photoieduction with cell-free preparations of Anabaes?a variabilis. Biochim. Biophys. Acta 120: 65-72. 16. TAGAWA. K. AND D. I. ARNON-. 1965. Oxidation-reduction potentials and stoichiometry of electron transfer in ferredoxins. Biochim. Biophys. Acta 152: 602-613. 17. YAMASHITA. T. AN-D W. L. BUTLER. 1968. Photoreduction and photophosphorylation w-ith tris wvashed chloroplasts. Plant Physiol. 43: 1978-1986. 18. YAMASHITA. T. AND IV. L. BUTLER. 1968. Inhibition of chloroplasts by UIV irradiation and heat-treatment. Plant Physiol. 43: 2037-2040.
