INVESTIGATIONS ON PHOTOSYNTHESIS; THE HILL … Bound... · reduction of ferric oxalate to ferrous...

Philips Res. Rep. 9, 140-159, 1954R 240

INVESTIGATIONS ON PHOTOSYNTHESIS;THE HILL REACTION *)

by J. S. C. WESSELS - 541.144.7

SummaryThe photochemical reduction of various quinones and dyes byisolated chloroplasts is studied by measuring the redox potentialduring illumination. The experimental details are described inchapter 1.In chapter 11the possibility of the formation of free radicalsin the Hill reaction is discussed. Neither polymerization of acrylo-nitrile nor formation of phenol in the presence of benzene couldbe demonstrated. From this it is concluded that free radicals areeither not present or only present in very low concentrations inthe solution during this reaction. The results of an investigation intothe factors which are essential for the ability of a redox compoundto undergo photochemical reduction by illuminated chloroplastsuspensions are described in chapter Ill. Whether a redox substanceis able to serve as a Hill oxidant appears to be determined mainlyby its E~**). The maximal reducing power of chloroplasts in vitro is,to a high degree, dependent on the oxygenconcentration. In an oxygenatmosphere, final potentials lower than about 230 mV were neverattained whereas the lowest final potential observed upon exclusionof oxygen amounted to about 0 mV. Redox systems with a negativeEó value, the reduction ofwhich requires more energy than correspondsto one light quantum, are hardly reduced at all by chloroplasts,The theoretical and biochemical aspects of these results are discussed,particularly in connection with the fact that recent investigationsgive the impression that D.P.N. also is able to act as a substratein the Hill reaction. The primary formation of an intermediatehydrogen donor (XH2) is considered as probable, on the basis of whichthe following simplified scheme is proposed for the Hill reaction

h.X +H20 ~ XH2+ t02'

XH2 + quinone ~ X + hydroquinone,hydroquinone + t02 -* quinone + H20.

In chapter IV it is pointed out that some characteristic featuresof the reaction can be interpreted satisfactorily by this scheme.The discrepancies in, literature in the field of inhibitors induced aninvestigation on the influence of these substances upon the Hillreaction by means of the redox potentiometric method. The resultsof this investigation are described in, chapter V. The concentrationof various inhibitors which has an inhibitory effect is i.a. dependenton the nature of the redox compound used as a hydrogen acceptor.TIllS is caused by a difference in reoxidation rate of the reducedcomponents and sometimes by the occurrence of a reaction betweeninhibitor and substrate, o-PhenanthrÓline, 2,4.-dirJitrophenol,thymol,phenylurethane and hydroxylamine completely inhibit the Hillreaction at relatively low concentrations « 0·01 molar). Sodiumfluoride, sodium azide, iodoacetamide and potassium cyanide, onthe other hand, inhibit only at higher concentrations. The fact that

"') "Investigations into some aspects of the Hill reaction", Thesis, University of Lcyden,January, 1954.

**) For definition of Eó see first page of chapter I.

INVESTIGATIONS ON PHOTOSYNTHESIS; THE HILL REACTION 141

the reaction rate is not affected by p-chloromercuribenzoate and p-aminophenyldichlorarsine indicates that free sulphydryl groups arenot essential for the activity of the chloroplasts. Someantibiotics andother biochemically important compounds like oxidized glutathione,dehydroascorbic acid, riboflavin, D.P.N., A.T.P., and dihydroxy-phenylalanine, havc likewise no influence upon the Hill reaction,

RésuméLa rëduction photo-chimique de diverses quinones et matièrescolorantes par des chloroplastes isolés est ëtudiëe par la mesuredu potentiel rédox pendant l'iIIumination. Les dêtails expérimentauxsont exposés dans Ie chapitre I. Lc chapitre II traite Ia possibilitéde formation deradicaux libres lors de la réaction de Hill. L'additiond'acrylonitrile ou de bcnzène aumélange de rëaction ne permet pas dedëceler une polymérisation, respectivement une formation de phënol.On en conclut que dans cette réaction il n'existe pas deradicaux libres,ou s'il en' existe, ce n'est que dans une eoncentration extrèmementfaihle. Les résultats d'une recherche des facteurs qui déterminent si-une substance de rëdox peut être réduite ou non par des chloronlastessont mentionnés au chapitre lIl. IIs'avère que c'est principalementle E~ qui détermine si une telle substance convient comme oxydantde Hill. Le pouvoir réducteur maximum de chloroplastes in vitrodépend, en une mesure importante, de la concentration en oxygène.Dans une atmosphère d'oxygène, on n'obtient jamais des potentielsfinaux infërieurs à ~ 230 mV, tandis que Ie plus bas potentielfinal relevé lors de I'exclusion d'oxygène est de ~ ° mV. Lessystèmes rédox à valeur Eó nëgative, dont Ia réduction requiertnotablement plus d'énergie que celle correspondant à un quantumde lumière, ne sont pratiquement pas rëduits par les chloroplastes.Les aspects théoriques et biochimiques de ces rësultats sont discutës,spécialement en rapport avec Ie fait que des recherches récenteslaissent I'impression que le D.P.N. peut également agir comme substratdans Ia réaction de Hill. La formation primaire d'un dormeurd'hydrogène intermédiaire (XH2) est estimée probable, et sur cettebase, pour la réaction de Hill, le schêma simplifiësuivant est proposé:

hvX+ H20~XH2+ i02'

XH2 + quinone ~ X + hydroquinone ,hydroquinone + i02 -+ quinone + H20.

Le chapitre IVmentionne que lestraits charactéristiques de la rêactionpeuvent être interprétés d'une façonsatisfaisante à I'aide de ce schéma.L'absenee de coneordance dans les publications relatives aux in-hibiteurs a incité à étudier ces substances, par voie de déterminationdc potentiel rëdox, en ce qui concerne leur influence sur la réactionde HilI. Les résuItats de cette étude sont mentionnés au chapitre V.La concentration active de divers inhibiteurs est -dëterminëe entreautres par la nature de la substance rëdox utilisée comme accepteurd'hydrogène. La cause de ce fait doit être cherchée dans unc différenceen vitesse de réoxydation des composants rëduits et parfois dansI'existence d'une réaction entre I'inhibiteur et le substrat. 1'0-phénanthroline, le 2,4-dinitrophénol, le thymol, le phényluréthaneet l'hydroxylamine freinent complètement la réaction de HiII enconcentration relativement faible « 0,01 molaire). Le fluorurede sodium, l'azide de sodium, l'iodoacétamide et le cyanure depotassium, par contre, ne freincnt qu'aux fortes concentrations.Le fait que la vitesse de réaction n'est pas influencëepar le benzoatep-chloromercurique et par le p-aminophényldichloroarsine prouve queles groupes SH libres ne sont pas essentiels pour I'activité des chloro-plastes. Quelques antibiotiques et autres substances biochimiquesimportantes telles que Ie glutathion oxydé, l'acide dëhydroascorhique,

142 J. S. C. WESSELS

la riboflavine, le D.P.N., l'A.T.P. et le dihydroxyphénylalaninen'exercent pas non plus d'influence sur la réaction de Hill.

ZusammenfassungDie photochemische Rcduktion vcrschiedener Chinone und Farb-stoffe durch isolierte Chloroplaste wird durch Messung des Redox-potentials während der Belichtung untcrsucht. Die experimentellenEinzelheiten der MeBmethode werden in Kapitel I bcschricben.In Kapitel 11wird die Möglichkeit einer Bildung von freien Radikalenbei der Hill-Reaktion diskutiert. Wurde dem ReaktionsgcmischAcrylonitril oder Benzol zugesetzt, so war kcine Polymerisationbzw. Phenolbildung nachweisbar. Hieraus wird der SchluB gezogen,daB bei dieser Reaktion freie Radikale nicht oder nur in äuûerstgeringer Konsentration in der Lösung vorhanden sind. Die Ergebnisseeiner Untersuchung nach den Faktoren, welche bestimmend dafürsind, ob ein Redoxstoff durch Chloroplaste reduziert werden kannoder nicht, werden in Kapitel III mitgeteilt. Es zeigt sich; daB vor

• allem das Eó bestimmt, ob der betreffende Stoff sich dazu eignet,als Hill-Oxydant zu fungieren. Diemaximale reduzierende Kraft vonChloroplasten in vitro ist in beträchtlichem Mallevon der Sauerstoff-konzentration abhängig. In SauerstofIatmosphäre werden niemalsniedrigerc Endpotentiale als ca. 230 mV erreicht, wogcgen beiAusschlieBungvon Sauerstoff das niedrigste beobacht.eteEndpotentialca. 0 mV beträgt. Redoxsysteme mit einem negativen Eó-Wert,deren Reduktion erheblich mehr Energie erfordert, als einem Licht-quant entspricht, werden praktisch nicht durch Chloroplaste reduziert.Die theoretische und biochemische Bedeutung dieser Resultate wirdeingehend besprochen, speziell im Zusammenhang mit der Tatsache,daû in jüngster Zeit durchgeführte Untersuchungen den Eindruckhinterlassen, daû auch D.P.N. als Substrat bei der Hill-Reaktiondienen kann. Die primäre Bildung eines intermediären \Vasserstoff-donors (XH2) wird als wahrscheinlich erachtet und auf Grund davonfolgcndes vereinfachtcs Schema für die Hill-Reaktion vorgeschlagen:

h.X+ H20~XH2 +t02'

XH2 + Chinon ~ X + Hydrochinon,lIydrochinon + i02 ---?>- Chinon+ H20.

In Kapitel IV wird auseinandergesetzt, daB die Kinetik der Heaktionmit Hilfe diesesSchemas in befriedigender \Veiseinterpretiert werdenkann. Diemangelnde Einheitlichkeit der Auffassungenin der Literaturauf dem Gebiet dcr Inhibitoren bildete den Anlall, diese Stoffc aufredoxpotentiometrischem Weg hinsichtlich ihres Einflusses auf dieHill-Rcaktion zu untersuchcn. Die Ergebnisse dieser Untersuchungwerden in Kapitel V beschrieben. Die Konzentration verschiedenerInhibitoren, welchc einc hemmendeWirkung ausübt, ist u.a. abhängigvon der Art der Redoxverbindung, die als WasserstofIempfängerverwendet wird.· Dies ist auf Unterschiede in der Reoxydations-geschwindigkeit der reduzierten Komponenten zurückzuführen, undmanchmal auf das Auftrcten einer Reaktion zwischenInhibitor undSubstrat. o-Phenanthrolin, 2,4-Dillitrophenol, Thymol, Phenyluretanund Hydroxylamin hemmen die Hill-Reaktion vollständig bei relativniedrigen Konzentrationen « 0,01molar). Natriumfluorid, Natrium-azid, .Todacetamidund Kaliumcyanid hemmen dagegenerst bei höhe-ren Konzentrationen. Die Tatsache, daB die Rcaktionsgeschwindigkeitnicht durch p-Chlormercuribenzoat und p-Aminophcnyldichlorarsinbeeinfluût wird, weist darauf hin, daB freie Sulfhydrylgruppen nichtwcsentlich für die Aktivität dcr Chloroplaste sind. Einige Antibiotikaund andere biochemisch wichtige Verbindungcn, wie oxydiertesGlutathion, Dchydroascorbinsäure, Hiboflavin, D.P.N., A.T.P. undDihydroxyphenylalanin haben gleichfalls keinen EinfluB auf dicHill-Reaktion.

INVESTIGATIONS ON PHOTOSYNTHESIS; THE HILL REACTION

INTRODUCTION

The term "photosynthesis" is usually applied to the process by whichgreen plants are, under the influence of light, able to synthesize organiccompounds from CO2 and water. This could be said to he the mostfundamental reaction of life on earth. Although all living organismshave the capacity of synthesizing complicated organic molecules, theyare almost entirely dependent upon starting material which originated

. from photosynthesis. 'The overall result of the process of photosynthesis may be represented

by the equation

It involves an increase in free energy by about 120 kcal per mole ofreduced CO2• The annual accumulation of energy by photosynthesisamounts to 3.1018 kcal, which corresponds to the fixation of 3.1011 tonsof carbon in organic compounds. To illustrate more clearly what a yieldof 3.1011 tons per year means, we may compare it with the yearly worldproduction of the chemical, metallurgical and mining industries, whichis of the order of 109 tons 1).It is now generally agreed that the specific photochemical process of

photosynthesis may be described as a decomposition of water under theinfluence of light absorbed by chlorophyll. This photolysis of water involvesthe production of reducing compounds, which accomplish - directly orindirectly _.:._the reductive transformation of carbon dioxide to carbo-hydrates in a series of dark reactions. This idea is supported by manyexperimental data; we mention, for instance, the character of the CO2

uptake in the dark after illumination of algae 2), the fact that chemo-autotrophic bacteria are able to convert CO2 into organic materialwithout light energy 3) (in this process the required energy is furnishedby oxidation of inorganic substrates), fluorescence and redox potentialmeasurements 4),6),6), and the experiments with isolated chloroplasts 7).

The photochemical process of photosynthesis takes place in the chloro-plasts, the green granules in the cells containing chlorophyll. Chlorophyllis not evenly distributed in the chloroplasts; it is located in very smallgranular particles, called grana. Upon exposure to light chloroplasts are,in the presence of ferric oxalate, able to produce O2 under simultaneousreduction of ferric oxalate to ferrous oxalate ("Hill reaction"}, Thisphenomenon may be interpreted as a photolysis of water, ferric oxalatetaking the place of CO2 as hydrogen acceptor. The increase in free energy(LlF) of the reaction

2 Fe3+ + H20 -+ 2 Fe2+ + 2 H+ + t O2

143


amounts to 32 kcal so that actually light energy is converted into chemicalenergy.

Also in connection with the fact that several known carboxylases arelocated in the cytoplasm 8), it is now generally accepted that the reductiveincorporation of CO2 takes place in the cytoplasm (see also 9)). As thephotolytical splitting of water, however, takes place in the chloroplast,one may conclude that a photochemically formed reducing agent diffusesinto the cytoplasm. The question of whether the primary photochemicalprocess produces a relatively stable and highly reducing intermediate is,however, one of the most disputed problems in the field of photosynthesis.

Although the process of CO2 reduction (the path of carbon) is notyet elucidated, we have, largely through the work of Calvin and hiscoworker~ 10),11),12),13),14),obtained a much better insight into this matter.In these experiments green algae are allowed to fix C1402 in the light forshort periods, after which the cells are killed and extracted with alcohol. Theextract is analysed by paper chromatography for C14-labelled compounds.

After a short exposure to C1402 in the light radioactivity is found inalanine, serine, phosphoglyceric acid, hexose phosphate, etc., i.e. in inter-mediates of hexose and amino acid synthesis in general. After a Jongexposure lipoids, carbohydrates and proteins also appear to contain C14.A very short exposure to C14-labelled carbon dioxide (a few seconds)results in only phosphoglyceric acid, phosphopyruvic acid, and malic I

acid becomin~ active.Phosphoglyceric acid, the first active product, has to be considered '

as the primary stable photochemical intermediate. This conclusion wasarrived at only after a thorough experimental research and a highly criticaldiscussion of the results. So, of course, the fact has to be taken into accountthat the occurrence of a C14-containing compound in the light. and not !

in the dark indicates this compound to be a product, though not necessarilyan intermediate of photosynthesis.

The identity of a number of phosphate esters with those involvedin glycolysis suggests that the synthesis of the hexoses is accomplishedthrough a reversal of glycolysis. A support of this hypothesis is foundin the identity of the distribution of C14in the C3, C4, the C2, Cs and theCl' Caof hexose with that in the carboxyl, a and fJ carbon atoms, respective-ly of phosphoglyceric acid.

In the dark only a very small C1402 uptake takes place while in thiscase the active products are malic acid, fumaric acid, succinic acid, alanine,aspartic acid and glutamic acid. This is probably due to reversibility of thewell-known decarboxylation reactions of the respiration. On the other hand,when C1402 is supplied to algae in the dark immediately after a period ofillumination in the absence of carbon dioxide, the same products are found


as in short exposures in the light. Calvin, therefore, concludes that inthe dark after pre-illumination a photochemically produced, nonspecificreducing agent survives, which in. the dark also is able to reduce CO2,

The fact that phosphoglyceric acid and alanine appear to contain thelargest percentage of C14in the carboxyl groups, suggests that phospho-glyceric acid is formed by carboxylation of a C2 compound. A similar rea-soning holds for malic acid and aspartic acid, so that - also in connectionwith the observation that phosphopyruvic acid and malic acid successivelybecome active after phosphoglyceric acid in an early stage of the process -one may tentatively conclude that phosphoglyceric acid is converted intophosphopyruvic acid, which then is carboxylated as in the Wood-Werkmanreaction to give oxalacetic acid, from which malic acid may be formedby reduction.

According to Calvin the CO2 reduction process thus includes two carbox-ylations. First, an addition of CO2 to an unknown C2 compound, by whichphosphoglyceric acid is formed; secondly, the carboxylation of a Cacompound, probably phosphopyruvic acid, to oxalacetic acid. The questionremains which one is the C2 compound and how this compound is formedin the plant. In connection with the fact that oxalic acid, formaldehydeand formic acid have not been found labelled, and that C14appears onlylater on in glycolic acid and glycin, Calvin originally thought the splittingof a C4 dicarboxylic acid into two C2 molecules the most probable.The, hypothesis that during exposure to light a stable reducing inter-

mediate is formed, is disputed by Gaffron who, in contradietion to Calvin,observed that after preliminary illumination only phosphoglyceric acidis formed 15). Besides; according to the former, an after-effect, i.e. reductionof quinone, should be detectable also for the Hill reaction after pre-illumi-nation, which was not observed however. Gaffron suggests, inter alia,the following scheme for the CO2 redll;ction .

3C2H402 _ __.:_:3C",,0,--2 -- __ 6CH20H,CHOH,COOH

1.t (phosphoglyc.ric acid)

3trtos. l,2(H) (light)

t hexose _ ~trios~ , trios. -1 triose78172

The photochemical step is the reduction of phosphoglyceric acid to triose.Of the six triose molecules formed one is used for' the synthesis of cellconstituents, three are needed as hydrogen donors for the reductive,carboxylation of three acceptor molecules, and two are used to reproducethe three acceptor molecules. This relatively simple scheme thus impliesa strict specificity of the photochemical part which here, in contrast with

145


Calvin's scheme, is coupled only with one reduction step. The hydrogentransferring agent for the dark: reduction by triose, however, may be acoenzyme of the diphosphopyridine nucleotide (D.P.N.) type, playing apart also in many other redox reactions.The C4 dicarboxylic acids do not, according to Ga:£fron,participate in

the photosynthetic cycle but originate from secondary reactions. Thatmalic acid, at any rate, is no intermediate in photosynthesis ha~ beenconfirmed by Calvin, because he was able to show that inhibition· ofthe formation of malic acid by malonate does not change the activityof other intermediates 16). The recent identification of sedoheptulose andribulose as products of short period photosynthesis 17) has now broughtCalvin to the following scheme

(rlbulos<phosphate) (sec!Oh.ptulosephosphate). 78173

Investigators of the Berkeley and Chicago groups, however, agree thatthe first stable product of the CO2 fixation in photosynthesis is phospho-glyceric acid, which is formed by carboxylation of a C2 compound. Theorigin and identity of this C2 compound and the mechanism of itscarboxylation form some of the most important problems which arebeing submitted to investigation at the present time. In this connectionit is important to note that Fager 18) has obtained a cell-free spinachmacerate which appeared to be able to fix CO2 under phosphoglycericacid fo;mation.Extremely interesting are the observations by Vishniac and Ochoa 19),

Arnon 20) and Tolmach 21), proving that, exposed to the influence of light,chloroplasts + triphosphopyridine nucleotide + Mn2+ ions + "malic en-zyme" are able to carboxylate reductively pyruvic acid to malic acidaccording to the equation

chloróplasts. H20+T.P.N. . ~ T.P.N.2H+t02'

h1'

."malic enzyme"T.P.N.2H+pyruvicacid+C02 +'" malicacid+T.P.N.,

Mn2

H20 + CO2 + pyruvic acid -.--"" malic acid + t O2, •


Thus for the first time this is a realization of the photochemical reductiveCO2 fixation outside the living cell, so long looked for. The importanceof these investigations can hardly be overestimated although they donot imply that photosynthesis in vivo would proceed m: quite the same way.

Vishniac and Ochoa 22) found that also other enzyme systems likeisocitric acid, lactic acid and glutamic acid dehydrogenase, are able' tofunction in a manner analogous to that of the "malic enzyme" system.The carboxylation of the C2 carbon dioxide acceptor in photosynthesismight take place in a similar way.

Vishniac and Ochoa thus come to the conclusion that, upon exposureto light, chloroplasts are able to reduce T.P.N. (the E~ of both T.P.N.and D.P.N. is about ,- 0·30 V at pH 7,0), and that reduced T.P.N.is present in a quantity sufficient to effect the reductive carboxylationof pyruvic acid, which proceeds with an increase in free energy. Theyhave also succeeded in detecting the rednetion of D.P.N. by the formationoflactate when pyruvate, D.P.N. and lactic dehydrogenase were incubatedwith chloroplasts in the light.

As mentioned before, Hill was the first who observed the photochemicalreduction of ferric to ferrous salts by a chloroplast suspension '). Warburgand Lüttgens 23) found that p-benzoquinone, too, can be reduced by illumi-nated chloroplasts, whereas Aronoff 24) observed that at a high lightintensity the rate of O2 evolution in the Hill reaction decreases in theseries benzoquinone, K-naphthoquinone-sulfonate, Kvanthraquinone-sul-fonate. Holt and French 25) obtained good results also 'with some indo-phenols such as phenol-indophenol (E~ = 0·254 V at pH = 6·6), 2,6-dichlorophenol-indophenol (E~ = 0,24·7V), and o-cresol-indophenol (E~=0·217 V). With thionine (E~ = 0·074 V) the results were doubtful, whereasl-naphthol-2-sulfonate-indophenol (E~ = 0,147 V), methylene blue (E~ =0·024 V) and indigodisulfonate (E~ = -0·104 V) showed no reduction.From these experiments the impression is obtained that a simple relationbetween the redox potentialof a compound and its photochemical reductionby chloroplasts does not exist.

Besides manometry the following methods were used for the studyof the Hill reaction: photometry (especially with dyes; Holt, Smith andFrench 2.6)), measurement of the pH (Holt and French 27), Clendenningand Gorham 28)), and redox potentiometry (Spikes, Lumry, Eyring andWayrynen 2.9), Van der Veen 30)).

Mehler 31) showed that O2 also can act as hydrogen acceptor in theHill reaction, being itself reduced to H202• This may indicate that photo-autoxidations in vivo take place under the influence of O2, which thentakes the place of CO2 as .hydrogen acceptor in photosynthesis. Of thebiological redox compounds cytochrome c appeared to be reduced by

147


chloroplasts, but this was not the case with D.P.N.31). As mentionedbefore, Vishniac and Ochoa, Arnon and Tolmach, on the other hand,observed reduction of D.P.N. and T.P.N. by coupling with appropriateenzyme" systems. Quick reoxidation of the formed D.P.N.2H might bethe reason why no reduction could he demonstrated in systems containingonly chloroplast material and D.P.N.In connection with the foregoing it seemed very important to investigate

which factors are essential for the ability to undergo photochemicalreduction by chloroplasts. For this reason a number of quinones and dyeswas examined by the redox potentiometric method, and special attentionwas paid to the redox potential as a possible decisive factor 328). Also,the influence of some biochemically important co:rp.pounds like D.P.N.,A.T.P., dehydroascorbic acid, riboflavin, etc., upon the Hill reactionwas investigated. Mixtures of two quinones or dyes, too, were submittedto an investigation; besides, the kinetics of the Hill reaction were studiedmore closely 32b).

That the Hill reaction has, indeed, to be considered as the part of photo-synthesis containing the photochemical reaction in which the naturalH-acceptor is replaced by e.g. quinone, becomes clear from the fact that·the evolved oxygen actually originates from the water. Holt and French 33)were able to demonstrate this by means of 018. Clendenning and Ehrman-traut 34) found that the kinetic characteristics of the Hill reaction closelyresemble those of photosynthesis. Hill reaction and photosynthesis contain,therefore, the same primary photochemical process.

A further confirmation of this view is found in the fact that severalinhibitors of photosynthesis inhibit the Hill reaction as well, although theliterature enumerates some contradictory results. Hill and Scarisbrick 35)and Macdowall 38) observed that the Hill reaction is not inhibited bycyanide, which indicates that in photosynthesis cyanide is a specific poisonto the carboxylation process and does not affect the primary photochemicalprocess. Inhibition of the Hill reaction by azide and hydroxylamine isreported by Arnon and Whatley 37)and Macdowall êê), but not by Hill ê")and Aronoff 24). Clendenning and Gorham 28) found that sodium azideand hydroxylamine inhibit the photochemical reduction of ferricyanide-ferric oxalate and quinone to various degrees. The Hill reaction is inhibitedvery strongly by o-phenanthroline 23),24),37),38),Cu2+ and Hg2+ ions 38),and 2,4-dinitrophenoI38), less by narcotica like phenylurethane 24),36),38).

The difference of opinion on the active concentrations of various inhibi-tors ànd in some cases even on the question of whether a compound isan-Inhibitor or not, induced us to investigate these problems again bymeans of redox potential measurements. The .effect of some other knownenzyme poisons was also submitted to investigation.


Efforts to separate .reversihly some essential organic compoundfrom thechloroplast system, i.e.rto restore the activity by. mixing two' individuallyinactive fractions, were fruitless. However, a very fine disintegration ofchloroplast material could be effected whilst retaining activity 39),40),41 ),42),although 90% of the material still had a particle weight of 6-7 millionsand the activity was less than that of intact chloroplasts.

Concentration of the active material by salt precipitation, loweringof the pH, etc., was quite unsuccessful.. This suggests that the activityis due to the co-operating of several components in such a way thatpurification of anyone of these components automatically brings about adecrease of activity owing to the loss of other factors needed for the reaction.Isolated chloroplasts may be considered, in a sense, as intermediate

between the living cell and chlorophyll preparations. This material re-sembles the cell in its capacity to sensitize oxidation-reduction reactionsin which light energy is converted into chemical energy, and to use H20as hydrogen donor in these reactions. Chlorophyll has lost the latter propertybut is still able to sensitize oxidation-reduction reactions. This can bea "physical" sensitization, in which the photocatalyst has the functionof transferring energy only, or a "chemical" sensitization, in which thecatalyst undergoes a reversible oxidation-reduction.A reversible photoreduction of chlorophyll was observed by Krasnov-

sky 43),44),45),46). When exposed to light in pyridine solution, ascorbicacid, cystein, phenylhydrazine and H2S all give reduced chlorophyll withan absorption maximum at 525 m[L. In the dark reoxidation of the chloro-phyll takes place. He also found that chlorophyll, upon illumination, isable to transfer hydrogen from ascorbic acid to D.P.N. or riboflavin,which involves a considerable increase in free energy of the system. Herealso reduced chlorophyll with an absorption maximum at 525 ~[L acts asan active intermediate. It is reported 47),48), that chlorophyll can also beoxidized reversibly and that t~is reaction is affected by light.The great difference with the Hill reaction, however, is the fact that

chlorophyll in contrast to chloroplasts is no longer able to use H20 as 'H-donor for photochemical reductions. The way in which H20 is splitin photosynthesis still constitutes an unsolved problem. It seemed possible,in connection also with the investigations of Evans and Uri 49),50) andWeiss 51) on the photolysis of water by metal ions, that free radicalsmight function as intermediates in the Hill reaction. We have thereforeinvestigated whether (OH) radicals could be demonstrated in systemsin which the Hill reaction was actively proceeding.

In the following chapters we will deal successively with:Chapter I Methods and materials used for redox potential measure-

ments.


,,' II : Investigation into the occurrence of radicals as intermediatesin the Hill reaction.

" Ill: Relation between redox potential and photochemicalreduction by chloroplasts.

" IV: Kinetics of the Hill reaction.

" V : Inhibitors and activators.

REFERENCES

1) E. I. Rabinowitch, Photosynthesis and related processes, vol. I; InterseiencePublishers, New York, 1945.

2) 1\1. Calvin arid A. A. Benson, Science 107, 476, 1948.3) C. B. van Niel, Photosynthesis in plants, ed. by J. Franck and W. E. Loomis;

Iowa State College press, 1949.4) E. C. Wassink, Advanc. EnzymoI. XI, 91, 1951.5) E. C. Wassink, Leeuwenhoek Ned. Tijdschr. 12, 281, 1947.G) E. C. Wassink, Symp. Soc. expo BioI. V, 251, 1952.7) R. Hill, Nature, Lond. 139, 881, 1937.8) E. R. Waygood and K. A. Clendenning, Canad. J. Res. C 28, 673, 1950.9) A. W. Frenkel, Plant PhysioI. 16, 654, 1941.10) M. Calvin, J. chem. Educ. 26, 639, 1949.11) M. Calvin, J. A. Bassham and A. A. Benson, Fed. Proc. 9, 524, 1950.12) M. Calvin, J. A. Bassham, A. A. Benson, V. H. Lynch, C. OuelIct, L. Schou,

W. Stepka and N. E. Tolbert, Symp. Soc. expo BioI. V, 284., 1951.13) S. Kawaguchi, A. A. Benson, M. Calvin and P. M. Hayes, J. Amer. chem.

Soc. 74, 4477, 1952.14) M. Calvin and P. Massini, Experientia 8, 445, 1952.15) H. Gaffron, E. W. Fager and J. L. Rosenberg, Symp. Soc. expoBioI. V, 262, 1951.lG) J. A. Bassham, A. A. Benson and M. Calvin, J. bioI. Chem. 185, 781, 1950.17) A. A. Benson, J. A. Bassham and M. Calvin, J. Amer. chem. Soc. 73, 2970, 1951.18) E. W. Fager, Arch. Biochem. Biophys. 37, 5, 1952.19) W. Vishniac and S. Ochoa, Nature, Lond. 167, 768, 1951.20) D. J. Arnon, Nature, Lond. 167, 1008, 1951.21) L. J. 'I'o lm a ch, Nature, Lond. 167, 946, 1951.22) W. Vishniac and S. Ochoa, J. bioI. Chem. 195, 75, 1952.23) O. Warburg and W. Lüttgens, Naturwissenschaften 32, 161, 301, 1944.24) S. Aronoff, Plant Physiol. 21, 393, 1946.25) A. S. Holt and C. S. French, Arch. Biochcm,' 19, 368, 1948.20) A. S. Holt, R. F. Smith and C. S. French, Plant Physiol. 26, 164, 1951.27) A. S. Holt and C. S. French, Arch. Biochem. 9, 25, 1946.28) K. A. Clendenning and P. R. Gorham, Canad. J. Res. C 28, 78, 102, 114, 1950.29) J. D. Spikes, R. Lumry, H. Eyring and R. E. Wayrynen, Arch. Bioehem. 28,

4.8, 1950.30) R. van dcr Veen, unpublished.31) A. H. Mehler, Arch. Bioehem. Biophys. 33, 65, 1951.32a) J. S. C. Wessels and E. Havinga, Rec. Trav. chim. Pays-Bus 71,809,1952.32b) J. S. C. Wessels and E. Havinga, Rec. Trav. chim, Pays-Bas 72, 1076, 1953.33) A. S. Holt and C. S. French, Arch. Biochem. 19, 429, 1948.34) K. A. Clendenning and H. C. Ehrmantraut, Arch. Biochem. 29, 387, 1950.35) R. Hill, Proc. roy. Soc. B 127, 192, 1939.30) R. Hill and R. Scarisbrick, Proc. roy. Soc. B 129, 238, 1940.37) D. I. Arnon and F. R. Whatley, Arch. Bioehem. 23, 141, 1949.38) F. D. H. Macdowall, Plant PhysioI. 24, 462, 1949.

INVESTIGATIONS ON PHOTOSYNTHESIS; THE HiLL REACTION

30) H. W. Miln er, N. S. Lawrence and C. S. French, Science 111, 633, 1950.40) H. W. Milner, M. L. G. Koenig and N. S. Lawrence, Arch. Biochem. 28, 185,

1950. . . .'41) H. W. Milner, C.S. French, M.L. G. Koenig and N. S. Lawrence, Arch..Blochem.

28, 193, ,1950.42) C. S. French and H. W. Milner, Symp. Soc. expoBioI. V, 232, 1951.43) A. A. Kr as novsk y, C. R. Acad. Sci. U.R.S.S. 60, 42, 1948.44) A. A. Krasnovsky, C. R. Acad. Sci. U.R.S.S. 61, 91, 1948 .:45) A. A. Kr as novsk y and G. P. Brin, C. R. Acad. Sei. U.R.S.S. 67, 325, 1949.40) A. A. Krasnovsky and G. P. Brin, C. R. Acad. Sci. U.R.S.S. 73, 1239, 1950.47) D. Porret and E. Rabinowitch, Nature, Lond. 140, 321, 1937.48) E. Rabinowitch, Annu. Rev. PI. Physiol. 3, 229, 1952.40) M. G. Evans and N. Uri, Nature, Lond. 164, 404, 194·9.50) M. G. Evans and N. Uri, Symp. Soc. expo BioI. V, 130, 1951.51) J. Vleiss, Symp. Soc. expo BioI. V, 141, 1951.

CHAPTER I

METHODS AND MATERIALS. .USED FOR REDOX POTENTIAL MEASUREMENTS

1. Introduetion

If a chemically inert metallic electrode, such as platinum, is immersed ina solution of a reversible oxidation-reduction system, a potential differencewill be set up between the electrode and the solution. This potentialdifference appears to be dependent on the ratio of the concentrations(activities) of the oxidized and reduced forms of the system. The morehighly oxidized the redox substance is the higher will be the electrodepotential. At constant pH the redox potentialof the system

R~ Ox+ ne

is represented by the equation

I RT (Ox)E=Eo +-ln--,

nF (R)

in which (Ox) and (R) are the concentrations (activities) of the oxidizedand reduced forms respectively. The quantity E~.is a constant specific to agiven oxidation-reduction system, and is defined, as the potentialof thehalf-reduced system at the pH under consideration.If two' redox systems are present in the same solution, the system of

higher E~ will generally be in the reduced form for a greater percentagethan the system oflower E~.For the sake of brevity, systems of lower E~

151


are said to be oxidized by systems of higher E~.This reaction will proceeduntil equilibrium has been reached, that is to say, until both systems havearrived at the same potential. The equilibrium potential can be describedby the equation

As the reduced form defined above is often a base which takes upoprotonsto a certain extent in the system under consideration, the pH of the so-lution should usually he taken into account. The reduction of quinonesand of many dyes may be represented in a convenient way by an equationof the following type

in which the reduced form H2R is a (mostly weak) dibasic acid, charac-terized by two ionisation constauts. The redox potentialof such a reversil ..leoxidation-reduction system is given by the equation

Here (R) is the total concentration of the reduced form; Kl and K2 arethe ionisation constants. It is easy to see that if Kl and K2 are very smallwhen compared to the hydrogen ion concentration, the equation reduces to

RT (Ox) RTE=Eo+ 2pln (R) +pln(H+).

In this Eo is the normal (standard) potentialof the redox system underconsideration. Only in this simple case may the relationship between E~and Eo be represented by

I RT +u; = u,+ FIn (H ).

The photochemical splitting of water, in the presence of chloroplastsand a suitable redox substance as hydrogen acceptor, was achieved for thefirst time by Hill "). This "Hill reaction" which has been the most im-portant subject of study in our investigation, can be represented by theequation (benzoquinone has been chosen as an example of a hydrogenacceptor) .

H 0 + benzoquinone chloroplast •• ". '- hydroquinone + .i, 02 "7 2 2'

According to this equation it should be possible to follow the reaction bymeans of redox potential measurements. The advantage of this with


respect to other methods, such as manometrio. or photometric measure-ments, is that the reduction ofslightly soluble and uncoloured substancescan also be observed. The redox potential can still be measured when theredox substances are present in low concentrations (6.10-5 molar).

2. Isolation of the chloroplasts *)300 g of fresh lettuce or spinach leaves are disintegrated in a waring

bleudor for one minute in 250 ml of 0·025molar phosphate buffer (KH2P04-

NaOH) of pH 6·5, the latter containing 0·25 g of KCI per litre. Theobtained suspension is filtered through cloth and centrifuged for a veryshort period of time in order to remove fragments of cells. The chloroplastsare then separated by centrifuging the supernatant for at least 4.5 to 60minutes at 2000 g. The green sediment is washed twice by suspending inphosphate buffer followed by centrifugation. The washed chloroplasts aresuspended in 100 ml of phosphate buffer and then stored in the dark atabout 2°C. To obtain a highly active suspension all operations have tohe performed at a low temperature (0-3 °C).

3. Measurement of the redox potential

The redox potential was measured by means of a Philips potentiometer(GM 4491). Figs 1 and 2 give an impression ofthe arrangement used by us.The front and the back of the cuvette holder consist partially of glass

(drawn as a shaded area in fig. 1) and are provided with a. metallicshutter which is removed to start illumination. The temperature duringmeasurement is maintained at 16 to 17°C by cooling with tap water.

c

so

-

78138 5

Fig. 1. Cuvette holder (cross-section).L = sodium-vapour lamp,S = shutter,C = cuvette..

*) Here the indication chloroplasts does not only refer to-Jntact chloroplasts hut alsoto fragments of chloroplasts and grana. .

154 J. S. C. WESSELS'

In the cuvette, containing for instance 50.ml of a 6.10-5 molar 2,6-di-chlorophenol-indophenol solution in phosphate buffer (pH 6,5), a platinumand a saturated-calomel electrode are immersed. Pure, practically oxygen-free nitrogen is passed through the solution in order to stir as well as tocounteract the reoxidation of the reduced component. The influence ofoxygen' upon the Hill reaction will be described in detail in the chaptersIII and IV, but it is worth mentioning already here that photoreductionof many oxidants (for instance benzoquinone and 2,6-dichlorophenol-indophenol) occurs to a considerable percentage even when oxygen ispassed through.

~=I 1=~- -- - -_

~

potentiometer-

78139

Fig. 2. Cuvette with electrodes.Dimensions of the cuvette: 6·5 X 5·5 X 2 cm.

As soon as the redox potentialof the solution has become constant 2 mlof chloroplast suspension is added; this normally causes little or no changein potential. The solution is then exposed to the light of a Philips sodium-vapour lamp (SO 140W) by removing the shutter and the redox potentialdrop is observed until a constant level has been reached. In our experi-ments the distance between sodium-vapour lamp and cuvette amountedto 4 cm; this corresponds to a light intensity of 15000 lux.A characteristic example of the change of the redox potential as a func-

tion of time is the curve of 2,6-dichlorophenol-indophenol represented infig. 3. -The slope of the redox potential curve is determined by the activity

of the chloroplasts, the latter being strongly dependent on the season andon the age of the chloroplasts used. The rate of the photochemical rednetionof 2,6-dichlorophenol-indophenol by a 1, 2, 6, 8 and II days old chloro-plast suspension is shown in fig. 4.


. Gorham and Clendenning 2) reported that chloroplasts can be stored at-40°C for a whole year without distinct decrease of the photochemicalactivity. If chloroplasts are heated, however, at a temperature of 50°Cfor a few minutes the activity is lost completely. In this connection itseems probable that the decrease of activity during storage, representedin fig. 4, is caused by inactivation of one or more thermo-labile componentsessential for the Hill reaction.

£ in mV

I 330

310

addition of chloroplasts,290

270

250

230

78140 _ time in minutes

Fig. 3. Change of the redox potential as a function of time for 2,6.dichlorophenol.indophenol.

addition of ch/orop/as'sE in mV3301---....J..----.

310

290.....

270

250

7814120 25 3D 35 40 45

-~ time in minutes

Fig. 4. Change of the redox potentialof 2,6·dichlorophenol.indophenol by the samechloroplast suspension after 1 (-), 2 ( ), 6 (•.•.•... ), B(•..•..•.. ), and II ( ) days,

The .redox potential drop of 2,6-dichlorophenol-indophenol usuallyamounts to about 100 mV-, which in this case corresponds to nearlycomplete reduction. A redox potential drop of 100 mV may he caused, forinstance, by an increase of the percentage of the reduced form from 2%to 98%, as appears from the equation

155


L1E = E~ + 0·03 lo.g 928 - E~ - 0·03 log -if1f = 0·06 log 49 == 0·1 V.

The E~ then is half-way between the initial and final potentials, As isshown in fig. 3 this is actually the case for 2,6-dichlorophenol-indophenol.The E~ of this compound under the experimental conditions is given bythe inflexion point of the redox potential curve. Unfortunately, an in-floxion point is not found for every substrate so that in many cases aquantitative interpretation of the potential curve presents difficulties.As an example of a curve lacking an inflection point the redox potentialcurve for benzoquinone is represented in fig. 5.

oddi/ion of ehloroplos/s!

360

320

300

280

7814230 35 '0 '5

~ time in minutes

Fig. 5. Change of the redox potential as a function of time for benzoquinone.

Besides, the E~ values under the conditions of our experiments neednot be exactly equal to the literature data referring to more concentrated I

solutions. Thus in the case of benzoquinone, for instance, (E~ = 332 mVat pH 6·5 and at 16°C) a potential drop from about 380 to about 280 mVwould he expected. Our experience was, however, that at low concen-trations a too high redox potential is found with this compound. Thedeviation usually amounts to about 10 mV. Moreover, various platinumelectrodes give somewhat different values. For 2,6-dichlorophenol-indophe-nol, on the other hand, the deviation is at most a few mV whereas themutual differences between various platinum electrodes are much smallerthan for benzoquinone.

In table I the redox potentialof a quinhydrone solution in 0·025M phos-phate buffer is given as a function of the concentration. From this tableit appears that the redox potential rises on decreasing the concentrationof the quinhydrone while the deviations between the various platinum i


TABLE I

Redox potential (mV) of quinhydrone solution as a function of ,theconcentration (pH 6·5; nitrogen atmosphere) *)

Concentration of Platinum electrode 1 Platinum electrode 2quinhydrone

6.10-4 M 78 773.10-4 M 81 • 78

1.2.10-4 M 85 816.10-5 M 90 863.10-5 M 99 97

1,2.10-5 M 117 1116.10-6 M 124 123

*) The redox potentials are given with reference to the saturated-calomel electrode inthese tables.

electrodes become larger upon dilution. Enlargement or reduction of tbeplatinum area and purification of the nitrogen by leading the gas overheated copper gauze do not effect improvements (see table II). Whenany contact with atmospheric oxygen in these experiments was preventedby the use of a cuvette as represented in fig. 6 the results were not better.

The increase of the redox potential upon dilution of a quinhydronesolution is apparently not caused by oxidation of the system by traces ofoxygen, possibly still present in the nitrogen used.

No increase of the redox potential was observed polarographically at

oxygen-frnitrog~

potent;-e~

R ~ I1<fl"'t~

"'t

- - - ----- -

water trap

~.

ometer

78143

Fig. 6. Cuvette with electredes.


TABLE II

Redox potential (mV) of quinhydrone solution (pH 6·6).

Platinum electrode 1 I Platinum electrode 26.10-5 M quinhydrone; 86 82purified nitrogen . --

passed through.6.10-5M quinhydrone; 86

I

81 ,unpurified nitrogenpassed through

6.10-6M quinhydrone; 116 108purified nitrogenpassed through

6.10-6 M quinhydrone; 116 106unpurified nitrogenpassed through

6.10-6M quinhydrone; 154 128no nitrogen passedthrough

saturated quinhydrone 72 72solution (with or with-out passing through ofnitrogen)

the lower concentrations of quinhydrone (see table Ill); this phenomenonis undoubtedly due, therefore, to the platinum electrodes. The deviationof the redox potential from the literature data may well he attributed to along time of adjustment of the electrodes, since it had decreased afterseveral hours. (The readings, given in the tables, were taken when thepotential has become practically, although not completely constant, whichwas usually the case after about 30 minutes.) Biilmann and Jensen 3) havestated already that the quinhydrone electrode gives rise to an erroneouspH measurement if too small concentrations of quinhydrone are used.

In spite of these difficulties the application of the redox potentiometricmethod to the Hill reaction appears to lead to quite reproducible results.

lNVES'i'IGATIONS ON PHOTOSYNTHESIS; THE HILL REAL'TION 159

TABLE Ill.

Polarographically determined no~mál potential of benzoquinone (pH = 0;24°C)

Concentration s; (mV)of benzoquinone

5.10-4 M 475474

2,5.10-4 M 47610-4 M 472

4778.10-5 M 4696.10-5 M 462

The polarographical measurements were performed in phosphate buffer of p Tl 6'5,containing 0·05 molar KCI.

In order to compare reduction rates, however, it is recommendahle to usealways the same platinum electrodes. For a quarrtitative interpretationof the potential curve 2,6-dichlorophenol-indophenol is preferred tobenzoquinone, as only for the first-mentioned substance the E~ canreadily he determined from the curve itself, owing to the occurrence of aninflexion point. A further advantage of the use of this compound is thefact that not only the redox potential drop but also the decolorization ofthe dye indicates photochemical reduction.

(To be continued)

REFERENCES

1) R. Hill, Nature, Lond. 139, 881, 1937.2) P. R. Gorham and K. A: Clendenning, Canad. J. Res. C 28, 513, 1950.3) E. Biilmann and A. L. Jensen, Bull. Soc. chim. Fr: 41, 151, 1927.

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