Carbon Dioxide & the Hill Reaction'Norman E. Good 2

Research Institute, Canada Agriculture, University Sub Post Office, London, Ontario, Canada

Recently a i uniber of investigators have reportedthat the Hill reaction is mior-e ral)i(l in the plreeliceof CO., thiani in its absence. However., thle phenome-1on1 (it CO., dependence can only Ie (lemonstrated un-(ler certain conl(litioiis and it is thlerefore regrettablethat there hlas beenino agreement as to the nlattire ofthese cond(litions. \Warburg an(l Kiippahl ( 9) haxvestresse(l the iniportaince of preillumiinatinig the chlioro-pllasts. whereas Abeles et al. (1) foulid the effectprimarily in unbufferedl nedia. Stern anid Vennes-land, on the othier han(l, observe(l large CO., effectsin butfered niie(lia cand( without preillumination: inthieir stu(dies the composition of the buffer xvas imii-portant ( 7 ) \Ve have con fir-mie(d and extendledl theoblservations of the last-named authors. \\e haveailso invxestigate(l the relati,on of CO., elpendlence tolighit intensity, the state of uncouplilig of the plios-iPhorvlation system, ain(l the niattul-e of the Hill re-atctionl oxidant. The new information l)resenitedl her-eprovides little support for \Warburg's contenitioni thatCO, pllaYs a role in the Hill reaction atkin to its roleini pihotosynthesis. In fact it is inconsistent withsomile of the (letails of Warburg's p)ostulate(l miechiai-isin. Ho ever, the crucial (Iuestioln reniais Un-iswere(l: Is CO.) a metabolite in tile Hill reaction

(Or are We (lealiig with ind(lirect and possibly ulispecificeffects of the bicar-b-onate ion ?

Materials & Methods

(Illoroplasts vere isolated frolni voting pea plants(Pi'sum1 stivizum L. var. Alaska ) 1b the following

proce(ldure: Thle leaves xxwere g round ill a cliillediiortar with a buffer containing 135 g sucIose, 2.5 g'bovine serum adbumiin, and 0.05 milole tris (hN'droxv-iethl\l)miiethi-lglNcine-NatOH per liter, )H 7.5. The

lioniogenate was filtered thlroughi glass wvool inito ice-encaseld centi-ifuge tubes. It was tileni centrifugedait 4000 X q for 3 miinutes and the supernatant flui(ddistcardedl. The chloroplasts xvere resus.pended in thles;ame buffer., recenltr-ifuge(l, takemi tl) ill (a Smllall vol-tuinme of the buffer all(n store(d in anl ice bath. Simall.aliqIuots of tthe (lense suslemisiosi were ad(l(le(l to tllereaction Vessels.

Oxygen lpro(luction was nmeare(lau ediaiometricallyvin tand(lar(l Warl-urg vessels. P'otassiunm ferricva-

Receivedl Aug. 31, 1962.2 Present address: Department of Botainy anid Planit

Pathology, Michigan State lUnliversity, East Lansing,M ichi-aii.

iiide, if used, was a(l(le(l fromil the side a.rimi toxwardsthe end( of the 2 to 3 hour incubation pelrio(l. Thepreilncubatioll took lplace in the dim light of a shut-tere(l roolim. \hlite lighit frlomii six 300-xv incan(les-cenit r-eflector lamps xx as use(l (tlu-ing the reactionl.The intensity of the lighit, xvhich was iiot measured,fell appreciably sliort of saturaiting thle miiol-e activeHill reactions (see fig 5). The CO.) colicentrationiof the gas phase vas regulate(d 1y the addition ofCO., buffers to the center well. Tlle CO., pressuresin equilibrium wxith several of thle buffersx%vere mneas-ure(l \xvitlh a miiass spectrometer. These observedpressures agree(l reasonably x-ell xv ith the plressuresrel)orte(l by \Warburg anid Krippail (8) and \x-itlmthe l)pressures p)re(licte(l by the lax of mass action.

Because ferricvanide redluction in the FHill reac-tiOli leads to hvdrogeI ionl formation, a(le(qtIate CO.,buffering and pH bufferinig are essential if the re-actioli is to be folloved nianonietricallv. Ini order toiimake the CO., buffer capacity as large as possib)le.the CO., buffers wxere very concentrated (usuallyabout 2.0 m) a(ind 0.4 niil instea(l of the usual 0.2 nilxvas use(l. Spilling oxer of this larger x'olunie x-vasplrexenite(l 1v the use of extra large paper fans illthe center wells. Equilibration of tile CO., hufferwith the gas philse xvas catalyzed by the inlusioni of0.1 _m so(liuiii arsemiite. [Arsenite catalyzes the lh-dlratioll of CO., (6( ). In sonile experiments CO,piressue xwa5s miaintaine(l by- a 2.0 xi KHCO 2.0 Mso(diumii arsenite soltution milixedl with 1.0( m (diethaiaiol-aminie. This grave 0.6 c, CO., in the ga lphiase ani(lxveriy rmapi(l equilibration. \Wlien sucll plrecautionsxxere takeni. it xv-as possible to assXigil practically allof tihe pressure changes in the vessels to 0...

Simice there Nxvere somiie aniomlalies ili the reactionlof the chiloroplasts to thle available 2,6-dichiloroplhelnol-in(lopheiiol (DCPIP) purcliase(l frolii the EastmiialiOrganic Chemicals, it xvas suspectedl that the (hIvemiglit contain imlpiurities. Conse(quenitly it xas purl-fie(d in the folloxviigi manner: by caieftul regmulationof tile pH! the (lye xvas transferred repeatedly be-tweeni chilol-oforiii (aci(d fornil ) ami(l x ater ( salt form-).theni betxveen ether and xxater. TIle last traces ofether- xere remoxve(l fromi the filial xvater plhllse byaeration ani(l the sodliumli salt of thle (lye xvas precipi-tate(d by tile a(l(itioni of colitrolle(d aniouiits of NaC1.This saltinig out l)roce(lure xvas repeated. The pUri-fie(d material and the original sampimile had the samileoptical (denisity aiid tile sanime effects oil the Hill re-acti mll.

Tris(lh\d(r-oxniiietli\l )niiethvlgl-ciine ( tricinie) xwas

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GOOD-CARBON- DIOXIDE & HIILL REACTION2

prepared by reacting tris ( hvdroxymethyl ) amiiiomoieth-anie (tris) with nmonochloroacetic aci(d as dlescribe(delsewhere (3).

Results

Initertactionis of Bicarbonate with Oticr-A nions.The Hill reaction is influencedI very ilmuch by thepresence or absenice of a number of different aniions.

\\arburg first demlonstrate(l that chloride could beessential to the photochenmical activities of isolatedchloroplasts (11). Subse(ouentlv, it was showl thatclhlori(le interacts with other anionis in (liverse ways,somletimes stimiulating an(d sometimes inhibiting theHill reaction, anid that anionis other thaln chloridle re-

act in a sinmilar complex manner (3, 5). Consequent-ly it seemed that the dependlence of chloroplasts on

CO., (which is miiostly in the fornm of bicarbonate ionat the pH's used) miight be very much miiodified byother anions. Perhaps some other aniioIn could fuil-fill the function of bicarbonate in CO. (leplete(lcliloroplasts, thereby restoring the Hill reactioin po-

tential.The results of a large and heterogeneous assort-

menit of experimiients, in which chloroplasts were pre-

incubated for two to three hours witlh a variety ofanions or niiixtures of anions, nmay )e summarize(l

as follows:The smaller mionofunctional ionls such as formiate,

acetate, chloride. and fluoride, have the effect of in-creasing the dlepen(lence of the chloroplasts on CO..The saiiie ions are only slightl) inhibitory, if at all.when the CO., has not been removed. Fornmate andlacetate are effective at much lower concentratiolnsthan chloride: miiaxinmunm chloride enhancement of theeffect of CO., reilmoval is at concentrations wvhich are

osnmotically unfavorable (0.3-0.5 a), wlhile very sig-nificant acetate enhancenment can be detected at con-

centrations below 10-2 r. Furthermore, there issyVnergism aiiioing these anions. For instanlce, acetateand chloride together clepress the rate in the CO..depleted system more than wouldI be predicted fromlltheir separate activities. Figure 1 illustrates somleof the effects of chloride and chloride with acetate.

Glycolate anid mlalate are the only two ions wve

have yet founcd to have any action in relieving theinhibition caused by CO., removal. Perhaps thereis some significance in the fact that tlhey resemblebicarbonate in having a hy(lroxyl group close to thecarboxyl group. However, the stimulations cause(dby these two iolns are har(ly spectacular and (1o notnearly restore the full Hill reaction capacity observedin the presenice of CO... Thus in an acetate-chloridemedium the rate is often more than 90 % inllibitedlby the removal of CO_. Glycolate (10- 2 ) in-creases this very low rate, but by not mlor-e than40% instead of 1000%W.

Glycolate also can be powerfully inhibitory whenthe Hill reaction oxi(lant is DCPIP, wlhether or nCotCO., is present. The anions of two other hydlroxyacids with hydrox Is close to the carboxyl are always

inhliibitor, whlether or niot eitlher CO., or DCPIP ispresenit; salicvlate is strongly inhibitory at concentra-tions (loNvn to 10-3 AI, while lactate is slightly in-hiibitorv but only at iimuch higher concentrations.Predictably, many other anions are also indiscrimi-natelv inlhibitory, especially at high concentrations:io(li(le, arsenite, nitrate, fatt)y acids, etc.

A great many anions do not significantly affectthe Hill reaction under the conditions of our study.This groul) consists mainly of large or polyfunctionalion1s suchi as citrate, oxalate, miialonate, maleate,arseniate. phosplhate, anid pyrophosphate. It may heof somle interest that the bulky trimlethylacetate ionis inert although the slimmliler propionate andI butyrateion1s resemiible acetate in enllhancing the effect of CO.,removal. Substances with hydroxyls renmote fromthe carboxyl group, such as p-hydroxybenzoate andD,L-,8-hydroxybutyrate also hiave Ino effect on theHill reaction. The samiie is true of the zwitterionsglyNcine and tris ( hdroxymiletll) methylglycine.

It shoul(d be emlphasize(d that the above observa-

25

20-

oS-

EO deed'cfth ilrato.+O Pea Mhorolat

x~0

0~.-

IL~~~~~

Z.16°~in- 0.26 U Nodi0-

0 0IS2

TIME. MINUTES

FIG. 1. The inifluence of chloride and acetate on theCO2 depenidenice of the Hill reaction. Pea chloroplasts(126 gg chlorophyll) were incubated for 2'/2 hours at160 in 0.05 m phosphate buffer containing the indicatedconcentrations of sodium chloride alnd sodium acetateplus 1.35 X 10- moles DCPIP. Final pH 6.6, finalvolume 1.9 ml. The center wells of the vessels containedeither KOH or CO., buffer. The concenitration of CO.,in the gas phase in equilibrium with this CO, buffer (65parts 2.0 M KHCO3: 35 parts 2.0 M K2CO3) was ap-proximately 1.1 %. Twenty micromoles of potassiumferricyanide in 0.1 ml water were tipped into the vesselfrom the side arm after the incubation period and be-fore the period of illumination. The three lower curves

illustrate the progress of ferricyanide reduction in theabsence of CO2. The three upper curves are their coni-trols with CO9.

299

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PLANT PHYSIOLOGY

go-

+CO,

0:

':1""10I 30

ox. 40O-

^,; e 30\

20~~ ~ 2 8

INCUBATION TIME. MINUTES

FIG. 2. The dependence of the chloroplasts on exoge-nous CO, as a function of the time of incubation. Peachloroplasts (130 ,ug chlorophyll) were incubated at180 for the indicated times under air containing 0.0 or0.6 % CO., (KOH, or 50 parts 2.0 M KHCO3 and 50parts 2.0M KC03 containing 0.1 M sodium arsenite, inthe center wells). The reaction medium consisted ofphosphate (0.05 M), sodium chloride (0.2 M), and sodiumacetate (0.01 M) and contained 1.35 X 10-7 molesDCPIP. Final pH 6.55, final volume 1.9 ml. Twentymicromoles of potassium ferricyanide in 0.1 ml waterwere tipped into the vessel from the side arm immediatelybefore the period of illumination. The time of incuba-tion is taken as extending to the middle of the 15 minuteperiod of illumination.

tions are applicable to pea chloroplasts incubatedwith shaking for at least two hours, usually underconditions designed to give the largest CO. depletioneffects. Under more favorable conditions or withshorter times of exposure these anions might evokequite different responses. Perhaps the use of otherchloroplast sources and other preparative methodswould lead to different results. However, there isgood reason to believe that the spinach chloroplastsof Stern and Vennesland responded in the same wayto chloride. These authors found that the dependenceon CO., was greater with pyrophosphate buffer thanwith phosphate buffer (7). Since pyrophosphatebuffer is ordinarily made by adding HCI to sodiumpyrophosphate, their pyrophosphate buffer was al-most certainly inadvertently high in sodium chloride.

Preincubation & Preillumination of Chloroplksts.Although the removal of CO.2 causes some inhibitionin the shortest experimental periods consistent withconventional manometric techniques, a prolongedperiod of incubation under a CO, free atmosphere isnecessary to bring about a maximum reduction in theHill reaction rate (fig 2). The required time ofabout two hours is not shortened appreciably by con-tinuous illumination at high light intensities and nogreater CO2 dependence develops as a result of such

illumination. In these respects we cannot duplicatethe observations of Warburg and Krippahl (9).

Under our conditions of incubation carbonic an-

hydrase in the medium does not shorten the neces-

sary incubation period. Consequently the require-ment for a long preincubation probably reflects theslow dissociation of some CO2 or bicarbonate com-

plex, rather than the slow removal of the last tracesof bicarbonate from the medium.

Reversibility of Inhibition Caused by CO2 Re-moval. On first inspection the decline in Hill re-

action capacity shown by the CO2 depleted prep-

arations in figure 2 might be interpreted as a grad-ual, irreversible inactivation of the chloroplasts underthe influence of an unfavourable mixture of salts.Similarly the much smaller decline in the presence

of CO2 might be attributed to a protective effect ofbicarbonate ion through some kind of ion antago-nism. However, as Stern and Vennesland have re-

ported (7) and we have confirmed, the effects ofCO., removal are reversible if CO, is restored beforethe period of illumination. Therefore, the progres-

sive decline illustrated in figure 2 cannot be due toa process which damages the chloroplasts over thefull 2 hours. Nevertheless, it still might be arguedthat CO2 removal sensitizes the chloroplasts to lightinjury, and Warburg's claim that preillumination isnecessary to bring out the full effect of CO2 removalwould tend to support this argument. That such isnot the case can be seen from the experiment shownin figure 3. In this experiment CO. depleted chloro-

H O U R S2 5- n ARK

,,

35

MINUTESnl A D .

z _;v 2 0-

o-

z

es o

>- E_k0

- CO, THROUGHOUT

2 0 2 5 3 0

ILLUMINATION TIME. MINUTES

FIG. 3. Reversibility of the inhibitory effects of CO2removal. Pea chloroplast (130 ,og chlorophyll) were

preincubated and illuminated in the reaction mixturedescribed for figure 2. Some vessels (labeled + CO2throughout) were incubated and illuminated with a CO,buffer in the center wells (bicarbonate-arsenite-diethanol-amine as described in text). Other vessels were incu-bated with KOH in small removable cups. These cups

were left in the vessels (-CO2 throughout), removedand replaced by CO2 buffer after the incubation but be-fore illumination (CO2 added at zero time), or removedand replaced by CO2 buffer after 15 minutes of illumina-tion (CO2 added here). Solid lines represent the pres-

ence of CO2, broken lines its absence.

300

30-

I I"I,C,otZ/Z/-"

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GOOD-CARBON DIOXIDE & HILL REACTION

(5 - --- --ARSURG SKRIPP-- L

30

6loi * **---.---4. GOOD

0

° o -0-4STRO k VENNESLAN4

%C0. IN GAS PHASE

FIG. 4. The requirements of various Hill reactionsystems for CO,. The data of Stern and Vennesland(7) and Warburg and Krappahl (9) are replotted withour data using arbitrary scales designed to make thecurves coincide at high CO9 pressures. In all of theseexperiments the CO., concentration wNas established bybuffers containinig potassium bicarboniate and potassiumcarbonate. The concentrations of CO., are either takendirectly from the table provided by Warburg and Krip-pahl (8) or are computed from a constant derived fromthat table. Our reaction medium was the phosphate-chloride-acetate system described for figures 2 and 3,whereas the other data were obtained with media devoidof acetate and considerably lower in chloride.

l)lasts lost all ability to prodluce 02 after thev hadbeen illuminate(d in the presence of ferricvanide for afew minutes. Yet these chloroplasts recovere(l theirfull Hill reaction capacity when CO., as admIlitte(dto the gas phase.

Chloroplasts Actizvity as Fuinction of CO., Conl-centration1. The influence of various concentrationsof CO., on the rate of ferricyanide reduction is shownin figure 4. In this figure the data of WAarburg andKrippahl (9) for kohlrabi chloroplasts and the (lataof Stern and Vennesland (7) for spinaclh chloro-plasts have been replotted along with our data forpea chloroplasts. The data of these two groupsagree remarkably well, but we find a lower rate inthe absence of CO., and a somewhat higher require-ment for CO,. Both differences are probably relatedto the differences in the me(lia: the chloroplasts usedby us wvere exposed to acetate and high concentrationsof chloride. Perhaps acetate and chloride compete,with the bicarbonate ion for bicarbonate-dependentreaction sites on the chloroplasts.

Effects of pH & Buiffer Composition o07 CO., De-pendence. The chloroplasts used by Abeles et al.(1) exhibited a requirement for CO., only in un-buffered media where the pH was rather low (about6.0). From their observation one might be temptedto attribute a critical local buffering role to CO..Certainly around pH 6.0 the CO.,-carbonic acid-bi-carbonate shifts could provide the chloroplasts -with

a very small amount of a highly mobile hydrogen ionbuffer. However, the observations of Abeles et al.do not seem to be universally applicable. We usual-ly find that the CO, requirement is independent ofthe composition, concentration and pH of the bufferif the active small anions described above (acetate,chloridle. etc.) are avoidedl (see table I).

Table IEffects of Various Btffers & pH on CO.,

Dependence of the Hill ReactionPea chloroplasts (140 ,ug chlorophyll per vessel) were

incubated in semi-darkness in the presence or absenceof CO., (0.0 & 0.6 % in the gas phase) for 2 hrs at 180.The inicubation mixture contained the inidicated buffer,2 X 10--, moles NaCl, and 1.35 x 10- moles DCPIP.Potassium ferricyanide (2 x 10-5 moles in 0.1 ml HO)was added from the side arm after the incubation period.Final volume 2.0 ml. Phosphate buffer was preparedby mixing KH.,POv and Na,HPO4 solutions. Triciniebuffer was prepared by adding NaOH to tricinie.Tris buffer was prepared by adding H.SO4 to tris. Theinhibitory nature of tris buffers & the superiority of tri-cine buffers is emphasized by the long incubation period.

Rate of O., productionpH Buffer Buffer (,umoles/mg chljhr)concentration

-CO., +CO,

0.05 M 92 108phosphate 0.2 M 74 906.8 tricine 0.2 Ni 74 89

tris 0.2 i%f 3 6phosphate 0.2 Mr 39 49

7_5 tricie*0.05 M 74 897.5 tricine 0.2 NM 70 81

tris 0.05M 3 3tris 0.2 M 3 4

Nature of Oxi-dant and Degree of Uncouipling.Flavin reduction is much less sensitive to CO., re-moval than is the reduction of ferricyanide. (Seetable II.) This difference could be the expressionof a fundaimiental difference in the nature of the elec-tron transfer in the two types of Hill reaction. or itcould be simply the result of a profound difference inthe reaction conditions. We varied the conditionsof preincubation, using air or nitrogen in the gasphase and small amounts of ferricyanide or ascorbatein the incubation medium. Then the effect of CO,removal on ferricyanide and FMN reduction wvas dle-termined for each condition of preincubation. Theonly outcome of these experiments was the obser-vation that ascorbate under a nitrogen atnosphereimproves the stability of the chloroplasts somewhat.However, as has already been pointed out, the effectof CO., removal is fully reversible and therefore oneshould not expect to find critical differences asso-ciate(l wvith the incubation history of the chloroplasts.

40

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Table II

CO, Dependentcc of Hill Reaction as FIunction of Oxidant & Degree of UncoulplinigPea clhloroplasts (144 ,ug chlorophyll/vessel) were incubated for 2 hrs at 180 in semi-darkness, in a medium-l

coInsistiIng of phosphate (0.05 M), NaCl (0.20 sr), sodium acetate (0.01 i), pH 6.55. The celnter well containe(deither KOH or the potassium bicarbonate-sodium arsenite-diethanolamine buffer described in the text. Ferricyalnide(2.0 X 10- moles in 0.1 ml H,0) was added from the side arm after the incubation period. FM1N\ (2.5 X 10-moles) anid DCPIP (1.35 X 10- moles) were added before the inicubation perio(l. Final volumiie 2.0 mIl.

Reaction conditions

Rate of O. piroduction(,umoles 'iilg chlorophyll/hr)

-Co., + CO.,

Ferricyaniide as oxidant. Chloroplasts' electron transport system spontaneously &only partially uncoupled from phosphorylationFerricyanide as oxidant. Electron transport fullytuncoupled from phosphlorylationwith 0.045 -m methylaminieFerricyanide & DCPIP as oxi(lants. Reduction of DCPIP is niot coupled tophosphorylationFIIN as oxidanit. Electroni tranisport uncoupled from phosphorylation with 0.045 Mmethylamine*

12, 16

12, 13

10, 12

33, 44

61, 66

61, 69

-36, -35 -62, -(5

* Since the reduce(d flavini reacts with niolecular 0, to form hydrogen peroxide, the Hill reaction results in a netul)take of 0, equal to the amount of 0. actually produced. Catalase anid 2.0 % alcohol were added as a hydrogenl)eroxi(le trap) in tlle flavin system (4). The amounts of catalase an(d alcohlol use(d had inisignificanit effects onthe ferricyanii(le containiing systemus.

The pertinenit experiments ouldl be to test the actualre(luctioiif0 ferr-icyani(le in a reducinig ime(liunm andre(lucitioni of flavinis in an oxidizing mile(liumli. Suchexperiments a,ire not technically feasilile.

\When the Hill reaction is severely limlitedlbv theabsenice of CO.. the rate is not influience(d lb agentssucih a-s amines- and DCPIP wvhich unlcouple electrontr-aiispor-t fi-omil thle phosph-orylatio(n of ADP. Thlesinplilest interl)retation of this pheniolimenionl presuip-poses a liniear se(quenice of reactions wxith pllosphory-lationi anld the CO., senisitivxe reaction occurring atdifferent sta-ges. Thlts \x e mliust )ostullate that thephosphorvlation step is rate-limiiting- wx-lheni CO., ispresenit sinice w-e know- that uincoupleis of phosphory-latioln stimulate the overall r1ate. \Vre suggest thatthe overall rate in the absence of CO. is uneffectedby the preseince of uncoupler-s sinice the phosphoryla-tion stel) no longer presents a bottleneck: leakage byit is inuore tlhan suifficielnt to htandle the trickle of elec-trons g,ettinlg past the steli inhibited by CO., removal.If thisx interphretation is corl-ect, it followx that theprocess iniihibited by CO.. removal is not a reactionlwliich is directlv oi obligator ily couple(d to phos-plhor-vlationl.

(O. DTepenidecice & I ighlt Intensitv. As w-e haveseen, CO., influienices the Hill reaction rate when thelighlt intellsity is saturatin-g or nearly saturating.However. CO.. also influenices the amlount of lightrequired to aclhie-e saturation (see fig 5h).\henthe photochemical process (leternuiniies thle rate, theHill reaction rate ix imunclh less CO. (lepeni(lent. an(dmayvsx ell be completely in(lel)en(lent. If this is so.the process inihibited by CO., removal is neitlher- thepriimiary photochemical reaction nior any reactionNx hI clii i 1d1rectlhv affects the inaxinimim photochemiicalefficiency of the lTill reactioni.

°' 402

o./E 20

30C

E~~~~~200-0 8

LIGHT INTENSITYarb units )

FIc,. 5. Light intenisity and the CO., (lelien(ldence ofthe Hill reaction. Reaction conditionis were as in figures2, 3, and 4. The loxer light intensities xwere obtainedly wrappinig the vessels with increasing numibers of layersof permanganate dyed cheese-clotlh. The light trans-Inissioin of each multilayer was determiine( with a light-miieter while the cloth was immersed in water. The factthat the transimiissioni of light diminiislhed in a preciselylogarithmllic fashion with increasing numbers of layersxas taken as proof of the accuracy of the imieasuremelntsof relative inteinsities.

Discussion

In the conventiommal licture of p)hotosynthesis, CO.,fixatioin and redluctionl a-e thermochemical pirocesses

302 PLANT PHYSIOLOGY

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GOOD-CARBON- DIOXIDE & HILL REACTION

wlhich utilize TPNH and ATP to elaborate a varietyof reducednmetabolites, via reactions which are at

least analogous to the reversal of respiratory delev-drogenations and decarboxylations. This concept issupported by a wealth of evidence: Labeling experi-ments employing C1402 have shown that most of theinternmediates of photosynthetic CO, fixation are alsowell known respiratory intermediates; the intercon-versions of these interme(liates demonstrably requir-ed TPNH anid ATP: finally, illuminated chloroplastsreduce pyridine nucleotides while simultaneously

phosphorylatinig ADP to ATP. It is implicit in theconventional theory that the photochemical reductionof TPN or other oxidants by chloroplasts precedesand is fundamlentally independent of CO, metabolism.

Warburg does not agree that the oxido-reductionreactions of chloroplasts are, in fact, independlent ofCO, metabolism. Indeed it is almost impossible toreconcile the above widely accepted interpretation ofthe Hill reaction with WVarburg's schemiie of photo-synthesis, which postulates a photochemical process

yielding one molecule of 0., and consuming one

molecule of CO., per quantum followed by a thermo-chemical back-reaction consuming the greater partof the released 0. and releasing the greater part ofthe consumed CO.2. (2). Therefore Warburg pro-

poses the following radically different interpretationof the Hill reaction: the oxidant. CO,,, water, phos-

plhoric acid. and illuminated chlorophyll collaboratein somie mainner to form phosphorylpercarbonate.The phosphorylated peroxide of carbonic acid ishvdlrolysed and decomposed into oxygen, formic acid,an(d phosphoric acid and a second molecule of oxidantthen oxidizes the formic acid to CO,. Thus it isheld that CO., functions as a metabolite and yetcataly tically (9. 10).

As evidence in support of this complex and ex-

plicit new hypothesis, Warburg presents two re-

lated observations: A. the Hill reaction is inhibit-ed by the exlhaustive removal of COO, and B. theconcenltraitioni of CO., required for the Hill reactionis the samiie as that required for photosynthesis.While suclh evidence scarcely seems adequate as thesole exxperimental basis for an elaborate and detailedhypothesis. it is certainly legitimate to ask broaderquestions: Is CO., actually an intermediate in theHill reaction? If so, does it play the same role inthe Hill reaction as in photosynthesis? Or doesCO., hav-e, fortuitously, two unrelated functions inphotosynthesis. a catalytic or stabilizing function as

a cofactor in the oxido-reduction reactions of thechloroplasts anid a non-catalytic function as the ul-timate electron acceptor and the major substrate forautotrophic mletabolism? Before attempting to an-

swvAer these questions, we must consider Warburg'stwo pieces of evidence carefully.

The indispensibility of CO., cannot be establish-ed unless it can be shown that no other substancecan be substituted for CO2 and that the conditions re-

quired to produce CO., dependency are the conditionsnecessary for CO, removal or neutralization. No

systematic attenmpt to dlemonstrate either of thesepoints has been reported to date. But even if we

were to grant the indispensibility of CO. it by no

means follows that CO.2 must be a metabolite. Spe-cific ions are frequently necessary for the mainte-nance of structural integrity in biological systems

and for the activities of many enzymes. One mightreasonably expect that bicarbonate ions would have

some unique interactions with proteins; they have a

negative charge for ionic bonding to the numerous

positive sites on proteins juxtaposed to a hydroxylgroup available for hydrogen bonding to the even

more numerous carbonyl oxygens. In this connec-

tion it is interesting to recall that several otheranions with hydroxyls close to the carboxyl havesignificant effects on the Hill reaction.

WXarburg and Krippahl (9) have pointed out thatthe Hill reaction and photosynthesis require similarconcentrations of CO,, thereby inmplying a common

role for CO.,. Indeed, their data for photosynthesisby kohlrabi leaves and for ferricyanide reduction bykohlrabi chloroplasts are strikingly similar. How-ever, there is some doubt as to the significance ofthe comparison. When photosynthesis proceeds atan appreciable rate, the concentration of CO., at thesite of its utilization must be lower than at thesurface of the leaf. With decreasing external CO,concentrations this difference between the observedexternal CO.2 concentrations and the pertinent inter-nal concentration will become a larger and largerproportion of the total. Although Warburg has at-tempted to nminimize the complication introduced bydiffusion lags, using very low light intensities with a

consequent low rate of CO, uptake. there is still no

assurance that the concentration of CO., inside theleaves bore any predictable relation to the knownconcentration outside. Consequently, the fact thatthe processes in the chloroplasts and in the leavesseem to saturate at about the same level of externalCO.2 may not be meaningful. M1oreover, there isanother complication in interpreting this comparisonof the Hill reaction and photosynthesis. It is truethat the samie levels of CO., will be required tosaturate a common site whether it be in chloroplastsor in leaves, but saturation of photosynthesis by CO.,rarely indicates saturation of the CO2 binding en-

zyme. Ordinarily, photosyinthesis rates fail to in-crease with rising CO2 concentrations because some

reaction subsequent to CO2 bindling becomes saturat-ed. This is shown by the fact that the CO., satura-tion level of photosynthesis is usually a function oflight intensity. Indeed, it is very probable that thesimilarity of CO., requiremlents notedl by WVarburgand Krippahl applies only to the light intensitiesthey chose.

Considering the primitive nature of all our ideasconcerning the processes going on in illuminatedchloroplasts. how should we martial the argumentsfor and against W;'arburg's conception of the roleof CO2 in the Hill reaction? No single argumentis conclusive and, assemnbled, the arguiments are not

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4PLANT PHYSIOIOGY

very colvliiicing on eitller side. Probably the bestreason for countenancinig interpretations of the kind(put forwar(d by \VXlarburg is a proper abhorrence ofrelving on explaliationis whici itnvoke coilici(lentalrelationships. CertailyIbelievinig that CO.has twotinrelated ftuiictiolis ini photosvuthesi s-or onle func-tion in photosynthesis anld another trivial funictionin isolated chlioroplasts-p-resents uxs with a truly(lisconcerting coincidlence. Tn adl(lition. solime of themiiore oblvious explalaatiolns xvIhich w-oul(l assign atr-iv-ial function to CO in the Hill reactioni fail toaicconmmodate the fact thlat the effect of CO., removalis completely reversible. Howvever. there ar-e alsogocrod reasonis for n1ot vet acceptilng \Warburg's l)ic-tul-e of the Hill reactio.li The fact tilat CO. (lelen(-enice seemiis to be corr-elate(d ith the presence of small

oiions imlilies that bicarbolnate ion is the importanltsubstailce, not CO., If thiis is so, the phellomielioresembles ioln antagoniism. lnot l)hotosvlithetic C(Ollletabolismll. Schemes suchi as \Warburg's. wh-lichipostulate the reduction-i of CO., followed 1w its re-oxi(lationi bv tile Hill reaction oxi(llnt, predict equwlo1- greater CO., (lepell(lellce wheni the oxi(lant is aweak oxidant suchi as FMA\N: actualiFMv re-Iluctioni is very miluchi less (lepeni(lenit on the pr-eseniceof CO.. thaii is ferricvanide re(luction. The specificsccheme lut forward by W\arbur- et al. (10) suiggetsthait a photochemical reaction is the process wvhichiis inhibited 1w CO., rem.,wval: on this noint the autithor,sari-e expldicit "Dabei iiiiissen alle Z\viischenprio(luktemit (ler Chlorophyll verbuilnden sei ." Yet wvehave seen tlhat the plhotochemical efficiency of theHill reaction islprobablyinot impaired 1y CO., 1re-miioval. Finally. 'Warburg w-outild have us believethat photophosphorvlation is initimately associateclw ith CO., imletabolisimi. OUi (lata stluggest that theeffect of CO., removal inivolves a reaction remotefr'om01 phosphorvlation.

Summary

1. Pea chloroplasts incubated for twN-o to threellouIIrs Uni(lelr a CO.. fr-ee atillosl)llere may lose tllegreater part of theil- Hill rieaction caplacitv.

Tl. This loss of Hill reactioll capacity inl bicar-boilate-depleted chloroplast suspenision is correlatedl\with the presence of othler slmlall aanions. Fairlylyiglhconcenitrations of formliate, acetate. fluoridle. orclhlori(le miiust be pIresenit before removal of CO.. re-suilts in a major reduction in the activity of thechl oroplasts.

TTI. Phosphate. p)yroplhosphate. arselnate, citratemaleate. malonate, tri metlhvlacetate. l)-hy(lroxyhben-zoate, glyci le. and tri S ( hvd(roxvm-ntheyl ) mliethvlglv cine(tricine) (1o not affect the Hill reactioni riate. eitherini the presence or absence of CO._.

TI. Aniolls of alpha- anIld orthohvdroxv acidlssuch as glvcolate aiml salicvlate have a variety ofeffects. usllyv inllibitiing the Tlill reaction whether

or not CO, is present but somietimiies partly relievnllgthe inhibitory effects of CO.. removal.

V. The effect of CO, removal is collpl)letelvreversible. Chloroplasts v hiclh have been incuiebatedwith chlori(le acnd( acetate in the a.bsence of CO._ theniillumlinated with ferricVa di(le, rapidly lose all abilityto pro(luce oxygen. \Vhlien suclh inactive chloroplastsare exposel to CO. they fully recover their -Jill re-aIction0 caplacitv.

V 1 FFerricy-anidle reductioni is mlucl mlore de-pend(leit oln the presence of CO., than is the re(ductionof FMTN.

VII. There is evidence whichl suggests that thereaction inhliibite(d 1b CO., renloval is ineither a plhoto-chemical reaction nor a reaction which is (lirectlycoouple(d to phosphoryl ation.

AcknowledgmentsThis work was ma(le possible by the excellent tech-

niical assistance of 'Mr. S. T. Bajuira. I would also liketo thank Drs. F. B. Aheles and B. C. Mfavne for deter-miininig the equilibrium carbon dioxi(le pressuires of theCO., buiffers ilse(l.

Literature Cited1. A\EILES, F. B., A. H. BROW N, & B3. C. IMAYNE.

1961. Stimulationi of the Hill reaction bv carbondioxi(le. Plant Phvsiol. 36: 202-07.

2. BURNK I). & 0. W\ARBURG. 1950. 1-Quialnten-Mechanismius und Energie-Klreisprozcss bei (lerPhotosvnthese. Naturwissenschaften 37: 560.

3. (,cK)X), N. . 196,2. Uncoupling of the Hill reactionfrolm photophosphorylation by anions. .\rch. Bio-chemili. Biophvs. 96 653-61.

4. GOOD, 'N. & R. HIm.I 1955. Pliotochem11ical re(Ihuc-tioIn of oxygen in chloroplast p)reparations. II.MIechanlisms of the reaction with oxygen. Arch.Biochemn. Bioplhs. 57: 355-66.

s. (;OR11AMI, P. R. & K. A. (GL.EL)Ex-N\N(. 1951.Anionic stitmutilationi of the Hill reactioin in isolatedchloroplasts. .\rch. Biochemi. Biophys. .7199-223.

6. RO(7(;11TNo, F. S. WV. & A. 'M. CI.\RK. 1951. In:The Enzymes, J. B. Sumnler & K. Mv rbfck, eds.Aca(lemiic Press, New York. V'ol. 1. 1) 125(4.

7. STFRN, B. K. & B. VEXNE.SIAND). 1962. The effectof carboIn dioxi(le On the Hill reactioln. J. Biol.Chemt1. 237: 596-(02.

8. WARBURG, 0. & ('. KRIPP1AII.L. 1960. X citeren-twicklung der imianotmetrischen 'Methoden, (Carbon-atgemisclhe). Z. Naturforschg. 15b: 364-67.

9. WARBURG, 0. & G. KRIPPAHI. 1960. Notwendig-keit (ler Kohlenlsajire fuir (lie Chiinoni- und Ferri-cyaniid-Reactioineln in grunen (rana. /. Natuirfor-sclhg. 15b: 367-69.

10. WNARBURG, O., G. KRI1PAHL, H. S. GE\vIT/. & XV.V6LKER. 1959. tlber den chemiiischeni Mechanis-nntis der Photosynlthese. 7. Naturforschg. 14h:712-24.

11. \VNARI31 RG, 0. & WX. LlUTTGENS. 1944. Experillmentzur Assimilation (ler Kohlenisaiire. Naturwissen-schiaften 32: 161.

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