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Plant Physiol. (1966) 41, 937-945

Photoreduction of Pyridine Nucleotide by SubcellularPreparations from Rhodopseudomonas spheroides1Joseph A. Orlando, Dennis Sabo, and Constance Curnyn

Department of Biology, Boston College, Chestnut Hill 67, MassachusettsReceived January 24, 1966.

Summary. We have investigated the photoreduction of pyridine nucleotides by crudeextracts and ehromatophores of Rhodopseuidomonias spheroides.

Our findings are as follows:NADP is preferentially photoreduced by crude extracts (3/7,000 X g supernatant

fraction) and there is no requirement for the addition of exogenous substrates. Crudeextracts also catalyze a nonphotosensitive reduction of NAD.

NADP photoreduction is completely inhibited if an NADH trapping system is pres-ent and indicates that NADH is required for NADP photoreduction.

Washed chromatophores (150,000 X g pellet) do not catalyze NADP photoreductionunless the supernatant fraction is added. The restoring effect of supernatant fractionis lost upon boiling and dialysis. However, supernatant materials can be replaced byan NADH generating system. There is no requirement for anaerobic conditions.

Evidence has been presented which suggests that 1?hodopseudoanonas spheroides con-tains an energy-link-ed transhydrogenase that can be driven by a high energy intermediategenerated by light or ATP. This intermediate may also be functional in ATP synthesis.The synthesis of ATP and the ATP-supported transhydrogenase is inhibited by oligomy-cin. This inhibitor does not affect the light-mediated reaction.

The light-dependent reduction of pyridine nucleo-tides by cell-free preparations of photosynthetic bac-teria is well established. Frenkel (6, 7) has (lem-onstrated that Rhodospirillurm ruibruti chromatophorescatalyze the photoreduction of NAD if FMNH. orsuccinate is added. These res,ults have been con-firmed and extended by other researchers (11, 18. 19.20). Recently, Hood (8) showed that a small par-ticle preparation from Chromiiatiumii suipports a suc-cinate-dependent photoreduction of NAD. In ex-periments reported thus far, there is little indicationithat photosynthetic bacteria photoreduce NADP.Nozaki et al. (12) have shown that NADP is photo-reduced by chromatophores of Rhodospirilluinii rubrumn,however, only if catalytic amounts of NAD are pres-ent. They suggest that NAD is photoreduced andNADPH is formed via transhydrogenase. This in-terpretation may also explain the photoreduction ofNADP 'by Rhodospirillmn ruibrumiii reported by Ver-non in an earlier communication (20). Recently,Yamanaka and Kamen (22) demonstratecd that NADand NADP are reduced nonphotosynthetically by fer-redoxin and NADP-reductase of Rhodpscudonionaspaluistris. They 'found that NADP was a better elec-tron acceptor than NAD and suggest that NADP

1 This iinvestigation was supported by a research granit(NSF-GB 3908) from the National Science Foundation.

m'ay be the electron acceptor in photoreduction inthis organism.We have investigated the photoreduction of pyri-

dine nucleotide by cell-free preparations of Rhodo-pseuidomiionas sphcroides, and in this paper we reporta light-dependent reduction of NADP.

Experimental

Six-liter batches of Rhodopseuidornonas spheroideswere cultured in 2-liter Roux culture bottles in Hut-ner media modified according to Cohen-Bazire et al.(4). After inoculation, the bottles were filled withsterile media and illuminated from both sides by 2banks of 40-w showcase laimps with an intensity of200 ft-'c. Cells were grown at a temperature of 300and harvested during the logarithmic phase of growth(48-52 hr). After washing twice with 0.1 M Tris-HCl buffer-10 % sucrose (pH 8.0), the cells weresuspended in the same buffer (1-5, w/v) and dis-rupted in a pre-cooled French Pressure CdllI at 20,000psi. All operations were 'conducted at 0 to 20 unlessotherwise stated. The broken cell suspension wascentrifuged at 18,000 X g for 15 minutes and the re-sultant suipernatant material was recentrifuged at37,000 X g for 1 hour. The heavily pigmented 37.000X g supernatant fraction is referred to as crudeextract. Chromatophores were prepared by centri-fuging the crude extract at 150,000 X g for 90 min-

937

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

PLANT PHYSIOLOGY

utes. The pellet was washed twvice with the Tris-HCl-sucrose buffer, then resuspended in a smallvolume of the same buffer. T'he supernatant frac-tion was also recentrifuged at 150,000 X g to removesmall chlorophyll-containing particles. All fractionswere stored in argon-filled Thunberg tubes in dark;ness at 0 to 20. Bacteriochlorophyll concentrationswere determined by the method of van Niel andArnold (17).

The photoreduction of pyridine nucleo,tide wasfollowed by measuring the increase in CD at 340 mu.with a Beckman DU spectrophoton;.eter. Reactionswere carried otit anaerobically in Thunberg-typecuvettes in a waiter bath mlaintailled at 300, and illum-ination was from a single photoflood lamp at 1200ft-c. The standard reaction mixture contained thefollowing components in the main chamber of thecuvette: 50 ug bacteriochlorophyll; 100 xmoles Tris-sucrose buffer, pH 8.0; 10 Au'moles MIgCl.. The sidearm contained 5 junmoles of NADP and the final vol-ume was brought to 3 ml with distilled water. Toeffect anaerobic conditions, Thunberg cuvettes weresubjected to prolonlged evacuation uintil Ino bubblingoccurred when the tubes were agitated. The reactionmixtures became extrenmely turbid with excessivefrothing. Thus, care was taken to keep frothing at amninimum. Evacuated tubes were filled with argonand the cycle was repeated 5 times. At 7ero time,the contents of both chamibers were mixed and theOD at 340 mIt was recorded against an appropriateblank. Reaction mixtures were then illuminiated andOD measurements were recor(led at (!esignated timeintervals. Each light reactioin was comlpared againistan identical dark coiitrol. The experimental proced-tire was the same for aerobic experimeints and it wasconvenient to use open Thunberg cuvettes. ,]-LI-droxybutyrate reductase was assayed according toCarr and Lascalles (3). Transhydlrogenase was as-sayed by the method of Kaplan et al. (9), with theexception that NADPH formationi was moniitored at340 m)u by addinig GSSG and glutathione re(luctaseinstead of the NADP-specific cytochrome c reductaseemployed by the authors cited. Phosphorus was de-termined by the imietlhod of Tausskv andl Shorr (16).

Yeast alcohol dehydroggenase. lactic dehyclrogenase.glutathione reduictase anld GSSG were obtainled from,-the Boehringer Mannheim Corporation, New York.The oxidized and reduced forms of NAD anId NNADP,ATP an(d thyroxinie were products of Calbiochem,Los Angeles, California, aind quiiiacrilne-HCI. anti-mycin A an(d 2-N-heptvl-4-lhydroxy-quinioline-AN-oxideand oligomnycin were obtained from the Sigma Chem-ical Compaly, St. Louis, Mlissouri. All other re-agents were con.mmercial preparations.

Results

The investigation of pyridiine lnucleotidle photore-cluction in Rllodopsicidoiiioiias splier-oi(cs wxvas prOiMnpt-ed by the report that this organismn has the capacitN

to catalyze numerous dye-linked photooxidations anidreductions but does not photore(luce NAI) (21). \Vrefound this to be truie dIuring the early phases of thiswork, and attempts to demonstrate succinate-depeln-dent pyridine nucleotide photoreduction were uni-successful. However, we noted that oln some occa-sionis, cell-free preparations of Rl odopseuldo;iioui(asspleroides could photoredluce py-ridliine inucleoti(le.It seemed possible that the inconsistancv could havebeen due to lack of strinigent control of growth con-ditions and culture age. Thus, a series of experi-ments was performed to investigate the effect of cul-ture age on the ability of extracts to photoreducepyridine nucleotide. Onie liter of culture was pre-pared photosynthetically, andcl durinig middIle lo, phasegrowth (ca. 35 hr) a volutne of inioculumii wras re-moved and an idlentical amount was added to a seriesof 2-liter culture bottles containing sterile liledia.The growth rate under photosynthetic coniditionis wasidentical in each case. At designiate(d timiies tlle cul-tures were harvested alnd crude extracts prepared asdescribed earlier. The crude extracts were assayed forpyridine ntucleotidle photoredluctioni activity anid theresults presenited in table I were obtained. There is

Table I. Effect of Cutltuire- . e on Photoreductioni ofPi-ridinc .ctc/cotidc

Each cutvette conitainied cru(le extract that lhad beenprepared from cells of x-arious ages as inidicatecl. Re-actioni mixttires conitaine(d 50 t,g bacteriochlorophl 11 anid thefollowing in ,umoles: Tris-sucrose buffer (I)H 8.0), 100:MIgCl,, 10; pyridinie niucleotide, 5. The final volumelesw as brougllt to 3 nml -,vith distilled v, -acer and the reactionw as carried out at 300 unider aniaerobic coniditionis. Thereductioni rates are (Ine to eindogelnouis substrates alonie.

A OD 340 in,u in 20 mili

Culture age

38 hrsNADNADP

48 lhrsNADNADP

72 hrsNADNADP

132 lhrsNADNADP

Li',-ht

0.4400.490

0.3100.345

0.2650320

0.215(.195

Dark Liglt milll-s dark

0.3750.130

0.2900.105

0.2500.200

0.2050.125

0.0650.360

0.0200.240

0.0150.120

0.0100.070

a significalnt din.inishiing effect of cuiltuire age olnpyridiine nucleotide photoreduction ancd log plhase cells(38-48 lhr) possess the highlest activity. Hoxvever,the molst strikinlg observation is that NAD)P is a muchbetter electron accepitor in pli)otoredtictioln than N AL).With preparations fromii 38-hour cells, a light-imus-(lark absorbalncv differelnce of 0.36 is readily obtainedafter 20 minutites wlheni NAN)P is the electroln acceptor.

938


ORLANDO AND SABO-PYRIDINE NUCLEOTIDE PHOTOREDUCTION

This photoreduotion rate is comparable to the ratesof succinate-dependent NAD reduction demonstratedin Rhodospirilluiin ruibrutm (18). On the other hanid,light-minus-dark differences obtained with NAD(table I) are too low to be considered as the majorphotoreduction pathway in this organism. Althoughrates diminish with culture age, the preferential pho-toreduction of NADP is maintained.

In order to deimonstrate that the data in table Irepresented the reduction of the pyridine nucleotideindicated, the experiments were repeated, and at theend of the incubation time, an appropriafte pyridinenucleotide trapping system was added. In figure 1,results are presented of a typical experiment withNAD as the electron acceptor. After 15-minutesincubation there is a significant reduction, but noappreciable light-minus-dark difference. At the timeindicated by the arrow, 0.05 mg lactic dehydrogenaseand 2 umoles of sodium pyruvate were added to themain chamber of the Thunberg cuvette. There wasan immediate drop in absorbance. This NADH trap-

0O

0,

0

:L0

1'

0

0

0

0

-0

FIG. 1. Reduction of NAD by crude extracts. Eachcuvette contained crude extract equivalent to 50 Au ofbacteriochlorophyll and the following in Atmoles: Tris-sucrose buffer (pH 8.0), 100; MgCl.., 10. The reactionwas started by tipping in 5.0 ,tmoles of NAD containedin the side arm. The final volume was 3.0 ml anid noadditional exogenous substrates were required. The re-action was carried out anaerobically at 30°. At the timeindi-ated by the arrow, the NADH trapping svstemdescribed in the text was added.

0.1 * dark

~L.0 8E0

o.066

.04

.020

0 5 10 15

Time (minutes)FIG. 2. Photoreduction of NADP by crude extracts.

Each cuvette contained crude extract (50 jug bacterio-chlorophyll) and the followving in ,umoles: Tris-sucrosebuffer (pH 8.0), 100; M--Cl,, 10. Th-e reaction wasstarted by tipping in 5.0 umoles of NADP containe(l inthe side arm and the final volume x as 3.0 ml. No addi-tional exo-encus subsirates were re {uired aid the plioto-reduction was carried out anaerohi!aflv at 300. At thetime indicated by the arrow the NADPH trappin- s-stemdescri'. ed in the text N-as added.

ping system had no effect on1 a similar reaction mllix-ture containing NADP as the electron acceptor. Toconfirm that NADPH was formed during the courseof this reaction 2 iumoles of GSSG and 0.05 mg of glu-tathione reductase were added at the end of the ilncu-bation time. The ablsorbaline imin:c diately droppedto zero (fig 2). The GSSG-gl.atathione reductasesystem is not specific for NADPH, but does oxidizeNADH at a verv slow rate (13). However, t'hcrewas no detectablle oxidation of NADH ly the con-centrations of enzyme and( substrate used in the ex-periment of figure 2.

The data presented indicate that crude extractscatalyze the reduction of pyridine nucleotide withotitthe requirement of exogenous substrates. With NAD,the high endogenous reduction rates may be due tothe presence of substrates and oxido-redcuctases inthe crude extracts used. This seen's possible, sinceRhodopseudomn onas sphleroides contains high conceni-trations of such enzymes, including 8-hydroxybutyratereductase (3). Some of the preparations used in ourexperiments contained 425 units of this enzyme pernil of extract. In addition, Stanier et al. (15) havedemonstrated that under certain conditions, the con-

939


PLANT PHYSIOLOGY

centration of poly /3-hydroxybutyrate may comprise25 % of thie dry weight of photosynthetically growncells. An enzyme system of this type might accotuntfor the higlh endogenous rates with NAD, since itseems possible that the (lilution of substrate incurredduring the preparation of the extract might not havebeen sufficient to eliminiate endogenious activity.

Similarly, it seems reasonable to expect that suchendogenous oxidizable substrates could be the elec-troni source for the light-dependent reduction ofNADP. To study this problem, the 37,000 X g crudeextract was resolved ilnto a chromatophore fraction(150,000 X g pellet) aii(d a particle-free supernatantfraction. The washed chromatophore fraction doesnot have any photoreduction activity even if oxidiz-able substrates (e.g. succinate) are added. The chro-matophore fraction does have the capacity to photo-reduce NADP if the supernlatanit fraction is added(fig 3). The restoring power of supernatant mate-rials is lost ul)on dialysis anid boiling for 5 minutes.WN'hen, however, the boiled fraction is added to dia-lyzed supernatant materials and chromatophores, thephotoreduction of NADP is partially restored. Theseresults are presented in table II. These data stuggestthat the photoreduction of NADP by chromatophoresrequires soluble heat labile protein factors andI heat

I I I I

0.5 _o light

° dark0.4~~~~~~~

E/

E

° 0.3 _

0It

to

a 0.2_1

0 0.2 0.4 Q6 0.8

Supernatant (ml)1Ffi(,. 3. The effect of the suiperinatanit fraction on1 the

pihotoreJuction of NADP b! washe I chromatoI)hores.In a(lditioln to the standard reactioni mlixture, eacih reac-tionl vessel contained a measured volumile of particle-freesuI)er1atant fraction (5.7 mg protein/nil) To start thereaction, 5 unmoles of NADP was tippe 1 into the mnlinreactioni chamber and liniear rates wer-e meassured over aleriod of 20 miniutes. Coniditionis -x-erce anaerobic and thetemperature w as 300.

E0

to 0.1

o

0.05

0 5 10 15 20Time (minutes)

FIG. 4. Inhlibitioni of the photoreduction of NADP b-ani NADH trapping system. The reaction mixtuire ani(lexperimental I)rocedure xx-as the same as described in thletext itlh the exception that reactioln mixtures repre-sented by A, A containied 5 tg of lactic dehldrogenaseand 3 Amoles of sodium I)ruvate. The control reacticusare represented by 0, 0. At the time designated by thearrow, 2 gimoles of GSSG an(d 0.05 ng glutatllionie re-duictase were added.

stable substrates or cofactors present in the super-natant fraction. The amount of supernatanit fluiduse(l in these experimenits (loes inot catalyze the re-(luction of NADP butt reduces NAD at an en(log-enious rate of A OD 340 n,.u per 20 minutes e(ual to0.2.

Another possibility is that the electron donor forNADP photoreductioin is NADH. If so. the enzymesand substrates in the supernatanit fraction couldcc n5titute an NADH generating system. The .ADHthus formed nonphotosynthetically could serve as theelectron source for the photoreduction of NADP. Ifthis is true, then it would follow that NADP photo-re(luction is (lepenldenit upon NAMD. \Ve teste(d thishypothesis by carrying out the NADP photoredIuctiolnreaction in the presence of an NADH trapping svs-tem. The results in figture 4 show that NADP photore-(luction is completely inhibited in the prese


Table II. Effect of Dialysis and Boiling on the Capacityof the Supernatant Fraction to Restore NADP

Photoreduction by ChromatophoresThe particle-free supernatant fraction containing 5.7

mg protein/ml was boiled for 5 minutes and centrifuged.Dialysis was against several changes of 0.1 M Tris-HCl-10 % sucrose (pH 8.0) overnight at 0 to 40. The mainchamber of each Thunberg cuvette contained the amountof supernatant material indicated below and the usualstandard reaction mixture and experimental procedurewas employed with the exception that chromatophores(50 ,ug bacteriochlorophyll) were used in place of crudeextract. The final volume was 3.0 ml and the reactionswere carried out anaerobically at 300.

A OD 340 m,A/20 minSupernatantmaterial added Light Dark Light minus dark

None 0.00 0.00 0.000.8 ml 0.360 0.060 0.300.8 ml, boi'ed 0.00 0.00 0.000.8 ml, dialyzed 0.09 0.00 0.0008 ml, dialyzed

+05 ml, boiled 0.195 0.070 0.125

natant fraction was examined by repeating the ex-periment with washed chromatophores. In this case,supernatant fluid was replaced by an NADH generat-ing system. The data presented in figure 5 indicatethat under these conditions, 3 X washed chromato-phores support NADP photoreduction at a rate sig-nificantly greater than that observed in earlier ex-

1.0 I I I

o light

0.8 * dark

E 0.6 -0

0.4 -

0.2

0 5 10 15

Time (minutes)FIG. 5. The effect of an NADH generating system

on the photoreduction of NADP by chromatophores. Inaddition to the standard reaction mixture, each tube con-tained 3 X washed chromatophores (50 /,g bacterio-chlorophyll), 0.1 ml of 95 % ethanol, 0.1 m- of alcoholdehydrogenase, a catalytic amount of NAD (0.125,umole)and 2.5 ,moles of NADP. Conditions were anaerobicand the temperature was 300.

periments. Thus, it appears likely that the sole func-tion of the supernatant fraction is to supply NADH.

It is apparent that much greater rates can beobtained if an NADH generating system is presentin the reaction mixture. In addition, a photoreduc-tion of NADP can be demonstrated aerobically andthe rate is almost identical to the reduction rate underanaerobic conditions. In a typical experiment aerobicand anaerobic rates were compared over a time periodof 5 minutes by measuring the absorbance at 340 m,u.For the light reaction the absorbancy change was1.357 under anaerobic conditions and 1.282 in air.All rates were linear over the time period and -thelight-minus-dark difference was 0.863 and 0.812 ab-sorbancy units for anaerobic and aerobic conditions.respectively. Thus, subsequent experiments wereroutinely performed aerobically and reaction mix-tures were supplemented with a catalytic amount ofNAD, ethanol and yeast alcohol dehydrogenase.

The results obtained here are similar to those ofNozaki et al. (12), who demonstrated that chromato-phores of Rhodospirilluiml. rubrurn will photoreduceNADP at a rate comparable to succinate-c'ependentNAD photoreduction if a catalytic amount of NADis present in the system. They suggest that the pho-toreiduction step involves NAD alnd NADP is reducedvia transhydrogenase. The preparations of Rhodo-pseuidomlonas spheroides used in these experimentswere found to be high in transhydrogenase activity(240 units/ml). Thus, it might be argued that asimilar mechanism for NADP photoreduction is op-erative in this organisn. This mechanism is notconsistent with our observations, since we have notedthat a significant NAD photoreduction cannot bedemonstrated with Rhodopseuidomiionias spheroides ex-tracts when either succinate or the 2,6-dichlorophenol-indophenol-ascorbic acid couple is supplied as theelectron donor. In addition, if the sole purpose oflight was to photoreduce NAD, it would seem reason-able to expect that light could be replaced by theNADH generating system. The reduction of NADPwould then be a dark reaction via transhylrogenaseand there should be no light-minus-dark differencesunder these conditions. This is not in evidence fromthe data of figure 5. The dependence of the photo-reduction of NADP on NADH might, however, beexplained on the basis of an energy-linked transhv-dregenase similar to that of mitochondria (5). Inthe mitochondrial system, NADP is reduced byNADH and there is a requirement of ATP.

To test this hypothesis, an experiment was per-formed in which ATP was substituted for light. Theresults presented in figure 6 show that when ATE'is present, the reduction of NADP prozeeds at thesame rate as the light reaction. This suggests thatRhodopseuidomiiontas spheroides contains an energy-lilnked transhydrogenase that can be driven by ATP.It seems possible that a similar enzyme system mayrbe operative in the light-dependent reaction. In thiscase, the energy needed to drive the reaction is sup-plied by light.

941


PLANT PHYSIOLOGY

Table III. Effect of Tlhvroxine on NADP Photore-duction (nd Photophosphorylation

The main clhamber of the cuvette contained an amountof crude extract e(quivalent to 29.4 ,ug bacteriochlorophyll,0.1 ml of 95 % ethanol, 0.1 mg alcohol dehydrogenase andthe follow'ing in /Lmoles: gIg , 10; Tris-sucrose buiffer(pH 8.0), 180; anid NAD, 0.1. The test cuvette coIn-taiael 0.18 umo'e of thyroxine. At zero time 5 umolesof NADP was tipped ilnto the main clhamber and reac-tions were carried out at 30° under ana'erobic conditions.For the photophosphorylation reaction, each tube coIn-tained crude extract (29.4 ,u bacteriochloroplB 11) anidthe following in Aimoles: Tris-sucrose buffer (pH 8.0),180; MgCl., 10; ADP, 10; and KH.,PO4, 10. The testreaction contained 0.18 umiole of thyroxine and the reac-tion mixtures were incubated at 30° under anaerobic coni-ditions. The final volume for all experiments Nx-as 3.0 ml.

0 2 4 6 8 10

Time(minu tes)

Fmri;. 6. lEffect of ATP oni the 'NADH-depenident re-(lIuctioni of NAI)P. The mialhin chaimber of eachi cuivetteconitainiedwashed chromatoplhores (30.6 ,ug bacteriochloro-p)hll), 0.1 ml of 95 % etlanol, 0.1 mg of alcolhol dehv-dlro-teiase and the folloxw ing in /Lmloles: Tris-suicrosebuffer (pH 8.0), 100; MgCl.,, 10; NTAD, 0.125. The reac-tiolns were started b\ tip)p)ilg 2.5 uimoles of _NADP con-talio.ed in ti.e si le armii. The fi ial volumie was 3 0 iIl.For the experimeiits labe'ed ATP, tihe reactioni miiixture-was the samiie as above, except that 10 ulimoles of ATPvere aldde 1. Thle latter experimenmt xwas co-iducte(l at 300in the dar'\-. Coinditioiis for all exl)erimiieats wvere aerob)ic.

Aniother line of evidenice in supl)ort of an energy-linke2d transhydrogenase w-as obtained by studyingthe effect of thyroxine on the photoredluction reactioln.Kiester et al. (10) haxe rep)orte(l that spiniach tranis-hNvdrogeenase is inhibited 33 % by 3.3 X 10-5 'I thy -

roxii'e. \\Wel have obtlained a simllilar resultl- WithRlioopseoldoboohi(aS Shlicr(oidCs l)reparatimios ( table

1). These data show that both the light aln(d thelark reactionis l)roceedl at sloxver rates and the lig,ht-mins-d(lark (lifference is diiniiiishled by 30 % in thlepresence of 6 X 10-5 a\ thxroxine. It is noteworthythat this colncentrationi of thyroxine (loes lnot signifi-calntlv affect photophosp)horvlation.

In a(Ilition, oligoomycin did niot have any effecton the light-(lel)endent reactiol (fig 7) When oli-gomycin xxas added to a reaction miixture in wx hichlight xvas rel)laced by ATP, the re(uction of NADPxvas comI)letely inhibited (fig 8). This con, entra-tioIn of oligomnycin also completely inhibited photo-phosplhorylation.

'lThese findinigs raise the initeresting (luestioll ofwhether or niot the NADP photore(dtictioin reaction is

A OD 340 mnu/10 min,mnoles Pi/hr per

-ight Imillus mg bacterio-Additions Li-ht Dark Dark chlorophx ll

Nonie 1.050 0.450 0.600 565.06 X 10-5 -%

thvroxine 0.650 0.235 0.425 501.0

0

0

0.8

0 4

0 2 4 6 8 10

Time (minutes)

FEn.. 7. 1E'ffe-t of olio'mc'1ia On lightmediate(l NADP1C ,I,.ctiOCl. Ti e .il chamiiVcr of each cuvette contaei:le

xashed chrom-natophores (33.6 /,l bacteriochlorophy)ll), 0.1n1l of 95 % ehlianol, 0.1 milg alcohol (ledhcrogeniase anidthe followinpig in uiimoles: Tris-sticrose btuffer (pH 8.0),100; MIgCI.,, 1i); NAI), 0.125 and as ilndicated, 10 yg ofO' i'nOl'cm. The reactions v-er-e started by tipping inI

2.5 ui-moles of NADP fr( ii the si(lde arm. Concditions xvereanaerobic at 300 anid the f inal oltumiie wxas 3.0 nml.

942

14

1.2

1.0

0.84-E0

.' 0.60

0.4

0.2



ments containing none of these inhibitors photore-duced NADP at a rapid rate. These concentrationsof inhibitors had the same effect on photophosphory-lation activity. The results indicate that the pho-toreduction of NADP does not proceed unless thereis a cyclic flow of electrons. Thus, it would appearthat the photoreduction reaction would be enhancedunder conditions favorable to photophosphorylation.However, we noted that there is a slight inhibition ofNADP photoreduction when ADP and inorganicphosphate are present (table IV). The most pro-nounced effect is on the dark reaction which is mar-kedly enhanced. A similar result obtains with ADPalore. Inorganic phosphate has little effect on thelight rate but stinm.ulates the dark reaction.

Discussion

0.2 /]/ _ In the past, attempts to demolnstrate photoreduc-0.2 /ion reactions with cell-free preparations of Rhodo-psetdoncinas spleroides have been unsuccessful (21).In view of the (lifficulties that we encountered dluring

D0 2 4 6 8 10 the early phases of this research, some comments onltie irethod are in crder. It has been demcnstrated

Timeo(minutes) that active preparatioins could be obtained if the cellsFIG. 8. The effect of oligomycin ATP-supported were grown under carefully controlled conditions and

NADP reduction. The reaction mixture and experimen- harvested during log phase. Routinely, inocula weretal conditions were as described in figure 7 with the

following exception: were indicated, 10 moles of ATP prepared in small volumes in screw cap vials containi-wN-ere added to the side arm of the cuvette and the reac- ing a i-edium of 0.2 % proteose peptone and 0 3 %tion was carried out in the dark. The amount of oligo- of yeast extract. Our observations have been thatmycin used was 10 ,ug. the inoculum must be transferred to sterile Hutner

media during log phase if best results are to be oh-coupled to a cyclic flow of electrons. We have ap- tained. Under these conditions growth proceededroached this qlestion studying var- without any lag phase. It was also apparent that dur-

ions photophosphorylation inhibitors on the photo- ing the growth peried, variations in temperature morereduction reaction. Our findings were that the light- than degree 2 more or less than 300 could notdependent reaction was completelv inhibited when be tolerated.10 ,Ut of antimycin A or 100 jug of 2-N-heptvl-4-hy- 'The result presented here indicate that the phnto-droxy-quinoline-N-oxide were added to the basic re- reduction reaction in Rhodopseudomoas spheridesaction mixture. Quinacrine-HCl (3 X 10-4 M) requires NADP and not NAD as the electron ac-markedly inhibits the light reaction. Control experi- ceptor. This is in contrast to what has been ohserved

in other photosynthetic bacteria where NAD is pref-erentially photoreduced. 'The results of Yamanaka

Table IV. Effect of ADP and InorgADnc Phosphate on and Karr-en (22) are of interest, since it appears thatNADPthe photoreduction reaction in the related photohet-

The main chamber of each cuvette contained an amountof crude extract equivalent to 24.4 ,g of bacteriochloro- erotroph, Rhodopseudornonas paluistris, may be simi-phyll, 0.1 ml of 95 % ethanol and 0.1 mg of alcohol lar to that in chloroplasts (14). Arnon (1) hasdehydrogenase and the following in ,umoles: Tris-sucrose stuggested that the mechanism is that of an electronbuffer (pH 8.0), 100; MgCl9, 10; NAD, 0.125; and as flow from the donor system to NADP via ferredoxinindicated below, ADP, 10; and KH,PO4, 10. The re- and NADP-reductase. The researches described inaction was started by tipring 2.5 ,umoles of NADP into this communication do not suggest that a similarthe main chamber and the photoreduction was carried mechanism is operative in Rhodopseutdonizonzas spher-out aniaerobically at 30°. The final volume was 3.0 ml. oides. However, this possibility cannot be discounted

and n-ust be tested experimentally.A OD 340 mu/5 min We have suggested that NADP may be photore-

Additions Light Dark Li,ht minus dark duced via an energy-linked transhydrogenase similarto that in mitochondria (5). It should be pointed

None 1.165 0.262 0.903 out that we do not regard the data presented hereADP and Pi 1.028 0.541 0.487 as rigorous enough to constitute conclusive evidence.Pi 1.090 0.355 0.735 The elucidation of such an enzyme system will have

to await a more intensive investi,gation. However, our

.2

1.0

0.8

0.6

E0

0

0.4

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PLANT PHYSIOLOGY

findings appear to be suggestive enough to permit adiscussion of a mechanism for NADP photoreductionbased on an energy-linked transhydrogenase. An at-tractive hypothesis is that light is required to gen-erate ATP which can theii serve as the energy sourceto (Irive the transhydrogenase reaction. This hypoth-esis would be consistant with the requirement forcyclic electron flow, but (loes not appear likely forthe following reasonis: A) washed chromiiatophoresdo not support photophosphorylation uiiless exogenoussubstrates are added, but do catalyze photoreductionat a rapid rate; B) ulnder conditions favoring photo-phosphorylation, the photoreduction reactioni is slightlyinhibited; C) the ATP-supporte(l reaction is inihilbitedby oligomycin but this inhibitor (loes not have aniysignificanit effect on the light-mediated reaction. Apossible mechanism that appears consistanit w'ith ourdata is presenited in figure 9. A mechanlismn similar

NADH + NADP NADPH +NAD

oligomycin

ADP+ Pi+ [--X] ,+ATP

( cl ic el ectron f lo

1ightFI(.. 9. Hypothetical scheme for the NADH-depeni-

dent photoreduction of NADP in RhodopseudonionassPhcroides.

to this has been advance(l by Bose and Gest (2) forbacterial photosynthesis aild by Daanielsoni and Frns-ter for the mitochondrial tranishydrogeinase reaction(5). According to the sclheimne in figure 9. light isrequired to generate a high energy intermiiediatethrough a cyclic flow of electrons. The high energyinterilie(liate can then drive the energy-dependenttran.shydrogenase reaction. Under conditions of cyclicphotophosphorylation, the same intermiedliate may berequiredl for the synthesis of ATP. A comlimoni inter-mediate for ATP synthesis and NADP photoreduc-tion is indicate(d by the inhibition of photoreductionaffor(ledI by ADP and iniorganiic phosphate (tableIV). \Ve have obtained preliminary evidence whichindicates that Rhodopseuidoinli0oas spheroides containsATPase. Under concditionis favoring ATPase activ-ity, the light-supported transhydrogenase is com-pletely inhibited by ADP and inorganic phosphate.The schen-e presented in figutre 9 is further suip-

ported by the experimlents perfornred with oligo01 yciii. Danielscn and Ernster (5) have found( thatsuccinate-mnediatedc NADP reduction is not inhibitedby oligomycin. \NVe have obtain-ed a similar r2siltfromii Rl odopsct(ol/io0ioas spheeroidcs preparationis.The experiments preselnte(l here suggcsL that oligo-nycin does not affect the formation of the light-generated intermediate requiredl to drive the trans-hydrogenase reaction. However, subsequent re-actions leading to the formation of ATP are inhibitedby oligomycini. The inhibition of the ATP-supportedreaction may indicate that the interme(liate funictioinalin the light reaction can be generated by ATP aswsell. The formation of this interme(liate from ATI'Pis inihibitedl by oligomycinl.

Literature Cited

1. ARNON, D. I. 1965. Ferredoxill and photosynthesis.Science 149: 1460.

2. BosE, S. K. AND H. GEST. 1963. Relationships be-tw een energy generation anid net electron transferin bacterial photosynthesis. Symp. oni Energy-Linked Functions of Mitochonl1ria. AcadeimiicPress, NeN%- York. p 207.

3. CARR, N. AND J. LASCALILES. 1961. Some enzymicreactioils concerned in the metabolism of aceto-acetyl-coenz3 me A in .4 thiorhzodaceac. Biochemii. J.80: 70.

4. COHEN-BAZIRE, G., WA. R. SJSTRONI, AND R. Y.STANIER. 1957. Kinetic studies of pigment syn-thesis by nonsuilfur purple bacteria. J. Cell.Phvsiol. 49: 25.

5. DANIEISONN, L. AND L. ERNSTER. 1963. Energy-dependent reductioni of TPN by DPNH. Symp.on Energy-Linked Functions of Mitochonidria.Academic Press, New York. p 157.

6. FRENKEL, A. W. 1958. Simuiltaneous reduction ofdiphosphoryridine nucleotide and oxidation of re-duced flavin mononucleotide by illuminate l bac-terial chromatophores. J. Am. Chem. Soc. 80:3479.

7. FRENKEI, A. W. 1958. Lighlt-induced reactions ofchromatophores of Rhodospirillhon ru1)rlan. Brook-haven Symp. Biol. II: 276.

8. HOOD, S. L. 1964. Photoreductioin of nicotiniamide-adenine dinucleotide by a cell-free system fromChromnatiuw?. Biochimni. Biophys. Acta 88: 461.

9. KAPLAN, N. O., A. P. COLOWICK, AND E. F. NEU-FELD. 1952. Pyridine nucleotide transhvdrogenase.11. Direct evidence for and mecliaiii-imi of thetranshydrogenase reaction. J. Biol. Chemii. 195:107.

10. KEISTER, D. L., A. SAN PIETRO, AND F. E. STOLTZ-ENBACII. 1960. Pyridine nucleoti de transbydrogenasefrom spinach. J. Biol. Clhem. 235: 2989.

11. NOZAKI, M., K. TAGA\VA, AND D. I. ARNON. 1961.Noncyclic photophosphorylation in photosvntheti'-bacteria. Proc. Natl. Acad. Sci. U. S. 47: 1334.

12. NOZAKI, NI., K. TAGANVA, AND D. I. ARNON. 1963.Metabolism of photosynthetic bacteria. II. Certainaspects of cyclic and noncyclic phosphorylation inRhodospirillum rutbrumii. Symp. on Bacterial Pho-tosvinthesis. Anltioch Press, Yellow\ Springs, Ohio.i) 175.

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13. RACKER, E. 1955. Glutathione reductase from baker'syeast and beef liver. J. Biol. Chem. 217: 855.

14. SAN PIETRO, A. AND H. M. LANG. 1958. Photosyn-thetic pyridine nucleotide reductase. I. Partialpurification and properties of the enzyme fromspinach. J. Biol. Chem. 231: 211.-

15. STANIER, R. Y., M. DOUDOROFF, R. KUNISAWA, ANDR. CONTOPOULOU. 1959. The role of organic sub-strate3 i bacterial photosynthesis. Proc. Natl.Acad. Sci. U. S. 45: 1246.

16. TAUSSKY, H. H. AND E. SHORR. 1953. A micro-colorimetric method for the determination of in-organi- phosphorus. J. Biol. Chem. 202: 675.

17. VAN NEIL, C. B. AND W. ARNOLD. 1938. The quan-titative estimation of bacteriochlorophyll. Enzy-mologia 5: 244.

18. VERNON, L. P. AND 0. K. ASH. 1959. The photo-reduction of pyridine nucleotides by illuminated

chromatophores of Rhodospirillum rubrum in thepresence of succinate. J. Biol. Chem. 234: 1878.

19. VE?NON, L. P. 1959. Pliotooxi latio:ns catalyzed b;chromatophores of Rhodospirillum rubrum underanaerobic conditions. J. Biol. Chem. 234: 1883.

20. VERNON, L. P. 1958. Photoreduction of pyridine nu-cleotides by cell-free extracts and chromatophoresof Rhodospirillum rubrum. J. Biol. Chem. 233: 212.

21. VERNON, L. P. 1963. Photooxidation and photore-duction reactions catalyzed by chromatophores ofpurple photosynthetic bacteria. Symp. on BacterialPhotosynthesis. Antioch Press, Yellow Springs,Ohio. p 235.

22. YAMANAKA, T. AND M. D. KAMEN. 1965. Purifi-cation of an NADP-reductase and ferredoxin de-rived from the facultative, photoheterotroph, Rho-dopseudomonas palustris. Biochem. Biophys. Res.Commun. 18: 611.

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