Laser-Activated Electron Transport in a Chlamydomonas Mutant' · Plant Physiol. (1968) 43, 303-312 Laser-Activated Electron Transport in a Chlamydomonas Mutant' W. W. Hildreth Johnson

Plant Physiol. (1968) 43, 303-312

Laser-Activated Electron Transport in a ChlamydomonasMutant'

W. W. HildrethJohnson Researoh Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received July 31, 1967.

Abstract. Following laser activation of electron transport in the pale green mutant ofChiamydomonas reinhardii, the following kinetics are observed: 1) A rapid absorption decreaseat 421 mu (half-time < 2 X 10-6 sec) recovering with a half-time of , 7 X 10-3 sec.2) Oxidation of cytochrome f at 554 mIA with a half-time of 1 X< 10-4 sec. 3) Oxidation ofcytochrome of type b, at 432 and 564 myu, with a half-time of - 6 X 10-3 sec, following a2 X 10-3 sec lag.

The results are interpreted according to a linear electron transport sequence: system Itrap v- cytoohrome f v- v- cytochrome b with an additional molecule of cytoohrome b in thecyclic photophosphorylation pathway. Experiments with unoouplers provide evidence for asite of photophosphorylation between cytochrome f and cytochrome b.

Additional studies involve inhibitors of electron transport, the temperature dependenceand quantum efficiency of cytochrome oxidation, and the effect of oxygen and pre-illuminationon the laser-induced absorption changes.

The pale green mutant of Chlamttydontonas rein-hardii 'has been previously studied by means ofcontinuotus ac,tinic ililumination (5) and the normalmode ruby liaser wi'th a pulse duration of 1 msec(6). These studies demonstrated that more accu-rate cytochrome absorption changes could be re-corded by using the pale green mutant rather thanthe wild type Chlamydomonas. In particular, cyto-chrome of type b was oxidized w'ilth a h1alf-time ofless than 20 msec, fol:lowinog a laser flash (6). Therole of cytochrome f in the electbron transport chainof the Chlartydornonas pale green mu'tan't was leftin question by these ini-tial inveistigations. In thepresent wo'rk, the shorter pulises from a Q-switchedruby laser have been emiployed to activate absorptionchanges 'in the pale green mutanit. By tuse of thelaitter technique, the responses of cytoc:hromes andother electron carriers have been separaited on botha kinetlic and spectral basis. Preliminary reportshave been presen'ted elsewhere (3, 15).

Materials and Methods

T'he pale green mutanit resulted in 1951 fromultra-violet irradiiation of t'he green ailga Chilany-domonas reinhardii (23). I,t has been subsequentlycultutred in the dark on an acetaite medium and, inthese expeniments, has been harvesited near the endof the log phase. Tlhe cellls were centrifuged and

1 Supported in part by research grant number 5151-26from the Office of Naval Research.

303

resuspended in 2 to 3 ml off growth mediunm.Rapid absorption changes in the photosynthetic

sample have been observed with a single beamspectrophotometer an'd accompanying fast-responsetiming circuitry developed iby DeVaullt and Chance(10, 11). Briefly, the technique con'4i'sts of reflect-ing the Q-swi'tched liaser pulse (pulse duration - 30n'sec) on to a cuvette containing a ituirbid suspenisionof algae. The optical path lenglbh is 0.16 cm wi-tha typical sample concentration of 50 jg chloro-phylil/ml. For 'low 'te'm'perature studies, the pro--jecting atlum;inum fin of the cuveltte can be immersedin an ice water balth within an unsillvered Dewarfilask. The '694.3 m/ 'laser flash can 'be attenuatedbefore striking ithe cuvette to incident initensities of103 to 107 w/cm2 (measured with a TRG bolomeiter),by means of negatbive len's and neutral density filiters.

The spectra;l initerval of -the spectrophotometeris 3 mix, and the 'monoc'hroma'tor lamp volitage canoperate in the boosted (_. 0.1 mw/cm2) or un-boosited mode (- 0.03 mw/cm2) (10). The boostedlamp vol't'age is used only for observation of rapidkinetics, that is for sweep rates faster 'than 0.5msec/division (cf. figs 5D and H). In the boostedmode, the lamp voltage is increased by a factorof - 1.5 to give a constanit 'initensity for - 33 msec,during which ti'me the laser is flasghed. The photo-multiplier (EMI 9592B or 9524B) is guarded fromthe laser filash by a Corning 4-96 blue-green filter.T'he output signal is amplified, balanced by a DCoffset vol't'age, and dispqayed on an oscilloscope.

In the pre-il'lutmin'ation experiments, continuousilluminaftion wa,s provided during the laser flash bya Unitron mlicroscope lamp pluts a Corning 2-64

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red cut-of f filter (2 MnW/CM2) . The aerobic-an-aeroblic stuidies employed the moist chamber andspirral platinum 02, electrode of Chance and Strehler(7). In the latter apparattus, O. or N2 gas ispassed over the surface of tihe sample to provideaIn aerobic or anaerol)ic, state o,f the cells. In theabsence of an external flow of gas, the algal cellsrespire to an anaerobric state in the dark prior toeach laser flash (~ 2 mmii between successive laserflashes). The (increase in 02 tensioni clue to peri-odic iFlllumination with the measuring light pluts laserflash was less thain 4 % of the 0, tenision in thefully- aerobic suispension.

The quant,titative data presented lin tables I, II,aind III were meastured (lireotly from Xerox copiesof the photographed oscilloscope traces, after hand-

smoothing of the traces. All of t1he controll datarefers to anI average of 10 or monre traces, and thetabtulated uincertainities are stanidard deviiations. Forother entries in the tables where les,s than 10 tracesare averaged, the tabulated uncertainty refers tothe maximuim deviation.

Results

Loser-iMjducctd Kiicties. In figutre 1 ar-e shownthe mo.st prominent featulres of tihe kinetics induticedbv a laser flasih in the pale greein mutant oif Chllamvy-dom1lonl(ts. A rapid absorption decrease occulrs at421 mtx and 554 rn/k, folloedl by a lag of 1 to 2 msecand a first ordler recovery (figs 1A ancd 1C). Therecovery at 421 mi is more complete andli'as a

tAbsorption Decrease

-

Laser 5msec

421 m,L

0.002

432mb

0.004

EI

0.008

. . .-A .

1111I IILaseI 5 msec

Laser 5rmsec

554m,L

0.001

564 m

0.001

i it t I

Laser 500msec

I---- .I1t-- -. 1.7.....~~~~~~~~(6~~~~~~~~~~~~(

Laser 200msec

Laser 5 msec Loser 500msecFIG. 1. The time course of absorption chaniges in the pale greeni ilmutant of C/l/am vdoiwonas following a flash

from the Q-switched ruby laser. 3X 10- einsteins/cm2, at 694.3 ni;: 1.6 mim opticall path; 50 ,ug chlorophyll/mI.Instrumenit time constants - 10-4 -sec (fig 1 A, B, C, and 1)), 10-I ( fi I E. F (G. anld H).

0

m

I

304



HILDRETH-ELECTRON TRANSPORT IN A CHLAMYDOMONAS MUTANT

half-time of 7.5 msec. On the other hand, therecovery alt 554 mp, is more variable in both extentand half-time. In different samples, the h'alf-timevaries from 1 to 10 msec, whille the extent of re-

covery varies from 50 % to no recovery within theindicated time span.

Ait the faster sweep rate of fiigure 2, a furtherdifference is observed between the kinetics at 421

Absorption Decrease t432min

Al

Laser Loser

FIG. 2. Rapid kinetics following a laser

10-9 einsteins/cm2) in the pale green

Chaiaydontonas. 50 ,ug chlorophyll/ml.time constant , 3 X 10-5 sec.

flash (3 Xmutant ofInstrument

and 554 m,u. T'he 421 m,u absorption c'htange hasa rapid phase w'hliclh occurs wiith a half-timetll.2 < 2 jmsec, (estimat'ed from recordings at a

sweep rate of 10 Musec/division), that is, less thanthe response time of 'the apparatus under the presentexpenimentail conditions. The slower ph'ase of therise kinebics at 421 mu h'as a half-time of 170 usec(fig 2A) and can be correlaited Wiith 'the first orderabsor,ption decrease at 554 m,u (fig 2B). Th-us, itaippears that cytoc'hrome f is responsiible for theabso,rption ohange at 554 mMu (cf. fig 3) and a

o

* Aerobic } Rapid Absorption Changes

AnaerobicISteady State Difference

Aerobic Spectrum

410 420 430 440 450 540 550 560 570 580Wavelength (m,ol

FIG. 3. Spectra of rapid absorption changes (within1 msec of the laser flash) and spectra of the oxidizedsteady stalte of cytochrome of type b in the C//lamiydo-m11onias pale green mutant (30 jgtg chlorophyll/ml; spec-

tral halfwidth = 3 mg). Spectra are shown underanaerobic and aerobic conditions.

portion of tihe kinetics at 421 mu; 'however, anotherfaster componen,t is allso contribulbing 1to the absorp-tion change at 421 m,u.

The final recovery at 421 mp, (fig IE) alsoshows biph,asic kinetics, the slower recovery phasehaving a hallf-time, tj/2 = 150 msec (avg of 15recordings). The latter half-time can be correlatedwiith the final recovery of cytochrome f at 554 mpu(fig 1G) with a hallf-time, t1/9 = 180 msec (avg of25 recordings).

Figures 1B and 1D show absorption changescharacteristic of the So,ret and a-bands, respectively,of cytochrome of type b. FoIll,owing the laserflash is a lag of - 2 msec, then a ftirst orderabsorption decrease, interpreted as an oxida,tion ofcytoc'hrome b '(tl/2 - 6 rnsec). The cytochrome bkinetics can be correlated wi,th respect to lag andhialf-time to the recovery at 421 m,u, buit not to themo,re variable, partial recovery at 554 m,u. Thefinal recovery of cytochrolme b i,s sihown in fligulres1F and H (t1/ - 0.7-1 'sec).

Tihe lag in tihe cytochrome b kinietics is shownin more detaiil at a 10 times faster sweep rate infigures 2C and 2D. At 432 mM, (fig 2C) a rapiddecrease in absorption occurs, probably dule to thetail of the 421 m/A band. A posiltive transientfolilows with a hailf-time of - 300 ,usec. At 564 mp(fig 2D) a rapid increase in absorption occulrs,followed by a posiitive itranisienit with a half-time of- 250 MAsec. The slowness of the 432 mp transient

in com,parison with that at 564 m,u is most probablydue to distortion of the kineltics ait 432 mMu by thenearby, relaitivdly large 421 mM banid. The readerwill notice the laser artifact in figtu,res 1 and 2

occu-rring as an upward spike at the instanit of thelaser flasih, that is, in the direction of increasedlighit to the photomtlnltiplier.

The above-described kinetics are summarized intables I, II, and III wi'th the designation "Controls".

Differenlce Spectra. Figure 3 demonstratesspectra of the previously described absorptionchanges. The cuirves labeled "'steady sitate d,iffer-ence spectra" represent the oxidized steady state ofcyto'chrome b wi,th the sample in the aerobic or

anaerobic state. The oxidized steady stalte of cvto-chrome b fol'l'owing a laser flash is decreased inextent by more than 50 % tinder aerobic condi!tions,indicaiting that cytoch,rome b is partialily oxidizedbefore the laser flash. The cturves labeled "rapidabsorption change" represent changes occurringwi,thin 1 msec of the laser flash. Neiither the exten'tof cvtochrome f oxidati'on 'at 554 m,u nor the 421 m/L

absorption change is affecdted by the anaerobic-aerobic transiti,on. (The so-cal,led "421 mix absorp-tion change" 'peak's at 421-422 'mM in (lifferentsamples).

The relative magniitudes of tihe peaks at 421 and554 m,u are consiFitent wi(th tihe possibility that theenitire 421 m,u absorption chan'ge is duie to the Soretband of cytochro'me f. However, from the pre-

ce,ding section, the rise and recovrerxv kinetics at

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PLANT PHYSIOLOGY

421 m,u (figs 2A and 1E) are biphasic. Therefore,the exact protportion of the 421 m, absorptiondhange, which is dule to tihe S!oret baniid of cvto-chrome f, is still in question.

The positive tro,ughs in the cytochrome bkinetics are shifted from 432 and 564 mnu to thevicinity of 435 and 565 m/, respectively, prestumablydue to the proxi,mity of the 421 and 554 mjt bands.Furtrhermore, tihe extents of the posivtiv-e trotighsare enhanced in the transitbion from the anaerobicto the aerobic state. Similar increases in the ex-tents of the troutgh,s are produiced at 00, uipoinaddition of HOQNO2 (cf. fig D-5, ref 3) oruncoutplers of photophosphorylaition, and wvith con-tinulouis red illutminaition during the recorrding of thelaser-inducedl kinetics. The latter phenomena w-illbe more ftully trea'ted in the Dis,cussion.

In the region of 518 my no absoirption increase,characteristic of wild itype photosynthetic samples.has been detercted uindei r the presenit experimentalcon,diftion.s. This null result is mosit probably re-lated to the deficiency of fl-carotene in the palegreen mutant of Chlamnydonmonas (5, 16).

The Response (it o0. The rel,ativel- larg,e un-certainties in the kinetics ait 00 (tables I andc II)are dule I)a;rtiall4y to bicological \-ariabilitx in theresponse of the cel,ls ait 00. In addition, the dis-tance betxween ithe ctivette and photonitoltiplier isincreasedi duie to the initervening Dewar flask; henice,the signal-,to-noise raitio of the trace is clecreased.In general, the oxidation h'al f4times for cvtochromef (table 1, co;luimn 3, denoted table 13) and cyto-ch,rome b (t,able 112 and 114) are increased l)yfactors of 4 and 5, respectively, at 00. 'T'he initiallag before ox,idation of cyto,hrome b (figs 1B and(iD) is increased by a factor of 6 at 00. h'l'e lagbefore oxidation of cytochrome b can again bedisplayed at a fasiter sweep rate (cf. figs 2C and2D) anid descilibeid as a transient half-timie at 00(table 12 and 14). Also, at 00 the slox p)hase ofthe rise kinetlics at 421 m,u (table I) can b)e ap-proximately correrlated wi,th the oxidation of cvto-ohrome f at 554 m,u (table 13).

Inhibition by HOQNO. The rise kinetics ob-served uinder in,hibiition of HOQNO are illustratedin figtire 4 and summarized in tables I atid II.W\ithin the dTimits of experimental error, the kineticsof oxidation of cytochrrome f are unchanged bIy theaddit.ion of 40 KM H,OQNO (ifig 4A anid 4C:table 13). Hiowever, both the initial lag and oxida-tion half-itime for cytoc-hrome b are tlouibled byaddition of 40 dusi H,OQNO (fig 4B and 4D; tableI12 anid I14). Asstsining a linear arrangement of

2 Abbreviations: HOQNO, 2-n-heptyl-4-hydroxyqui-noline-N-o,xide; CCCP, carbonyl cvanide m-ch4orophe-nylihydrazone; FCCP, carbonyl cyanide p-trifluorometh-oxyphenylhydrazone; DC\ItT. 3- (3,4-(lichlorophenyl)-1J1 -diniethylurea.

554m,uControl

.1,

105msecLoser

4 x 10-5 M HOQNO.. .

r.0

0 5msec.oaser

0

Loser 5msec Loser 5msec

FIG. 4. The effect of HOQNO oln the laser-inducedkinetics in the pale green miiutanit of Chlamnydownomis.50 ug chlorophyll/mil. Instrumiienit time constanits - 3X 10-5 sec (fig 4\. 40 atnd - 10-4 sec (fig 4B, 41)).

these carriers, it is clear that the site of inhibitionby HOQNO is betNx-eein cvytochrome f and cyto-chrome b.

T'he concentration of HOQNO reqeuired to pro-lutce the above effects is closer to the chlhorophyllconceintraition of the sample than to the concentra-tioIn of cyotoohromens. As discussed by Niishimuira(21), it is likely that the local concentratlion ofHOQNO in the electron transport systerm is closeto the concenitration of cvtochromes dtue to a Der-meability barrier in these intact cell experiments.

Inhibition by Salicvlaldoximne. As pre,sented inreference 3, figuire D-6, the inhibitor salicyllald.do2cimeproduced an inslignificant effec(t oIn the cytochromef oxidation kinetics (table I1 aind 13). Howeverthe additi,on olf 4 mnM salicylaldoxime increases theinitial lag and oxidation half-btime of cytochrome bby factors of 4 and 3, respectively (;table 112 andII4). Thus, the site of inhibition by salicyl,aldoximeis between cytochrome f aind cytocwh,rome b. ReceIntexperiments by Katoh and San Pietro (18) provideevidence that salicyla,ldoxime is not a specific in-hibiitor of plastocyanin; hence, the site of inter-acbion of sa,licylaldoxime in the eilectron translpoSrtchlain cannot be fuirther specified (see also refer-ences 20 and 22).

Inhibition by DC1IU. The possibillity must beconsidered that the observed cytochrome b occuirsonily in the mitochondria of the in,ta,ct algal celtls.According to this infterpretaition, oxygen evolved bythe flash would mligrate to the mitochond,ria andprodtice the observed oxidation of cytochrome bfollowing a 2 msec lag. It was, therefore, impor-tant to inhiibiit oxygen evolution by DCMU andobserve the effect oIn the cyitochrome b kinetics.In order to obtain a nelt oxygen evoilutibon of zerounder red lighit illtimiinatioin, it was necessary toadd the relatively htigh concentration of 300 1iMDCMU. T'he need for a high concentraltion ofDCMU is prestimabl-ly duie to a permeability barrieracross the intact cell menibraine.

306

Ii M-Ill



HILDRETH-ELECTRON TRANSPORT IN A CHLAIMYDOATONAS AlUTANT

Table I. Sumnuumary of Rapid Kinietics, Following a Laser Flash, in [7itole Cell of the ChlamnydomonasPale Green Mutant

The absorption change at 421 mni consists of an unidentified rapi(d phase, and the tabulated slow phase, dueto the Soret band of cytochrome f (fig 2A). The absorption changes at 432 mg and 564 mIA are due to the Soretand a-bands, respectively, of cytochrome b (figs 2C and 2D). The absorption chanige at 554 m,L is due to thect-band of cytochrome f (figs 2B and 5D).

421 ini 432 m,u 554 m, 564 m,

rise t1/2 transient t1/2 oxidation transient t1/2(slow phase) t1/2

,IscC A.sec gscC AsecControls 170 ± 50 300 ± 70 140 ± 50 250 ± 100Temperature = 0° 400 ± 200 1200 ± 400 600 ± 200 1200 ± 400HOQNO (40 ,m) ... 600 ± 200 190 ± 50 650 ±200Salicylaldoxime 200 ± 50 1400 ± 400 200 ± 70 1800 ± 500

( 4 mM)DCMU (1mM) 170 -'- 50 300 ± 100 150 ± 50 300 ±100CC'CP (10 JAM) 150 ± 100 250 ±+ 550 100 ± 50 250 ± 50

Talble II. Sunimzcry of Kinetics in the 1 to 40 insecond Range, Follozwing a LaseIr Flash, in the ChlamydomonasPale Green Mutant

At each wavelength, a lag is observed. followed by the tabulated first-order kinietics, as illustrated in figure 1

421 ipiArecovery t1 2/(fast phase)

ControlsTemperature = 00HOQNO (40 tiM,)Salicylaldoxime

(4 mM)DCMU (1 IlM )CCCP (10 AiM)

mnisec7.5 ± 2

35 + 2511 ± 324 -4- 6

19 ± 67 + 3

432 m,

oxidationt1/2msec

6.7 ± 2.030 ± 2014 ± 516 + 5

15 ± 42.8 ± 0.6

554 nim

recovery t(fast phase)

11isec4 -

15 4-3 -+-13 -+-

526

. .1.0_-4 0.6

564 mni

oxidationtl/2

5.7301119

wiisec

-4-+-

-+--

2.02038

15 + 52.8 + 0.6

For laser experiments an excess of 1 mm DCMUwas ad-ded which gave only a 20 % decrease in theextent of oxidattion of cytochrome b. For thelatter reason and due to the observed first orderkinetios of oxidation of cytoch-rome b, it was con-cluided that the fla,sh-oxidized cytochrome b exisgtedontly in the photosynthetic electron transport chain.However, from table I12 and II4, the oxidationhalf-4time for cytochrome b is increased by a factorof 2. The latter inhibitory effect is probably asecondary inhilbi!tion due to the excessive concen-tration of DCMU and is in addlition to the primaryinhibittion of photosystem II by DCMU (13). Noeffect on the laser induced kinetics was observed atlower concentrations of DCMU (< 100 Mm).

T.he rapid rate of the control reaction, t,/2- 6 msec, is independent eVidence that the observedoxidatbion of cytochrome b occur,s in the photo-syn,thetic electron transport chain rather than inthe mitochondriia of the allgal celits. The pho-tosyn-thetic oxidation of cytoohrome b is 5 to 10 timesfaster than that observed in mitochondnria and wholecdlls of an(imal onigin (1,4). In particular, Chance(2) observed a hallf-time of 70 msec for the oxida-

tion oif cytochrome b in the State 4 to 3 transitionin rait live,r mitochondria. More recenitly, a half-bime of 30 msec has been observed for the latterreaotion (B. Chance, personal communication).

The Effect of Uncouplers. The effect on thelaser-induced kineltics from adding 5 /LM CCCP isdemonstrated in figure 5 and summarized in tablesI and II (10 im CCP). The most significanteffect is an incretase in the ra.te of cytoch,rome boxidaition by a factor of more than 2 (figs 5F and5G, table I12 and II4). The most immediate initer-pretation is thlat a site of photophosphorylationexis'ts beitween cytochrome f and cytochrome b,asistuming a linear sequence of these 2 ca,rriers (9).It should be noted ithait the 421 mu recovery half-time (itable II1 and fig 5E) is not correlalted withthe uincoupled cytochrome b oxidabion h,alf-time.This fact willil be discussed in the final section.

Referring to table I3,.the cytoehrome f oxidationrate is increased by 30 % tipon addition of 10 ,LMCCCP. However, the experimenital utncertain,tiesare larger compared with the cytochrome b case, sothat a site of photophosphorylation between thephotosynithetic trap and cytoch-rome f is not so

307



PLANT PHYSIOLOGY

Controls-65x0 M CCCP

Absorption Decrease

zA _T ~~IT-7

- 5msecLaser

0.421m421m/i

LlI.I7. 7H~~~~~~~.... ....

Laser-1 t-5 msec

[pULLJ

Laser

432 ml

0.002

II

-w S5msecLaser

564 mp.

f -+ ~-5msec H 05msecLaser

D

Laser554mp.

+ 100gsecLaser Laser

FI(;. 5. The effect of the uncoupkIlr CCCP' on the laser-ilnduced kinetics in the C/laoidomol(InaS p.ile grcLilmiuitant. 440 Ug chlorophyll/nil. Iistrument tiuine Constalts I 0 e, (fig 5 , C, an1d K F, G ) and _ 10 C(figr S|) and H).

__-

t+ --a.- .

I

A11 .11, I'll H H H H

..I r

I

I

-i I I

308

t

5 msec

F7i

. -I



HILDRETH-ELECTRON TRANSPORT IN A CHLAYIYDO1O.N-AS MUTANT

firmly establiished. The diff.iclt4ty is illustrated infigures 5D and 5H w,he-re the signal-to-noise ratiofo,r cytochroome f kinetics is considerably lower thanfor the cytoch'rome b kinetics.A shhort lag can be detected between the insitanrt

of tfhe la,ser filash and the start of first-orderoxid,ation of cytoch,rome f in figure 5D. Averagedover 20 recordings, the lag amiotinited to 30 ,usec,and may be duie to a slighit undershoot in the re-covery of the photomultiplier from the laser flash.The baseiline preceding the flash in figures SD and5H is insufficient in length; hence, for an accuratecomiparis,on, tihe trace is reset and an entire baselineis shown.

The effect of FCCP uiponl the laser indtucedkinetics was essenitiallily identbical to that for CCCP.These uncoulplers in h'igher concentra.tions, produtcedan inhibitory effect upon the cytocihirome f andcytochhroime b oxidation rates. For example, thelaitter r-ates are decreased by - 30 % with theaddiition of 140 jsM CCCP.

Final Decay Rates of Laser-induced Kinetics.Table III lisits tihe final recovery h,alf-times forthe previously described laser-induced absorptionchanges. These decay t-races are observed at sweep

rates of 200 to 500 m'sec. /cm and are, therefore,sul)ject to instabilities and drifting in the single-be,am o,ptica,l system (cf. fig 1G).

Under inhiibition by HOQNO or salicylaldoximethe final recovery ralte o,f cytocohrome f is increased(table III3), apparently in contradiction witih our

previouis conclusion that these inhib-itors act at a

site between cy,toclhrome f and cytochrome b. How-ever, the increased recove.ry rate most probablyindicates that the normal decay pa,thway for cyto-chrome f is interrupted by the inhibiftor and some

alternalte reductanit causeis the recovery of cyto-chrome f. This is in agreement wit,h the con-clusionof Fork and Urbaclh (12, see allso reference 14),tihat the recovery of electron carriers in the darkoccurs via electron sources independent of photo-system II when the norma,l reduotion pathway isinhijibited. According to this interpretation, the in-hiibitor HOQNO or salicylaldoxime produces a

greater reducing potentiall in the viciin,iity of cyto-chirome f, olbserved as an increa,sed recovery raltein the dark.

Tihe recovery half-4times observed at 432 m/uanid 564 m/L (italble III2 and II4) are ch,aracteristicof the first order decay of the oxiddized steady stateof cytochrome b. Under DCMU inhfibi'tion ofphot,o,system II, the cytochrome b recovery rate isnott signif(icanitly different from th,at oif th;e control,again indicating recovery vi,a electron stources inde-pendent olf system II.

Quantum Requiremuents for Cytochrome Oxida-tion. Figure 6 shows the extents o,f the oxidizedsteady staltes of cytochrome f and cyttoclhrome b inthe light 1limited region. That is, the ]laser bea,mha's been aittenuated by means of a negative lensand neutral densilty filters, t,o the incident energiesindica.ted on figure 6.

From the initiall slopes of 12 curves sim,ilar tothose shown on figure 6, the following quantumreqtuirements have been cailculaited:

1/Df = 4.0 0.6 for cytochrome f;li/b = 1.7 + 0.5 for cytochrome b;

where (Df and Db are the quan(ttim efficiencies for

oxidation of cytochrome f and cytochrome b re-

16

0c0

-0.004

00D03- Cytochrome b

x~ ~ ~~~564miL

-0.002-

-0.001- k Cytochrome f

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7Incident Loser Energy (mJoule)-

FIG. 6. The extents of the oxidized steady states ofcytochrome f and cytochrome of type b in the Chlamny-domtonias pale green mutant as a funiction of incidentlaser energy. 95 ,g chlorophyl,l/mdl.

Table III Suinniary of Finial Recovery Kinetics, Following a Laser Flash, in. the ChlamydomonasPale Greent Mutant

The tabulated data for each wavelengtlh refers to the halftime for final recovery of the absorption change,to the baseline, (cf. fig 1). For futrther details, see text.

421 nim, 432 m, 554 m,u 564 m,u

recovery t1/2 recovery t, / recovery t1/2 recovery t1/2('low phase) (slow phase)

insec msec miisec msecCointrols 150 ± 70 900 ± 150 180 ± 60 800 + 150Temperature = 0° 400 ± 100 4000 ± 1000 400 150 4000 1000HOQNO (40tm) 80 + 40 1200 ± 400 120 + 40 1200 ± 300Salicylaldoxime 1700 + 400 60 ± 20 . . .

(4 mm)DCMU (1 m-m) 100 ± 50 900 ± 150 200 ± 100 900 ±300CCCP (10 /I) 100 -4- 50 1200 -4- 200 150±i 50 1200 + 150

309



PLANT PHYSIOLOGY'

spectively. Extinction coefficients of 20 mM-1 cm-'have been used for both cyltochrome f at 554 m/.tand cytoohrome b at 564 mEu. Independent observa-tions showed th(at the percen!taige of laser lighitabsorbed by the allgal suispension ait 694.3 mI. fol-lowed a logariithmic cuirve from 81 % for 35 tugchllorophylil/ml to 99 % for 95 utg chlorophvll/mIl(0.16 cm optica,l path length). The laitter range ofvalues for a,bsorption wvas uised in the calctulationof quiaanttum reqtuliremenits and Nxvas corrected forscaftterinig oif the sample by a Beer's Law deter-mnination under similar experimental condiltions.The qulant,um requiremenits of equtaitionl I are rela-bively higlh compared to 1, (mte in part to the factthait routghly one-half of the acitinic quanta at694 mp are absoribed by system II, aind thereforea,re inef!feebive for cxitochrome oxicdation. Only therelaltive values of (I) and (1Fh, are needed to draw theconclusi,on that the ratio of cytochrome b to cvto-chrome f, oxidized by the laser fla,sh, is of theorder of 2.

From the extents of the oxidized steady statesof cvtoclhrome f ancd cylto,chrome b uider saturatinglight coniditions (maximutim laser outpta of 400mi,lllijoutles) the f(olllowing ratioos were calculated:(chilorophylil)/(cyitochrome f) -- 1/70 +4- 50( c,h,lorophyl,l) / (cytocthr,ome 1 ) =70,() 30Aga,in, fo,r satuiraiting lighit, tihe ratio o,f cytochrome1' to cytochrome f is of the order- of 2.

The f,aclt that the ratios of equation II aresmaliler by a facitor of - 3 than the uisual chloro-phNlll to cytochrome ratios in wild type greein algaeis related to the chflorophyll dleficiencv, per phoito-sx-nltihetic uinit, of the pale greeni muitant. The latterchilo,rophyll deficiency is observed as a photocsyn-thetic rate which is higher bv a facitor of 3, on ach'loro?phyll basis, in the pale greeni muttant, whencompared with the wild type Chlainydomnonras (5).

Discussion

Variouts portions of the preceding results wereiinterpretedl according to a linear sequtenice of cyto-chronie f anld cytochrome b in the photosyntheticelectron transport chain as originally postulated byHill and Benidalil (17). The follow-illg scheme canbe plroposed1 as a working hypothesis:

--Cyt b (i)Systeml Cyclic Pathway \Trap ForExcitation D 24- Cyt. f C- Cyt. b- E- System II

Energy

An electron carrier D hetwxeen the trap andIcytochrome f has not beeni firmly established. Ifthe carrier D exists, it musit react \within the 30jusec lag-time in the kinetics of cyitohrnome f (figSD). On the other hand, at least 1 carrier C mlustexist between cytochrome f and b duie to a lag inthe cyitochrome b kine-tics wthich is greater than thecxtochrome f half-time by more thani a factor of 10

(figs 1 B anid D). T'he latter lag periodcl caiL beclearly observ-ed as a 2 msec interval between thein1stant of the laser fl,ash anfd the start of a first-order oxidationi of cytochrome b. TI'he recovery ofcytochronme f from its in1itial flash-oxidized stateoccurs in 2 diistinct phases, (figs IC an'd 1G). Thenature of the rapid phase (fig IC) indicates theredox level of the ciarrier C prior to the laser flash.No overshoot, or a rapidily recovering over4hoot(t11/, - 1 nisec) in(licates that C was in a relativelymore redlucedI sta,te lprior to the laser flash. Aslowly recovering overshoot (tI/'22 10 msec) indi-cates that C wvas relatively more oxidized. In thiscase, cytochrome f becomes oxidized beyond itssteady state level anid partially recovers followsingdonation of electron.s from cvtochro-me b. Fig0ure1 C with a recovery hal fstime of 2 to 3 milsec isintermeciate betw-eenl the latter 2 cases.

A further point for- discussion is the lpositivetransient in the cvtochrome b kinetics, observed asa trouigh iii the regioni of 435 mny and 565 mu (fig 3,anid reference 3. fig D-5). As previously descri,bed.the extent of thuls trotugh is increased at 0° and ulpon1inhibition by HOQN'O.

One hvoAtheosis (3) is that the p)ositive trans_ientsat 435 mnit and 565 mif are portions of the cito-chrome f s-pectruinm, reacbhing their flll extent onlywhen electroni floxw from cytochrome b to f is in,hib-ited at 00 or by HOQN\'O. At room temperalture, orin the absence of HOQNO, cytochrome b w\\oulldiundlergo a rapid partial oxidation which woulddecre<ase the extent of the observed positive tranlsient(cf. fig 4B and 4D ). rhi, rappld partial oxidationwotild be folflowed 1)v tvhe slox\ er oxidation of c*to-chrome b to its steady state oxidlized leve(l.

Ho'wever, this latiter hypothesis is disproved bythe facit that the samne ilncrease in the 435 mfi and565 m, positive trans'ient is observedl in the ani-aerobic to aerobic transition (fig 3), in the IuIn-cotipled stiate (fig 5), aid tilder pre-illtimination.Unider the latter coilniItioIs, Ino inhibition in electronflow from cytochrome b to cvtochrome f is ob-served, as in the former case of 00 or addition ofHOQNO (talble IT).

An alternatixve hypo,thesis is that cvtochronie bis in the cyclic photophosphorylation p)athwvay assuggested b)y Levinie (R. P. Leviine, personial com-municationi). 'rhis hyipothesis is also supported byevidence obtaiined bv- Cramer anld Butler (8) tIsinigspinach chloroqplasts (cf. G. Hind and J. A. Olson,BNL Symp. Biol., No. 19: l) 188-94, 1966).

The present experimenits then provide evidencefor an iniitial flash ilnduceod redulection of cytochromeb, via System I. Uncouplers, the anlaerobic toaerobic tranisitioin, or Pre-illumin-ation all shift thered,ox equil,ibIrPiuim of cytochrome b toward a mo,reoxidi7ed staute prior to the laser fla.sh. Hence aflas;h gives a greater reduictioni transienit in tihecytiochrome b kinetics anid an oxidized steady statewhich is decreased in extentt. OIn the ofther hand,inhibition at 00 or by)v HOQNO also gives a greater

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HILDRETH-ELECTRON TRANSPORT IN A CHLAMYDOMONAS MUTANT

redutction trangient in the cyttochrome b kineticsdue to the inhibition of Oleetron flow from cyto-chrome b to cytochrome f. The slower rate ofreduction of cytochrome b under H'OQNO ('tableI4) is interpreted as due to a direct interaction ofHOQNO with cytoohrome b. That is, the site ofinhibition by HOQNO is not carrier C in scheme(ii), sinc,e both the iniltial redueti'on and the oxida-tion of cytochroime b are affedted.A sim)illar decrease in the ralte of cytochrome b

reduction is observed with sailicylaildoxime (table14). However, in this ciase, a conitinuous dec-reasein the extenit o,f all laser induced kinetics is observedunder incubation wiith 4 mm salticylaldoxime. Thus,cytochirome b is initiall!ly inhibi'ted by the additionof salicylalidoxime followed by a general in'hibitionof all laser-4nduced absorption ohanges.

The biphasic kindtics of the cytochrome b re-duction transient (fig 2D) are probalbly related tothe cytochrome b to f ratio of 2. Possibly bothcy.todhrome b molectules are in the cyclic path pro-ducing the observed biphaisic kinetics. Alternaitely,1 cytochrome b is in the cyclic pathway, and 1is between System II and System I (scheme i) inagreement with the resulits of Gr(amer aind Butler(8). Tihe eviidence is against a rapid reducition ofcytowhrome b via System II, due to the fact thatD'CMU did not decrease the extent nior the ra'te ofthe positive transienit in t-he cytochrome b kineltics.

An additionall poinlt to be discussed i.s the site ofphotophosphoryl,ation be,tween cytochrome f andcytochrome b (cf. fig 5).cytochro,me f - C - cytochrome b (ii)

A BIf photophosphorylation occurs at site B, then theaddiltion of uncotupller shifts the reidox level ofca,r,rier C towa,rd a m'ore reduced sta.te prior to thelaser flash. The more reduced stalte of C is ob-served by meaniis of an acceleraited paritial recoveryof cytochrome f folilowing a laser flash (table II3).If photophosphoryllaition occurred at site A, then theuncotupler would shi(ft the red'ox levels of both Cand cytochrome b toward a more oxidized stalteprior to the laser fliash. The parltial recovery ofcytochrome f would then be slower than in theconitrol sample, contrary to observation ('table 113).Therefore, the photophosphorylaition site is placelbetween carrier C and cytochrome b in scheme (i).Extending the' line of reaasonling, if more than 1carrier exisits between cyt'och'rome f and cytochromeb, we conclude that the photophosphoryl'a'tion sieis not adjacent to cytodh'rome f.A finail problem to be discussed is the component

respon'si,le for the rapid phase of the 421 m,uabs,orpti,ou change (t1/2 < 2 ,usec; fig 2A). Ahypothesis to be considered ius that the a'bsorptionChange is dtue to the rapid oxidaition of the SvstemI trap, P700 (3, 15). T'he shifit in the P700 Soretband peak from its tusu,al locaition ait 430 to 435 m/Awould then be ascrilbed to the alitered pigmenlt ratiosin the palle green mutttanit of Chlamitydomitona(is. Evi-

dence for similar shilfts in the Soret bland of P700in various photosynthetic samples can be olbservedin spectra taken by Kok (19). In order tio verifythe idenftiflication of P700, attempts have been madeto observe flash-induiced absorption changes in thevicinity of 705 m/%. No concilusions can be re-ported at present from these experiments, whichhave inv-olved pulised laser wavelengt'hs other than694 m,u.

Acco,rding to the above hypothesis, the rapidrecovery ph.ase at 421 m,u woulld be correlated wi,thtihe oxidation of cytochrome b ('figs 1A and 1B;table II). An additional problem then arises inthe uncoupled stalte, where the 421 m,u recoverykinetics, ( fig 5E) do not correlate with the cyto-chrome b oxidation kinetics (figs 5F anid SG). Itis clear th,at the finial interpretation of the kineticsobserved at 421 m,u must await further experimen-tation.

Acknowledgments

The author is greatlv indebted to Dr. B. Chance forproviding the original motivation for this work and forcontinued guidance throughout the investigation. Theauthor acknowledges valuable discussions with Drs. R.P. Levine, W. Parson, H. Schllever, D. DeVault, M.Avron, J. Biggins, and 'M. Nishimura. Thanks are duealso to Dr. Ruth Sager who supplied cultures of thepale green mutant. Assistance in the growth of theChlainydornonas pale green mutant, provided by Dr.Jane Gibson, is gratefufll acknowlcdged.

Literature Cited

1. CHANCE, B. 1955. Intracellular reaction kinetics.Faraday Soc. Disc., No. 20: 205-16.

2. CHANCE, B. 1965. T,he energy-4linked reaction ofcalcium with mitochondria. J. Biol. Chem. 240:2729-4.

3. CHANCE, B., D. DEVAULT, W. W. HILDRETH, W. W.PARSON, AND A. NISHIMURA. 1966. Earlychemical events in photosynthesis: kinetics ofoxidation of cytochromes of types c or f in cells,chloroplasts, and chromatophores. In: EnergyConversion by the Photochemical Apparatus.Brookhaven Symp. Biol., No. 19. Upton, NewYork. p 115-3.1.

4. CHANCE, B. AND B. HESS. 1959. Metabolic controlmechanisms. I. Electron transfer in the mam-malian cell. J. Biol. Chem. 234: 2404-12.

5. CHANCE, B. AND R. SAGER. 1957. Oxygen andlight induced oxidations of cytochrome, flavo-protein, and pyridine nuoleotide in a Chlamydo-monas mutant. Plant Physial. 32: 54-61.

6. CHANCE, B., H. SCHLEYER, AND V. LWALLAIS.1963. Activation of electron transfer in a Chlamy-domnonas mutant by light pulses from an optical1-aser. In: Studies on Microalgae and Photo-synthetic Bacteria. (Special issue) Plant CellPhysiol. p 337-46.

7. CHANCE, B. AND B. STREHLER. 1957. Effects ofoxygen and red light upon the absorption ofvisible light in green plants. Plant Physiol. 32:536-48.

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8. CRAMER, \\. A. AND WV. L. BUTLER. 1967. Onthe existence of two cvtochrome b components inthe electron transport chain of spinach chloro-plasts. Abstracts, Biophys. Soc. WE 6.

9. DE KIEWIET, D. Y., D. 0. HALL, AND E. L. JEN-NER. 1965. Effect of CCCP on the photochemi-cal reactions of isolated chloroplasts Biochim.Biophvs. Acta 109: 284-92.

10. DEVAULT, D. 1964. Photochemical activation ap-paratus with optical maser. In: Rapid Mixingand Sampling Techniques in Biochemistry. B.Chance, R. H. Eisenhlardt, Q. H. Gibson, andK. K. Lonberg-Holm, eds. Academic Press, NewYork. p 165-73.

11. DEVAULT, D. AND B. CHANCE. 1966. Studies ofphotosynt,hesis usinig a pulsed laser. I. Tempera-ture dependence of cytochrome oxidation rate inC/raoiatiuni. E-idence for tunneling. Biophys.J. 6: 825-47

12. FORK. D. C. A_ND NV. 'URBACH. 1965. Evidence forthe localization of plastocyanin in the electron-transport chain of photosynthesis. Proc. Natl.Acad. Sci. 53: 1307-15.

13. GINGRAS. G., C. LEMASSON, AND D. C. FORK. 1963.A study of the molde of action of CMU on pho-tosynthesis. Biochim. Biophys. Acta 69: 438.40.

14. GOEDHEER, J. C. 1963. A cooperation of two pig-ment sYstems and respiration in photosvntheticlumzinescenlce. Biochim. Biophys. Acta 66: 61-71.

15. HILDRETH, WV. NW. 1967. A site of photophos-phorx-lationi in the pale -reeni mutaLnt of C/alaiy-

dom)onatis. Abst. Seventh Intern. Congr. Biochemi..Tokvo. H-59.

16. HILDRETH, W. W., M\. AVRON, AND B. CHANCE.1966. Laser activation of rapid absorption changesin spinach chlioroplasts an d Chilor/l1a. Plant Phy--iol. 41: 983-91.

17. HILL, R. AND F. BENDALL. 1960. Functioni of thet-o cytochrome components in chioroplasts: aworking hypothesis. Nature 186: 136-37.

18. KATOH, S. AND A. SAN PIETRO. 1966. Inhibitoryeffect of salicylaldoxime on chloroplast photo-oxidation-reduction reactions. Biochem. Biophys.Res. Commun. 24: 903-08.

19. KoK, B. 1957. Light induced absorption changesin photosynthetic organisms. Acta Botan. Neerl.6: 316-36

20. KROG-MANN, D. \N`. AND J. J. LIGHTBODY. 1966.PlastocYaniIn froimi A abacoa .arIa bi/is. PlantPlhvsiol. xxii.

21. NISHIMURA, L. 1963. Studies onl the electron-tranisfer systems in photosynithltic bacteria. II.The effect of HOQNO and antimycin A on thephotosynthetic and respiratory electron-tranisfersv,tems. Biochim. Biophys. Acta 66: 17-21.

22. RENGER. G., J. VATER, AND H. T. WITT. 1967.Ef fect of salicylaldoxime on the comiiplete electrontransport system of photosyntkhesis anid onl theisolated reaction cycle II Biochemll. Biophys. Res.Communi. 26: 477-80.

23. SAGER. R. AND M. ZALOKAR. 1958. Pigmntits andphotosynthesis in a carotenoid deficient multantof Ch/a iividoinoiOas. Nature 182: 98-1l(%.

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Laser-Activated Electron Transport in a Chlamydomonas Mutant' · Plant Physiol. (1968) 43, 303-312 Laser-Activated Electron Transport in a Chlamydomonas Mutant' W. W. Hildreth Johnson

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