oxygen and light induced oxidations of cytochrome

PLANT PIIYSIOLOGY

conmponent of respiratory enzymes. Biophys. Bio-chim. Acta 12: 289-298. 1953.

14. CHANCE, B. Spectrophotometry of intracellular re-spiratory pigments. Science 120: 767-775. 1954.

15. CHANCE, B. and SAGER, R. Oxygen and light inducedoxidations of cytochrome, flavoprotein, and pyri-dine nucleotide in a Chlamydomonas mutant.Plant Physiol. 32: 548-561. 1957.

16. STREHLER, B. L. Some energy transduction prob-lems in photosynthesis. In: Rhythmic and Syn-thetic Processes in Growth, D. Rudnick, ed. Pp.171-199. Princeton Univ. Pr ess, Princeton, NewJersey 1957.

17. DUYSENs, L. N. M. Methods for measurement andanalysis of changes in light absorption occurringupon illumination of photosynthesizing organisms.In: Research in Photosynthesis, Gaffron, ed. Pp.59-67. Interscience Publ., New York 1957.

18. DUYSENs, L. N. M. and SWEET, G. Fluorescencespectrophotometry of pyridine nucleotide in photo-

synthesizing cells. Biochim. Biophys. Acta 25:13-16. 1957.

19. CHANCE, B. and STREHLER, B. L. Effects of oxygenupon light absorption by green algae. Nature180: 749-750. 1957.

20. SMITH, LucILE Abstract of paper to be presentedbefore Biophys. Soc., Ann. Meeting, February,1958.

21. LUMRY, R. Reaction patterns in photosynthesis,discussion. In: Research in Photosynthesis, Gaf-fron, ed. P. 83. Interscience Publ., New York1957.

22. SIEKEVITZ, P., Low, H., ERNSTER, L. and LINDBERG, 0.Effect of Redox dyes and inhibitors on mitochon-drial oxidative phosphorylation, P32-ATP exchangeand ATP-ase. Abstr., p. 44 c, Amer. Chem. Soc.,Ann. Meeting, New York, Sept. 8-13, 1957.

23. FUJIMORI, E. and LIVINGSTON, R. Interactions ofchlorophyll in its triplet state with oxygen, caro-tene, etc. Nature 180: 1036-1038. 1957.

OXYGEN AND LIGHT INDUCED OXIDATIONS OF CYTOCHROME,FLAVOPROTEIN, AND PYRIDINE NUCLEOTIDE IN A

CHLAMYDOMONAS MUTANT 1,2

BRITTON CHANCE AND RUTH SAGERTHE JOHNSON FOUNDATION FOR MEDICAL PHYSICS, UNIVERSITY OF PENNSYLVANIA,

PHILADELPHIA, PENNSYLVANIA AND DEPARTMENT OF ZOOLOGY,COLUMBIA UNIVERSITY, NEW YORK, NEW YORK

In green cells direct spectroscopic studies of therespiratory pigments involved in oxidation-reductionreactions are difficult because their absorption bandsare obscured by those of the photosynthetic pigments.Visual spectroscopy has been used by Hill and byDavenport (1, 2) on etiolated leaves and on cyto-chromes extracted from them and from green algae,but no studies comparable to the classic ones of Keilinon non-photosynthetic systems have yet been carriedout. Spectra representing absorbancy changescaused by illumination have been reported by Duy-sens (3, 4, 5) and by Chance and Strehler (6) forChlorella. Spectra representing changes immediatelyfollowing cessation of illumination have been reportedby Strehler and Lynch (7), and long persistentchanges following a previous illumination are reportedby Lundeg'ardh (8). Witt has studied in detail thekinetics of spectroscopic changes at 520 and 480 m,u(9). Inconsistencies in the results so far obtained in-dicate the necessity for a comprehensive study ofspectroscopic effects in the intact cell. Duysens, who

1 Received March 30, 1957.2 This research was partly supported by grants from

the National Science Foundation and U. S. PublicHealth Service.

first found increased absorbancy at 515 mu upon il-lumination of Chlorella, later found the effect to belacking in Porphyridium. He further reported thatcytochrome f was oxidized upon illumination of Chlor-ella on the basis of an absorbancy decrease at 420 mu,an observation which was later verified in Porphy-ridium, but, in that case, he found, corresponding tothe Soret band at 420 mtu, an a band at 555 mu.This a band agrees with that of purified cytochromef, but the Soret band differs by 4 m,u, a discrepancybeyond the experimental error.3 This inconsistencyin the identification of the cytoehrome involved alsoapplies to the question of whether pyridine nueleo-tide has been observed to be affected by illumination(4). Broad and non-specific increases of absorptionin the ultra-violet region were observed for Porphy-ridium (4) and were attributed to increased reduc-tion of pyridine nucleotide even though no 340 m,upeak was observed. Lundegafrdh, using slower meth-ods than any of the other authors, finds that oxi-dation of cytochrome f following illumination musthave persisted (according to our estimates) for at

3 A close reading of Davenport and Hill's graph (19)gives 424 mg as the correct wavelength and this value isused by Duysens (4).

548

CHANCE AND SAGER-SPECTRA OF RESPIRATORY CO'MPOUNDS

least 30 seconds.4 The cause of some of these dis-crepancies may be the failure to recognize aerobi-osis or anaerobiosis as a factor affecting the natureand extent of the spectroscopic effects caused byillumination, even though this had already been shownto be of importance in the studies of Rhodospirillumrubrum (10). The possible importance of anaerobio-sis was suggested by Duysens' recommendation thatthe cells be allowed to stand in the dark for "half aday" in order to enhance the spectroscopic effect ofillumination and by his later note regarding the en-hancement of the light-induced increase of absorptionat 515 mu by anaerobiosis (5).

The cytochromes that might participate in res-piratory or in photosynthetic electron transfer ingreen and etiolated plants have been studied inten-sively by Hill and his collaborators (1, 2, 11). Cyto-chromes c, b3, f, and b6 have been isolated and in-terest has centered about the possibility of cyto-chromes f and b6 participating in the photosyntheticprocess. The relative values of their oxidation-re-duction potentials led Hill to speculate that b6 wouldbe completely reduced even in the presence of oxygenand that cytochrome f is oxidized in the illuminatedleaf. An absorption band has been observed in theleaves of the golden varieties of certain plants in theposition appropriate to reduced cytochrome b6, al-though this absorption band was not observed in thechloroplast preparation unless dithionite was added.

It is apparent that spectroscopic data on the re-spiratory chain of the green cell together with theaerobic and anaerobic effects of illumination are nec-essary in order to give a comprehensive picture of theinteratetion of the respiratory and photosynthetic proc-esses. While the oxidized minus reduced spectrum ofChlorella shows chiefly the 515 mu pigment and verylittle indication of cytochrome, the studies on a palegreen mutant of Chlamydomonas having low concen-

4 Lundegairdh's recorder (8, 22) is a Leeds and Nor-throp device that plots one point every 2.2 seconds, andwould require at least 30 seconds to record, at intervalsof about 2 m,u, a spectrum from 540 to 570 m,u. Thescanning mechanism described (22) requires an intervalof 4 seconds between readings or a total interval of aminute. A personal communication from Lundeg'ardhv-erifies that by "a few seconds" (8) he meant 10 to 20seconds. In this time interval after cessation of illumi-nation, our rapid recordings show that the oxidation ofcytochrome f directly caused by illumination would havefallen to a small fraction of its initial value, to less than10 %. One could propose that Lundeg'ardh recorded thislast 10 % of the light reaction, but his figure 3 (8) showsthat ascorbate reduction gives "completely reduced"bands of cytochromes c and f and that these absorbancychanges are very nearly identical in magnitude to thoserecorded upon illumination. Thus, the hypothesis thatLundegardh observed the same phenomenon as Duysensand we, appears to be untenable and the hypothesis pro-posed here that he observed the persistence of oxygena-tion of a previously anaerobic Chlorella suspension fitsnicely with his own data. This confusion was caused bylack of adequate controls on oxygen concentration dur-ing experimentation with living cells.

trations of carotenoids and chlorophyll (26) showclearly difference spectra of cytochrome, flavoprotein,and pyridine nucleotide components that are involvedto varying extents in respiratory and photosyntheticreactions. The response of these components to aero-biosis and to anaerobiosis suggests their participationin the respiratory chain and their response to illumi-nation under aerobic and anaerobic conditions mayidentify those components involved in photosynthesis.The importance of oxidation reactions caused by il-lumination is emphasized and the relative speeds ofresponse of the components to illumination gives pre-liminary indication of the sequence of their reactions.

METHODSAMeasurements were carried out with a double-

beam differential spectrophotometer (12) fitted withan especially designed moist chamber described in thepreceding paper (6). Since a wide range of wave-lengths was covered in these experiments, a Wratten39 and a Corning 9781/2 filter was used, the latterserving to eliminate the infra-red transmission of theformer. This filter combination permitted measure-ments down to 320 mu with an acceptable signal-to-noise ratio, and an excellent signal-to-noise ratio from340 to 480 m,u. The filter combination also minimizedstray light effects in the grating monochromators. Forobservations in the ultra-violet region, the tungstensource was operated at a considerable over-voltage inorder to give a better emissivity. For studies of thevisible region of the spectrum, the most satisfactoryfilter combination was Corning 430 plus 9781/2. Forred illumination, Corning 2030 filter provided adequateintensity for saturating effects which were readilycontrolled by the insertion of neutral filters.

As in the previous paper (6), the oxygen concen-trations were altered by passing either nitrogen oroxygen mixtures over the algal suspension. Although5 % CO2 was present in the nitrogen and oxygen, itwas apparently not necessary for the effects uponcytochromes b and f. In one test, pure nitrogen waspassed over the cells for two hours with no evidenceof CO2 lack, but it is possible that a longer intervalmight have produced a demonstrable effect.

The moist chamber was filled as described in theprevious paper (6). The mutant cells settled to thebottom of the chamber in a uniform film of severaltenths of a millimeter thickness. The wild typeformed an irregular film. The respiration of the sus-pension was measured by the two types of platinumelectrodes described previously (6). The Chlamydo-monas cells respired so rapidly that records of theiroxygenation and disoxygenation resemble those ob-tained with yeast (cf fig 2 of (6)). Prolonged oxy-genation of the suspension was required to oxygenatecompletely the respiratory system and to eliminatethe "anaerobic light effects."

Carbon dioxide utilization by the cells was meas-ured in terms of the decrease of acidity. A suitableapparatus for such changes was described previouslyfor simultaneous measurements of respiration and fer-

549

PLANT PHYSIOLOGY

mentation in suspensions of yeast cells (12) andSpruit and Kok have applied similar methods to 02and CO2 exchange in Chlorella (27, see also 13).Suitable controls (fig 2) provided direct calibration ofthe sensitivity of the bicarbonate-buffer cell suspen-sion system. The effects of heating the solution bythe lamp as well as loss of CO2 to the atmospherewere found to be negligible compared to the changescaused by photosynthesis of the algae. The effect ofelectrode polarization upon the potential of the glasselectrode was controlled by periodically removing suchpolarization voltage and noting the effect upon theelectrode potential. The same 100-watt tungstenprojection lamp was used to illuminate the moistchamber as was used to illuminate the cuvette inthese studies.

MATERIALSThe pale green mutant (strain no. 95) was ob-

tained in 1951 after ultra-violet irradiation of thegreen alga Chlamydomonas reinhardii and has beenmaintained suibsequently on an acetate medium in thedark. This strain has never back-mutated to the nor-mal green. For these experiments cultures were grownin the dark on the acetate medium (25) and harvestedat the end of the growth cycle. The cells were centri-fuged and resuspended in their own medium or in0.01 M NaHCO3. The moist chamber requires 1.5 mlof a cell suspension the density of which was ad-justed to approximately 20 micromolar chlorophyll.

For comparison of photosynthetic activity, thenormal green algae were grown in the presence ofacetate in the light and in the dark, and the cells wereprepared as described above and suspended in a bi-carbonate buffer. The normal green strain used wasthe one from which the pale green mutant had beenobtained.

-rE

c- =

ER K

0

c DU)

C>

oj o00

0.

0

RESULTSEXTRACTABLE PIGMENTS: The carotenoid and

chlorophyll content of the mutant is indicated by thegraph of figure 1, which is a recording of the absolutespectrum of an ether solution of pigments extractedby treatment of the cells with 80 % acetone. It isseen that there are major absorption peaks near 660and 430 mu. At the sensitivity used in recordingthese peaks, no absorption bands can be detected thatmight be attributed to carotenoid, but if the scale ismultiplied ten times, a small shoulder is seen on the430 mp peak at about 480 m, which is probably dueto carotenoid. In a study of pigment content of thepale green mutant (26), it was found to contain onlv1/500 the total carotenoid of normal green dark-grown cells, and about 1/10 the chlorophyll. Theonly carotenoids detected in extracts of a one gram(dry wt) sample of pale green mutant cells werealpha and beta carotene. In the normal green cells,these two pigments account for about 65 % of thetotal carotenoids. The mutant has a chlorophyll tocarotenoid ratio of 180 :1 (mole basis) while thenormal cells have a ratio of 15: 1 when grown in thelight and only 4: 1 when grown in the dark. On thebasis of beta carotene only, these ratios are respec-tively 240: 1, 24: 1, and 7: 1. Thus the mutant hasless than 1/30 the a-carotene relative to chlorophyllof the normal green dark-grown cells.

An approximate value of the cytochrome contentrelative to that of chlorophyll is provided by a meas-urement of the ratio of the absorbancy change at430 and 405 m,u caused by the transition from aero-biosis to anaerobiosis to the chlorophyll absorption at430 mu in 80 % acetone. The ratio is 1: 12 and hasroughly the same value when converted to a molebasis. This value indicates the relatively favorable

I -I

Eu

CE

C-

>% 4,

a.0

360 400 440 480 520 560 600 640 680A (myl )

FIG. 1. The absolute spectrum of an extract of the total amount of Chlamydomonas mutant used to obtainthe spectrum of figure 10. Curves A and C are recorded at the same sensitivity, and B is amplified 10-fold. (674 d).

550

CHANCE AND SAGER-SPECTRA OF RESPIRATORY COMPOUNDS

pHpH

200

CO2

A B CFIG. 2. A quantitative determination of the rate of CO2 uptake by 3 different types of Chlamydomonas. The

C02 uptake is measured by the pH change in a 0.01 M NaHCO8 solution in a 2.8-ml cuvette. The electrode sensi-tivities and time scales are included in the diagram as well as the initial values of pH. The upward deflection ofeach of the three traces marks a calibration by the addition of 200 micromoles C02. The interval of illuminationby 110 ft-c tungsten light is also indicated. A. Normal cells are grown in the light in acetate medium. B. Normalcells are grown in the dark in acetate medium. C. The pale green mutant is grown in the dark in acetate medium.(710 f).

conditions provided by the pale green mutant for ob-servation of the cytochromes.

PHOTOSYNTHETIC ACTIVITY, Carbon Dioxide Fixa-tion: The carbon dioxide fixation by three types ofChlamydomonas is illustrated by figure 2. The car-bon dioxide uptake of the illuminated cells is meas-

ured in terms of the change of pH of 0.01 M NaHCO3solution by the glass electrode inserted into an opencuvette. As contrasted with the recent method ofRosenberg (13) whereby the conversion from pHchange of carbon dioxide concentration was calcu-lated for a solution free of interfering substances, wecalibrate directly by the addition of a known volumeof a saturated solution of carbon dioxide as do Spruitand Kok (27). Thus, the three records start withthe addition of the calibrating solution to the dark cellsuspension. This causes a decrease of pH (an upwarddeflection of the traces) and the amplitude of the de-flection is indicative of the buffering capacity of thevarious suspensions. In the case of both the dark-grown green cells (B) and the pale green mutant(C), some acetate was present from the growth med-ium. The differences of these calibrations illustratethe importance of using this method (cf Rosenberg13, table I).

As soon as the pH change due to addition of thecalibrating solution is completed, the light is turnedon and CO2 uptake begins, prior illuminations havingreduced the induction effects to a negligible value.The illumination is maintained until an amount of CO.,

equal to the calibration solution has been taken up.Then the light is turned off, and as shown by thetraces, the CO2 uptake comes to a halt. The rate ofCO2 uptake is simply the quotient of the concentrationof CO2 added in the calibrating solution (200 micro-molar) and the number of seconds required to utilizethat amount of CO2. After the activities have beendetermined, the samples are treated with 80 % ace-

tone, centrifuged, and the chlorophyll estimated in theBeckman spectrophotometer at 660 mu. These valueswere divided by the molecular extinction coefficientfor chlorophyll a (82 cm-1 x mM-1 (14)). In thenormal cells, chlorophyll b is also present and theconcentration found for chlorophyll a is multipliedby 1.7 to give the total concentration. In the mu-tant cells, the correction factor is only 1.1. Thephotosynthetic activity is given in terms of chloro-phyll turnover-,uM CO2 sec-1 chlorophyll-'. Thedata are summarized in table I, and it is seen that theturnover number of the chlorophyll increases fromthe normal, to the dark-grown green cells, to the palegreen mutant cells. These turnover numbers forchlorophyll are the reciprocal of the "assimilationtime" and an inspection of Rabinowitch's table 28 V(15) shows that the values of table I agree very

closely with those obtained for a wide range of leavesand algae; the best value that he gives for Chlorellais 0.03 and agrees very well with the values obtainedfor the light- and dark-grown Chlamydomonas.

Since the ratios of chlorophyll to 8 carotene con-

21

551

PLANT PHYSIOLOGY

TABLE I

COMPARISON OF CO2 FIXING ACTIVITIES OF THREE FORMS OF CHLAMYDOMONAS

CONDITIONS CNITILXCCHL/SROD RATIO OF CO2 X CARO-DESCRIPTION OF MEASURE- INTA /MC2/SE DewsOXNPHYLOR- CHLORO- TENIXAR-1

ME-NT PH IN LIGHT (CM-') (CM-'MM-') XSEC-P PHYLL TO X SECB

MENT X SEC ~~~~~~~~~~~~~CAROTENEB XSC

Light grownnormal Aerobic 7.7 - 0.5 1.9 82 - 0.02 24 - 0.48

Dark grownnormal Aerobic 7.6 -0.5 1.4 82 - 0.03 7 - 0.21

Dark grownpale greenmutant "Anaerobic" 7.4 - 1.0 1.1 82 - 0.10 240 - 24.00

Cells resuspended in 0.01 M NaHCO3. White light, 1000 ft-c. (710 f).

centration in the three types of cells are available, itis possible to calculate the turnover number of 18 car-otene, assuming that it is participating in photosyn-thesis. It is seen that the value for the pale greenmutant would have to be 100 times greater than thatfor the dark-grown normal cells. The significance ofthis result is discussed below.

Oxygen Evolution: Due to the relatively high re-spiratory activity of the pale green mutant under theconditions of figure 2, these cells were anaerobic atthe time of illumination and the increase of oxygenconcentration in the medium was too small to bemeasured by the platinum electrode. By passing a

on off

2.0-

1.0- I

N

0- 430 - 450 mylog Io/I =0.001

k-50 sec -Cytochrome b oxidation+

FIG. 3. Evidence for oxygen evolution caused byillumination of the pale green mutant. The cells arecontained in the moist chamber described in the textand the oxygen concentration (lower trace) is measuredby a spiral platinum electrode located in the lucite bot-tom of the chamber and polarized at -0.6 volts. Thecalibration for the oxygen concentration is given in thediagram and the upper trace represents a spectroscopicrecording of the simultaneous absorbancy change by thedouble beam spectrophotometer. (702b).

1.2-

1.0-

0.8-0

"4(ai) (A)

0.4-

0 .

400 420 440 460

\( mJJ)480 500

FIG. 4. The relative effectiveness of wavelengths oflight appropriate to chlorophyll and carotenoik in photo-synthetic oxygen evolution in the pale green mutant.The method used is that of Castor and Chance (17).(Experiments carried out in collaboration with Mr.Selwyn Ramsay.) (754).

mixture of 5 % oxygen and 95 % nitrogen over thesurface of the washed cells in the moist chamber, theoxygen concentration in the medium was raised tothe point where the spiral platinum electrode could beused to demonstrate evolution of oxygen upon il-lumination. A typical record of both platinum elec-trode and spectrophotometer traces is shown in figure3. The record begins at the left-hand edge with thecell suspension in the dark and both traces horizontal.Upon illumination, there is a disturbance of the spec-trophotometric trace and then an abrupt upward de-flection which corresponds to a decrease of light ab-sorption at 430 m,u measured with respect to 450 mtu.This corresponds to an oxidation of cytochrome of

r-'i

CP

0

I_

7F

552


type b. Shortly after illumination, the platinum elec-trode trace rises indicating an increased oxygen con-centration due to photosynthesis in mutant cells. Uponturning off the light, an abrupt reduction of cyto-chrome b occurs and is followed by a fall of oxygenconcentration due to dark respiration. The increaseof oxygen concentration in the light and its utiliza-tion in the dark, together with the correlation ofthis change with the oxidation and reduction of in-tracellular cytochrome b, give further support to theidea that the mutant cells have an intact photosyn-thetic system.

RELATIVE EFFECTIVENESS OF CHLOROPHYLL ANDCAROTENOID PIGMENTS IN PHOTOSYNTHESIS: In orderto determine the extent to which carotene pigmentsof the pale green mutant contribute to the photosyn-thetic activity, we have used the apparatus describedby Castor and Chance (17) for measurement of theaction spectrum for photosynthesis in the region ofabsorption maxima for chlorophyll and carotenoidpigments (fig 4). The wavelength of the peak ofthis spectrum indicates the participation of chloro-phyll in oxygen evolution by the mutant cells. Thesharp decrease of the effectiveness of longer wave-length of light indicates that carotenoid is not impor-tant in photosynthesis in the mutant cells.

SPECTROscoPIc EFFECTS OF AEROBIOSIS AND AN-AEROBIOSIS: The spectrum representing the absorb-ancy changes that occur in the transition from thesteady state aerobic condition to the steady state an-aerobic condition of the algal suspension is given infigure 5. It should be noted that the scale is brokenat 500 m, and the absorption bands in the visibleregion are plotted at twice the scale of those in theSoret and ultra-violet regions.

A number of components similar to those of the

CPc)

0

C,

Dc0.0

0En)n

4.010 -

0

-.010

Reduced PyridineNucleotide

Cytochrome bft

Flavoprotein

400

mammalian respiratory chain are immediately recog-nizable from the data.5 Reduced pyridine nucleotideshows a symmetrical absorption band centered atabout 345 mju. Flavoprotein shows a distinctivetrough at 470 mu; the trough is caused by the disap-pearance of the oxidized form of flavoprotein. Cyto-chrome b shows a distinctive a band at 563 mixand its corresponding Soret band at 430 to 431 m,u.An a-band attributable to cytochrome of type c showsclearly at 551 to 552 m,u. The Soret band of cyto-chrome c does not show clearly because it is rela-tively less distinctive. The cells show very unusual

5 In order to clarify what may seem to be arbitraryrelationships between the positions of the absorptionbands of components of the electron transfer systemsand the nature of the enzyme systems involved, we havemade the following list:

a. Isolated diphospho- and triphospho-pyridine nu-cleotide have the absorption peaks of their reducedforms at 340 m, and our spectra present the sumof the reduced forms of such pigments.

b. 430 and 563 m,-these absorption peaks are attrib-uted to cytochrome of type b without an attemptat this point to distinguish among cytochrome bof the respiratory ehain, cytochrome bs and cyto-chrome be.

c. 424 and 555 m,u-absorption bands having theirpeaks at these wavelengths will arbitrarily be at-tributed to cytochrome f, although this may notbe an unique or a final designation.

d. 420 and 550 m,u-pigments having their absorptionbands at these wavelengths are identified withcytochrome c of the respiratory chain.

e. 450 to 470 m, is the region where the oxidizedform of flavoprotein absorbs and the pigmentstudied here is assumed to be that involved notonly in the respiratory chain but also in thephotosynthetic proeesses.

Cytochrome ctochrome b

7

600500% (m,u)

+.005

cuC0

0 c

0

0-o-0U,

-O.00

FIG. 5. The reduced minus oxidized difference spectrum for the respiratory components for the Chlamydomo-nas mutant. The open circles represent data taken by alternately flushing oxygen and nitrogen over the cell sus-pension with consequent oxidation and reduction of the pigments (for an example of a recording at a particularpair of wavelengths, see fig 6). A preliminary identification of the cytochrome components is included in the figure(ef footnote 3). Note that the magnification of the scale has been doubled in the visible region of the spectrum.The effects of alternately flushing CO and 02 are illustrated by the closed circles in the region of the Soret band.(672).

553

PLANT PHYSIOLOGY

TABLE IIRELATIVE ABSORBANCIES AND CONCENTRATIONS OF

RESPIRATORY ENZYMES IN PALE GREENMUTANT CHLAMYDOMONAS (672)

WAVE- ABSORBANCY CONCEN_LENGTHS PROBABLE CHANGES AE(cM-1 TRATIONSUSED IDENTITY RELATIVE X MM') RELATIVE(M.") TOC TOC

430-445 a8CO (?) < 0.7 90 0.15 (?)562-575 b 0.8 20 0.8551-540 c 1 20 1470-510 Flavo-

protein 1 11 1.8370-374 Pyridine

nucleo-tide 2.8 6 9

absorption characteristics with respect to cytochromea, for in the region of the a-band of this pigment,about 605 mM, there is a scarcely distinguishable peak.Furthermore, no absorption band attributable to cyto-chrome a3 appears in the Soret region at 445 ma.Since there is no evidence in favor of a terminal oxi-dase of the a + a3 type, other possibilities for the ter-minal oxidase of these algae were considered; for ex-ample, the carbon monoxide binding pigment foundin so many micro-organisms (16). If the carbonmonoxide binding pigment were the terminal oxidase,a distinctive absorption band would be expected toappear at 415 m, upon the addition of CO. Thedashed portion of the trace in figure 5 shows that thisis not the case; no distinctive band appears at 415m/M, in fact, the absorbancy changes for CO additionare very nearly identical to those caused by nitrogen.There is, however, a small diminution of the intensityof the absorption band in the region of 445 m, anda slight intensification in the region of 425 m,u. Theseeffects suggest the presence of cytochrome a3-CO(16). Thus, the small effects in the region of 615 muin the oxidized minus reduced spectrum, and at 445m,p and 425 mM in the oxidized minus carbon-monox-ide-treated spectrum suggest the presence of a ter-minal oxidase system more nearly similar to cyto-chrome a+ a3 than to the CO-binding pigment. Anunequivocal test of this conclusion would require astudy of the photochemical action spectrum for reliefof CO-inhibited respiration in these micro-organ-isms according to the method of Castor and Chance(17), but it is unlikely that this can be carried outsatisfactorily because of the rapid oxygen evolutioncaused by illumination of the mutant.

It has been customary in studies of various celltypes to represent the components of the respiratorychains as a sequence of optical density changes rela-tive to a particular member of the chain, and alsoas a sequence of concentrations. This has been donefor the pale green cells and the results are given intable II. The relative concentrations of cytochromesb and c are about equal as has been observed in manyother types of intact cells (18), but pyridine nucleo-tide and flavoprotein are observed in somewhat lower

concentrations relative to cytochrome than in othercells; for example, ratios of 20 and 3, respectively, areobserved in isolated mitochondria. A partial ex-planation for the low concentration of pyridine nu-cleotide relative to cytochrome is that the spectro-scopic measurements are based on the transition fromthe aerobic steady state to the anaerobic steady state,and it is probable that only a portion of the pyridinenucleotide is affected by this transition; substrate-free cells are desirable for measuring the total con-centration of pyridine nucleotide.

The relative concentration of cytochrome a3, asassayed from the very small spectroscopic effectscaused by the addition of carbon monoxide, is so lowthat it is questionable as to whether this componentwould function together with the other cytochromesas a part of the respiratory chain. In other cells therelative concentration of cytochrome a3 to c has nevershown such a disparity as has been observed here.We conclude, therefore, that it is unlikely that wehave yet identified the terminal oxidase of these cells.

SPECTROSCOPIC EFFECTS OF ILLUMINATION: A typ-ical record illustrating the effect of light comparedwith that of oxygen upon cytochromes of type b isgiven in figure 6. The record begins after anaerobi-osis of the cell suspension has been established. Thecells are then illuminated and an absorbancy decrease

4eming

02+C02 g , Platinum-microelectrode/ trace kF

Cellsinitiallyin N2+(

FIG. 6. The effects of light and oxygen concentrationupon the absorbancy of the Chlamydomonas mutantsuspension. A cell suspension, the difference spectrumof which is given in figure 7, is illuminated with red lightat the moment marked "on" and the illumination isterminated at the moment marked "off." Such illumi-nation causes an upward deflection of the trace whichcorresponds to a decrease of absorbancy at 430 mu withrespect to 450 m,u. The increase of oxygen concentrationon changing the gas stream from N2 + C02 to 02 + C02 isindicated by the platinum micro-electrode trace. Thecorresponding oxidation of the cytochromes of the cellsis indicated by the upward deflection of the spectro-photometric trace which corresponds to a decrease ofabsorbancy at 430 m,u with respect to 450 m,. Illumina-tion of the aerobic algae gives a small, further decreaseof absorbancy at 430 m,u. (669 c).

554


+.toaL)

0

-c

-0L-

-0

350 400 500 550

aL)-tool 01

0

a

I >%

-0-.001 o

D).0

FIG. 7. Effect of aerobic and anaerobic illumination of the Chlamydomonas mutant compared with the effectsof oxygenation. Repetitions of experiments similar to those of figure 6 give the difference spectra for aerobic andanaerobic light effects, as well as the aerobic minus anaerobic difference spectrum. The absorption peaks shownclearly are those due to pyridine nucleotide in the ultra-violet region and cytochromes in the region of the Soretband. In the visible region, only the aerobic and anaerobic light effects are shown because the amplification of thescale is such that the oxidized minus reduced spectrum would be far off scale. The color filter combinations approp-priate to the wavelength regions are discussed in the text. (673).

occurs as is indicated by the upward deflection of thespectrophotometric traces. This decrease correspondsto an oxidation of cytochrome b. On cessation of il-lumination, the absorbancy increases towards the an-aerobic value. The cells are then oxygenated with amixture of 02 aind C02 as is indicated by a downwarddeflection of the patinum electrode trace and the ab-sorption of 430 m,u decreases as the oxygen concentra-tion inereases. After the cells have been oxygenated,illumination causes a smaller absorbancy decrease.Thus an exhaustive study of the light effect requiresrecordings of the spectra in both the aerobic and theanaerobic states. Figure 7 illustrates such an experi-ment and, in addition, gives for comparison the oxi-dized minus reduced spectrum for the region of 330m,u to 520 m/L. The absorbancy changes are plottedwith the aerobic state as the base-line for the oxi-dized minus reduced spectrum and the dark state asthe base-line for the light-on-light-off spectrum.Thus, the congruence of the traces shows that oxyge-nation and illumination or nitrogenation and darken-ing cause similar oxidation-reduction changes.

In the case of pyridine nucleotide, illumination ofthe anaerobic cells causes about half the oxidation thatoccurs with oxygenation. Nevertheless, illuminationof the aerobic cells causes a further small increase ofoxidation of pyridine nucleotide.

Studies have also been made on the effect of redillumination upon absorbancy changes in a Chlorellasuspension placed in the moist chamber in the man-ner (lescribed above, other details of the experimentaltechnique being approximately the same as those de-scribed above. In Chlorella the absorption of the

material in this region of the spectrum is so large thatthe amount of measuring light falling on the photo-tube is small compared to the leakage of the red lightthrough the filter combination. As shown in figure 8,illumination causes a transient downward deflectionand cessation of illumination causes a transient up-ward deflection. These deflections represent the re-sponse of the resistance-capacitance coupling circuitof the amplifier to the square pulse of photocurrentcaused by turning on and by turning off the light anddo not represent spectroscopic changes. Followingthis transient deflection, true spectroscopic effects canbe recorded and an absorbancv decrease at 340 mu isobserved upon illumination. Upon turning off thelight, the absorption increases towards the initial leveland the phenomenon may be repeated upon illumi-nating for a secondl time. Thus, in Chlorella we find

340-374m-i

- _ _Ot e log Io/I 0.002

increasing absorbancy at 340 muFIG. 8. The absorption spectra changes caused by

red light illumination of the Chlorella sispension. Fordetails see (6). (623 e).

555

PLANT PHYSIOLOGY

confirmation of the phenomenon demonstrated clearlyin the Chlamydomonas mutant.

While illumination causes qualitatively similar ef-fects in both aerobiosis and anaerobiosis in the ultra-violet region, the effects are distinctly different in theregion of the Soret band. Illumination under an-aerobic conditions primarily affects cytochrome b asseen by the peak at 430 m,u, while illumination underaerobic conditions primarily affects cytochromes oftype c or f as shown by the peak in the region of425 m/A. The difference between the anaerobic andaerobic light effects is more clearly distinguished inthe visible region of the spectrum where peaks at-tributable to cytochrome b at 563 m,u and to cyto-chrome f at 555 m,u are found. Flavoprotein, inter-estingly enough, although it shows a relatively largechange of absorption in the oxidized-reduced spectrum,shows little or no effect upon illumination. This re-markable discrepancy is made clear by reference tofigure 9, especially under anaerobic conditions wheresuch a large oxidation of cytochrome b is observed.Effects upon cytochrome a3, if they occur, are toosmall to measure.

It is found that the effect of light compared withthat of oxygen varies with the characteristics of thecell suspension (possibly age and endogenous substratecontent) and figure 9 represents a suspension whichshowed a very large anaerobic light effect. In fact,the oxidation of cytochrome b caused by illuminationvery nearly equals that caused by oxygen. This spec-trum also clearly shows the distinction between aerobicand anaerobic light effects both in magnitude and inposition of the peak; the Soret peak of the anaerobiclight effect lies 4 mu below that of the aerobic effect.

The data of both figures 7 and 9 indicate that nomeasurable absorbancy change has occurred at 515m,u, a wavelength at which distinctive effects are ob-served in algae containing their full complement ofpigments. Since these organisms carry out the photo-synthetic process, the lack of the 515 mMu absorptionband is of some significance.

The rather distinctive effects of oxygen upon theresponse to illumination at 554 mjA and 563 mMu is il-lustrated by figures 10 A and 10 B, respectively.

Aerobiccells'

N2 N2A erobic P 4

cells -

554-540mp

+.010.

0L)C"a

C-)

-0-o0A.0

0

440

A (m,J)

FIG. 9. Light and oxygen effects in an algal suspen-sion in which the anaerobic light effect nearly equals theeffect of oxygen. The difference spectra are obtained byrepetition of experiments with the cell suspension of fig-ure 6. Noteworthy is the very small effect of light uponthe oxidation-reduction state of flavoprotein measuredin the region of 470 m,u. (669 c).

These are the direct photographic recordings of thespectrophotometer output when the monochromatorsare set at wavelengths 554 and 540 m/A in figure 10 Aand 563 and 540 mM4 in figure 10 B. The trace beginswith the cells in the aerobic state. Upon illuminationthe upward deflection indicates the decrease of ab-sorbancy at 554 mu. This absorbancy decreases rap-idly and returns to the initial level upon cessation of

563-540m,uT

log Io/I=0. 001

\ ~Tlog Io/I= 0.001

± Anaerobic+ l, cellson off

off cells

1 50 sec-- 51O50 sec-iA B

FIG. 10. The kinetics of the effects of illumination and disoxygenation measured at wavelength appropriate tocytochrome f (fig 10 A) and cytochrome of type b (fig 10 B). Other details of the experiment are included on thefigure. (669 c).

556


the illumination. The gas passing through the moistchamber is then changed to nitrogen and the traceshows the absorbancy increase caused by the reduc-tion of the cytochrome. Some seconds after anaero-biosis has been established, illumination is repeatedand no significant change other than a brief transientis recorded. The record at 563 m,u differs in thatthere is a slight response to illumination in the aero-bic state and a larger increase in anaerobiosis. Theserecords also give some indication of the clarity of re-cording of these small absorbancy changes in the vis-ible region of the spectrum. The noise fluctuation ofoptical density is less than 1iQ and yet the speed ofresponse is considerably less than 1 second.

A detailed spectrum of the effects in the visible re-gion is shown in figure 11. The anaerobic light effectshows the distinctive and sharp peak at 563 mu. Thesubsidiary peak lies very close to 554 m,u (this peakshows somewhat less clearly in figure 7 and its magni-tude relative to that of 563 mu depends upon the in-terval of illumination as illustrated by figure 12 be-low). The aerobic light effect has a peak around 556m,u which is the appropriate wavelength for cyto-chrome f.

KINETICS OF THE ANAEROBIC LIGHT EFFECTS: Fig-ure 10 A showed that no appreciable absorbancychange occurs at 554 mu for an illumination lasting afew seconds. Nevertheless, a definite peak shows inthe spectra of figures 7 and 11. This is due to thelonger interval of illumination used in those experi-ments and the two apparently divergent results mayreadily be correlated by reference to figure 12. This

+.001

C

C.)

-00

530 550

,A (MU)570

FIG. 11. A detailed study of the effects of light upon

the a-bands of the cytochromes under aerobic and anaer-

obic conditions. The sensitivity used in this experimentis adequate to detect the small shoulder on the peak of

cytochrome b in the anaerobic light effect. (674).

____ ~ sec.

-log Io/I =1,ooff ~ o0.001 of

on-

554-540 mp 563-540 mpFIG. 12. A comparison of the kinetics of the light

effect at wavelengths appropriate to cytochrome f (left)and cytochrome of type b (right). The same cell sus-pension was used in this experiment as in the experi-ment of figure 10. (674).

figure shows absorbancy changes in response to longperiod illumination of the anaerobic suspension. Thetypical rapid response measured at 563 m,u is shownin figure 12 and this agrees with that of figure 10 Bwhich shows this response to occur in less than a sec-ond. The response measured at 554 m,u is muchslower; immediately upon illumination there is no re-

sponse at all (cf fig 10 A). After 30 seconds illumina-tion, a considerable absorbancy decrease has occurred.Upon cessation of illumination, this change dimin-ishes abruptly. Thus, the behavior of the two cyto-chromes upon illumination differs, that of type breaches its steady state and returns therefrom sym-metricallv, while cytochrome f shows a lag in responseto illumination of the anaerobic cells and a rapid re-sponse to cessation of illumination.

DISCUSSIONAs stated in the introduction, there is little con-

sistency of the various experimental studies on thespectroscopic changes caused by illumination of thegreen cell. Nevertheless, various conclusions havebeen drawn, and we propose to examine such conclu-sions in the light of the data obtained in these experi-ments.

1. THE OXIDATION OF CYTOCHROME f UPON ILLU-MINATION: In Duysens' first paper he attributed adecrease of absorption based on a single point at 420m,u to the oxidation of cytochrome f upon illuminationof Chlorella (3). More points were obtained whichconfirmed the existence of such a peak at 420 mu inlater work on Porphyridium cruentum (4). However,424 m,u is the correct location (see footnote 3) for thepeak of the Soret band of cytochrome f (19), andDuysens' data can therefore be used only to supportthe supposition that cytochrome c is oxidized upon il-lumination of both Chlorella and Porphyridium. InPorphyridium, however, Duysens' data show the dis-appearance of a 555 m,u peak on illumination whichhe attributes to cytochrome f oxidation, and this is thecorrect wavelength for the band of cytochrome f (19).

557

PLANT PHYSIOLOGY

The data of Strehler and Lynch (7), taken from the"negative overshoot" phase of their spectroscopic rec-ord, suggest that cytochrome f is reduced upon illu-mination. An alternative interpretation of their re-sults, which is in no way inconsistent with the datathey present, is that the "negative overshoot" phe-nomenon applies only to the 518-m,u band and doesnot apply to the 555-mjA band. We suggest that theywere observing an oxidation of cytochrome f that per-sisted about 1 second after cessation of illumination, asupposition that is not ruled out by our data on cyto-chrome f (cf fig 10).

Lundleg'ardh (8) states that he measured an oxida-tion of cytochrome f "at illumination," but due tohis slow recording method this result must (a) dependupon an extremely long persistence (30 seconds) ofthe oxidation of cytochrome f upon the illumination ofChlorella or (b) be related to indirect effects of illu-mination (see footnote 4). That the spectroscopic ef-fect persists at any reasonable magnitude for approxi-matelv 30 seconds is a result contrary to our recordsof cytochrome f kinetics (see fig 10). Thus, it mayonly be a fortuitous circumstance that Lundegoardh'sdata agree with other experimental results.

In summary, Duysens showed that both cyto-chrome c and cytochrome f are oxidized upon illumi-nationtunder his experimental conditions, a re-inter-pretation of Strehler and Lynch's result is also in fa-vor of oxidation of cytochrome f, and the confirma-tion afforded by Lundegardh's results remains of du-bious value in view of the large interval between il-lumination and measurement. The results of this pa-per show that cytochrome f is oxidized upon illumina-tion under aerobic conditions.

The lack of a measurable oxidation of cytochromef under anaerobic conditions can be attributed to twofactors: 1) that relatively more reductant is presentanaerobically than aerobically and 2) that cytochromeof type b has a higher affinity for the oxidant than f.Both these factors are consistent with the considerabledelay between the oxidation of cytochrome of type band cytochrome f that is illustrated by figure 12. Itis possible that Duysens' oxidation of cytochrome coccurred under anaerobic conditions, especially in hisstudies of Porphyridium where he allowed the cell sus-pension to stand "about half a day" (4) in order toenhance the spectroscopic effects.

2. THE OXIDATION OF CYTOCHROME OF TYPE b: Itis not possible for us to determine which one of thethree possible cytochromes of type b is actually un-der observation because of the similarity of their ab-sorption bands (cytochromes b6, b3, and b of the re-spiratory chain), and our observations suffer from thesame limitations as those of Hill on the leaves ofgolden varieties of certain plants. In such leaves,Hill observes cytochrome b6 to be completely reducedeven in the aerobic illuminated leaf. Our results onChlamydomonas mutant show that the cytochrome oftype b can show only a small further oxidation underaerobic conditions, suggesting that it is already largelyoxidized, especially under illumination. Thus, these

observations differ from those of Hill and do raisequestions about the magnitude of the oxidation-reduc-tion span between cytochrome of type b and cyto-chrome f in the illuminated cell. Our results suggestthat both cytochromes of type b and cytochrome fare more oxidized in the aerobic illuminated green cell.

3. THE OXIDATION OF REDUCED PYRIDINE NUCLEO-TIDE UNDER AEROBIC AND ANAEROBIC CONDITIONS:Duysens' experimental data, from which he has drawnthe conclusion that pyridine nucleotide is reducedlupon illumination of Chlorella and Porphyridium (4),are indistinct and are contrary to the results obtainedhere. His recordings cover the range from 370 to 320miu and show a general increase of absorbancy in thisregion upon illumination. When his curves are studiedlin detail, however, it is difficult to see how this gen-eral increase of absorption could be attributed specifi-cally to the reduction of pyridine nucleotide. Forexample, his curve for Chlorella clearly shows the ab-sorption at 350 miu to exceed that at 340 mu; the re-verse is true for authentic DPNH. In the case ofPorphyridium, the data show a small peak at 340 mybut there is an equally large peak at 360 m,u. A partof this change could be attributed to the 8-band ofcytochrome f which gives an absorbancy change uponillumination that is larger at 360 than at 320 mtL.Thus, the spectroscopic evidence does not support Duy-sens' interpretation in favor of pyridine nucleotide re-duction. On the other hand, Duysens' data surely didlnot show a change of absorbancy that could be attrib-uted to an oxidation of reduced pyridine nucleotide,and there is at the present time no explanation of thediscrepancies between his results and ours. In ourexperiments on Chlorella at 340 m,u, the net effect ofillumination was a decrease of absorbancv at 340 mjAmeasured with respect to 374 m,u (fig 8), indicative ofan oxidation reaction. In the Chlamydomonas mu-tant, clear-cut results in the region from 330 to 370mp, were obtained upon illumination under aerobicand anaerobic conditions. This result is adequatelycontrolled by the response of the anaerobic cells toaerobiosis, i.e., the effects of illumination show thesame sort of absorption maximum and are of magni-tude that is consistent with the effect of oxygen.Thus Duysens' theory (4) that oxidized pyridine nu-cleotide reacts with excited chlorophyll to give re-duced pyridine nucleotide lacks support.

One explanation of the oxidation of reduced pyri-dine nucleotide under aerobic conditions that is to beconsidered is that light-induced oxygenation of the in-terior of the cell causes further oxidation of pyridinenucleotide. If this were so, other respiratory compo-nents should also show further oxidations. Actually,cytochrome f is the only one that shows distinctivechanges under aerobic conditions and cytochrome f isnot considered to be a component of the respiratorychain. Thus, the pyridine nucleotide oxidized uponillumination of the aerobic cells is probably not thatassociated with the respiratory chain but with thephotosynthetic mechanism of the chloroplasts. Thisresult now appears to be opposed to the chemical

558


studies of the effect of illumination upon isolatedchloroplasts; for example, Ochoa and Vishniac (20)used enzymatic methods for demonstrating pyridinenucleotide reduction upon illumination of chloro-plast fragments, and more recently, San Pietro andLang have obtained spectrophotometrically detect-able amounts of reduced pyridine nucleotide at veryhigh DPN and chloroplast concentrations (21).One suggestion that is compatible with both types ofresults is that both oxidation and reduction of pyri-dine nucleotide occur simultaneously in the whole celland that the reduction reaction is completely over-

balanced by the oxidative one. One must postulatethat the chloroplast fragments are deficient in the oxi-dation reaction. In any case, the fact that the neteffect of illumination of the aerobic photosynthesizingcell is an oxidation and not a reduction needs to becarefully considered in mechanisms of photosynthesis.

Both Strehler (28) and Duysens and Sweep (29)have attempted to use fluorimetric methods to demon-strate DPN reduction in Chlorella, but no measurableeffects are obtained on red illumination of the cellsunder conditions suitable for photosynthesis. How-ever, Duysens suggests reduction can be demonstratedin Rhodospirillum rubrum, although a discrepancybetween this result and that of Chance and Smith(10) is apparent.

4. EFFECT OF ILLUMINATION UPON THE STEADYSTATE OXIDATION-REDUCTION LEVEL OF FLAVOPRO-TEIN: Whereas oxygenation of the anaerobic cellscauses a large oxidation of flavoprotein as evidencedby the trough at 460 m/M in the oxidized minus re-

duced spectrum, there is a striking lack of effect ofillumination on flavoprotein under both aerobic andanaerobic conditions. While this result might havebeen expected under aerobic conditions, as a portionof the flavoprotein might be auto-oxidizable, it cer-

tainly is not to be expected under anaerobic conditionswhere the flavoprotein associated with the respiratorychain is shown to be considerably reduced in othercells, and would be expected to be oxidized upon il-lumination together with the cytochromes. A work-ing hypothesis is therefore proposed, that flavopro-tein is a receptor of reducing equivalents and hencethe lack of measurable changes in the steady stateoxidation-reduction level of this component is due tothe combined action of reducing power derived fromthe photolysis reaction and oxidizing power derivedfrom photolysis and the oxidation of other respiratorycomponents. If this hypothesis is applied to bothpyridine nucleotide and to flavoprotein, the effect ofreducing equivalents upon the latter is much greater.

A related effect of reducing equivalents may befound in the lag in the oxidation of cytochrome f uponillumination of the anaerobic cells, such as illustratedin detail by figure 12. It should be noted that theoxidation of cytochrome f proceeds slowly and reachesits steady state value after an illumination interval ofabout half a minute. On cessation of illumination,the reduction of cytochrome f proceeds very rapidly.This would suggest that cytochrome f is being oxi-

dized against a preponderance of reducing substanceswhich have accumulated during the dark interval.We have no explanation for the fact that similarkinetics have not yet been found in the response offlavoprotein and pyridine nucleotide to illuminationunder aerobic conditions.

5. THE ROLE OF CAROTENOIDS IN PHOTOSYNTHE-SIS IN THE MUTANT CELLS: Three results of thispaper bear upon the participation of carotenoid inphotosynthesis in the mutant cell. They are:

1) that the turnover number of chlorophyll in themutant cell is higher than that of the normal cell inspite of a /8 carotene content relative to chlorophyllthat is 1/30th that of the dark-grown normal cell;

2) that / carotene would have to turn over about100 times faster in the mutant cell than in the normalto keep pace with the photosynthetic activity;

3) that the action spectrum for photosyntheticoxygen evolution shows a peak due to chlorophyll, butno detectable shoulder due to carotenoid, as would beexpected if it participated in the activation of chloro-phyll by light;

4) that the 515-m,u absorption band does not ap-pear upon illumination of the mutant cells. It hasbeen proposed elsewhere that this band is due to acompound of carotenoid and, on this basis, the lack ofthe 518-mu band is consistent with a lack of carote-noid participation. Since about equal amounts ofchlorophyll were used in experiments with normal andmutant cells, the 30-fold deficiency of /8 carotene rela-tive to chlorophyll in the mutant cells would not besufficient to prevent the detection of a 515-m,u ab-sorption band upon illumination.

These data indicate that the mutant cells activelyphotosynthesize without any physical evidence for theparticipation of the small amount of carotenoid thatthey contain. A possible function of carotenoid is dis-cussed below.

These studies with the mutant also shed consider-able light on the nature of the chemical change thatcould give rise to the 518-mMA absorption band in thenormal cells. From an observation of several compo-nents of the respiratory chain, it is shown here thatthe predominant effect of illumination of the aerobicor anaerobic cell is an oxidation reaction; reductionreactions can be identified only by a lag period in anoxidation of cytochrome f or the absence of an oxida-tion of flavoprotein: no direct reductions have beenobserved of any components of the whole cell. Thepresence of an oxidant and the lack of a reductantsupports the hypothesis presented in the paper ofChance and Strehler (6) that the 518-m/u absorptionband (phase 3 reaction) (6) results from an oxidationreaction rather than a reductipn reaction.

Further evidence for the oxidation reaction may beinferred from the growth conditions for both this mu-tant and that of Rhodopseudomonas spheroides; theyare sensitive to prolonged illumination to such an ex-tent that the cells are killed. It is possible that theappearance of the 518 mjL absorption band in thenormal type Chlamydomonas or in Chlorella is an in-

5159

PLANT PHYSIOLOGY

dication of a protective reaction in which excess oxi-dizing equivalents produced by the photosyntheticprocess react with a pigment, presumably carotenoid,in order to prevent damage to the cell, and such ahypothesis has recently been presented on the basis ofwork with R. spheroides (23). The possible natureof the reaction was discussed by Calvin (24) and it isprobable that the oxidant involved is not molecularoxygen but rather an intermediate in the photolysisof water, since neither the Chlamydomonas nor the R.spheroides mutant is sensitive to oxygen itself.

6. POSITION OF CYTOCHROMES b AND f IN THESEQUENCE OF ILLUMINATION REACTIONS: The muchmore rapid oxidation of cytochrome b than f underanaerobic conditions in response to the illumination ofthe anaerobic cells suggests the manner in whichthese two cytochromes may react with the oxidizingand reducing equivalents produced by the photolysisof water. The reaction kinetics suggest that the oxi-dizing equivalents are received first by cytochrome band then, with considerable delay, by cytochrome f.But such a sequential action of cytochromes b and fis unlikely in view of the difference in their oxidation-reduction potentials and it is possible that they areoxidized by different systems; b by the terminal, oroxygen, oxidase, and f by photolysis intermediates.A plausible hypothesis is that the oxidizing systemsaffect cytochromes b, and, under anerobic conditions,f, but f can respond only slightly because it is alreadvreacting with reducing equivalents. Under aerobicconditions, cytochrome b responds only slightly toillumination, since it is already supplied with ade-quate oxidizing power from the terminal oxidase un-der these conditions. Under these conditions, the ef-fect of light upon cytochrome f is rapid, presumablybecause less reducing power is present, for example,less reduced pyridine nucleotide is available (see fig7) cytochrome b is already oxidized. Such a reactionsequence is in no way in accord with the oxidation-re-duction potentials of the isolated pigments from whichthe opposite results would have been expected upontheir reaction with oxidizing equivalents. Whetherthe cvtochromes in the intact cell have different oxi-dation-reduction potentials from those obtained uponisolation, or whether some unknown factor is affectingthe reaction kinetics is a point which cannot be de-cided at the present time, and further studies areneeded.

7. PATHWAYS FOR RESPIRATORY AND PHOTOSYN-THETIC ELECTRON TRANSPORT AND PHOSPHORYLATION:Since the components of the respiratory chain ob-served in the oxidized minus reduced spectrum for theChlamydomonas mutant resemble, with the exceptionof the terminal oxidase, those of mitochondria capableof oxidative phosphorylation, we can presume thatthe respiratory chain of the mitochondria of the greencell consists of a similar series of components:02 -+ oxidase cytochrome c -> cytochrome b -+ flavo-

protein pyridine nucleotide -* substrateWhile Hill has proposed that cytochrome f is more

likely to react with the photo-produced oxidant than

is cytochrome b, the delay in the oxidation of cyto-chrome f observed upon illumination of the anaerobiccells together with the rapid response of cytochrome bmust be taken into consideration in any mechanismfor photosynthetic electron transport and phosphoryl-ation. Our data on flavoprotein suggest that thiscomponent is nearest to the source of the reducingequivalents. At the present time our inability to dis-criminate between components of the respiratory andphotosynthetic chains is limited by the fact that ourobservations are based upon the whole cell. Furtherexperiments on particles isolated from these cells willbe necessary to distinguish between the enzymes thatare involved in the pathways of photosynthesis andrespiration.

SUMMARYUtilizing senstive spectrophotometric techniques

and a pale green mutant of Chlamydomonas whichcarries out an active photosynthesis with a low chloro-phyll content, the components of the respiratory chainhave been investigated. It is found that pyridinenucleotide, flavoprotein and cytochromes b and c arepresent in amounts typical of other micro-organisms.The terminal oxidase is present in such a low concen-tration relative to the other cytochromes that it hasnot been surely identified.

Pyridine nucleotide is affected by light aerobicallyand anaerobically; flavoprotein is scarcely changedunder either condition. Cytochrome b responds mostsensitively to illumination anaerobically but respondsvery little to illumination aerobically. Cytochrome fresponds rapidly to illumination under aerobic condi-tions and sluggishly under anaerobic conditions. Noevidence of increased absorption at 518 mu upon il-lumination is recorded. Since the mutant has a highphotosynthetic activity per unit chlorophyll content,it is probable that the compound absorbing at 518m,u and observed upon illumination of the normal cellsis not an essential intermediate in photosynthesis.

LITERATURE CITED1. HILL, R. Cytochrome components and chlorophyll

in relation to the carbon cycle of Calvin. Intern.Congr. Biochem., 3rd Congr., Brussels, Belgium,Proc. Pp. 225-227. Academic Press, New York1956.

2. DAVENPORT, H. E. Cytochrome components inchloroplasts. Nature 170: 1112-1114. 1952.

3. DUYSENS, L. M. N. Reversible changes in the ab-sorption spectrum of Chlorella upon irradiation.Science 120: 353-354. 1954.

4. DUYSENS, L. M. N. Role of cytochrome and pyri-dine nucleotide in algal photosynthesis. Science121: 210-211. 1955.

5. DUYSENS, L. M. N. Studies on catalysts in thephotosynthesis of Chlorella by means of sensitiveabsorption spectrophotometry. Intern. Congr. Bio-chem., 3rd Congr., Brussels, Belgium Abstr., p.43. 1955.

6. CHANCE, B. and STREHLER, B. Effects of oxygen andred light upon the absorption of visible light ingreen plants. Plant Physiol. 32: 536-548. 1957.

7. STREHLER, B. and LYNCH, B. Some relationships be-

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tween light induced absorption spectrum changesand chemiluminescence during photosynthesis. Bio-phys. Biochim. Acta (In press.)

8. LUNDEGXRDH, H. On the oxidation of cytochrome fby light. Physiol. Plantarum 7: 375-382. 1954.

9. WITT, H. T. Rapid absorption changes in the pri-mary process of photosynthesis. Naturwiss. 42:72-73. 1955.

10. CHANCE, B. and SMITH, LuCILE Respiratory pig-ments of Rhodospirillum rubrutm. Nature 175:803-806. 1955.

11. HiLL, R. The cytochrome b component of chloro-plasts. Nature 174: 501-503. 1954.

12. CHANCE, B. Spectrophotometry of intracellular re-spiratory pigments. Science 120: 767-775. 1954.

13. ROSENBERG, J. L. Use of a glass electrode for meas-uring rapid changes in photosynthetic rates. Jour.Gen. Physiol. 37: 753-774. 1954.

14. MACKINNEY, G. Absorption of light by chlorophyllsolutions. Jour. Biol. Chem. 140: 315-322. 1941.

15. RABINOWITCH, E. I. Photosynthesis. Vol. 2, pt. 1,p. 991. Interscience Publ., New York 1951.

16. CHANCE, B., SMITH, LUCILE and CASTOR, L. N. Newmethods for the study of the carbon monoxidecomponent of respiratory enzymes. Biochim. Bio-phys. Acta 12: 289-298. 1953.

17. CASTOR, L. N. and CHANCE, B. Photochemical actionspectrum of carbon monoxide inhibited respira-tion. Jour. Biol. Chem. 217: 453-465. 1955.

18. CHANCE, B. and WILLIAMs, G. R. The respiratorychain in oxidative phosphorylation. Advances inEnzymol. 17: 65-134. 1956.

19. DAVENPORT, H. E. and HML, R. Preparation andsome properties of cytochrome f. Proe. Roy. Soc.(London) B 139: 327-345. 1951.

20. VISHNIAC, W. and OCHOA, S. Phosphorylationcoupled to photochemical reduction of pyridinenucleotides by chloroplast preparations. Jour.Biol. Chem. 198: 501-506. 1952.

21. SAN PIETRO, A. and LANG, H. M. Accumulation ofreduced pyridine nucleotides by illuminated grana.Science 124: 118-119. 1956.

22. LUNDEG.IRDH, H. On the cytochromes b and dh inroots of cereals. Physiol. Plantarum 8: 142-163.1955.

23. GRIFFITHS, MARY, SISTROM, W. R., COHEN-BAZIRE,GERMAINE and STANIER, R. Y. Function of carote-noids in photosynthesis. Nature 176: 1211-1214.1955.

24. CALvIN, M. [Comments on "Function of carote-noids in photosynthesis," by Mary Griffiths et al.]Nature 176: 1215. 1955.

25. SAGER, RUTH and GRANICK, S. Nutrition studieswith Chlamydomonas reinhardii. Annals NewYork Acad. Sci. 56: 831-838. 1953.

26. SAGER, RUTH and ZALOKAR, M. Pigments and photo-synthesis in a carotinoid-deficient alga. (In prepa-ration.)

27. SPRUIT, C. J. P. and KOK, B. Simultaneous observa-tion of oxygen and carbon dioxide exchange duringnon-steady state photosynthesis. Biochim. Bio-phys. Acta 19: 417-424. 1956.

28. STREHLER, B. Absence of reduced pyridine nucleo-tide (D(T)PNH) changes during photosynthesisin Chlorella. Abstr. 1098, Federation Proc. 16:256. 1957.

29. DUYSENs, L. N. M. and SWEEP, G. Fluorescencespectrophotometry of pyridine nucleotide in photo-synthesizing cells. Biochim. Biophys. Acta 25:13-16. 1957.

MALONYLTRYPTOPHAN IN HIGHER PLANTS 1 2

NORMAN; E. GOOD AND W. A. ANDREAESCIENCE SERVICE LABORATORY, CANADA DEPARTMENT OF AGRICULTURE,

LONDON, ONTARIO, CANADA

During the course of our study of indoleacetic acidmetabolism (1, 6), we investigated the effect of exoge-

nous tryptophan on the synthesis of indoleacetyl-aspartic acid in excised pea epicotyls. Tissues bathedfor 24 hours in solutions containing 50 to 200 mg/ltryptophan accumulated considerable amounts of a

substance which closely resembled indoleacetylasparticacid in chromatographic mobility in several solvents,in acid strength, and in color reactions with the Ehr-lich (p-dimethylaminobenzaldehyde) and Salkowski(acid-ferric chloride) reagents (fig 1). Consequently,in discussing metabolic precursors of indoleacetic acidat the 1956 Annual Meeting of the American Societyof Plant Physiologists at Storrs, Connecticut, we re-

ported erroneously the conversion of tryptophan intoindoleacetylaspartic acid. Differences soon became

1 Received April 12, 1957.2 Contribution No. 103, Science Service Laboratory,

Canada Department of Agriculture, University Sub PostOffice, London, Ontario.

apparent. The unknown substance, which occurred inseveral families of higher plants, had a slightly higherRf in most solvents than had indoleacetylasparticacid. Furthermore, although the color produced onpaper with the Salkowski reagent was the purple ofindoleacetylaspartic at very low concentrations, thecolor at higher concentrations was brown; in this re-spect the substance resembled acetyltryptophan.Basic hydrolysis of the unknown yielded tryptophan,not indoleacetic acid.

ISOLATIONIn order to assign a structure to the tryptophan

derivative its isolation in a relatively pure state wasnecessary. This isolation was a somewhat laboriousprocedure since the solubilities of the derivative weresimilar to those of the bulk of the plant acids. How-ever, it was possible to take advantage of the factthat it was a very strong acid and a considerablepurification was achieved by partitioning the plant

561

oxygen and light induced oxidations of cytochrome

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