ACTION SPECTRA FOR THE APPEARANCE OF DIFFERENCE ABSORPTION BANDS AT 480 AND 520 mμ IN ILLUMINATED CHLORELLA CELLS AND THEIR POSSIBLE SIGNIFICANCE TO A TWO-STEP MECHANISM OF PHOTOSYNTHESIS

In the difference absorption spectrum of thin, actively growing* aerobic suspensions of Chlorella pyrenoidmu, both the 480 mp (negative) and the 520 rnp (positive) bands are produced by light absorbed in chlorophyll b and chlorophyll a; the ratio of absorption changes caused by equal number of incident quanta of 650 mp light and those of 680 mp light, is about 1.2. Both effects are partially inhibited by DCMU. Upon replacing air with argon, the effects are increased several fold and become relatively insensitive to DCMU. The increase is stronger in the absorption region of chlorophyll a, than in that of chlorophyll b; the ratio of the absorption changes, caused by equal numbers of 650 mp and 680 mp quanta decreases to about 0.8, for both effects. Variable (as regards the exact ratios of absorption changes), but parallel results for 480 and 520 mp bands were obtained with cultures having low quantum yield of photosynthesis. This parallelism in the behavior of the 480 mp and the 520 mp band suggests that at least part of these two bands have a common origin. However, many observations suggest that both difference bands may have a multiple origin; as a working hypothesis, this origin is discussed in terms of three reactions: Reaction A-Photoreduction of chlorophyll a in system 11; Reaction B-Photooxidation of chlorophyll b in system 11; and Reaction CPhotooxidation (perhaps of a carotenoid) in system I. I . INTRODUCTION THE RECENT hypothesis that photosynthesis involves two successive light reactions, I and 11, sensitized by two different pigment systems (l) leads to the question: Which of the two systems is responsible for the difference absorption bands at 480 mp (negative) and at 520mp (positive), discovered by Duysens@) in illuminated green cells ? Rubinstein and Rabino[cf. also Kok, et aZ.(4)] measured the action spectrum for the appearance of only 520 mp band in thick and supposedly anaerobic Chlorella cells in low, steady light; the results suggested the predominant role of system I, containing the long-wave forms of chlorophyll a. Miiller et aZ.(b) measured the 520 mp band in flashing light, using a suspension of chloroplasts treated with 2,6 dichlorophenol indophenol and excess ferryicyanide (reagents supposed to supress reaction I), the action spectrum was clearly that of system IT, containing chlorophyll b and chlorophyll a 670. The action spectrum for the 480 mp difference band hasnever been described before. We describe here new measurements of the action spectra? for the appearance of both 480 mp and 520 mp bands in thin suspensions of Chlorella cells, suggesting a variable participation of both pigment systems I and I1 under both aerobic and anaerobic conditions. *Having high (0.12) quantum yield of photosynthesis at 670 mp. ?These results were first mentioned in a discussion session of the IV International Photobiology Congress, 793 July, 1964, Oxford, England. 104 GOVINDJEE and R. GOVINDJFE 11. MATERIALS A N D METHODS Chlorellu pyrenoidosa (Emerson’s strain 3 ) was grown in inorganic culture media, with a continuous supply of 5% CO, in air, over a bank of incandescent and fluorescent lamps [see Govindjee(6) for details]. Almost all our samples were selected from actively growing cultures; the quantum yield of 0, evolution in these samples was of the order of 0.12 a t 670 mp as measured by Emerson’s manometric techniques.@) In optically dense (O.D. 2 1.0 at 680 mp) suspensions, as used by Rubinstein and Rabin~witch,(~) the action spectrum can be distorted, because most of the light in the peak of the absorption band does not reach the region of the cuvette traversed by the measuring beam. Instead of correcting for this distortion, we preferred to use relatively thin suspensions (optical density was about 0-5 in the peak of the red absorption band of chlorophyll a). The measuring beam, modulated at 400 cps by a rotatipg disc, was obtained from a 6 V, 18 A ribbon filament lamp and passed through a Bausch and Lomb monochromator (focal length, 250 mm; dispersion, 6.6 mp per mm of slit width). The actinic beam was obtained from a similar lamp, and passed either through a large Bausch and Lomb monochromator (focal length, 500 mm; dispersion, 3.3 mp per mm of slit width); or through ;t Farrand interference filter (half band width, 10-15 mp). The actinic beam (10 mmx 20 mni) uniformly illuminated one side of the cuvette at a right angle to the measuring beam (about 2 mm x 10 mm). A blue glass filter (Corning C. S. 4-72) was placed in front of a photomultiplier (RCA 6217) with S20 response to eliminate fluorescence and scattered red actinic light [see R~binstein(~) for details]. The absorption spectra of the cell suspensions were measured in a Bausch and Lomb Spectronic 505 spectrophotometer, equipped with an integrating sphere attachment. The light intensity was measured by an Eppley thermopile. The changes in absorption were recorded on a Brown recorder. The sample was illuminated for 30 sec after 2-1/2 min dark intervals ; the shutters were automatically controlled. The results were normalized by dividing the a’s (changes in absorption measured in relative units), by the thermopile readings, E (to reduce them to equal light intensity), and by the wavelength of actinic light, X (to reduce them to equal quantum flux). ln our experiments with the monochromator (band width: 3.3 mp), the actinic light was SO weak that one could assume being within thelinear part of the light curve, A ==f(E), and normalize the data, by dividing A by E. In experiments with interference filters, where a sufficiently wide range of actinic light intensity was available, a few action spectra were obtained by actually plotting the light curves, A =f(E), determining their initial slopes and plotting them as a function of wavelength: (d A/dE),=f(h). No significant difference was noticed between the results obtained by the two methods. An approximate correction was attempted for the absorption of the actinic beam in the front layer of the suspension, not traversed by the mearuring beam (an error not quite eliminated even by the use of optically thin suspensions). No precise correction was possible, because the measuring beam is scattered in the suspension, but curves 3 and 5 in Fig. show that this correction could not have changed the general picture. 111. E X P E R I M E N T A L RESULTS 1. Change in absorption as function of light intensity Figure 1 shows the dependence of the absorption change, A , on light intensity, E, with the 650 mp actinic light (obtained with a Farrand interference filter). These results confirm Action spectra for the appearance of absorption bands