Be r t s c h said that delayed light emission was due to the imperfect operation of the photosynthetic machinery. In Chlorella, no matter whether the long or short wavelength was excited, the emission spectrum was the same and was identical to fluorescence, implying that all the light which had been stored as energy and had then re- excited the chlorophyll was emitted from chlorophyll-673. If chloro- phylls-α and b are associated within the same system they must be orientated in such a way that there is no normal Boltzman distribution between the various energy levels.
Bi g g i n s wished to emphasize the techniques used in the determina- tion of the action spectrum and quantum yields for N A D P reduction by spinach chloroplasts. The relationship between requirement and incident intensity had been measured between 550 nm and 730 nm and the previously reported linearity confirmed. T w o systems had been used, one poisoned, in which reduced dichlorphenolindophenol was the donor, and a control, in which water was the donor. The initial velocity of N A D P reduction in each system was measured at each actinic wavelength. Extrapolation gave the zero intensity quantum requirements which could then be plotted as an action spectrum. This was interpreted in terms of two pigment systems, the control con- taining light-reactions I and II and the poisoned system containing only reaction I. In both systems the quantum requirement approached the theoretical minimum but at longer wavelengths light-reaction II (in the control) became limiting and hence the quantum requirement increased.
Quantasome aggregates reduced N A D P with about half the efficiency of whole chloroplasts.
Bu t l e r discussed the appearance of light-induced fluorescence
yield changes and the transformation of chlorophyll-α into chlorophyll- a 670 and a 680 as dark grown leaves were allowed to green. After 2 or 3 h of greening, freezing and thawing of the leaf destroyed the
279
D . A . Wa l k e r Queen Mary College, London
28θ E N E R G Y C O N V E R S I O N
light-induced fluorescence yield changes and the asymmetry of the chlorophyll-α band, but at a later stage in greening, freezing and thawing was less effective.
Fr e n c h drew attention to the main features of a scheme which had enabled him to calculate the time course curves for oxygen evolution.
First came a reaction driven by system ι in which some precursor was converted to an activated material. Then, in order to fit observed experimental results such as the overshoot in the oxygen uptake curves, it was necessary for oxygen first to be taken up by the activated complex and then liberated again. This would be consistent with
Wa r b u r g ' s finding of a requirement for oxygen in photosynthesis.
Equilibrium between the precursor and the activated complex would account for the initial spike. In the second-light reaction the product of the first-light reaction was utilized and finally there was the con- tinuing evolution of oxygen (after exposure to light) in what might turn out to be a bimolecular reaction.
Using slides he demonstrated the correspondence between experi- mental data (obtained with Ulva and Porphyridium) and theoretical predictions using the model scheme.
Go v i n d j e e stated that although his present experiments could be explained in terms of both ' spill-over ' and ' separate-package ' models, recent data of Ho c h and of Jo n e s and My e r s could apparently be explained only by the 'separate-package' scheme. He was yet to be fully convinced but he was undertaking similar experiments at the moment.
Ke wished to make three points: (i) The composite P700 and cyto-
chrome reactions were suggested by absorption-change transients with identical kinetics at 430 and 705 and at 430 and 405 nm. (2) The biphasic actinic intensity dependence suggested a sequential nature of the two reactions. (3) The 430 nm absorption change could be eliminated by titrating the aged chloroplasts with ferricyanide. At a ferricyanide concentration of 1-7 χ i o- 6 m, the 430 nm absorption change disappeared completely.
Mi c h e l- Wo l w e r t z referred to work with Madsen which indicated that the absorption band at 684 nm was that of lipoprotein complex with chlorophyllide-a while the 672 nm band corresponded to chloro- phyll-ö. The shift in absorption in vivo from 684 to 672 was caused by the enzymatic phytylization. Other results showed that the pigment was more closely linked to the protein in the water-soluble form than in the fat-soluble form and that the phytylization therefore tended to
detach the pigment from the protein and to solubilize it, more or less, in the lipid phase. She was tempted, therefore, to visualize two principal fractions in the green leaf. The first (680), in which chloro- phyllide-a was in intimate association with protein and the oxidation reduction system, and the second (672) consisting of chlorophyll-α which transferred light energy to the first.
Finally, speculating about the nature of the pigment absorbing at 695 and 700 nm, she referred to a supplementary band at 688 in isolated chlorophyll which she and her colleagues had called a3. This chlorophyll could account for the absorption at 695 in vivo.
Ru m b e r g said that flash induced light (minus dark) difference
spectra in Chlorella were measured using weak detecting light plus background light at 728 to stimulate reaction I or, at 650, to stimulate reaction II. Flash duration was i o- 5 sec and the half-life of the changes observed was i o ~2 sec at 200.
The flash-induced absorption changes of chlorophyll-α appeared only in the presence of 728 background light so when reaction I was not continuously stimulated a difference spectrum could be obtained which consisted of bands at 478, 513 and 648 nm. The absorption changes in the difference spectrum were measured as a function of the intensity of actinic light and at all three wavelengths the changes were identical. It follows that the same substance (probably a chlorophyll-i) is involved and that in light it gives a product at 513. From the mag- nitude of the changes the concentration of this chlorophyll-i (which they formerly called X) was about 1/1000 of the bulk of chlorophyll-α and b together.
Fr e n c h said that this was a very important observation which
brought changes observed in living cells into line with the observations
of Kr a s n o v s k i .
Vr e d e n b e r g expanded the summary of his work given by Wi t t .
He emphasized that it was possible to have P700 in the oxidized state following the addition of P M A (phenyl mercuric acetate) but that the fluorescent yield of H720 was unaltered. Upon illumination by light- stimulating system II, which reduced P700, the fluorescent yield of H720 was again unaltered. This suggested that the fluorescent yield of this pigment is not correlated with redox changes of substances lying between the two photochemical systems but is associated with the reducing side of system I.
We a v e r gave some additional information about the Scenedesmus
mutant (No. 8) which was green but could not photosynthesize because
282 E N E R G Y C O N V E R S I O N
it is blocked in some way in system I. It could be used to give a Hi l l
(benzoquinone) reaction but could not photoreduce carbon dioxide with hydrogen. It appeared to have the normal complement of cyto- chromes, plastoquinone, plastocyanin and ferredoxin. Its fluorescent behaviour indicates the presence of orientated chlorophyll but it did not show certain transients which were characteristic of the wild type and it did not give the narrow EPR signal.
Cell free preparations from both the wild type and the mutant could carry out an electron-transfer reaction from added cyt-c to ubiquinone but the additional ability of the wild type to transfer electrons from cyt-c to oxygen was missing in the mutant.
The narrow EPR signal had been ascribed to P700 but she was not prepared to say with certainty what the source of the narrow EPR signal was, or if P700 was missing, what it consisted of.
Ga f f r o n said that the good quantum yield found by Bi g g i n s for his ascorbate reaction in the near infra-red was reminiscent of the similar result obtained by Ho c h and Κ ο κ for T P N reduction. But such efficient utilization of near infra-red light was not restricted to work with chloroplast preparations. Intact cells would reduce carbon dioxide with hydrogen very efficiently in the infra-red if they con- tained an active hydrogenase. Another complete metabolic process was Wi e s n e r ' s photo-assimilation of acetate in Chlamydobotris. This was an aerobic process rather insensitive to D C M U poisoning.
Quantum yield measurement showed that, throughout the visible, about 14 quanta were absorbed per acetate converted into sugar; but only about 8 were needed at 720 nm. The reaction apparently could not make use of a great part of light energy which the chloroplast absorbed at shorter wavelengths, a rather rigid separation it seemed, from the true photosynthetic system. The latter did work to some extent in the same organism.
Ba n n i s t e r said the work of Jo l i o t, Du y s e n s and others strongly suggested that the oxygen in the ' burst ' originated in the same process as did steady state photosynthetic evolution. Since the burst was finished in about 0-4 sec or less, the oxygen evolution step must be tightly coupled to the photochemical step of system II. It was to be expected then that no evolution of oxygen could continue after a fraction of a second.
Whereas Fr e n c h and Go v i n d j e e had mentioned that evolution of oxygen continues for some tens of seconds after darkness begins, his own results suggested that the continuing evolution of oxygen in
dark was apparent rather than real. It was composed, in fact, of (1) an instantaneous stopping of oxygen evolution, (2) a gradual decrease in light-induced respiration (the magnitude of which could attain as much as 5 cell vol/h), and (3) a more or less pronounced respiratory oscillation which can begin with a marked negative spike. The net transient curve made up of these three super-imposed processes was, further distorted by the response characteristic of the electrode.
Fr e n c h admitted the possibility that there might be some electrode response to be taken into account but Go v i n d j e e said that red flashes on red background gave no prolongation of oxygen evolution whereas red flashes on green background did give a prolongation. If an electrode artifact was involved the response should be the same in both cases.
Fr e n c h added that if Scenedesmus is given light at 650 and then put into darkness the oxygen evolution response was again quite different from that experienced in the presence of background light at 700. This indicated that the last step in oxygen evolution used the products of both reaction and also that the electrode could go down rapidly. Similar responses were obtained with teflon covered elec- trodes.
Ga f f r o n said that if it were true that two light qualities were necessary to give the phosphorylated sugars which then became respiratory substrates it might be expected that, if light of only one quality was used, respiration would be slow to develop. A gradual increase in respiration would look like a slow decline in oxygen evolution.
Ho c h asked Ru m b e r g if the absorption change of cytochrome-ô responded to different wavelengths of actinic light as Du y s e n s had shown for cytochrome-/.
Ru m b e r g replied that cytochrome-è stayed in the oxidized form in the dark and was irreversibly reduced by actinic light at 650 and irreversibly re-oxidized by far-red light at 720.
Th o m a s asked Be r t s c h if it was not possible to explain his failure to find a trapping centre in terms of Wi t t ' s scheme. He replied that he was not aware of anything analogous to P700 of system I which could be used in system II but if Wi t t had such a reaction centre he would be glad to hear about it. Wi t t said that there was X or chlorophyll-ft but, in fact, they now had too many compounds in this area and that this part of the scheme would probably be modified in the future.
Questioning Mi c h e l- Wo l w e r t z about her evidence that both chlorophyllide-a and chlorophyll-α functioned in photosynthesis
Th o m a s asked if her views were comparable to Fr a n c e ' s notion that chlorophyll occurred in a protected as well as an exposed form. She agreed that they were.
Go v i n d j e e agreed with Ru m b e r g that the action spectrum of the 515 and 480 effects were related and probably due to chlorophyll-^
but said that the light intensity versus changes in optical density curves at 480 and 515 were not identical. He asked Ru m b e r g if these became identical in the separated condition and Ru m b e r g was able to assure him that they did.
Du y s e n s remarked that in normal intact green cells cytochrome-è changes had not been observed on moving from actinic light mainly absorbed by system I to that absorbed by system II and vice versa.
If cytochrome-έ was an intermediate between the two systems as suggested by Ru m b e r g and Wi t t oxidation and reduction of cyto- chrome-δ would be expected. He therefore believed that cytochrome-è (which had been shown by Hi l l and Da v e n p o r t to be present in the chloroplast) participated in a side reaction, such as protein synthesis.
Wi t t answered that it was possible to see the oxidation and reduc- tion of cytochrome-è in his system provided that there was a Hi l l
reaction with a good electron flow. Du y s e n s, on the other hand, felt that if it really occurred in intact cells (that is in the absence of a Hi l l
reaction) then it should be possible to see it there also.
Fo r k said that in relation to the assignment of the 513 nm absorb- ance change to chlorophyll-è there was no detectable 513 nm absorb- ance change in Tribonema (xanthophyceae) which lacks chlorophyll-è yet evolves oxygen.
He then asked Wi t t if the kinetics of the 478, 513, 648 nm absorb- ance changes were identical and whether the change at 254 nm exhibited identical kinetics with the 513 nm change.
Wi t t answered that at i o ~2 sec flash the kinetics were identical but that at i o ~5 sec the kinetics divided and that therefore plastoquinone was not producing the change at 513.
Following a question by Se s t ä k in answer to which Mi c h e l -
Wo l w e r t z confirmed that the spectral change 684 to 672 was
associated with phytylization Bu t l e r pointed out that, following aggre- gation, chlorophyll previously absorbing at 672 then absorbed at 680 and that this latter form should not be identified with non-phytyllated chlorophyll. Consideration of the time courses involved also showed that in a 6-day-old dark grown leaf the shift from 685 to 672 occurred in 10 min whereas Wo l f and Pr i c e had shown that phytylization
required about ι h. Furthermore, freezing and thawing was sufficient to effect the 685 and 672 change, though this could clearly not bring about phytylization.
Replying, Si r o n v a l said that in his experiments with aetiolated barley the time course of the Shibata shift and of the phytylation of chlorophyllide coincided. This was shown both by difference spectro- photometry and by paper chromatography methods. The Shibata shift and the phytylation of chlorophyllide both took about 20 min.
Working with 'normal aetiolated bean leaves', Shibata found that the shift occurred in about 15 min. It is true that, in the experiments of Wo l f and Pr i c e, the formation of the ' isoctane soluble chlorophyll- like pigment' took some 40 min. It would, however, be necessary to follow simultaneously in aetiolated bean leaves the in vivo Shibata shift and the phytylation of chlorophyllide (as he did it in aetiolated barley) before drawing a conclusion on the time courses in the bean. On the other hand, he was not prepared to say that a given physical treatment, such as a low temperature treatment, cannot produce a shift. He stated that, in vivo, in the normal culture conditions (in the air, at 200 C, etc.), the Shibata shift (684 nm->672 nm) results from the phytylation of the chlorophyllide produced by light. Any physical treatment which, like phytylation, will tend to modify the relations between the pigment molecule and the lipoprotein carrier would be expected to produce a similar shift.
Kr a s n o v s k i said that his results of some years ago agreed with what had been said by Bu t l e r. There were two types of protochlorophyll, one active, one inactive. The active one had a low temperature fluores- cence maximum at 655. After brief illumination this went to chloro- phyllide 690 which, after phytylization, aggregated, causing another spectral shift.
Th o m a s was curious to know if the 672 form produced by freezing and thawing went on to the 680 form. Bu t l e r said it did not and added that freezing and thawing would also convert protochlorophyll at 650 to protochlorophyll at 635 which would then go directly to the 672 form.
Referring to different kinetics in the fluorescence of H720 and P700 Wi t t asked Du y s e n s if it was permissible to compare two different kinetic measurements made under quite different conditions. Measure- ments of fluorescence yield were made in the absence of background light but P700 kinetics were always measured in the presence of background light (even if only the measuring beam). Ru m b e r g had shown that the change in absorption of P700 fell to zero without
286 E N E R G Y C O N V E R S I O N
background light. Replying, Du y s e n s said that with longer flashes there was no requirement for background illumination and that P700 was directly oxidized. While it was not true, however, that different conditions were used it was an interesting paradox, for which he had no good explanation that the increase in fluorescence was not correlated with a bleaching of P700.
Because of the discrepancy between the fluorescence kinetics and kinetics of the absorption at 515 nm he could not accept that chloro- phyll-δ was the primary reductant of system II.
Speaking about We a v e r ' s contribution Ol s o n said that Bu t l e r
and he had observed that Mutant 8 was particularly susceptible to photoxidation. We a v e r replied that since it had no system I it was conceivable that the products of system II had no outlet and might be concerned in the photoxidation. In reply, Fr e n c h said that they were all still basically using the scheme proposed some years before by
Hi l l and Be n d a l l which they modified to suit their own purposes.
He then asked Hi l l if he would care to comment.
Hi l l said that he was rather concerned with some dark reactions
and was anxious to remain somewhat in the dark. Cytochrome-ά was very queer in mitochondria as they all knew and in the leaves which he had examined he had obtained no evidence that it changed in the light. He had observed it in the reduced state when cytochrome-/was in the oxidized state. That was the wrong way round for the thermo- chemical gradient—a fact which had contributed to the formulation of the original scheme. Now it seemed more likely that plastoquinone might be the substance which was reduced in reaction II. One difficulty was that chlorophyll participated in both reactions (assuming that there were two reactions), accepting an electron from a cyto- chrome in the first case and donating an electron to a cytochrome in the second. This was very difficult to visualize in terms of electron transport.
Hi l l added that the gap between the measured oxidation reduction
potentials relative to oxidized and reduced chlorophyll was insuf- ficient to cover the whole range which they had to consider in photo- synthesis. They did not have available anything as strongly oxidizing as they would wish.
Experiments which show that the biosynthesis of chlorophyll-α in green leaves is represented by :
protochlorophyllide — c h l o r o p h y l l i d e p h y t Q> chlorophyll-α were
presented by Vl a s e n o k, Fr a d k i n and Sh l y k. Light produces an equimolecular amount of the intermediate from the precursor, and the use of C1 402 shows that this reaction precedes chlorophyll forma- tion. The second reaction, apparently brought about by chlorophyllase, was demonstrated on repeatedly darkened leaves; neither reaction appears to go to completion, indicating the possibility of different pigment states. The alternative reaction course whereby phytol is added first was eliminated by spectro-luminescence analysis on extracts from green seedlings of barley.
R E F E R E N C E
SHLYK A . A . et al (i960) Vesti Akad. Ν auk. Beloruss. SSRy ser. biol. nauk 2 , 133 ; (i960) Dokl. Akad. Nauk USSR 1 3 3 , 1472; (1961) Biokhimiya 2 6 , 259, (1962) 2 7 , 599, (1963) 2 8 , 57.
