CHARACTERIZATION OF THE PHOTOTRAP IN PHOTOSYNTHETIC BACTERIA* Paul A. Loach,? Mayfair (Chu) Kung, and Brian J. Hales$ Division of Biochemistry Department of Chemistry North western University Evanston, Illinois 60201

INTRODUCTION Of the three porphyrin analogs (porphyrin, chlorin, and corrin) that are known t o play important biological roles, two of these (porphyrins and chlorins) are essential components of all photosynthetic systems. Chlorophyll (or bacteriochlorophyll) plays the dual role of providing an efficient light-absorbing antenna, and it is also the molecular species which serves as the primary electron donor unit in the phototrap (reaction center). These roles make use of t w o different properties of the macrocycle molecule. Subsequent to the primary photochemistry, several heme proteins are involved in electron transport reactions, and probably in coupling to photophosphorylation the energy released from their oxidation-reduction reactions. Over the past five years much progress has been made in understanding the primary photochemical events in bacterial photosynthesis. Continued progress in this area seems assured as photosynthetic membrane systems are yielding t o systematic dissolution and reconstitution. In this paper, we describe the results of some studies with photoreceptor complexes prepared from the membranes of photosynthetic bacteria. The availability of such purified complexes has allowed further characterization of the primary electron donor complex, tentative identification of the primary electron acceptor molecule, and has given some insight into the structure of the photosynthetic membrane. MATERIALS AND METHODS Preparation of chromatophores from Rhodospirillum rubrum and Rhodopseudomonas spheroides were by standard procedures that made use of sonication and differential centrifugation. Dissolution of chromatophores and preparation of photoreceptor complexes were performed using the AUTS m e t h ~ d . ~ Bacteriochlorophyll -~ was prepared from R . spheroides and R .

* This investigation was supported by research grants from the National Science Foundation (GB 18420) and the National Institutes of Health (GM 11741). t Recipient of a Research Career Development Award from the United States Public Health Service (5 KO4 GM-70133). $ Postdoctoral Fellow of the U.S. Public Health Service (2 F02 GM-51397).Present address: Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803. 8 Abbreviations: AUT particles, the phototrapcontaining fraction prepared by the combined alkaline, urea. Triton-X-100 method for membrane dissolution; AUT-e, electrophoretically-purified AUT particles.

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rubrum by methods previously described. Bacteriopheophytin was prepared by adding HCl (final concentration = 0.25 N) t o a solution of bacteriochlorophyll in an acetone/methanol (7/2) at room temperature and recovering the desired product after 30 minutes by evaporating the solvent under reduced pressure. The bacteriopheophytin was dissolved in the acetone/methanol solvent (7/2) before adding it in an experiment. Chlorophyll a was purchased from the Sigma Chemical Company (St. Louis, Mo.). The preparation of Mn(II1) hematoporphyrin IX was previously reported.6 All glass apparatus for redox studies in the absence of air were patterned after those of Harbury7 and Loach. r9 Electron paramagnetic resonance signals were measured with Varian E-3 and E-4 spectrometers (Varian Associates, Palo Alto, Calif.). Low temperature measurements were conducted with a liquid helium or liquid nitrogen accessory. The light used t o excite t h e sample was provided by a Xenon Corporation Model B Spectroscopic Micropulser employing a Suntron-6A lOOJ Flashtube (Xenon Corp., Medford, Mass.). The experimental arrangement was similar t o that previously described for measuring kinetic transients by epr. The redox buffers used were purchased from K and K Laboratories, Jamaica, N.Y. (indigotetrasulfonic acid, Janus green B, 2,6-dichlorophenol indophenol), Allied Chemical Co., Morristown, N.J. (sodium hydrosulfite), Mallinckrodt Chemical Works, St. Louis, Mo. (potassium ferrocyanide), or Metal Hydrides Inc., Beverly, Mass. (sodium borohydride). pH buffers used were either 1,3-propanediol (tris potassium phosphate or 2-amino-2-hydroxymethyl(hydroxymethyl)aminomethane), pH 7.5. F o r exchange experiments in which bacteriochlorophyll was added to an aqueous solution of chromatophores or AUT-e, the light used t o excite the sample was first passed through a water filter and then a Baird Atomic B-9 narrow band pass filter with peak transmission at 850 nm (for wild type R. spheroides systems) or 880 nm (for R. rubrum and the R-26 mutant of R. spheroides). The bacteriochlorophyll t o be added was dissolved in methanol and added t o the degassed solution of the biological material in .01 M tris buffer, pH 7 . 5 , typically containing 0.05% Triton X-100. The final methanol concentration was between 5 and 10%. In each case, a control experiment was first conducted in which the bacteriochlorophyll was added t o the buffer-Triton solvent in the absence of biological sample and the solution tested for dark or light-produced epr signals. N o such signals were observed for any of the experiments reported here. Instrumental parameters typically used were modulation amplitude = 1.6 gauss and microwave power = 2 mwatt. Most of our experiments involving deuterium-containing material were performed using bacteria and/or bacteriochlorophyll prepared from bacteria, which were grown in 80 to 95% 2 H - H 2 0 with hydrogen-containing substrates (malate and casein hydrolysate). Thus, the “fully deuterated” samples d o not have epr signal half-widths as narrow as those. reported for bacteria grown o n a 100% deuterium-containing ~ n e d i a . ~ ~ ’ ~ Typically, our “fully deuterated” chromatophores had half-widths from 4.7 to 5.3 gauss. The corresponding isolated bacteriochlorophyll had half-widths of 6.0 to 6.5 gauss. These latter values were measured in each experiment and the resulting exchange data compared with the appropriate limit case. In several experiments we found that the bacteria growing on the fully deuterated media gave results comparable t o those grown as stated above, although they were very slow t o grow (2 t o 4 weeks), the cultures were not as dense, and the absorbance of the culture was somewhat changed from that of the normal photosynthetic bacteria. However, when they were grown on, for example, 90% 2 H - H 2 0 with y8

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hydrogen-containing substrates, they grew rapidly ( 3 - 5 days) and appeared much more similar to the normal bacteria. An additional factor in determining the conditions under which we grew our deuterated cultures was the cost. Fully deuterated media would have been prohibitive for the volume of cells we have grown over the past two years.

RESULTS AND DISCUSSION The Primary Electron Donor It was shown long ago that the longest wavelengths of light absorbed by photosynthetic systems were among those most efficiently used.' From this fact alone i t could have been reasonably concluded that either the primary electron acceptor species or the primary electron donor species of the phototrap was composed of chlorophyll (or bacteriochlorophyll). For the two photosynthetic systems that we know the most about, i.e., the bacterial system such as is found in R. rubrum and system I of green plants, we now know that the primary electron donor is composed of chlorophyll (or bacteriochlorophyll). The first evidence that this was so came after Duysens' was able to characterize the absorbance changes in photosynthetic bacteria and algae that occurred as a result of light absorption. Shortly thereafter, Goedheer' provided evidence that the absorbance changes caused by light in photosynthetic bacteria could also be produced in the dark by oxidation. Kok14 showed a similar relationship in green plant chloroplasts. From Kok's studies with chloroplasts and Loach's studies it was also shown that the light-induced epr with bacterial chromatophores,' signal that had been observed in photosynthetic systems' was attributable to photo-oxidized chlorophyll (or bacteriochlorophyll). Loach and Sekura demonstrated the coincidence of the rise and decay kinetics of the light-induced absorbance changes and epr signal under a variety of experimental conditions.'O They also first demonstrated the high quantum yield (0.95 .lo) for photooxidation of the donor unit by light absorbed in the far-red band of antenna bacteriochlorophyll.38 Further chemical studies of the absorbance changes for oxidized porphyrins, and bacteriochlorophyll in particular, were carried out by Mauzerall and coworker^^^^^ and allowed detailed comparison of the cation radical of these porphyrins with the oxidized primary electron donor species. The bacteriochlorophyll that makes up the primary electron donor unit has been shown t o be distinctly different from the rest of the antenna bacteriochlorophyll. The antenna bacteriochlorophyll can be more readily destroyed by pheophytinization' or by chemical oxidation with K21rC16.1*8The bacteriochlorophyll of the primary electron donor species may be reversibly oxidized by one equivalent of oxidant per donor unit and has a lower midpoint potential than that for the antenna bacteriochlorophyll. Reaction center preparations may also be made in which only the donor unit bacteriochlorophyll is isolated with a protein complex after causing physical separation of the antenna bacteriochlorophyll. 8 - 2 0 When bacteriochlorophyll of the primary electron donor unit is extracted from an unmasked phototrap2' o r a purified reaction center preparation,22 it appears t o be identical chemically with ordinary bac teriochlorop hyll. That several bacteriochlorophyll molecules interact to form a donor unit was suggested by Sauer, Dratz, and C ~ y n from e ~ ~ C.D. measurements and by Norris, Druyan, and KatzZ4 from a quantitative comparison of the epr signal properties with those of monomeric and aggregated chlorophyll models. The latter workers

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suggested that the donor unit was actually a special pair of bacteriochlorophyll molecules. This last conclusion has received additional support from endor measurements made o n monomeric bacteriochlorophyll in frozen organic solvent compared with those in in vivo system^.*^>^^ We have recently applied another approach t o show the uniqueness of the donor unit bacteriochlorophyll. Since the photosynthetic bacteria may be readily grown in a media containing deuterium instead of hydrogen, the bacteriochlorophyll of the donor unit of such bacteria will contain deuterium instead of hydrogen and the epr signal of its photo-oxidized form will be only about 4.0 gauss in width rather than 9.5 gauss.27 Such epr signals for equal concentration of free radicals are shown in FIGURE 1. We have sought to use this difference in signal width as a tool t o probe whether it was possible t o exchange the bacteriochlorophyll in the donor unit of the phototrap with bacteriochlorophyll added in solution. If this proved possible, we thought we might be able t o probe the binding site for the donor unit in the protein and also determine how specific the requirement for bacteriochlorophyll might be, or indeed, if the donor pigment is identical in all respects with bacteriochlorophyll.

COMPUTER SIMULATION 'H-Chromatophwas

I I\ \ I \ 1 1

/

2H-Chromotophorer

\

H(GAUSS)

~

~

-----

+

FIGURE 1. Simulated derivative epr signals of equal %pinconcentration for gaussian curves with half-widths of 9.5 gauss ('H-chromatophores) and 5.5 gauss (2H-chromatophores).

Our first experiments were conducted with preparations of photoreceptor complexes because they have a molecular weight of about 100,000 and we thought the membranes o f chromatophores o r whole cells might impede the desired exchange. We were pleased to find good apparent activity with both 'H-R. rubrum and 'H-R. spheroides AUT-e when exchanged with 2H-BChl prepared from R. spheroides. By conducting a series of experiments at different percentages of deuteration, it is possible to gain some insight into the number of molecules of bacteriochlorophyll that share the unpaired electron in the oxidized form of the donor unit. FIGURE 2 shows the theoretical expectations

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FIGURE 2. Dependency of the half-width of the epr signal on the numbers of molecules sharing the unpaired electron as determined by computer simulation. See text for full description.

for the variation of the half-width of the epr signal with the percentage of deuteration for several possible cases. In the simplest case, in which the unpaired electron rests o n only one molecule (labelled curve 1) the half-width rapidly decreases even at very low percentages of deuteration. For plotting each of the curves shown in the figure, it has been assumed that the fully hydrogencontaining and the fully-deuterium containing phototraps have a half-width of 9.5 and 5.5 gauss, respectively. In the first case, this would correspond t o the monomer, and the fact that it is significantly narrower than the half-width of the monomer in methanol would be ascribed to specific interactions with the protein. Known mixtures of H-BChl and 2H-BChl in methanol oxidized with one equivalent of iodine follow this relationship closely when the theoretical curve was simulated from 12.8 gauss t o 6.0 gauss. The nearly linear dependency o n t h e percentage of 2H-BChl at very low 2H-BChl percentages was used to measure unknown concentrations of 2H-BChl in the experiments reported in TABLE 1. Also following the dependency represented by curve 1 of FIGURE 2 are mixtures of H-chromatophores and H-chromatophores in the absence of added Triton X-100 or bacteriochlorophyll; in this case it would be expected that only fully protonated or fully deuterated phototraps contribute t o the epr signal regardless of the number of bacteriochlorophyll molecules that share the unpaired electron in the donor unit. T h e other theoretical curves drawn in FIGURE 2 are for the cases in which more than one pure donor species may be present in the mixture. F o r example, curve 2 represents the dependency of the half-width o n the percentage of deuteration when the unpaired electron is shared by two bacteriochlorophyll molecules and these molecules have a certain statistical probability of both being hydrogen-containing, deuterium-containing, o r where one member contains

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TABLE 1 BACTERIOCHLOROPHYLL EXCHANGE IN *H R. spheroides CHROMATOPHORES* Concentration

Concentration of 'H-BChl Added

of

Trap (M)

(MI 1.1

9.3 10-4 1.2 10-4

*

Assayed by

10-5

8.0 x 10-6 12

2H-BChl Released (Theoretical)

2H-BChl Released (Found)

(Crrnol)

(firnol)

21 18

32 16

oxidation of supernatant.

hydrogen and one deuterium. The last species would have twice the probability of occurring as either of the former two at 50% deuteration. For calculating the theoretical plot, the half-width of the mixed pair had t o be assigned since it cannot be measured. This was done o n the basis of a second-moment calculation. On the same bases, the hypothetical monomeric bacteriochlorophyll in this environment would have the same half-width as that of BChl' in a methanol solution. This model corresponds to that developed by Norris and associate^.^ Theoretical curves 3 and 4 were simulated similarly. For the case in which three or four bacteriochlorophyll molecules share the unpaired electron, the hypothetical monomer species would have a half width of 16.4 and 19.0 gauss, respectively. To understand how the hypothetical monomer might have a greater half-width than would the monomeric bacteriochlorophyll in frozen organic solvents, this would once again be ascribed t o interaction of the bacteriochlorophyll of the aggregate with the protein, and perhaps with other molecules in the aggregate. There is not yet any appropriate model t o guide our expectations as t o what the effect of aggregation and protein interaction might be on the spin density of the unpaired electron on each carbon within the bacteriochlorophyll molecule. Because of the assumption made, curves 1, 2, 3, and 4 of FIGURE 2 may not exactly represent the experimental expectation, but the trend toward a linear relationship a t higher numbers of bacteriochlorophyll molecules sharing the unpaired electron seems clear. The theoretical curves of FIGURE 2 are plotted somewhat differently in FIGURE 3. Plotting the dependency of AH,, in this way makes it easier to compare with our earlier experimental data. The experimental data given in FIGURE 3 are for R. spheroides chromatophores, but very similar results were obtained with R. rubrum and R . spheroides AUT-e. The experimental data are not consistent with those shown in curve 1 and appear most consistent with those of curve 4. The fact that good exchange is observed with chromatophores prepared from R. spheroides is somewhat surprising in view of the membranous nature of chromatophores, but it is comforting to observe the exchange with a more nearly in vivo system. Similar data have been obtained with chromatophores prepared from the R-26 mutant of R. spheroides, but interestingly chromatophores prepared from R. rubrum often show complete inertness toward exchange. We have found that one of the best exchanging of the biological systems are chromatophores prepared from the R-26 mutant of R. spheroides. Presumably the absence of carotenoids in this mutant allows these membrane fractions t o more freely interact with BChl added in the solvent. We have conducted a number of experiments with these chromatophores by adding increasing concentrations of a known mixture of 'H-BChl and 2H-BChl. The

Loach eta). : Phototrap in Photosynthetic Bacteria

*H-BCHII

3 03

T~OD

FIGURE 3. Dependency of the half-width of the epr signal on the number of molecules sharing the unpaired electron. The data of FIGURE 2 were used in constructing these curves. The ZH-BChl/trap plotted on the x-axis is the experimental ratio of the concentrations of added 2H-BChl to P-865. The phototrap was assumed to have five BChl molecules in it. The uppermost curve in the figure was calculated from the linear relationship in FIGURE 2 assuming that that data approximate a five molecule case.

resulting data could then be compared directly with the theoretical curves given in FIGURE 2. The data are again in best agreement with the curve representing a case in which all four bacteriochlorophyll molecules share the unpaired electron when the unit is oxidized by one equivalent. Although the experiments reported so far have been repeated many times with essentially identical results, there is an approximately equal number of experiments that have given data showing lesser exchange. However, H-AUT-e preparations from either R. rubrum o r R. spheroides are nearly always highly reactive. Systems which are reluctant t o exchange sometimes show much better exchange with a crude extract of bacteriochlorophyll o r with pure bacteriochlorophyll to which fresh phosphatidic acid has been added. The data of FIGURE 3 are representative of the greatest exchange observed for any particular concentration of added bacteriochlorophyll and therefore these data seem t o represent the limit case. One test for an exchange reaction is that it should proceed to the same final equilibrium for a given percentage of deuteration whether 2H-BChl is added t o 'H-chromatophores or if H-BChl is added to 2H-chromatophores. We have found good exchange with H-R. spheroides chromatophores or 2H-AUT-e when 'H-BChl is added. Again, only partial exchange is often observed, but those 2H-chromatophore systems that d o exchange well show good agreement with the expectations of a fully reversible system when compared with the most reactive H-chromatophore + H-BChI systems. In most experiments we have compared the product of the magnitude of the

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304

peak to peak change and (AH,,)2 before and after BChl exchange. This calculation would yield a number proportional t o the concentration of unpaired electrons provided the shape of all contributing curves was gaussian. By this calculation the total spin density seemed to be well conserved in most experiments and in many it is maintained t o within 10%. For AUT-e two complications were observed: ( 1 ) For R. spheroides AUT-e, phototraps seem to be regenerated by adding bacteriochlorophyll and ubiquinone. There is often about a 50% loss in phototrap activity on preparing AUT-e from R. spheroides and about that same amount can be regenerated. Little phototrap loss or regeneration is observed with R. rubrurn AUT-e. This problem could be taken into account by including a control in which IH-BChl was added t o 'H-AUT-e or 2H-BChl was added t o 2H-AUT-e and the increased trap activity measured. ( 2 ) A small amount of acceptor radical is often present in addition t o the donor epr signal. This problem was minimized by adding oxidized ubiquinone, which results in a very low concentration of the acceptor specie^.^ Since we realize that addition of BChl t o chromatophores or AUT-e could possibly give rise t o artifactual sites of epr signal production, it was important to demonstrate that BChl was actually released from the biological system in an amount that is consistent with exchange. An additional complication would occur if antenna BChl exchanged as well as the electron donor unit BChl. T o demonstrate that BChl is in fact displaced from the chromatophores during exchange we grew R. spheroides on 4C-malate, prepared chromatophores from them, and exchanged them with cold H-BChl. The chromatophores were then quantitatively separated from the supernatant by centrifugation, more cold BChl was added t o the supernatant, and the resultant BChl was then extracted from the detergent-containing supernatant by adding chloroform-methanol. The BChl-containing extract was purified by sugar chromatography and the resultant pure BChl assayed for I 4 C content. By comparing this latter value with the I 4 C content of purified I4C-BChl isolated from the same preparation of chromatophores, the amount of bacteriochlorophyll released could be calculated. The results are shown in TABLE 2. Although the quantitative correlation is only fair, significant radioactive BChl was found in the supernatant at about the concentration expected for exchange with the phototrap. TABLE 2 BACTERIOCHLOROPHYLL EXCHANGE IN R. spheroides CHROMATOPHORES Concentration of ZH-BChl Exchanging

Concentration

(MI

(M)

4 1.1

10-5 10-5

of

Trap 4.5 x 10-6 2.5 x 10-6

* 4C BChl

1%

Released (Theoretical) bmol) 1.2 2.9

I4C BChl Released (Found) bmol)

14.0 1.8

A second type of assay for released bacteriochlorophyll proved to be more reproducible. 2H-chromatophores of R. spheroides grown in H-H2 0 were prepared and pure 'H-BChl added to them in Tris-Triton buffer. The chromatophores were then quantitatively removed from the supernatant by centrifugation and after adding four volumes of methanol, the BChl of the

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*

supernatant was directly assayed for H-BChl by the iodine oxidation method. Excellent sensitivity for small concentrations of H-BChl could be obtained in this assay, as is illustrated by FIGURE 2. The results are shown in TABLE I . The data are in good agreement with BChl exchange at the electron donor unit, as was expected from the effect on the epr signal. The possibility of both the antenna and the electron donor unit BChl exchanging is easily ruled out by the data of both TABLES 1 and 2. In no case with wild-type R. spheroides and R. rubrum preparations have we seen evidence for such an extensive exchange of the bulk BChl. In view of the data of TABLES 1 and 2, which suggest complete exchange of the four (or possibly five) bacteriochlorophyll molecules in the phototrap, the computer simulation data needs to be corrected for the cases in which a smaller number of bacteriochlorophyll molecules than the total present in the phototrap share the unpaired electron when the unit is oxidized. In FIGURE 4 such a correction has been applied. In order t o take into account the maximal effect of such exchange, it has been assumed that there are a total of five bacteriochlorophyll molecules in the phototrap. We agree with most researchers2 that the likely number is four, but we wished to consider the maximum possible number that would still be consistent with the analytical measurements with reaction center preparations. A comparison of FIGURES 3 and 4 show that the effect of the additional exchangeable BChl molecules is t o move each curve up. Thus, our experimental data are more nearly compatible with the special pair case (labelled curve 2 + 3 in FIGURE 4). If an additional correction was t o be made for the possibility that a small percentage of phototraps may not be reactive toward exchange, the effect o n the special pair case would be t o again move the curve toward the experimental data. For the systems that we have

FIGURE 4. Effect of exchange of bacteriochlorophyll additional to those that share the unpaired electron. The upper curve is reproduced from FIGURE 3. Additional description is found in the text.

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treated quantitively, there cannot be many phototraps that are unreactive and therefore this effect should be small. However, even our best data can probably be regarded as being within experimental error of the special pair case. Confident the BChl exchange with the primary electron donor unit was occuring, we have initiated a probing of the structural requirements a t the presumed binding site. TABLE 3 shows the results of attempted exchange of H-AUT-e and H-chromatophores with various porphyrin analogs. Interestingly, only BChl or alkaline-hydrolyzed BChl appears t o be effective in causing this exchange. No effect was noted with BPheo in the absence or presence of BChl. Even Chl a was without effect. BChl prepared from either R. rubrum or R . spheroides was active. TABLE 3 EXCHANGE WITH BACTERIOCHLOROPHYLL ANALOGS -

Compound

Evidence for Exchange

BChl (R. rubrum) BChl (R. spheroides) Alkaline-hydrolyzedBChl BPheo BPheo (with BChl) Chl a Mn(1II)Hm 1X

+ t

+ -

-

In summary: (1) all four bacteriochlorophyll molecules in the phototrap can be readily exchanged with BChl in solution under appropriate conditions; (2) at least two BChl molecules must share the oxidizing equivalent and our data are perhaps more consistent with the conclusion that all four of the BChl molecules share it; (3) the protein which binds t h e BChl in the phototrap seems to show high specificity for this particular molecule. The Primary Electron Acceptor

Since it was discovered that the primary electron donor unit is composed of bacteriochlorophyll, it was thought unnecessary for the primary electron acceptor species t o be a pigment with an intense long wavelength absorbance band. Indeed, no absorbance changes or epr signals were detected that could be ascribed t o the primary electron acceptor molecule in photosynthetic systems until reaction centers and photoreceptor complexes had been prepared. The lack of an easily observable absorbance change or epr signal has made it very difficult t o identify this component of the phototrap. Some years ago we extended the controlled redox potential method t o anaerobic systems and attempted to learn something about the primary electron acceptor. The usual logic that has been a p p l i e d ' ~ ~to ? ~ interpreting the controlled redox potential measurements of primary events in photosynthesis is illustrated in FIGURE 5. P, and P, are defined as the primary electron donor and the primary electron acceptor species, respectively. Thus, as the environmental potential is raised sufficiently t o cause oxidation of the primary electron donor species, light-induced absorbance and epr changes are lost because the

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Red,

+

p2

+hu p2

-

Pi

nP;'

11

Ph

+

0x2 FIGURE 5. Scheme for explaining the redox dependency of phototrap activity. P1 represents the primary electron donor species and P2 the primary electron acceptor species Ox1 and Redl represent an added redox buffer such as K3Fe(CN)6/&Fe(CN)6 while Ox2 and Red2 represent a different redox buffer, such as oxidized and reduced indigotetrasulfonic acid.

donor has already lost an electron to the redox buffer. By systematically increasing the redox potential of the system, a midpoint potential may be determined, which can be assigned t o the P l + / P l equilibria.',* By using a similar logic, redox control experiments have been used to try t o reduce the primary electron acceptor, as suggested in the scheme of FIGURE 5. A reversible loss in phototrap activity was found t o occur with a midpoint potential of about -.02 volt.1*8y9 A major stimulus t o reassess the low potential titration data came from results obtained when a titration was attempted on purified photoreceptor complexes. These latter complexes were prepared by dissolution of the membrane using AUT condition^^,^ followed by column e l e c t r o p h o r e s i ~ ~ * ~ TABLE 4 summarizes properties of these complexes. Most samples contain less than 0.2 equivalent of iron per phototrap. The effect on the light-induced epr signal of lowering the potential is shown in FIGURE 6 and compared with a theoretical curve representing data obtained with chromatophores. The exciting light is from a continuous source employed at saturating intensities. The light was allowed t o fall on the sample until steady state was reached and then it was turned off. The epr signal observed under these conditions is that of an electron acceptor species which has been previously characterized in this p r e p a r a t i ~ n . ~ From the data, it is clear that there is n o significant quenching of the phototrap activity through the region of 0 to -.2 volt where the activity in chromatophores is reversibly quenched (solid curve in figure). As the light-induced epr

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Annals New York Academy of Sciences

TABLE 4 PROPERTIES OF PHOTORECEPTOR COMPLEXES PREPARED FROM R. rubrum COMPARED WITH CHROMATOPHORES

Molecular weight Prot ein/BChl (ms/ms) P-Lipid (%) BChllP-865 Car/P-865 C~/P-865 Mn/P-865 Fe/P-865 Polypeptides

Ubiquinone/P-865 hnax

AIIT-e

Chromatophores

100,000 to 125,000 3 to 4

About 30,000,000

< I 20 to 25 7 to 8 < .1

32 25 8

< .1 < .2

30,000 15,000 (24,000) (21,000) 4 878 802 5 89 546 51 4 483 377

13

... ... 6 Many

10

882 803 590 54 8 514

... 378

signal is finally quenched at very low potential (e.g., -.42 volt), a dark signal of the same magnitude, shape, and location replaces it. Whereas the difference in behaviour between chromatophores and purified photoreceptor complexes could be attributed t o an artifactual phototrap arising because of the somewhat severe conditions used for membrane dissolution, all physical data indicate that the phototrap of these complexes is little modified from its in vivo condition. While the photoreceptor complex has a molecular weight of about 100,000, i t still retains the light-harvesting bacteriochlorophyll and carotenoid pigments, which show n o significant shift in their max relative to chromatophores. The light-induced absorbance changes measured with AUT-e preparations show the same wavelength dependency as that of data obtained with chromatophores. The quantum yield for oxidation of P-865 remains very high (0.9). As a further check on the integrity of the photoreceptor preparation we have compared the decay kinetics and concentration of the light-induced epr signal as a function of temperature in chromatophores and AUT-e preparations. The data obtained are shown in FIGURE 7. The decay at low temperature is usually interpreted30 as a tunneling of the electron from the primary electron acceptor molecule back t o the primary electron donor unit. The half-life for decay in a tunneling process is known t o be a very sensitive gauge of barrier width and should thus be a good test of the integrity o f the photochemical site after preparation of AUT-e. Both sets of data follow the same temperature dependency all the way to liquid helium temperature. As may be seen in the Figure, the rate of decay increases with a decrease in temperature. Such a change

Loach et al. : Phototrap in Photosynthetic Bacteria

0 0

.

I

I

3 09

R rubrum

0,.

AUT-e Chromotophores

1 -.6 -.5

-.4

-.3 -.2

-.I

0

.I

.2

.3

FIGURE 6 . Potential dependency of the light-induced epr signal in AUT-e preparations from R. rubrum. o and 0 represent results with two different preparations of AUT-e. The experiment was conducted with background light in the room. The exciting light used was that from a 1000 watt tungsten lamp. The epr signal plotted is the light minus dark signal derived by first recording an epr signal under continuous illumination conditions, and then recording the signal with the exciting beam off. Anaerobic conditions. Redox buffer present was indigotetrasulfonic acid at 2 x 10-5 M. The pH buffer was .01 tris buffer, pH 7.5 with 0.2% Triton X-1 00 and 0.01% phosphatidyl ethanolamine. Phototrap concentration was approximately 5 x 10-6M. Reductant used was Na2S204.

is presumably due t o a small change in distance or orientation between the primary electron donor and the primary electron acceptor species. We consider that the close similarity of the data strongly supports our conclusion that the primary electron acceptor molecule and the phototrap, in general, are unchanged in the AUT-e preparation relative t o chromatophores. Furthermore, the magnitude of the light-induced epr signal remains unchanged as the AUT-e sample is frozen and the temperature lowered. The decay kinetics can be well fit with a single pseudo-first order equation throughout the entire temperature range by selecting the appropriate value for the rate constant. It should be noted that behavior of the chromatophore sample is somewhat more co2plicated. Often, when the temperature of chromatophores is lowered below -40 C, about half of the light-induced epr signal is irreversibly produced while the remaining signal is light-reversible t o liquid helium temperature. For the comparison shown in FIGURE 7 , we have plotted only the decay of the light-reversible portion. A similar pattern for decay of light-induced absorbance changes at low temperature has been reported for Chromatium chromatophores. 3 1 The fact that the kinetic dependency for the decay of the light-induced epr signal in Aut-e samples follows the same curve through the ice-melting point and t o room temperature further suggests that no secondary electron acceptor or

100

I

I

I

I

I

I

I

I

I T

R rubrum

80 -

J

0 Chromatophorns

/

/

i

AUT-e

70-

v

W

LL 60-

i

I

A

/

/

d

/

/'

-

-

/O

-1

9

I

I

/'

90 -

--2

I

50-

O//. /

8 40a

6'

0 /'O

30 20 m------w---

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-

10 -

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-

2'0

4b

i0

eb I d 0

d

ILO O:I IAO IAO 2bO 2h0 210 260 280 300 Temperalure ( O K )

FIGURE 7. Temperature dependency of decay kinetics of chromatophores and AUT-e prepared from R. rubrum. The dependency shown is reversible in that some measurements were first conducted at higher temperature, and then at lower temperatures, whereas with other samples the first measurement was made a t low temperature and then the temperature was increased for subsequent points. Anaerobic conditions. O.D. of samples at 880 nm was between 100 and 150.

donor molecules are present, unless they also accept the electron from the primary species at liquid helium temperatures. Esr measurements on purified photoreceptor complexes from R. rubrum and R. spheroides4 provided evidence for an acceptor species with a g-value, signal shape, and half-width consistent with a ubiquinone anion radical. Quantum yield, low temperature (FIGURE 7), and kinetic measurements o n the photoreceptor complexes were also consistent with the assignment of the signal t o the primary electron acceptor molecule. A similar radical species was subsequently observed in detergent-treated reaction center preparations3* and that radical was characterized as a ubiquinone anion radical. We have further examined the acceptor epr signal present in our AUT-e preparations by growing the cells on a deuterated media. Thus, whatever the electron acceptor species may be in the AUT-e prepared from these bacteria, it will have deuterium in place of hydrogens in its structure. This should have the effect of narrowing the epr signal and perhaps result in the observation of a more characteristic hyperfine structure. FIGURE 8 shows the epr signal of such a sample. Although we do not consider this evidence as conclusive proof, the signal is extremely similar to that expected for a deuterated ubiquinone anion radical and is also similar t o the ubiquinone anion radical observed at Q band by Feher, Okamura, and M~Elroy.~ Other evidence for the existence of a ubiquinone anion radical has been provided by Clayton and Straley for their reaction center preparations. They

-

Loach er al.: Phototrap in Photosynthetic Bacteria DONOR ( B Chi.* )

31 1

I G

T 25.C

.;1

AUT- a

ACCEPT OR

FIGURE 8. Donor and acceptor epr signals in AUT-e prepared from R. rubrum grown in dcutcrated media. T i p : light-induced epr signal of chromatophores. Middle: light-induced epr signal of AUT-e. Bortorn: AUT-e with 1 x l0-4M Fe2+ cyt c present. O.D. at 875 iim = 15; microwave power = 4 mW; modulation amplitude = 0.5 gauss.

have carefully documented an absorbance increase at 450 nm (P-450) which was associated with t he quenching of phototrap activity through the 0 t o -.2 volt region.33 S l ~ o t e nhas ~ ~pointed out the marked similarity of these absorbance changes t o those for the ubiquinone anion radical minus oxidized ubiquinone in polar solvents.35 The wavelength shift seen in vivo relative to the model system could be caused by a nearby iron at o m or could be due simply t o the absorbance change of ubiquinone in a nonpolar environment. Because of our evidence for high integrity of the AUT-e preparation, we felt the results of F l CUR E 6 provided a clue that the initial interpretation1y8,9 of low potential redox titrations was incorrect. Several alternative explanations of the low potential quenching of phototrap activity have been previously ~ u g g e s t e d . One ~ ~ >of~ these ~ is shown in FIGURE 9. For the scheme shown it is assumed that the primary electron acceptor is not reduced with a midpoint of -.02 volt but rather that the last equivalent of some secondary electron acceptor, P3, is reduced with this apparent midpoint potential. The reason that phototrap activity is quenched is then attributed t o the fact that the first quanta absorbed by each photoreceptor complex results in a reduced primary electron acceptor molecule that cannot decay because P I t has reacted more rapidly with another reductant in the system and the secondary electron acceptors are all reduced. This scheme also assumes that the added redox buffer is not readily reactive with the reduced primary electron acceptor complex. The quenching of the phototrap activity with a midpoint potential of about -.02 volt would then

-

Annals New York Academy of Sciences

312

p2

ps

+

Red,

11

PI

+hv

Pi

P;

-

P ; m ps

+

+

Red

0x2

P,

,

ver v

+ Ox,

slow ====

P, EZl PI

+ Red,

p3’ FIGURE 9. Variant scheme to explain the low potential (near zero volts) quenching of phototrap activity. PI and PZ are the primary electron donor and acceptor species, respectively. P 3 is a secondary electron acceptor species. 0x1 and Red1 represent the oxidized and reduced forms of a redox buffer such as indigotetrasulfonic acid. Further description of the scheme is found in the text.

reflect a titration of the last equivalent of secondary electron acceptors rather than the primary electron acceptor. To test this possibility we set out t o conduct really dark experiments using a single, very short light flash to excite the system. The difficulty in conducting this experiment properly is due in part t o the high quantum yield exhibited by the photosynthetic bacteria.38 Because they absorb visible light in all regions of the spectrum as well as near infrared energy, the phototrap may become charged and not fully recover in some regions of redox potential, as is suggested in FIGURE 9. Thus, even the very low intensity of a monochromatic detecting beam used in measuring light-induced absorbance change^'^*^^ could result in an unreactive phototrap. We, therefore, chose t o measure the phototrap activity by epr spectroscopy so that no detecting beam that could excite photochemistry was required. Another advantage in using the epr measurement is the fact that samples of much higher concentration can be used. This is desirable in that the minimal light used t o allow t h e human eye t o see in order t o operate the equipment, does not result in excitation of an appreciable percentage of the phototraps in the sample. These dark, single flash experiments were conducted in the following way. Phototrap activity was first measured in air using a single flash t o provide the excitation energy. Sometimes this signal size was compared t o the magnitude of the signal obtained with saturating, continuous light in order t o determine the

Loach et al. : Phototrap in Photosynthetic Bacteria

313

extent t o which the sample was light-saturated by the flash. For the experiments reported, the samples were between 85 and 100% light-saturated. While the sample was still at a high redox potential (above f.10 volt), the room was totally darkened and then the sample adjusted t o a lower potential under anaerobic conditions. Because of large amounts of reductant produced by chromatophores at high c o n ~ e n t r a t i o n ’ . their ~ environmental potential fell very quickly upon degassing. The electron transport components very rapidly respond t o this reductant whereas the electrodes that are reporting the redox potential are slower t o respond. We routinely added K3Fe(CN), to hold the redox potential above 0 volts in order to obtain a valid anaerobic reference point. FIGURE 10 shows a typical result for an experiment conducted in this fashion. Very importantly, the data indicate that there was still excellent activity at -200 mv which was far below the apparent midpoint potential of -.02 volt9 measured under “background light” conditions. Also shown in FIGURE 10 is the fact that no activity could be demonstrated in the sample after it was exposed to normal room light for a few seconds and flashed again. If after the

’I

R. rubrum CHROMATOPHORES I +100mV

6

2 -200mV;

4.-- 5 -

1st flash

3 -200mV; 2nd flash

c

at 4 K

ti 3> Q .c

=K 20

I-

0I

0

I 0.I

I

I

I

0.2

0.3

0.4

Time (s)

FIGURE 10. Light-induced epr signal caused by a single flash in chromatophores prepared from R. rubrum. Anaerobic conditions. O.D. of the sample at 880 nm = 200. Aliquots were taken for analysis from a sample that was adjusted to the potential shown under dark conditions. lndigotetrasulfonic acid was present at 2 x 10-5 M. A solution of NaZS204 was used as reductant. After the first flash, sample 2 was exposed to normal room light conditions for a few seconds while the sample was still in the epr cavity. Then the results of the third flash were recorded. Microwave power = 16 mW; modulation amplitude = 16 gauss; response time of instrument set = 3 msec; time of scan on Nuclear Data Enhancetron = 0.5 second. The scan was purposely initiated before the flash occurred. Temperature = 26”. .05 M phosphate buffer, pH 7.5.

Annals New York Academy of Sciences

3 14

first flash instead of exposing the sample t o room light, it is kept dark, the amount of activity remaining o n the second flash is typically less than 15% and a third flash shows no activity. Since the pulse width of the exciting flash is 10 psec, this result provides strong evidence that no secondary electron acceptors are functioning in the system and that the primary acceptor is initially a one-electron reduced species. It should be noted that the dark decay of P-865’ was about 20 times faster than t h e “tunneling” time at room temperature (see FIGURE 7). All of the redox control experiments were conducted at room temperature. The results of a series of experiments conducted as outlined for FIGURE 10 are shown in FIGURE 11. Because of t h e high concentration of chromatophores required and the difficulty in working in nearly total darkness, one experiment usually consisted of obtaining a reference point at higher potential, then a point or two at lower potential, and finally another point at higher potential. Good dark reversibility was found. The data of FIGURE 11 show that there is n o loss of first flash activity until quite low potential, where an apparent midpoint of about -.37 volt is observed.

I40 120

k I-

E W

60-

a

40-

/-

-Background Light

I

I

I

A/‘

I

L# f 1

-

a uarn, angle riasn

I

20 -

I

I

I

I

1

1

1

1

1

1

1

1

1

Loach et al. : Phototrap in Photosynthetic Bacteria

315

The results shown in FIGURE 1 1 were checked with a number of dye systems present. The first flash results were unaffected by the presence of DPIP, K4Fe(CN)6, indigotetrasulfonic acid, and J a m s green B. The latter is the only one which is positively charged. Sodium dithionite and potassium borohydride have been used as reductants and oxygen or K3Fe(CN)6 as oxidants. In addition t o chromatophores and whole cells from R. rubrum, similar experiments have been conducted with chromatophores from R. spheroides. In all cases the dark, single flash experiments showed complete phototrap activity down to potentials as low as -.30 volt, whereas experiments with “background illumination” showed quenching with a midpoint near -.02 volt.9 In interpreting earlier data1,s,9 obtained by the controlled redox potential method with “background light,” we suggested that the simplest interpretation of the results a t low potential was t o assume that the activity was quenched because the primary electron acceptor molecule was chemically reduced in the dark as the potential was lowered (e.g., FIGURE 5). However. we have also noted some other possible explanation^.^^.^' Other laboratories that have recently employed the controlled redox potential have found similar results and have also chosen to interpret them as a reduction of the primary electron acceptor molecule as suggested in FIGURE 5. We feel that as a result of the experiments reported here, all of the previous data published may have been conducted under “background light” conditions and may possibly need a reinterpretation. As an example of a puzzling feature in some of our earlier data which may now be explainable, the skewed shape suggested by the experimental points can be more readily understood. By assuming that the scheme suggested in FIGURE 9 is correct, the theoretical dependency of phototrap activity o n redox potential can be calculated for a variety of secondary acceptor possibilities. Some of these are summarized in FIGURE 12. For constructing the theoretical plots of FIGURE 12, it was assumed that the secondary electron acceptor was- not free to diffuse t o all phototrap sites on an experimental time scale, but was restricted in locale t o one phototrap. I t is interesting that a theoretical curve for such a localized secondary one-electron acceptor fits the experimental data9 particularly well when x = 2 or 3 and n = 1 (see below). An equation was developed t o express the relationships assumed in FIGURE 12: y=l(1

OnEh/-06+ l 1 r

where y = the fraction of the trap that is active; x = number of the secondary electron acceptor molecules associated with each trap; n = number of electrons transferred; and Eh = measured oxidation-reduction potential. F o r simplicity in developing the equation it was assumed that Eo‘ of the secondary electron acceptor = 0 volt. It should be noted that when x = 1 this equation reduces t o the Nernst equation. It was further assumed for the development of this relationship that only the last equivalent of the secondary acceptor pool t o be reduced will be reflected as a, loss of phototrap activity. Several types of molecules could have an Eo value in the region of potential near -.37 volt for a chemical change involving a one-electron reduction t o a free radical. A molecule which is particularly appropriate and known t o be present in chromatophores, photoreceptor complexes, and reaction center preparations is ubiquinone. One-electron reduction of this molecule is not observed in water-containing solvents because the free radical is quickly protonated and

Annals New York Academy of Sciences

316 100 -

1

1

1

1

-

90 -

00 r-

70 -

-

> 60k -

5> 5 0 a

-

lp 4 0 -

-

30 -

-

le, x 5 2 a le, x.3

20 -

-

2e, x=5 o

-

-

1

-.I0 -.08 -.06 -.04 -.02

1

0

1

1

1

.02 .04

,

1

.06

1

1

.08

1

1

.I0

1

1

.I2

€h (volt)

FIGURE 12. Theoretical redox potential dependency of the light-induced epr signal under background light conditions and assuming that activity is quenched due to a mechanism similar to that suggested in FIGURE 9. It was further assumed that the secondary reductant exists only within specific pool locations in communication with only one phototrap and does not diffuse to other phototraps in the time required to conduct the redox control experiments. The number of secondary molecules per phototrap is given by x in the figure. The number of electrons transferred in each case considered is also given in the figure. The dashed curve represents a one-electron transition where the secondary component is in a 1 : 1 ratio to the primary electron acceptor. The midpoint potential is arbitrarily set equal to zero. The solid curve represents a two-electron transition. Note that for each of the other cases considered, the apparent midpoint potential would be displaced to lower potential according the the relationship E,' = .06/n log[ (2)1/x- 1 1 . See text for a definition of terms. For the cases considered in the figure, the apparent midpoint potentials were arbitrarily set to zero in order to compare the shape of the curves.

disproportionates inty fully reduced (+2e and +2Hf at pH 7 ) and fully oxidized ubiquinone. The E, value for this reaction has been reported t o be near 0.1 volt.49 However, in anhydrous solvents - or in anhydrous environments such as one might expect t o find in the middle of a membrane or in a hydrophobic region of a protein - one-electron reduced ubiquinone is a very stable specie^.^ The polarographic half wave potential of p-benzoquinone in dimethyl formamide is -.29 volt vs. SHE and tetramethyl p-benzoquinone is -.Sovolt vs. SHE. Vitamin E quinone is -.49 volt and Vitamin K1 is -.SO volt vs. SHE.50 The experimental value of -.37 volt obtained in the data reported here for what may be the primary electron acceptor molecule is in the expected region of potential for equilibria involving ubiquinone and its one-electron reduced anion radical.

Loach er al. : Phototrap in Photosynthetic Bacteria

317

ACKNOWLEDGEMENTS We would like t o thank Dr. Brian Hoffman of the Department of Chemistry at Northwestern University for the use of h s liquid helium and liquid nitrogen accessories in making low temperature measurements. We also express our gratitude t o Drs. W. Svec and J. J. Katz of the Argonne Natihnal Laboratory for providing us with the first deuterated culture of R . rubrurn cells used in these experiments.

REFERENCES 1. LOACH, P. A., G. M. ANDROES, A. MAKSIM & M. CALVIN. 1963. Photochem. Photobiol. 2: 443. 2. LOACH, P. A., D. L. SEKURA, R. M. HADSELL & A. STEMER. 1970. Biochemistry 9: 724. 3. LOACH, P. A., R. M. HADSELL, D. L. SEKURA & A. STEMER. 1970. Biochemistry 9: 3127. 4. LOACH, P. A. & R. L. HALL. 1972. Proc. Nat. Acad. Sci. USA 6 9 786. 5. LOACH, P. A., R. A. BAMBARA & F. J. RYAN. 1971. Photochem. Photobiol. 13: 247. 6. LOACH, P. A. & M. CALVIN. 1963. Biochemistry 2: 361. 7. HARBURY, H. A. 1957. J. Biol. Chem. 225: 1009. 8. KUNTZ, Jr., I. D., P. A. LOACH & M. CALVIN. 1964. Biophys. J. 4: 227. 9. LOACH, P. A. 1966. Biochemistry 5: 592. 10. LOACH, P. A. & D. L. SEKURA. 1967. Photochem. Photobiol. 6: 381. 11. RABINOWITCH, E. 1. 1951. Photosynthesis. lnterscience Publishers, Inc. New York, N.Y. 12. DUYSENS, L. N. M. 1952. Ph.D. thesis. University of Utrecht. Utrecht, The Netherlands. 13. GOEDHEER, J. C. 1959. Biochim. Biophys. Acta 38: 389. 14. KOK, B. 1961. Biochim. Biophys. Acta 48: 527. 15. COMMONER, B., J. J. HEISE & J. TOWNSEND. 1956. Proc. Nat. Acad. Sci. USA 42: 710. 16. SOGO, P., N. G. PON & M. CALVIN. 1957. Proc. Nat. Acad. Sci. USA 43: 387. 17. CLAYTON, R. K. 1962. Photochem. Photobiol. I : 201-210. 18. REED, D. W. & R. K. CLAYTON. 1968. Biochim. Biophys. Res. Commun. 30: 471. 19. CLAYTON, R. K. & R. T. WANG. 1971. In Methods in Enzymology. S. P. Colowick & N. Kaplan, Eds. Vol. 23: 696. Academic Press, New York, N.Y. 20. FEHER, G. 1971. Photochem. Photobiol. 14: 373. 21. CLAYTON, R. K. 1966. Photochem. Photobiol. 5: 669. 22. STRALEY, S. C. & R. K. CLAYTON. 1973. Biochim. Biophys. Acta 292: 685. 23. SAUER, K., E. A. DRATZ & L. COYNE. 1968. Proc. Nat. Acad. Sci. USA 61: 17. 24. NORRIS, J. R., R. A. UPHAUS, H. L. CRESPI & J. J. KATZ. 1971. Proc. Nat. Acad. Sci. USA 68: 625. 25. NORRIS, J. R., M. E. DRUYAN & J. J. KATZ. 1973. I. Amer. Chem. SOC.In press. 26. FEHER, G., A. J. HOl:F, R. A. ISSAACSON & J. D. MC ELROY. 1973. Biophysical Society Meeting, Columbus, Ohio, February 27-March 2. Abstract No. WPM-H7. 27. MC ELROY, J. D., G. E’EHER & D. MAUZERALL. 1972. Biochim. Biophys. Acta 267: 363. 28. STRALEY, S. C., W. W. PARSON, D. C. MAUZERALL & R. K. CLAYTON. 1973. Biochim. Biophys. Acta 305: 597. 29. HALL, R. L., M. CHU KUNG, M. FU, 9. J. HALES & P. A. LOACH. 1973. Photochem. Photobiol. In press. 30. FEHER, G . 1970. Electron Paramagnetic Resonance with Applications to Selected Problems in Biology. Gordon and Breach, Inc. New York, N.Y.

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31. PARSON, W. W. 1969. Biochim. Biophys. Acta 189: 384. 32. FEHER, G., M. Y. OKAMURA & J. D. MC ELROY.1972. Biochim. Biophys. Acta 267: 222. 33. CLAYTON, R. K. & S. C. STRALEY. 1970. Biochim. Biophys. Res. Commun. 39: 1114. 34. SLOOTEN, L. 1972. Biochim. Biophys. Acta 275: 208. 35. LAND, E. J., M. SIMIC & A. J. SWALLOW. 1971. Biochim. Biophys. Acta 226: 239. 36. LOACH, P. A. & J. J. KATZ. 1973. Photochem. Photobiol. 17: 195. 37. LOACH, P. A. 1973. American Society for Photobiology Meeting, Sarasota, Florida, June 10-14. Abstract No. WAM-B2. 38. LOACH, P. A. & D. L. SEKURA. 1968. Biochemistry 7: 2642. 39. LOACH, P. A. & R. J. LOYD. 1966. Anal. Chem. 38: 1709. 40. CUSANOVICH, M. A. & M. D. KAMEN. 1968. Biochim. Biophys. Acta 153: 397. 41. CRAMER, W. A. 1969. Biochim. Biophys. Acta 1 8 9 54. 42. NICOLSON, G. L. & R. K. CLAYTON. 1969. Photochem. Photobiol. 9: 395. 43. CASE, G. D. & W. W. PARSON. 1971. Biochim. Biophys. Acta 253: 187. 44. DUTTON, P. L. 1971. Biochim. Biophys. Acta 226: 63. 45. DUTTON, P. L. & J. B. JACKSON. 1972. Eur. J. Biochem. 3 0 495. 46. LEIGH, J. S., Jr. & P. L. DUTTON. 1972. Biochem. Biophys. Res. Commun. 46: 414. 47. JACKSON, J. B., R. J. COGDELL & A. R. CROFTS. 1973. Biochim. Biophys. Acta 292: 218. 48. DUTTON, P. L. & J. S. LEIGH. 1973. Biophysical Society Meeting, Columbus, Ohio, February 27-March 2. Abstract No. WPM-H6. 49. MORTON, R. A. 1965. In Biochemistry of Quinones. R. A. Morton, Ed.: 1. Academic Press. New York. N.Y. 50. MANN, C. K. & K. K. BARNES. 1970. Electrochemical Reactions in Nonaqueous Systems: 190. Marcel Dekker, Inc. New York, N.Y. 51. KOHL, D., J. TOWNSEND, B. COMMONER, H. CRESPI, R. DOUGHERTY & J. J. KATZ. 1965. Nature 206: 1105. 52. FUHRHOP, J . H. & D. MAUZERALL. 1968. J. Amer. Chem. Soc. 9 0 3875. 53. FUHRHOP, J. H. & D. MAUZERALL. 1969. J. Amer. Chem. Soc. 91: 4174.

DISCUSSION

DR. NORRIS (Argonne Laboratories, Argonne, ZU): Since your model gives non-gaussian line shapes, can you get conservation of spin by just measuring peak height vs. peak width? I don’t understand your model. Second moment theory would give you 15 protons for a chlorophyll monomer in agreement with our very precise endor data. Therefore, it is impossible t o account for a line width of 9 gauss and have equal delocalization over 4 molecules of bacterie chlorophyll. That requires unequal delocalization, which is exactly opposite the assumption on which all those curves are based, but does agree with the endor data. Recent triplet state work suggests that chlorophyll minus could be the primary electron acceptor. Would you comment on these points. D R . LOACH: We assumed gaussian shapes for both the fully deuterated and fully protonated species. This would seem t o be safe for a fully exchanged system. Our data do not fit a single molecule case but could be consistent with equal delocalization over t w o molecules, although our error bars would have t o be increased by about 50% t o be compatible with the special pair interpretation. We see the ubiquinone anion radical first in these preparations, but that only

Loach et ~ l :.Phototrap in Photosynthetic Bacteria

3 19

shows it’s near the primary event. The primary acceptor could be bacteriochlorophyll anion radical o r bacteriopheophytin anion radical, but as long as it is so tightly associated with the ubiquinone, the transfer t o it might be very rapid. DR. NORRIS: By chlorophyll minus as the acceptor, I meant a chlorophyll plus side by side with a minus. It would be a triplet state not really like a n anion radical and you wouldn’t observe a single narrow width line. DR. FEHER: The difference between 2 and 4 in your curves is only 10% of the line width. That’s a rather small amount. Your model assumes all four bacteriochlorophylls contribute equally t o the line width. Our work shows that if the methyl stops rotating, you can get a 10% change in line width. Therefore perhaps all four bacteriochlorophylls d o not contribute equally t o the line width, especially if some of those that can rotate exchange with those that cannot; then you would easily be off by at least 10%. Therefore it could still be in agreement with dimers. DR. VERNON: Do you have any comments about bacteriopheophytin’s function as a component of the reaction center preparations? DR. LOACH: Yes, we tried exchanging the primary electron acceptor species with things like FMN, bacteriopheophytin, and bacteriochlorophyll, but all without any significant effects. Much more work is still required.