Received 2 April 1987; accepted in revised form 27 May 1987. Key words: pheophytin, photosystem II, PSII particles, P680-Pheo, energy storage, photo-.
Photosynthesis Research 14:71-80 (1987) © Martinus Nijhoff Publishers, Dordrecht
71 Printed in the Netherlands
Regular paper
Pheophytin-mediated energy storage of photosystem II particles detected by photoacoustic spectroscopy M. F R A G A T A , R. POPOVIC, E.L. C A M M 1 & R.M. LEBLANC Centre de recherche en photobiophysique, Universitd du Quebec ~t Trois-Rivibres, Trois-Rivi&es (Quebec) G9A 5H7, Canada; 1Permanent address: Botany Department, University of British Columbia, Vancouver, BC G6T 2B1, Canada Received 2 April 1987; accepted in revised form 27 May 1987
Key words: pheophytin, photosystem II, PSII particles, P680-Pheo, energy storage, photoacoustic spectroscopy Abstract. The photoacoustic (PA) characteristics (energy storage and heat dissipation) of photosystem II (PSII) core-enriched particles from barley were studied (i) in conditions where there was electron flow, i.e., in the presence of a combination of the electron acceptor K 3 Fe (CN)6, referred to as FeCN, and the electron donor diphenylcarbazide (DPC), and (ii) in conditions where electron flow was suppressed, i.e., in the absence of FeCN and DPC. The experimental data show that a decrease of heat dissipation with a minimum at ~ 540 nm can be interpreted as energy storage resulting from the presence of pheophytin (Pheo) in the PSII particles. On account of the capability of the PA method to measure the energy absorbed by the chromophores which is converted to heat, it is suggested that the PA detection of Pheo present in the PSII complex will permit to clarify the function of processes involving nonradiative relaxation of excited states in P680-PheO-QA interactions. Abbreviations: fl-Car- fl-Carotene, C h l - Chlorophyll, DPC - Diphenylcarbazide, E P R - Electron Paramagnetic Resonance, F e C N - p o t a s s i u m ferricyanide, HEPES-N-2-hydroxyethylenepiperazine-N'-2-ethanesulfonate, P680-reaction center of PSII, PA-Photoacoustic, Pheo-pheophytin, PSI-photosystem I, PSII-photosystem II, QA-primary electron acceptor of PSII.
Introduction Earlier work on reaction centers of photosynthetic bacteria [ 1-3] led Klimov and others [4, 5] to the discovery that pheophytin (Pheo) is an intermediate electron acceptor in photosystem II (PSII) prior to a quinone species Q which is now known to be QA (see recent reviews in [6] and [7]). Most of the evidence of the Pheo function comes from absorbance changes found in PSII particles of a number of green plants and algae but not in photosystem I particles (see e.g. [8]). As an example, light-induced changes in TSF-IIa
72 subchloroplast particles from spinach by continuous illumination showed absorption changes at 514 and 545 nm; also, PSII particles from Phormidium laminosum had absorbance changes at 512 and 541 nm [8]. In this respect, it is important to note that the observed maxima at ,-~ 510 and 540nm are consistent with absorption spectroscopy data of pheophytin in solution [9, 10] and linear dichroism of Pheo a incorporated into model membranes [11, 12] which in the case of the 540 nm maximum is attributed to a pure Qx (0,0) band whereas the 510 nm maximum is believed to be a composite of X- and Y-polarized absorptions [11, 12]. The light-induced changes of pheophytin in PSII particles were observed for the most part in light-minus-dark difference spectra, and lifetime and EPR studies [8, 12-14]. Following a different path, we applied photoacoustic (PA) spectroscopy to the measurement of energy storage (or "photochemical loss", see Materials and methods, and also [ 15-17]) of PSII core-enriched particles from barley in conditions where there was electron flow and when no electron flow was observed. We present here data which indicate that a decrease of heat dissipation between 510 and 540 nm can be interpreted as energy storage resulting from the presence of pheophytin in the photosystem II particles.
Materials and methods
Preparations of PSII core-enrichedparticles The particles were obtained from eight-day-old barley seedlings. First, PSII submembrane fragments were isolated by methods described in [18]. Then, PSII core-enriched particles were prepared by extraction with Triton X-100 and octyl-fl-D-glucopyranoside using the procedures described in [19]. Finally, the preparation was loaded on a sucrose gradient containing 30 m M octyl-fl-D-glucopyranoside (1 mg chlorophyll/gradient), and centrifuged in a IEC SB-283 rotor at 29,000 g for 16 h at 4 °C. SDS-gel electrophoresis of the fractions revealed a band near the bottom of the gradient that was enriched in the Chl a-containing complex associated with the reaction center of PSII and did not contain any chlorophyll-protein complexes of PSI (see in this respect [18]). This fraction was diluted to 240 #g Chl/ml in 50mM HEPES buffer (pH 7.6) containing 0.4 M sucrose, 10 m M MgCI2 and 10 m M NaCI, and used as such for further analyses.
73
Determination of energy storage and dissipated heat The photoacoustic spectrometer and methods used in the present study were described elsewhere [17]. PA measurements were performed from 400 to 720nm at a frequency of light modulation of 35 Hz. The intensity of the modulated light beam was 10Win -2 and that of the non-modulated actinic beam (white light) was 250 W m -2 . In contrast to conventional methods, PA spectroscopy measures absorption of photons by detecting heat generation (H) occurring inside the sample owing to nonradiative relaxation of excited states. Usually H is given in relation to a carbon black reference (Href) which is totally absorbing and not photoactive. The relative PA response, Q, which is proportional to H/H=r [15] is expressed by Q =
(4~AEp2) fct 1 Nhc
'
(1)
where f is an instrumental factor, ct is the fraction of light absorbed by the sample, ~ is the quantum yield for the photochemical reaction, AE v is the internal energy change per mole or unit of product formation in the photochemical reaction, 2 is the wavelength, N is Avogadro's number, h is Planck's constant, and c is the speed of light. The term t~AEp2/Nhc, henceforth designated ~ , is usually called "photochemical loss" [15]. ~b~ gives the effect of photochemistry which diminishes the maximum PA signal and is thereby a measure of energy storage in the biological system. To obtain qS~we must perform experiments in the following conditions: (i) the PSII core-enriched particles are incubated in conditions where there is electron flow, i.e., in the presence of a combination of the electron acceptor K3Fe ( C N ) 6, referred to as FeCN, and the electron donor diphenylcarbazide (DPC), in which case we obtain Qni, where ni stands for "non-inhibited"; and (ii) the incubation takes place in conditions where there is no electron flow, i.e., in the absence of both FeCN and DPC, which yields Qi, where i is for "inhibited". ~ is then calculated from the expression [17]
~;
=
Qi-
Qoi
(2)
Qi Note finally that, in contrast to ~b~, the term [1 - (thAEp2/Nhc)] is a measure of the dissipated heat [20] given by
74
Q/~ = f ( 1
q~AEPNhc 2).
(3)
Results and discussion
Energy storage and dissipated heat
Figure 1 displays the PA spectra of PSII core-enriched particles from barley obtained under conditions of electron flow (Qni), that is to say in the presence of FeCN and DPC which are required to support electron transport through PSII, and in the absence of FeCN and DPC (Qi). The figure shows that Qi < Qni at all wavelengths from 400nm up to ~ 710nm where Q~is nearly identical with Q~. We observe, in addition, that the Qi and Qni spectra are quite similar to the absorption spectrum of PSII particles represented on Fig. 2. This is to be expected if most of the pigments present in the PSII particles were photosynthetically active. The ~br vs. 2 data of Fig. 2 supports this contention. We see that ~b~ increases gradually from 400 nm to ca. 4 _
Pi
, - ( DPC
+
FeCN
Pni
, + ( DPC
*
FeCN )
)
..j 3 < Z ¢.-3,
P=
bl
W
0
I 400
I. I I 480 560 640 WAVELENGTH ( n m )
720
Fig. 1. Photoacoustic (PA) spectra of PSII core-enriched particles from barley obtained under conditions of electron flow (#hi) and when no electron flow was observed (Qi). Q is the PA response of the samples given in relation to a carbon black reference. The intensity of the modulated light beam (between 400 and 720 nm) was 10 Wm - : and that of the non-modulated actinic beam (white light) was 250Win -2. The frequency of light modulation was 35Hz. FeCN, potassium ferricyanide. DPC, diphenylcarbazide.
75
o.4t
0.8 •
ABS
0.3
0.6
,i
LU r~
z < m
U~ 0.2 >. O r~ U.I Z UJ O.1 !
O.0
I 400
0.4 u~
m
712nm (e.g., ABS at 720 nm ~ 0.002) the ~b~data are not represented in the figure on account of the high statistical uncertainty of the PA signals. Experimental conditions are as described in Fig. 1.
520-540 nm, then decreases up to 560 nm. This is followed by a new sharp increase of energy storage with a maximum between 600 and 640 nm, a shoulder at about 680 nm and a red-drop thereafter which is peculiar to PSII particles (see e.g. [17]). In this respect it is noteworthy that PA experiments performed in our laboratory with preparations of broken chloroplasts of barley which contain PSI in addition to PSII, showed also a red-drop of ~b~ but at longer wavelengths (data not shown) as is to be expected. This explains also the large increase of dissipated heat observed in Q/ct spectra at wavelengths > 670 nm (cf. Fig. 3). These data constitute therefore additional indication to support the view that the PSII core-enriched particles used in the present work are mostly, if not completely, depleted of PSI. An interesting feature of Fig. 2 is that it reveals the presence in the PSII particles of a photosynthetically active pigment absorbing at ~ 540 nm. This is best seen in Fig. 3 which represents the variation of dissipated heat, Q/~ (cf. equation 3), by the PSII particles as a fucntion of 2 in conditions of electron flow (Q=/~ vs. ,~) and in their absence (Qi/~ vs. 2). One observes a well-resolved dip at about 540 nm in the Oi/~ vs. 2 spectrum which does not disappear when the samples are irradiated in the presence of FeCN and DPC (cf. O,i/~ vs. 2 spectrum). It is remarked at first that this dip constitutes
76
o
¢1
/0i /(2 , - ( D P C
+ FeCN)
• Pni/CL , + ( D P C + FeCN)
q,. .7 I..I1.1 "r
-
"" 5 tl, I
oo
_
,:,o%O %a, OOOoOo qP
Oo
.....
% o•
•
3
I
o
o
1 400
I
•ee=e
I
I
480 560 640 WAVELENGTH ( n m )
I 720
Fig. 3. Heat dissipation spectra of PSII core-enriched particles from barley obtained under conditions of electron flow (Qni/ct) and when no electron flow was observed (Qi/o0. Q is the relative photoacoustic response and 0¢ the fraction of light absorbed by the sample. Experimental conditions are as described in Fig. 1.
a clear drop in the general trend of the progressively increasing dissipated heat between 480 and ,-~ 560 nm. For this reason we believe that the 540 nm absorbing species is more effective in photochemistry than, for instance, the species absorbing at 560 nm where the maximum O/~ is situated. This fact is well illustrated in Fig. 2 where it is shown that ~b~which is actually given by the difference between Qi/(X and Q.i/ct divided by Oi/o¢(cf. equation 2 above), does attain a maximum at about 540 nm as already referred to. These interpretations, furthermore, are corroborated by the data displayed in Fig. 4 where tkAEp is represented as function of wavelength. Note that each ~bAEp data point was calculated upon rearrangement of equation 2 which yields ~bAEp =
Nhc
,
(4)
where ~bAEpis given in kJmo1-1 and 2 in nanometers. Here, the interesting point is that the term ~bAEv is a measure of the quantum yield for the photochemical reaction provided that AEp (i.e., the internal energy change per mole or unit of product formation) is proved not to vary with 2. This assumption is reasonable since at every 2 we deal with a photochemistry
77 80
,,-.. 60
i-
•
.,
,.%,=,
...
- .,,.'-
."..,
-.,,
~40
u.i 62 kJmol-~ ) and above 580 nm up to about 630 nm (~bAEp between 60 and 68 kJmol -~ ). Figure 4 displays, in addition, a dip at about 560 nm and a sharp decrease of ~bAEp after ~ 660 nm as it is expected (see above for discussions of the data). A straightforward interpretation of the 540 nm band (Figs 2 and 3) is to attribute the energy storage at this wavelength to the presence of pheophytin in the PSII particles, precisely because this is the absorption maximum of the Qx (0,0) band of Pheo a (see Introduction and [11]). The 540 nm dip of the Q/~ vs. 2 plot (Fig. 3) would then be the equivalent of similar minima observed in light-minus-dark difference spectra (cf. Fig. 2a, b of [8]). Let us note, however, that close scrutiny of Fig. 2 shows significant contributions to the absorption spectrum of the PSII core-enriched particles of carotenoid pigments, presumably/;-carotene (fl-Car) (see [7]), and chlorophyll b; e.g., we detect easily maxima at 460-470 nm (Chl b and fl-Car), and shoulders at ,,~ 490 nm (E-Car) and 650 nm (Chl b). These spectral characteristics resem-
78 ble incidentally those of the CP29 chlorophyll a/b complex isolated by Camm and Green [21] which was shown to contain Chl b as well as r-Car (cf. our Fig. 2, and Fig. 3 of [21]). To try to determine the relative contributions of these pigments to the PSII absorption spectrum of Fig. 2, we performed a preliminary convolution calculation (data not shown) with the aid of spectra of pigments in organized matrices which we believe to be a more reliable approximation in the present case than solution spectra. We used, namely, absorption spectra of Chl a and Pheo a [11, 12], and Chl b (M. Fragata and B. Nordrn, unpublished data) incorporated into a lamellar matrix constituted of glycerylmonooctanoate/H20, and r-Car included in small, unilamellar vesicles constituted of dipalmitoylphosphatidylcholine (M. Fragata, unpublished data). The results showed that the contributions ofChl a and Pheo a to the spectral band at 540 nm are similar and account in the overall for ~ 75% of the total absorbance, whereas Chl b and r-Car contribute each one only ca. 10-12% of the total absorbance. We conclude thus that the decrease of Q/~ at about 540 nm is due at least partly to the function of pheophytin in energy storage of PSII particles. General considerations
It is reasonable to expect that the PSII core-enriched particles used in the present experiments contained the polypeptides which are thought to hold the binding sites for P680, Pheo and QA (see discussions in [7, 21]), and to assert by the same token the presence of pheophytin in the pigment-protein complexes. But, while the presence of Chl a, Chl b and r-Car is easily inferred from simple inspection of the absorption spectra (see above) the same cannot be said of Pheo a, specially of its red band which is qualitatively very similar to Chl a. It is therefore interesting to establish, as we have done hereinbefore, that PA spectroscopy enables the detection of the Qx(0,0) band of Pheo a at about 540 nm. This finding permits to supplement new information to studies of light-minus-dark difference spectra, as well as to lifetime and EPR studies (see Introduction). In short, given the characteristics of the PA methods, that is to say its capability to measure the energy absorbed by the chromophores which is converted to heat, it will be possible to clarify the function of processes involving nonradiative relaxation of excited states in P680-Pheo-QA interactions. For example, a plot of Q/~ vs. 2 (cf. equation 3) of data obtained carefully in the 540 nm range will permit to obtain the term q~AEp (cf. [20] and [23], and Fig. 4), and to evaluate eventually the quantum yield, ~b, or the internal energy change, AEp, of the photochemical reaction involving the pheophytin molecules of the PSII complex.
79 Our overall conclusion is that our hypothesis of an efficient photochemistry starting in pheophytin as the result of direct absorption of light in a Pheo molecule of the PSII complex is a novel suggestion which is supported by the experimental data displayed here. What is more, this finding could open a new avenue for the study of PSII photochemistry with photoacoustic spectroscopy.
Acknowledgements This work was supported by the NSERC Canada (grants No. A3047, A6357 and A6358), the Fonds FCAR du Qudbec (grant No. EQ-3186), and a FIR grant of the Universit6 du Qudbec ~ Trois-Rividres to two of us (M.F. and R.P.). We wish to thank Mr Claude Daneault and Dominic Bergeron for the drawings of the illustrations.
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