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Electron spin resonance and photoreaction of Mn(II) in lettuce chloroplasts
ARCHIVES
OF
BIOCHEMISTRY
Electron
AND
BIOPHYSICS
Spin Resonance Y. SIDERER,
179,
174-182 (1977)
and Photoreaction Chloroplasts
S. MALKIN,
The Weizmann
Znstitute
R. POUPKO, of Science,
Rehouot,
of Mn(ll)
AND
in Lettuce
Z. LUZ
Israel
Received June 21, 1976 Electron spin resonance (esr) of lettuce chloroplasts yields three types of signals: (i) a broad (-900 Gl signal around g = 2.22 (apparently due to Cue+ complexes); (ii) an Mnz+ spectrum aroundg = 2.003 consisting of six hyperfine lines (A = 94.5 G) of -30 G width, and (iii) a sharp signal at g = 2.00 due to photosignals I and II. The present work is concerned with the Mn*+ signal and its relation to the photosynthetic process. Intensity measurements were performed by comparing the intensities of the Mn2+ signals of two identical chloroplast preparations, one of which was slightly acidified. The integrated intensity of the signal in the normal preparation was approximately one-fourth of that in the acidified sample,. suggesting that only the-f e t fine structure band is observed in untreated chloroplasts. This indicates that the manganese in the chloroplasts is bound in an asymmetric environment, apparently in protein complexes. The Mn2+ signal is light sensitive, decreasing on illumination and reappearing in the dark. Typical values for the half-lives of the light and dark processes in normal chloroplasts are 0.25 and 2.1 s, respectively. The effect is interpreted in terms of the photooxidation of Mn2+ to higher oxidation states which are invisible to esr spectroscopy. In order to determine whether this process is related to photosynthesis the effect of certain reagents and treatments that are known to affect the photosynthetic system was studied. It was found that the oxygen evolution inhibitors 3-(3,4 dichlorophenyl)-l,l-dimethylurea (DCMU) and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) as well as the electron donors, phenylenediamine and sodium ascorbate, reduce or completely eliminate the light effect on the Mn2+ signal. Heat treatment and Tris washing caused deceleration of both the light and dark reactions. These effects indicate that the photooxidation of the Mn2+ is related to the photosynthetic cycle, the most probable site being the water splitting apparatus of photosyatem II.
of manganese in the process (3-5). All of them make use of the fact that manganese possesses several oxidation states, and therefore, can donate and accept electrons in the photosynthetic cycle. Specifically, manganese may be a likely candidate for the positive charge accumulator [S-states of Kok et al. (611 in the water-splitting enzyme . In this work, we used electron spin resonance (esr) as a tool to study the role of manganese in photosynthesis. Mn2+ complexes often give well-defined esr signals, while manganese in other oxidation states usually does not yield an esr signal in normal physiological solutions. Thus, the change in the steady-state concentration of Mn2+ as well as the kinetics of its oxida-
Extensive research in recent years has shown that manganese is required for oxygen evolution by plants and algae. The most accepted assumption is that it participates as an electron carrier in the electron transfer process between water and photosystem II (PSII)’ (1, 2). Several models were proposed to explain the involvement 1 Abbreviations used: PSI, photosystem I; PSH, photosystem II; esr, electron spin resonance; SNT, 0.2 Easucrose, 0.1 M NaCl, and 0.05 M Tris buffer, pH 7.8; Chl, chlorophyll; DPPH, diphenylpicrylhydrazyl; ZFS, zero field splitting; DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea; FCCP, carbonylcyanidep-trifluoromethoxyphenylhydraxone; PD, phenylenediamine; ADRY reagent, reagent Accelerating the Deactivation of the Reaction of the watersplitting enzyme Y; nmr, nuclear magnetic resonance. 174 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
ESR OF MN(H) IN CHLOROPLASTS
tion-reduction can, in principle, be studied by monitoring its esr signal. In the present work, we report the observation of esr signals due to manganese in lettuce chloroplasts. This signal was found to be light sensitive: decreasing on illumination and increasing again in the dark, suggesting that the process is associated with the photosynthetic system. We have studied the effect of light on the steadystate intensity of the Mn2+ signal as well as the kinetics of its disappearance and restoration as a function of a number of external parameters (light intensity, addition of electron donors and inhibitors, and inhibitory treatments). It is concluded that the manganese signal arises from bound Mn2+ ions associated with the inside of the chloroplast membrane. The results also suggest that the observed signal is due to manganese which participates in the electron transfer from water to PSII. The esr of Mn2+ in photosynthetic systems has been studied by other workers. Treharne et al. (7) studied the manganese signal in Chlorella pyrenoidosa and observed changes in its intensity between light and dark periods. The kinetics of this transformation was found to be quite slow, being of the order of 1 h. More recently, Lozier et ~2. (8) studied the Mn2+ signal in spinach chloroplasts. This signal could only be observed upon treating the chloroplasts with chaotropic agents. These agents are assumed to convert bound manganous ions, which are not observable by esr, to (“free”) hydrated ions which are readily detectable. The resulting signal was found to be light sensitive: decaying on illumination and recovering in the dark. Sauer and his colleagues extended this work on spinach chloroplasts and showed (9, 10) that reactivation of oxygen evolution in Tris-treated chloroplasts was associated with the disappearance of the Mn2+ signal. They concluded that Tris washing releases part of the bound manganese to the interior space of the thylakoid membrane, while reactivation is accompanied by reincorporation of the manganese . In our preparation of active lettuce chloroplasts, showing Hill activity, it is possible to observe Mn2+ esr signals even with-
175
out Tris washing or other inhibitory treatments. Our measurements are thus directly performed on the bound Mn2+ species. EXPERIMENTAL Materials. Lettuce chloroplasts were prepared as described by Avron (11). Typically, the chloroplast preparations contained between 1 and 4 mg of chlorophyll per 1 ml of solution. The exact chlorophyll concentration was determined spectrophotometrically according to Arnon (12). These samples, which we refer to as “normal” or “control” samples, also contained 0.2 M sucrose, 0.1 M NaCl, and 0.05 M TrisHCI buffer, pH 7.8 @NT). The chloroplast-free reagent mixture was checked by esr in order to make sure that it did not contain manganous ions. The different normal choroplast preparations gave Hill activities ranging from 180 to 390 wequiv/mg of Chl/h as measured by O2 evolution using (Fe[CNl,13- as an electron acceptor. In addition to the normal chloroplasts, a number of modified preparations obtained by special treatments or addition of reagents were also studied. These included: (i) Heat treatment, which was carried out by incubating normal preparation for 3 min at 50°C. This treatment almost completely inhibited the capacity to evolve oxygen. However, the reaction center complex was still partially active. This was shown by the ability of heat-treated samples to emit delayed light, measured in the >O.l s range after illumination, to the extent of one-third of that of the control sample. [The apparatus used to measure delayed light emission is described in Ref. (131.1 (ii) Tris washing, which was performed according to Yamashita and Butler (14) by incubating the chloroplasts in 0.8 M Tris, pH 8 for 20 min, centrifugation at 25OOg, and resuspending the pellet in SNT. (iii) Manganese extraction by magnesium. This treatment was performed according to Chen and Wang (151. The medium contained 0.1 M sucrose, 5 mM NaCl, 200 mM MgSO, and 0.01 M Tris, pH 7.8. Chloroplasts were incubated for 1 h followed by centrifugation at 10,OOOg for 10 min. The pellets were resuspended in a reaction mixture which contained 0.2 M sucrose, 0.01 M NaCI, and 0.02 M Tris, pH 7.8. The concentration of manganese in the resulting samples was 1:lOO Mn:Chl, i.e., one-third of its concentration in the control samples. Also, the oxygen evolution was reduced by a factor of 3 in these preparations. Electron spin resonance measurements. Electron spin resonance spectra were recorded on a Varian E12 spectrometer at X-band frequency (9.3 GHz) using a TE,,,, cavity (or a double cavity TE1,,) equipped with a slotted grid to allow irradiation of the sample during measurements. The manganese spectra were recorded with a lOO-KHz field modulation of about
176
SIDERER ET AL.
10 G amplitude and using a microwave power of 100 mW. Under these conditions the peaks due to the socalled chloroplasts’ signals I and II (16) were also observed around the center of the manganese multiplet. These signals did not interfere with our measurements since they span a very small portion of the whole manganese spectrum. Relative integrated intensity measurements of the Mn2+ signal were performed using a double cavity, TEIM, in which the measured solution was placed in one compartment of the double cavity and a reference diphenylpicrylhydrazyl (DPPH) sample in the second. In each series of experiments the same sample cell was used and placed in the same position in the cavity. The relative intensities were obtained by double integration of the derivative signal and normalized with respect to the spectrometer setting and the reference DPPH signal (17). Kinetic studies of the light-induced disappearance and restoration of the Mn*+ signal were performed by monitoring the signal of one of the Mn*+ hyperfine lines (usually the high-field or penultimate hyperflne component). In practice, the magnetic field was set at the peak of the derivative signal and its intensity was recorded as a function of time following a “light on” or “light off’ step. To improve the signal-to-noise ratio we used a signal averager, HP 5480A, in which a number of traces (usually 16 to 25) were accumulated. In these experiments the spectrometer settings were identical to those used for recording the spectra except that the filter time constant for the output signal was set at 0.1 s or less. The chloroplast samples were placed in a flat aqueous solution quartz cell of 0.4 mm internal width. The chlorophyll concentration (1 mg/ml) was chosen as a compromise between the requirement of a strong esr signal on the one hand, and homogeneous light intensity within the sample on the other. The light source was a 500-W projector. The light
I
I
I
was filtered using Corning 4-96 glasses which transmitted light between 400 and 600 nm. The intensity of the light impinging on the sample, estimated by setting a calibrated silicon photocell next to the sample cell in the esr cavity, was -5 x lo+’ Einsteins/cm2 s. The relative light intensity was controlled using Schott neutral density filters. The whole setup (i.e., cavity and light source) was covered with a black screen to prevent stray light from entering the cavity during the measurements. RESULTS
AND DISCUSSION
Description of the Mn2+ Spectrum
Figure 1 shows the esr spectrum of letr tuce broken chloroplasts, over a wide (4000 G) magnetic field span. Three types of signals can be discerned in the spectrum: (i) a broad, -900 G wide signal centered around g = 2.22. The origin of this signal is not clear; however, from its g value it may be assigned to Cu2+ complexes, (ii) a typical sextuplet hype&me structure due to Mn2+, and (iii) a weak light-induced peak at the center of the manganese multiplet which is generally referred to as signal I and signal II, representing the oxidized form of P,,, and a radical connected with system II, respectively. In this paper we are concerned with signal (ii) due to the Mn2+. This spectrum is shown on an expanded scale in trace a of Fig. 2. It is characterized by a spin hamiltonian, X = gj3H.S
-I- AI*S
with the following parameters: g = 2.003 + 0.001; hyperfme splitting, A = 94.5 +- 0.5
3380 G I
I
I
I
1. Room temperature esr spectrum of lettuce chloroplasta at X-band frequency (9.3 GHz) recorded in the dark over a wide-field sweep. The spectrum shows the three types of signals discussed in the text: (i) a broad (-900 G) resonance around g = 2.22 tentatively attributed to CuZ+, (ii) an Mn2+ sextuplet aroundg = 2.003, and (iii) a weak signal due to photosystems I and II at the center of the Mn*+ spectrum. The spectrometer settings are: microwave power, 10 mW; modulation frequency, 100 KHz; modulation amplitude, 10 G, time constant, 1.0 s; scan rate, 4 G/s. The chlorophyll concentration is 1.15 mg/ml, in a 0.2 M sucrose, 0.1 M NaCl, 0.05 M Tris-HCl, pH 7.8 medium. FIG.
Dl
ESR OF MN(H)
177
IN CHLOROPLASTS
G; and an average peak to peak linewidth of M = 30 G. From these results alone it is not possible to decide on the nature of the manganese species responsible for the esr spectrum. When Mn2+ is bound in an asymmetric environment its spin hamiltonian is augmented by a quadratic zero field splitting (ZFS) term, x ZFS= D[s: - B S(S + l)] El + E(Sz2 - Su2) This term may have a profound effect on the solution spectra; it may cause broadening of the spectrum and, under certain conditions, complete or partial smearing out of the resonances. When the ZFS interaction is small compared to the Zeeman energy, which is usually the case for manganese bound to proteins (l&20), the possible effects on the esr spectrum can be understood by the following considerations of the various dynamic ranges (21,22). In a single crystal (no motion) containing manganese ions with the hamiltonian in Eqs. 111 and [2], the spectrum consists of five fine structure bands due to the various iVf, - 1 + M, transitions (-% --, -% -4 + -& ’ + & $ + k %+ 8. The relative intensitT& of these transitions are 5,8,9,8, and 5, respectively, and their splitting is of the order of the ZFS interaction. Each of the bands is split into six with spacing A (Eq. [ll) due to the hyperfme interaction with the 55Mn nucleus. For a single manganese site each spectrum will thus consist of a total of 30 “allowed” transitions. These transitions are anisotropic, i.e., their resonance frequencies (fields) depend on the relative orientations of the manganous complex and the magnetic field. However, when ZFS/g@Y < 1 the anisotropy of the -4 + h transitions is much smaller (being of second order in the ZFS interaction) than that of the other transitions (M, # i) (which are of first order in this interaction). Consequently, in a powder sample, the resonances of the M, # 4 transitions are spread over a wide frequency range and are usually not detectable, while the -4 + $ transitions remain relatively sharp with a width of order (ZFS)21gfl. The integrated intensity of the observed resonances is therefore & of the total expected intensity.
w a
!
b
c
I I FIG. 2. The Mn*+ spectrum of lettuce chloroplasts in the dark recorded in an expanded scale. Spectra a and b correspond to the same preparation except that sample b was acidified by adding 0.05 ml of concentrated HCI per 1 ml of chloroplast suspension. The spectrometer settings are identical for the two spectra and are the same as in Fig. 1 except that here the time constant is 0.1 s. The chlorophyll concentration is 3.2 mg/ml and the medium is the same as in Fig. 1. The weak signal in the center of the Mn*+ spectrum corresponds to photosignals I and II.
Turning now to solution spectra, i.e., introducing molecular tumbling into the powder, it is convenient to consider the following two dynamic ranges: (i) l/7 < ZFS and (ii) l/7 > ZFS, where 7 is the correlation time for the molecular tumbling, and the ZFS interaction is in frequency units. In the first range, the tumbling rate is not sufficiently fast to average out the anisotropy of the ZFS and the M, # $ transitions will remain unobservable. The spectrum will consist of only the M, = % transitions with relative intensity &. A detailed discussion of the resonance lineshape in this limit is given in (21). At higher tumbling rates, when condition (ii) applies, there is more or less complete averaging of the ZFS anisotropy and the whole spectrum coalesces around the center frequency. Under this condition a single sextet with the full intensity will be detected. The esr spectra of manganous ions bound to macromolecules often correspond to the first dynamic range. In fact, in many manganous protein complexes, the widths of the 4 4 B transitions are so large that they completely smear out even these signals. On the other hand, small
178
SIDERER
complexes in nonviscous media often belong to the second dynamic range, exhibiting a sextet spectrum with full intensity. This is, e.g., the case for the aqua manganous complex [Mn(Hz0>,12+. In order to characterize the esr signal observed in our chloroplast preparation, we determined the signal intensity relative to that of total manganese in the sample. For this purpose we made use of the fact that acidification of the chloroplast preparation causes release of cell-bound manganous ions into the bulk water environment. Thus, by comparing the integrated intensities of two chloroplast preparations, of which one had been acidified, the relative intensity of the chloroplast esr signal can be determined. Figure 2 shows two spectra corresponding to a control chloroplast preparation and the same preparation after acidification with a few drops of concentrated HCl. The two spectra were recorded under identical spectrometer settings. By comparing the integrated intensities of the two spectra we obtained a ratio of control/acidified - 0.2. Similar values were obtained in a number of different chloroplast preparations. This result is close to the expected relative intensity of the -4 + 6 transitions and suggests that the observed signal in the (nonacidified) chloroplast preparation comes from the MS = $ transitions of bound manganese, rather than from free Mn2+ ions. A confirmation of this suggestion could, in principle, be obtained by a detailed lineshape analysis of the observed signal according to the theory of (21). However, the quality of the spectra is not sufficient for such an analysis. Also it is not possible to tell from the observed spectra whether they are due to a single type of bound manganese or to a mixture of several different manganous complexes. We have recently observed similar esr signals in chloroplast preparations from spinach. This system was studied previously (8-10) and it was reported that Mn2+ signals could only be observed after Tris treatment. These signals were assigned to free Mn2+ ions in the interior space of the thylakoid membrane. It is not clear why in our samples Mn2+ signals can be observed even without Tris treatment and in active
ET
AL.
chloroplasts. Perhaps it is due to different practices of preparation, although we have used the same “literature” method. We believe that the intensity ratio of approximately 0.2 between normal chloroplast preparations and acidified samples is not accidental and strongly suggests that the observed signal is due to bound manganese. Although on the basis of the appearance of the spectrum alone we cannot rule out the possibility that it is due to free manganese released from the chloroplasts. The above interpretation is further supported by dialysis experiments in which a chloroplast preparation was allowed to dialyze for 24 h against an SNT solution. During this period, only a small amount (7%) of the manganese ions passed the dialysis membrane. The Mn2+ signal of the dialyzate was also tested by acidification treatment: Addition of the acid resulted in an increase of about four- to fivefold the Mn2+ signal intensity. It therefore seems that the Mn2+ in the dialyzate was also bound. Testing the dialyzate by the Lowry et al. (23) method showed that it also contained proteins, apparently of low molecular weight, which penetrated through the dialysis membrane (Union Carbide dialysis membrane). Effect of Light
on the Mn2+ Signal
“Control” chloroplast preparations. Illumination of the chloroplasts in the cavity by visible light causes a reduction in the intensity of the Mn2+ signal. Turning the light off results in restoration of the signal to its original intensity. As an illustration, in Fig. 3 we compare two traces of the Mn2+ signals corresponding to “light on” and “light oft”’ periods. In the following we shall discuss the light effect in terms of the parameter R defined by R = hVh/,
E31 where h” is the signal intensity following the Yight on” step and is time dependent, and hOd is the steady-state intensity of the signal in the dark. R is thus proportional to the instantaneous concentration of Mn2+ ions and monitors’ its transformation to higher oxidation states. In experiments of the type shown in Fig. 4, R decreases from R = 1 to some steady-state value, R, dur-
ESR OF MN(H)
IN CHLOROPLASTS
ing the “light on” period, and increases again to unity after cessation of illumination. Within our experimental accuracy, the light and dark reactions follow exponential decay and rise kinetics. There are thus three parameters that describe the light response of the Mn2+ spectrum: kz, the rate constant for the light reaction, kd, the rate constant for the dark reaction,
179
and R,. The value of kd in the control sample was found to be about 0.35 -t 0.1 s-’ independent of the light intensity, I. On the other hand, both k’ and R, were found to depend on I. Typical results are shown in Fig. 5. It may be seen that R, decreases with I for low light intensities, leveling off to Rsminat higher values of I, while the rate constant kz increases monotonically with I in the range studied The values ofRsminwere not the same for r all batches and ranged from 0.2 to 0.3 in the different preparations used. In the quantitative measurements described dark i here, we have, therefore, always compared results of solutions prepared from the same batch of chloroplasts. In order to find the relevance of the light-induced change of the Mn2+ signal to the chloroplasts’ electron transport activity, we studied the effect of a number of treatments and reagents on the R values and the kinetics of the light response. The added reagents used (DCMLJ, PD, FCCP and ascorbate) did not change the steadyL state dark signal of MI?+. FIG. 3. Comparison of the chloroplast Mn2+ specEffect of DCMU. It is known that trum during dark and light periods. The same chloDCMU blocks electron transfer from PSI1 roplast preparation and spectrometer settings used to the plastoquinone pool and thus should in Fig. 1 were employed except that the time conof the Mn2+ stant was 0.3 s. In this particular example, the also inhibit the photoreaction if it is related to this photosystem. We reduction of the signal due to illumination is about 80%. Note also the increase in intensity of signal I in have, therefore, measured its effect on the the light period (exceeding the recorder range). light-induced reduction of the Mn2+ signal. off
FIG. 4. Kinetic traces of the Mn2+ signal following “light on” and “light off’ steps. The ordinate corresponds to the reduced amplitude R = hi/hod of the penultimate hyperfine line (counted from low field). During the experiments, the field was fixed at the peak of the derivative line and the time evolution of the signal monitored using an HP 5480A signal averager. Twenty-five scans were accumulated to obtain the above traces. Each cycle consisted of 4 s of illumination followed by 16 s in the dark. The spectrometer settings are: modulation amplitude, 10 G; microwave power, 100 mW; time constant, 0.1 s. Note that the time scales for the light and dark periods are not the same. The symbols & and e,2 correspond to the halflives of the light and dark reactions, respectively. The chloroplast preparations are as in Fig. 1.
180
SIDERER ET AL
still active in the electron transport chain, the ability to oxidize H,O and evolve 0, is lost. Effect of electron donors. As an electron donor phenylenediamine (PD) may compete with Mn2+ in the electron transfer reaction from water to PSII. PD may also affect the reaction sequence by catalyzing the reduction of the oxidized form of manganese to Mn2+. As may be seen in Fig. ‘7, addition of PD indeed affects the light response of the Mn2+ signal, increasing R, and increasing kd. The latter effect is most FIG. 5. Effect of light intensity on R, and kc. The probably due to reduction of the oxidized chloroplast preparation was the same as in Fig. 1. manganese form to Mn2+ by PD. Ascorbate at higher concentration abolResults of Rs versus DCMU concentration are summarized in Fig. 6. It may be seen ished the effect of light on the Mn2+ signal that addition of DCMU indeed inhibits the completely. Thus, in a suspension containeffect of light on the Mn2+ signal. The ef- ing 1 mM ascorbate there was no change in the intensity of the Mn2+ signal on illumifect increases with DCMU concentration up to 2 x 1O-4 M where the inhibition is nation (Fig. 7). Effect of FCCP. FCCP was used in the complete. At this concentration, the molar past as an uncoupler. However, it has an ratio of DCMU to chlorophyll is approximately 15. This result may be compared to additional effect as an ADRY reagent (Accelerating the Deactivation of the Reaction that obtained for blocking the Hill reaction of the water-splitting enzyme Y) (24). Its by the same reagent where the molar ratio effect as an ADRY reagent is shown by the for complete inhibition was found to be inhibition of the oxygen yield in consecuaround 1:lO (8). This simple experiment of both shows that Mn2+ oxidation involves PSI1 tive flashes (24) and the inhibition delayed light emission and the triggered activity. (25). Figure 6 shows that Effect of treatments which inhibit oxy- luminescences gen evolution. Further support for these addition of FCCP decreases the photooxiconclusions was obtained by studying the dation of Mn2+ and at around 3 x 10m4 M completely inhibits the reaction. This effects on the Mn2+ signal of treatments which are known to inhibit oxygen evolu- should perhaps be taken as evidence for since FCCP is tion. We have used the following treat- direct PSI1 participation, ments (see Experimental): (i) heat treatn I -7. ment (14), (ii) Tris washing (141, and (iii) incubating with Mg2+ (15). All of these treatments resulted in an increase of the dark Mn2+ signal apparently due to release of part of the bound manganese. The resulting signal was still affected by o----t DCMU light. However, the kinetics of both light A--+ FCCP and dark reactions were considerably slower after the treatments (see Table I and Fig. 7). In Tris washed chloroplasts, oxygen evolution is inhibited completely; however, delayed luminescence persists to 040-‘-’ 5x10 M the extent of lo%, indicating partial activ[DCMU] or [FCCP] ity of the reaction centers. It seems that FIG. 6. Effect of DCMU and FCCP on the light the above treatments affect the manganresponse of the Mn2+ signal. The chloroplast prepaous site in such a way that while PSI1 is rations are as in Fig. 1.
ESR OF MN(H)
TABLE KINETIC
PARAMETERS OF THE IN CHLOROPLASTS
k' (s-‘)=
181
IN CHLOROPLASTS I
MnZ+ esr SIGNAL SUBJECTED
AND OTHER DATA RELATED TO PHOTOSYNTHETIC TO OXYGEN EVOLUTION INHIBITORY TREATMENTS
kd (s-9”
O2 evolution (pequi;$)Vmg c
[Chll, [Mnl
of
Control Tris washed Heat treated Mg2+ replacement
ACTIVITY
Delayed luminescence intensitvd 100 10 33 -
2.8 k 0.3 0.35 a 0.1 30 390 0.9 0.12 50 4 1.1 0.07 30 -=S 100 130 0.8 0.05 (1The uncertainties in the treated samples are estimated at k--50%. b Molecular ratio of chlorophyll to manganese. Manganese concentration was determined by atomic absorption. c Determined by Clark-type oxygen electrode with [Fe(CN1613- as an electron acceptor. d The delayed luminescence intensity is given in arbitrary units. The figures correspond to the emission intensity -160 ms after cessation of ilhumination.
not expected to intervene in PSI reactions or to inhibit steady-state electron transfer at pH values used in our experiments -7.8 (26). Indeed, a check was made on the effect of FCCP on steady-state transport to [Fe(CN),13- at similar conditions to the esr measurements. As expected, the rate of electron transport was enhanced rather than inhibited. The results gathered so far lead us to think that the observed Mn*+ photooxidation is by electron donation to photosystem II by manganese, which is part of the photosynthetic machinery. There are several other alternatives. Direct oxidation of Mn*+ by HO, radicals known to be generated at the acceptor side of PS-I (27, 28) seems unlikely for the following reasons: (i) We tried to rule out this possibility by an experiment in which methylviologen was added as an acceptor. In this case the superoxide radical formation is expected to be enhanced considerably. However, addition of this reagent did not change any parameter of the Mn*+ photooxidation reaction. (ii) Although inhibitory treatments (DCMU, Tris treatment, etc.) could inhibit Mn*+ photooxidation due to PSI1 or PSI by inhibiting steady electron transport, the specific effect of FCCP cannot come under this category since it does not inhibit steady-state electron transport. FCCP seems to interact rather specifically with PSII. (iii) The back-reaction of the Mn*+ photooxidation is slowed down considerably after Tris treatment, which again may suggest an interaction with PSII.
0.4 I
i off
( 4ac
‘1
FIG. 7. Retracings of kinetic plots of the type shown in Fig. 4, exhibiting the effect of various reagents and treatments.
CONCLUSIONS
Although there are several routes that can account for the reaction of the Mn*+, we believe that the above results confirm the conjecture that the manganous species responsible for the esr signal is an integral part of the photosynthetic machinery. There are three main points that emerge from the above results: (i) Mn*+ ions participate in the electron transfer process of the photosynthetic cycle via oxidation-reduction equilibria involving +3 or higher oxidation states of manganese. (ii) The active manganous ions are bound in an asymmetric environment within the chloroplast structure. (iii) The effects of the various reagents on the light response of the manganous signal suggest that its photooxidation is associated with PSII. Whether this Mn is also associated with the positive charge accumulator needed for
182
SIDERER
oxygen evolution is still an open question. Further studies using flashes are undertaken in order to clarify this question. Very recently, evidence on the participation of Mn*+ in the S-state cycle has been obtained by nmr relaxation measurements (2%
ET
14. 15. 16. 17.
ACKNOWLEDGMENT We interest
wish to thank Professor M. Avron for in the work and for helpful discussions.
his
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24. 25. 26.
27. 28. 29.
AL. Melendri, A., eds.), pp. 253-269, W. Junk, The Hague. YAMASHITA, T., AND BUTLER, W. L. (1968) Plant Physiol. 43, 1978-1986. CHEN, K., AND WANG, J. H. (1974) Bioinorg. Chem. 3, 339-352. WARDEN, J. T., AND BOLTON, J. R. (1974) Act. Chem. Res. 7, 189-195. AYSCOUGH, P. B. (1967) Electron Spin Resonance in Chemistry, Appendix 5, Methuen, London. MEIROVITCH, E., Luz, Z., AND KALB (GILBOA), A. J. (1974) J. Amer. Chem. Sot. 96,7538-7542. MEIROVITCH, E., Luz, Z., AND KALB (GILBOA), A. J. (1974) J. Amer. Chem. Sot. 96,7542-7545. COHN, M., LEIGH, J. S., JR., AND REED, G. H. (1971) Cold SpringHarbor Symp. Quant. Biol. 36, 533-539. BARAM, A., Luz, Z., AND ALEXANDER, S. (1973) J. Chem. Phys. 58, 4558-4564. MANIV, S., REUVENI, A., AND Luz, Z. (1977) J. Chem. Phys., in press. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. RENGER, G. (1972) Physiol. Veg. 10, 329-345. SIDERER, Y., HARDT, H., AND MALKIN, S. (1976) FEBS Lett. 69, 19-22. AVRON, M. (1971) in Proceedings of the 2nd International Congress on Photosynthesis, Stress (Forti, G., Avron, M., and Melandri, A., eds.), Vol. 2, pp. 861-871, W. Junk, The Hague. EPEL, B. L., AND NEWMANN, J. (1973) Biochim. Biophys. Acta 325, 520-529. KONO, Y ., TAKAHASHI, M., AND ASADA, J. (1976) Arch. B&hem. Biophys. 174, 454-462. WYDRZYNSKI, T., ZUMBULYADIS, N., SCHMIDT, P. G., GUTOWSKY, H. S., AND G~VINDJEE (1976) Proc. Nat. Acad. Sci. USA 73, 1196-1198.
OF
BIOCHEMISTRY
Electron
AND
BIOPHYSICS
Spin Resonance Y. SIDERER,
179,
174-182 (1977)
and Photoreaction Chloroplasts
S. MALKIN,
The Weizmann
Znstitute
R. POUPKO, of Science,
Rehouot,
of Mn(ll)
AND
in Lettuce
Z. LUZ
Israel
Received June 21, 1976 Electron spin resonance (esr) of lettuce chloroplasts yields three types of signals: (i) a broad (-900 Gl signal around g = 2.22 (apparently due to Cue+ complexes); (ii) an Mnz+ spectrum aroundg = 2.003 consisting of six hyperfine lines (A = 94.5 G) of -30 G width, and (iii) a sharp signal at g = 2.00 due to photosignals I and II. The present work is concerned with the Mn*+ signal and its relation to the photosynthetic process. Intensity measurements were performed by comparing the intensities of the Mn2+ signals of two identical chloroplast preparations, one of which was slightly acidified. The integrated intensity of the signal in the normal preparation was approximately one-fourth of that in the acidified sample,. suggesting that only the-f e t fine structure band is observed in untreated chloroplasts. This indicates that the manganese in the chloroplasts is bound in an asymmetric environment, apparently in protein complexes. The Mn2+ signal is light sensitive, decreasing on illumination and reappearing in the dark. Typical values for the half-lives of the light and dark processes in normal chloroplasts are 0.25 and 2.1 s, respectively. The effect is interpreted in terms of the photooxidation of Mn2+ to higher oxidation states which are invisible to esr spectroscopy. In order to determine whether this process is related to photosynthesis the effect of certain reagents and treatments that are known to affect the photosynthetic system was studied. It was found that the oxygen evolution inhibitors 3-(3,4 dichlorophenyl)-l,l-dimethylurea (DCMU) and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) as well as the electron donors, phenylenediamine and sodium ascorbate, reduce or completely eliminate the light effect on the Mn2+ signal. Heat treatment and Tris washing caused deceleration of both the light and dark reactions. These effects indicate that the photooxidation of the Mn2+ is related to the photosynthetic cycle, the most probable site being the water splitting apparatus of photosyatem II.
of manganese in the process (3-5). All of them make use of the fact that manganese possesses several oxidation states, and therefore, can donate and accept electrons in the photosynthetic cycle. Specifically, manganese may be a likely candidate for the positive charge accumulator [S-states of Kok et al. (611 in the water-splitting enzyme . In this work, we used electron spin resonance (esr) as a tool to study the role of manganese in photosynthesis. Mn2+ complexes often give well-defined esr signals, while manganese in other oxidation states usually does not yield an esr signal in normal physiological solutions. Thus, the change in the steady-state concentration of Mn2+ as well as the kinetics of its oxida-
Extensive research in recent years has shown that manganese is required for oxygen evolution by plants and algae. The most accepted assumption is that it participates as an electron carrier in the electron transfer process between water and photosystem II (PSII)’ (1, 2). Several models were proposed to explain the involvement 1 Abbreviations used: PSI, photosystem I; PSH, photosystem II; esr, electron spin resonance; SNT, 0.2 Easucrose, 0.1 M NaCl, and 0.05 M Tris buffer, pH 7.8; Chl, chlorophyll; DPPH, diphenylpicrylhydrazyl; ZFS, zero field splitting; DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea; FCCP, carbonylcyanidep-trifluoromethoxyphenylhydraxone; PD, phenylenediamine; ADRY reagent, reagent Accelerating the Deactivation of the Reaction of the watersplitting enzyme Y; nmr, nuclear magnetic resonance. 174 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
ESR OF MN(H) IN CHLOROPLASTS
tion-reduction can, in principle, be studied by monitoring its esr signal. In the present work, we report the observation of esr signals due to manganese in lettuce chloroplasts. This signal was found to be light sensitive: decreasing on illumination and increasing again in the dark, suggesting that the process is associated with the photosynthetic system. We have studied the effect of light on the steadystate intensity of the Mn2+ signal as well as the kinetics of its disappearance and restoration as a function of a number of external parameters (light intensity, addition of electron donors and inhibitors, and inhibitory treatments). It is concluded that the manganese signal arises from bound Mn2+ ions associated with the inside of the chloroplast membrane. The results also suggest that the observed signal is due to manganese which participates in the electron transfer from water to PSII. The esr of Mn2+ in photosynthetic systems has been studied by other workers. Treharne et al. (7) studied the manganese signal in Chlorella pyrenoidosa and observed changes in its intensity between light and dark periods. The kinetics of this transformation was found to be quite slow, being of the order of 1 h. More recently, Lozier et ~2. (8) studied the Mn2+ signal in spinach chloroplasts. This signal could only be observed upon treating the chloroplasts with chaotropic agents. These agents are assumed to convert bound manganous ions, which are not observable by esr, to (“free”) hydrated ions which are readily detectable. The resulting signal was found to be light sensitive: decaying on illumination and recovering in the dark. Sauer and his colleagues extended this work on spinach chloroplasts and showed (9, 10) that reactivation of oxygen evolution in Tris-treated chloroplasts was associated with the disappearance of the Mn2+ signal. They concluded that Tris washing releases part of the bound manganese to the interior space of the thylakoid membrane, while reactivation is accompanied by reincorporation of the manganese . In our preparation of active lettuce chloroplasts, showing Hill activity, it is possible to observe Mn2+ esr signals even with-
175
out Tris washing or other inhibitory treatments. Our measurements are thus directly performed on the bound Mn2+ species. EXPERIMENTAL Materials. Lettuce chloroplasts were prepared as described by Avron (11). Typically, the chloroplast preparations contained between 1 and 4 mg of chlorophyll per 1 ml of solution. The exact chlorophyll concentration was determined spectrophotometrically according to Arnon (12). These samples, which we refer to as “normal” or “control” samples, also contained 0.2 M sucrose, 0.1 M NaCl, and 0.05 M TrisHCI buffer, pH 7.8 @NT). The chloroplast-free reagent mixture was checked by esr in order to make sure that it did not contain manganous ions. The different normal choroplast preparations gave Hill activities ranging from 180 to 390 wequiv/mg of Chl/h as measured by O2 evolution using (Fe[CNl,13- as an electron acceptor. In addition to the normal chloroplasts, a number of modified preparations obtained by special treatments or addition of reagents were also studied. These included: (i) Heat treatment, which was carried out by incubating normal preparation for 3 min at 50°C. This treatment almost completely inhibited the capacity to evolve oxygen. However, the reaction center complex was still partially active. This was shown by the ability of heat-treated samples to emit delayed light, measured in the >O.l s range after illumination, to the extent of one-third of that of the control sample. [The apparatus used to measure delayed light emission is described in Ref. (131.1 (ii) Tris washing, which was performed according to Yamashita and Butler (14) by incubating the chloroplasts in 0.8 M Tris, pH 8 for 20 min, centrifugation at 25OOg, and resuspending the pellet in SNT. (iii) Manganese extraction by magnesium. This treatment was performed according to Chen and Wang (151. The medium contained 0.1 M sucrose, 5 mM NaCl, 200 mM MgSO, and 0.01 M Tris, pH 7.8. Chloroplasts were incubated for 1 h followed by centrifugation at 10,OOOg for 10 min. The pellets were resuspended in a reaction mixture which contained 0.2 M sucrose, 0.01 M NaCI, and 0.02 M Tris, pH 7.8. The concentration of manganese in the resulting samples was 1:lOO Mn:Chl, i.e., one-third of its concentration in the control samples. Also, the oxygen evolution was reduced by a factor of 3 in these preparations. Electron spin resonance measurements. Electron spin resonance spectra were recorded on a Varian E12 spectrometer at X-band frequency (9.3 GHz) using a TE,,,, cavity (or a double cavity TE1,,) equipped with a slotted grid to allow irradiation of the sample during measurements. The manganese spectra were recorded with a lOO-KHz field modulation of about
176
SIDERER ET AL.
10 G amplitude and using a microwave power of 100 mW. Under these conditions the peaks due to the socalled chloroplasts’ signals I and II (16) were also observed around the center of the manganese multiplet. These signals did not interfere with our measurements since they span a very small portion of the whole manganese spectrum. Relative integrated intensity measurements of the Mn2+ signal were performed using a double cavity, TEIM, in which the measured solution was placed in one compartment of the double cavity and a reference diphenylpicrylhydrazyl (DPPH) sample in the second. In each series of experiments the same sample cell was used and placed in the same position in the cavity. The relative intensities were obtained by double integration of the derivative signal and normalized with respect to the spectrometer setting and the reference DPPH signal (17). Kinetic studies of the light-induced disappearance and restoration of the Mn*+ signal were performed by monitoring the signal of one of the Mn*+ hyperfine lines (usually the high-field or penultimate hyperflne component). In practice, the magnetic field was set at the peak of the derivative signal and its intensity was recorded as a function of time following a “light on” or “light off’ step. To improve the signal-to-noise ratio we used a signal averager, HP 5480A, in which a number of traces (usually 16 to 25) were accumulated. In these experiments the spectrometer settings were identical to those used for recording the spectra except that the filter time constant for the output signal was set at 0.1 s or less. The chloroplast samples were placed in a flat aqueous solution quartz cell of 0.4 mm internal width. The chlorophyll concentration (1 mg/ml) was chosen as a compromise between the requirement of a strong esr signal on the one hand, and homogeneous light intensity within the sample on the other. The light source was a 500-W projector. The light
I
I
I
was filtered using Corning 4-96 glasses which transmitted light between 400 and 600 nm. The intensity of the light impinging on the sample, estimated by setting a calibrated silicon photocell next to the sample cell in the esr cavity, was -5 x lo+’ Einsteins/cm2 s. The relative light intensity was controlled using Schott neutral density filters. The whole setup (i.e., cavity and light source) was covered with a black screen to prevent stray light from entering the cavity during the measurements. RESULTS
AND DISCUSSION
Description of the Mn2+ Spectrum
Figure 1 shows the esr spectrum of letr tuce broken chloroplasts, over a wide (4000 G) magnetic field span. Three types of signals can be discerned in the spectrum: (i) a broad, -900 G wide signal centered around g = 2.22. The origin of this signal is not clear; however, from its g value it may be assigned to Cu2+ complexes, (ii) a typical sextuplet hype&me structure due to Mn2+, and (iii) a weak light-induced peak at the center of the manganese multiplet which is generally referred to as signal I and signal II, representing the oxidized form of P,,, and a radical connected with system II, respectively. In this paper we are concerned with signal (ii) due to the Mn2+. This spectrum is shown on an expanded scale in trace a of Fig. 2. It is characterized by a spin hamiltonian, X = gj3H.S
-I- AI*S
with the following parameters: g = 2.003 + 0.001; hyperfme splitting, A = 94.5 +- 0.5
3380 G I
I
I
I
1. Room temperature esr spectrum of lettuce chloroplasta at X-band frequency (9.3 GHz) recorded in the dark over a wide-field sweep. The spectrum shows the three types of signals discussed in the text: (i) a broad (-900 G) resonance around g = 2.22 tentatively attributed to CuZ+, (ii) an Mn2+ sextuplet aroundg = 2.003, and (iii) a weak signal due to photosystems I and II at the center of the Mn*+ spectrum. The spectrometer settings are: microwave power, 10 mW; modulation frequency, 100 KHz; modulation amplitude, 10 G, time constant, 1.0 s; scan rate, 4 G/s. The chlorophyll concentration is 1.15 mg/ml, in a 0.2 M sucrose, 0.1 M NaCl, 0.05 M Tris-HCl, pH 7.8 medium. FIG.
Dl
ESR OF MN(H)
177
IN CHLOROPLASTS
G; and an average peak to peak linewidth of M = 30 G. From these results alone it is not possible to decide on the nature of the manganese species responsible for the esr spectrum. When Mn2+ is bound in an asymmetric environment its spin hamiltonian is augmented by a quadratic zero field splitting (ZFS) term, x ZFS= D[s: - B S(S + l)] El + E(Sz2 - Su2) This term may have a profound effect on the solution spectra; it may cause broadening of the spectrum and, under certain conditions, complete or partial smearing out of the resonances. When the ZFS interaction is small compared to the Zeeman energy, which is usually the case for manganese bound to proteins (l&20), the possible effects on the esr spectrum can be understood by the following considerations of the various dynamic ranges (21,22). In a single crystal (no motion) containing manganese ions with the hamiltonian in Eqs. 111 and [2], the spectrum consists of five fine structure bands due to the various iVf, - 1 + M, transitions (-% --, -% -4 + -& ’ + & $ + k %+ 8. The relative intensitT& of these transitions are 5,8,9,8, and 5, respectively, and their splitting is of the order of the ZFS interaction. Each of the bands is split into six with spacing A (Eq. [ll) due to the hyperfme interaction with the 55Mn nucleus. For a single manganese site each spectrum will thus consist of a total of 30 “allowed” transitions. These transitions are anisotropic, i.e., their resonance frequencies (fields) depend on the relative orientations of the manganous complex and the magnetic field. However, when ZFS/g@Y < 1 the anisotropy of the -4 + h transitions is much smaller (being of second order in the ZFS interaction) than that of the other transitions (M, # i) (which are of first order in this interaction). Consequently, in a powder sample, the resonances of the M, # 4 transitions are spread over a wide frequency range and are usually not detectable, while the -4 + $ transitions remain relatively sharp with a width of order (ZFS)21gfl. The integrated intensity of the observed resonances is therefore & of the total expected intensity.
w a
!
b
c
I I FIG. 2. The Mn*+ spectrum of lettuce chloroplasts in the dark recorded in an expanded scale. Spectra a and b correspond to the same preparation except that sample b was acidified by adding 0.05 ml of concentrated HCI per 1 ml of chloroplast suspension. The spectrometer settings are identical for the two spectra and are the same as in Fig. 1 except that here the time constant is 0.1 s. The chlorophyll concentration is 3.2 mg/ml and the medium is the same as in Fig. 1. The weak signal in the center of the Mn*+ spectrum corresponds to photosignals I and II.
Turning now to solution spectra, i.e., introducing molecular tumbling into the powder, it is convenient to consider the following two dynamic ranges: (i) l/7 < ZFS and (ii) l/7 > ZFS, where 7 is the correlation time for the molecular tumbling, and the ZFS interaction is in frequency units. In the first range, the tumbling rate is not sufficiently fast to average out the anisotropy of the ZFS and the M, # $ transitions will remain unobservable. The spectrum will consist of only the M, = % transitions with relative intensity &. A detailed discussion of the resonance lineshape in this limit is given in (21). At higher tumbling rates, when condition (ii) applies, there is more or less complete averaging of the ZFS anisotropy and the whole spectrum coalesces around the center frequency. Under this condition a single sextet with the full intensity will be detected. The esr spectra of manganous ions bound to macromolecules often correspond to the first dynamic range. In fact, in many manganous protein complexes, the widths of the 4 4 B transitions are so large that they completely smear out even these signals. On the other hand, small
178
SIDERER
complexes in nonviscous media often belong to the second dynamic range, exhibiting a sextet spectrum with full intensity. This is, e.g., the case for the aqua manganous complex [Mn(Hz0>,12+. In order to characterize the esr signal observed in our chloroplast preparation, we determined the signal intensity relative to that of total manganese in the sample. For this purpose we made use of the fact that acidification of the chloroplast preparation causes release of cell-bound manganous ions into the bulk water environment. Thus, by comparing the integrated intensities of two chloroplast preparations, of which one had been acidified, the relative intensity of the chloroplast esr signal can be determined. Figure 2 shows two spectra corresponding to a control chloroplast preparation and the same preparation after acidification with a few drops of concentrated HCl. The two spectra were recorded under identical spectrometer settings. By comparing the integrated intensities of the two spectra we obtained a ratio of control/acidified - 0.2. Similar values were obtained in a number of different chloroplast preparations. This result is close to the expected relative intensity of the -4 + 6 transitions and suggests that the observed signal in the (nonacidified) chloroplast preparation comes from the MS = $ transitions of bound manganese, rather than from free Mn2+ ions. A confirmation of this suggestion could, in principle, be obtained by a detailed lineshape analysis of the observed signal according to the theory of (21). However, the quality of the spectra is not sufficient for such an analysis. Also it is not possible to tell from the observed spectra whether they are due to a single type of bound manganese or to a mixture of several different manganous complexes. We have recently observed similar esr signals in chloroplast preparations from spinach. This system was studied previously (8-10) and it was reported that Mn2+ signals could only be observed after Tris treatment. These signals were assigned to free Mn2+ ions in the interior space of the thylakoid membrane. It is not clear why in our samples Mn2+ signals can be observed even without Tris treatment and in active
ET
AL.
chloroplasts. Perhaps it is due to different practices of preparation, although we have used the same “literature” method. We believe that the intensity ratio of approximately 0.2 between normal chloroplast preparations and acidified samples is not accidental and strongly suggests that the observed signal is due to bound manganese. Although on the basis of the appearance of the spectrum alone we cannot rule out the possibility that it is due to free manganese released from the chloroplasts. The above interpretation is further supported by dialysis experiments in which a chloroplast preparation was allowed to dialyze for 24 h against an SNT solution. During this period, only a small amount (7%) of the manganese ions passed the dialysis membrane. The Mn2+ signal of the dialyzate was also tested by acidification treatment: Addition of the acid resulted in an increase of about four- to fivefold the Mn2+ signal intensity. It therefore seems that the Mn2+ in the dialyzate was also bound. Testing the dialyzate by the Lowry et al. (23) method showed that it also contained proteins, apparently of low molecular weight, which penetrated through the dialysis membrane (Union Carbide dialysis membrane). Effect of Light
on the Mn2+ Signal
“Control” chloroplast preparations. Illumination of the chloroplasts in the cavity by visible light causes a reduction in the intensity of the Mn2+ signal. Turning the light off results in restoration of the signal to its original intensity. As an illustration, in Fig. 3 we compare two traces of the Mn2+ signals corresponding to “light on” and “light oft”’ periods. In the following we shall discuss the light effect in terms of the parameter R defined by R = hVh/,
E31 where h” is the signal intensity following the Yight on” step and is time dependent, and hOd is the steady-state intensity of the signal in the dark. R is thus proportional to the instantaneous concentration of Mn2+ ions and monitors’ its transformation to higher oxidation states. In experiments of the type shown in Fig. 4, R decreases from R = 1 to some steady-state value, R, dur-
ESR OF MN(H)
IN CHLOROPLASTS
ing the “light on” period, and increases again to unity after cessation of illumination. Within our experimental accuracy, the light and dark reactions follow exponential decay and rise kinetics. There are thus three parameters that describe the light response of the Mn2+ spectrum: kz, the rate constant for the light reaction, kd, the rate constant for the dark reaction,
179
and R,. The value of kd in the control sample was found to be about 0.35 -t 0.1 s-’ independent of the light intensity, I. On the other hand, both k’ and R, were found to depend on I. Typical results are shown in Fig. 5. It may be seen that R, decreases with I for low light intensities, leveling off to Rsminat higher values of I, while the rate constant kz increases monotonically with I in the range studied The values ofRsminwere not the same for r all batches and ranged from 0.2 to 0.3 in the different preparations used. In the quantitative measurements described dark i here, we have, therefore, always compared results of solutions prepared from the same batch of chloroplasts. In order to find the relevance of the light-induced change of the Mn2+ signal to the chloroplasts’ electron transport activity, we studied the effect of a number of treatments and reagents on the R values and the kinetics of the light response. The added reagents used (DCMLJ, PD, FCCP and ascorbate) did not change the steadyL state dark signal of MI?+. FIG. 3. Comparison of the chloroplast Mn2+ specEffect of DCMU. It is known that trum during dark and light periods. The same chloDCMU blocks electron transfer from PSI1 roplast preparation and spectrometer settings used to the plastoquinone pool and thus should in Fig. 1 were employed except that the time conof the Mn2+ stant was 0.3 s. In this particular example, the also inhibit the photoreaction if it is related to this photosystem. We reduction of the signal due to illumination is about 80%. Note also the increase in intensity of signal I in have, therefore, measured its effect on the the light period (exceeding the recorder range). light-induced reduction of the Mn2+ signal. off
FIG. 4. Kinetic traces of the Mn2+ signal following “light on” and “light off’ steps. The ordinate corresponds to the reduced amplitude R = hi/hod of the penultimate hyperfine line (counted from low field). During the experiments, the field was fixed at the peak of the derivative line and the time evolution of the signal monitored using an HP 5480A signal averager. Twenty-five scans were accumulated to obtain the above traces. Each cycle consisted of 4 s of illumination followed by 16 s in the dark. The spectrometer settings are: modulation amplitude, 10 G; microwave power, 100 mW; time constant, 0.1 s. Note that the time scales for the light and dark periods are not the same. The symbols & and e,2 correspond to the halflives of the light and dark reactions, respectively. The chloroplast preparations are as in Fig. 1.
180
SIDERER ET AL
still active in the electron transport chain, the ability to oxidize H,O and evolve 0, is lost. Effect of electron donors. As an electron donor phenylenediamine (PD) may compete with Mn2+ in the electron transfer reaction from water to PSII. PD may also affect the reaction sequence by catalyzing the reduction of the oxidized form of manganese to Mn2+. As may be seen in Fig. ‘7, addition of PD indeed affects the light response of the Mn2+ signal, increasing R, and increasing kd. The latter effect is most FIG. 5. Effect of light intensity on R, and kc. The probably due to reduction of the oxidized chloroplast preparation was the same as in Fig. 1. manganese form to Mn2+ by PD. Ascorbate at higher concentration abolResults of Rs versus DCMU concentration are summarized in Fig. 6. It may be seen ished the effect of light on the Mn2+ signal that addition of DCMU indeed inhibits the completely. Thus, in a suspension containeffect of light on the Mn2+ signal. The ef- ing 1 mM ascorbate there was no change in the intensity of the Mn2+ signal on illumifect increases with DCMU concentration up to 2 x 1O-4 M where the inhibition is nation (Fig. 7). Effect of FCCP. FCCP was used in the complete. At this concentration, the molar past as an uncoupler. However, it has an ratio of DCMU to chlorophyll is approximately 15. This result may be compared to additional effect as an ADRY reagent (Accelerating the Deactivation of the Reaction that obtained for blocking the Hill reaction of the water-splitting enzyme Y) (24). Its by the same reagent where the molar ratio effect as an ADRY reagent is shown by the for complete inhibition was found to be inhibition of the oxygen yield in consecuaround 1:lO (8). This simple experiment of both shows that Mn2+ oxidation involves PSI1 tive flashes (24) and the inhibition delayed light emission and the triggered activity. (25). Figure 6 shows that Effect of treatments which inhibit oxy- luminescences gen evolution. Further support for these addition of FCCP decreases the photooxiconclusions was obtained by studying the dation of Mn2+ and at around 3 x 10m4 M completely inhibits the reaction. This effects on the Mn2+ signal of treatments which are known to inhibit oxygen evolu- should perhaps be taken as evidence for since FCCP is tion. We have used the following treat- direct PSI1 participation, ments (see Experimental): (i) heat treatn I -7. ment (14), (ii) Tris washing (141, and (iii) incubating with Mg2+ (15). All of these treatments resulted in an increase of the dark Mn2+ signal apparently due to release of part of the bound manganese. The resulting signal was still affected by o----t DCMU light. However, the kinetics of both light A--+ FCCP and dark reactions were considerably slower after the treatments (see Table I and Fig. 7). In Tris washed chloroplasts, oxygen evolution is inhibited completely; however, delayed luminescence persists to 040-‘-’ 5x10 M the extent of lo%, indicating partial activ[DCMU] or [FCCP] ity of the reaction centers. It seems that FIG. 6. Effect of DCMU and FCCP on the light the above treatments affect the manganresponse of the Mn2+ signal. The chloroplast prepaous site in such a way that while PSI1 is rations are as in Fig. 1.
ESR OF MN(H)
TABLE KINETIC
PARAMETERS OF THE IN CHLOROPLASTS
k' (s-‘)=
181
IN CHLOROPLASTS I
MnZ+ esr SIGNAL SUBJECTED
AND OTHER DATA RELATED TO PHOTOSYNTHETIC TO OXYGEN EVOLUTION INHIBITORY TREATMENTS
kd (s-9”
O2 evolution (pequi;$)Vmg c
[Chll, [Mnl
of
Control Tris washed Heat treated Mg2+ replacement
ACTIVITY
Delayed luminescence intensitvd 100 10 33 -
2.8 k 0.3 0.35 a 0.1 30 390 0.9 0.12 50 4 1.1 0.07 30 -=S 100 130 0.8 0.05 (1The uncertainties in the treated samples are estimated at k--50%. b Molecular ratio of chlorophyll to manganese. Manganese concentration was determined by atomic absorption. c Determined by Clark-type oxygen electrode with [Fe(CN1613- as an electron acceptor. d The delayed luminescence intensity is given in arbitrary units. The figures correspond to the emission intensity -160 ms after cessation of ilhumination.
not expected to intervene in PSI reactions or to inhibit steady-state electron transfer at pH values used in our experiments -7.8 (26). Indeed, a check was made on the effect of FCCP on steady-state transport to [Fe(CN),13- at similar conditions to the esr measurements. As expected, the rate of electron transport was enhanced rather than inhibited. The results gathered so far lead us to think that the observed Mn*+ photooxidation is by electron donation to photosystem II by manganese, which is part of the photosynthetic machinery. There are several other alternatives. Direct oxidation of Mn*+ by HO, radicals known to be generated at the acceptor side of PS-I (27, 28) seems unlikely for the following reasons: (i) We tried to rule out this possibility by an experiment in which methylviologen was added as an acceptor. In this case the superoxide radical formation is expected to be enhanced considerably. However, addition of this reagent did not change any parameter of the Mn*+ photooxidation reaction. (ii) Although inhibitory treatments (DCMU, Tris treatment, etc.) could inhibit Mn*+ photooxidation due to PSI1 or PSI by inhibiting steady electron transport, the specific effect of FCCP cannot come under this category since it does not inhibit steady-state electron transport. FCCP seems to interact rather specifically with PSII. (iii) The back-reaction of the Mn*+ photooxidation is slowed down considerably after Tris treatment, which again may suggest an interaction with PSII.
0.4 I
i off
( 4ac
‘1
FIG. 7. Retracings of kinetic plots of the type shown in Fig. 4, exhibiting the effect of various reagents and treatments.
CONCLUSIONS
Although there are several routes that can account for the reaction of the Mn*+, we believe that the above results confirm the conjecture that the manganous species responsible for the esr signal is an integral part of the photosynthetic machinery. There are three main points that emerge from the above results: (i) Mn*+ ions participate in the electron transfer process of the photosynthetic cycle via oxidation-reduction equilibria involving +3 or higher oxidation states of manganese. (ii) The active manganous ions are bound in an asymmetric environment within the chloroplast structure. (iii) The effects of the various reagents on the light response of the manganous signal suggest that its photooxidation is associated with PSII. Whether this Mn is also associated with the positive charge accumulator needed for
182
SIDERER
oxygen evolution is still an open question. Further studies using flashes are undertaken in order to clarify this question. Very recently, evidence on the participation of Mn*+ in the S-state cycle has been obtained by nmr relaxation measurements (2%
ET
14. 15. 16. 17.
ACKNOWLEDGMENT We interest
wish to thank Professor M. Avron for in the work and for helpful discussions.
his
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