©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Difference Fourier Transform Infrared Spectroscopic Study of Chlorophyll Oxidation in Hydroxylamine-treated Photosystem II (*)

Gina M. MacDonald , Jacqueline J. Steenhuis , Bridgette A. Barry (§)

From the (1) Department of Biochemistry, University of Minnesota, St. Paul, Minnesota 55108

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In oxygenic photosynthesis, photosystem II is the chlorophyll-containing reaction center that carries out the light-induced transfer of electrons from water to plastoquinone. Fourier transform infrared spectroscopy can be used to obtain information about the structural changes that accompany electron transfer in photosystem II. The vibrational difference spectrum associated with the reduction of photosystem II acceptor quinones is of interest. Previously, a high concentration of the photosystem II donor, hydroxylamine, has been used to obtain a spectrum attributed to Q


INTRODUCTION

PSII() is found in plants, green algae, and procaryotic cyanobacteria and is one of two chlorophyll-containing reaction centers that cooperate to transfer electrons from water to NADPin oxygenic photosynthesis. PSII carries out the light-induced transfer of electrons from water to plastoquinone. This reaction center is composed of integral membrane proteins that bind the prosthetic groups required for efficient charge separation. Extrinsic polypeptides are also required for oxygen evolution under physiological conditions. The prosthetic groups associated with PSII include the primary chlorophyll donor, P, accessory chlorophylls, pheophytin, and two plastoquinone molecules, Qand Q. Upon absorption of light, P

Although there is no crystal structure of PSII, there is an atomic level structure of a related protein, the reaction center from purple, non-sulfur bacteria (3) . Comparison of the acceptor side of PSII to the bacterial reaction center reveals both sequence and functional homology (3) . However, the bacterial reaction center does not oxidize water and, thus, does not contain redox-active tyrosines or a manganese cluster.

Other electron transfer reactions occur in PSII that are not described above and do not occur in the bacterial reaction center. For instance, redox-active tyrosine D can be oxidized by P

Difference FTIR can be used to obtain dynamic structural information about PSII. Such ``reaction-induced'' infrared spectroscopy allows the study of the structural changes in cofactors and protein components that accompany function. In application to PSII, light-minus-dark difference spectra provide ``dynamic'' structural information concerning light-induced electron transfer events. For example, previous studies have identified vibrational modes associated with the oxidation of the two tyrosine radicals, D and Z (11, 12) . These spectra may contain contributions from the vibrational difference spectrum, Q

The contributions of the acceptor side of PSII to the light-minus-dark infrared spectrum are certainly of interest. A high concentration of hydroxylamine is sometimes used as a donor in steady state illumination experiments in order to accumulate Q

High concentrations of hydroxylamine have been used previously in difference (light-minus-dark) FTIR spectroscopy in an attempt to identify structural changes on the acceptor side of PSII (24) . PSII membranes were used in this work (25) . These membranes were treated with the inhibitor, DCMU, to block electron transfer from Qto Q. The resulting difference spectrum was considered to be the vibrational spectrum of Q

However, some FTIR studies of PSII are not consistent with these previous assignments to Q

Therefore, we have reinvestigated the redox state of PSII reaction centers in the presence of hydroxylamine. We have used EPR spectroscopy as a control for our difference infrared studies. Samples were prepared in the same way for both EPR and FTIR experiments, that is, dehydrated on solid substrates. Our work shows that steady state illumination in the presence of hydroxylamine results in the reversible production of a chlorophyll cation radical in approximately 5% of the centers. We present evidence that this level of chlorophyll contribution to the difference infrared spectrum is detectable. Further, we show that global labeling of photosystem II with N causes a 2 cmshift of the most intense spectral feature, the positive 1478 cmline. A 2 cmN shift is inconsistent with the previous assignment of the 1478 cmline to Q


EXPERIMENTAL PROCEDURES

Sample Preparation

PSII membranes were prepared as in Berthold et al. (25) and resuspended in a buffer containing 50 m M MES-NaOH, pH 6.0, 15 m M NaCl, and 25% glycerol. This preparation is not monodisperse, but is in the form of membrane sheets that can be pelleted by centrifugation. The purified PSII membranes evolved oxygen at rates of approximately 600 µmol of O/mg of chlh.

The membranes were manganese-depleted by the addition of hydroxylamine from a stock that contained 50 m M hydroxylamine, 1 m M EDTA, 50 m M MES-NaOH, pH 6.0, 15 m M NaCl, and 25% glycerol. This stock was made up immediately before use. The final concentration of hydroxylamine in the membrane sample was 10 m M. After addition of hydroxylamine, the membranes were agitated in darkness for 80 min at 0 °C. The membranes were diluted 1:4 by the addition of a buffer containing 50 m M MES-NaOH, pH 6.0, 15 m M NaCl, and 25% glycerol. After hydroxylamine treatment, the membranes were pelleted by centrifugation for 30 min at 19,000 rpm in a SS-34 rotor. The pellets were resuspended in a buffer that contained 5 m M MES-NaOH, pH 6.0. The membranes were pelleted again as described above and resuspended in 5 m M MES-NaOH, pH 6.0. The final chlorophyll concentration was 4.0 mg/ml.

PSII complexes were prepared, manganese-depleted, and concentrated as in (11, 31) . As purified, the PSII complexes had a specific activity of 1100-1200 µmol of O/mg of chlh. The final resuspension buffer was 5 m M MES-NaOH, pH 6.0. Unlike PSII membranes, this preparation is detergent solubilized and is monodisperse. The PSII complexes do not contain functional Q(11) . The final chlorophyll concentration was 1.3 mg/ml.

Cyanobacterial PSII particles from Synechocystis sp. PCC 6803 were prepared as previously described (35) and had a specific activity of 2300-3000 µmol of O/mg of chlh. PSII particles were hydroxylamine-treated as described elsewhere (11, 31) , except the incubation time was only 40 min. The manganese-depleted PSII particles were then concentrated as in MacDonald and Barry (11) . Global isotopic labeling with N was performed as previously described (31, 36) . Mass spectral analysis (37) of acetone extracted chlorophyll was used to verify isotopic incorporation. Mass spectra were obtained on a Kratos MS25 mass spectrometer. The sample was ionized by fast atom bombardment through the use of an 8-KeV xenon beam. The matrix was 3-nitrobenzyl alcohol, and positive ions were detected.

Immediately before EPR and FTIR spectroscopy, hydroxylamine was added to either the PSII membranes or PSII complexes to a final concentration of 10 m M. For the cyanobacterial PSII complexes, the final hydroxylamine concentration was 6 m M. In some cases, potassium ferricyanide also was added to a final concentration of 3 m M.

Oxygen-evolving PSII complexes used for cryogenic FTIR were prepared as previously described (11) and were in 25% glycerol, 50 m M MES (pH 6.0), 7.5 m M CaCl, 0.05% lauryl maltoside (Anatrace, Maumee, OH), and 260 m M NaCl.

PSI was purified from the cyanobacterium, Synechocystis sp. PCC 6803, by the procedure described in Noren et al. (35) .

EPR Spectroscopy

The manganese-depleted PSII membranes and complexes were dehydrated on mylar substrates as in MacDonald and Barry (11) . Membrane samples contained 280 µg of chlorophyll. The PSII complex samples contained 400 µg of chlorophyll. Spectra were recorded at -9 ± 2 °C as described by MacDonald and Barry (11) . Spectra were obtained at X band using the following conditions: modulation amplitude, 3.2 G; microwave power, 0.67 mW; scan time, 4 min; time constant, 2 s; gain, 3.2 10.

Spin quantitation was performed at -160 °C on samples that had been illuminated at -9 °C and then frozen in darkness. The amount of the light-induced radical was quantitated by double integration of the EPR signal using the program, IGOR (Wavemetrics, Lake Oswego, OR). Fremy's salt was used as a spin standard (38, 39) .

The g value was determined by using the chlorophyll radical in purified PSI as a standard; the PSI sample was dehydrated on a mylar substrate. The EPR spectrum was obtained at -9 °C. The g value of the chlorophyll radical in PSI is known to be 2.0025 (40) . FTIR Spectroscopy at -9 °C-Samples contained 50-70 µg of chlorophyll for spinach samples and 20-30 µg of chlorophyll for Synechocystis PSII particles. All PSII preparations were dried on a germanium-coated window as described by MacDonald and Barry (11) . Spectral conditions were as previously described (11) . The absorbance in the amide I band at 1655 cmwas always <0.9 absorbance units. Spectra were recorded at -9 °C. The spectral resolution was 4 cm, the mirror velocity was 1.57 cm/s, and 1000 scans were co-added for each interferogram. The difference spectrum was constructed by directly ratioing the interferogram obtained under illumination to the interferogram obtained in the dark and then converting to absorbance units. Five to seven difference spectra were averaged in order to obtain the final data.

Cryogenic FTIR Spectroscopy

The sample contained 30 µg of chlorophyll. Spectral conditions were as described above. The temperature was controlled to ±0.1 K with a High-Tran liquid nitrogen cryostat (R. G. Hansen & Associates, Santa Barbara, CA) and a temperature control unit (Scientific Instruments model 9620, West Palm Beach, FL). The absorbance of the amide I band was 0.7 absorbance units. Spectra were recorded at 80 K, and 1000 scans were co-added to construct each interferogram. Difference spectra were constructed by the method described above. Two difference spectra were then averaged in order to obtain the final spectrum.


RESULTS AND DISCUSSION

EPR Spectra of PSII Membranes at -9 °C-In Fig. 1, we present EPR spectra recorded on PSII membranes in the presence of hydroxylamine alone (Fig. 1 B) and in the presence of hydroxylamine and potassium ferricyanide (Fig. 1 A). The presence of hydroxylamine will lead to accumulation of Q


Figure 1: EPR spectra of manganese-depleted spinach PSII membranes at -9 °C in the presence of hydroxylamine and potassium ferricyanide ( A) or hydroxylamine alone ( B). The inset shows low temperature (-9 °C) EPR spectra of manganese-depleted PSII complexes in the presence of hydroxylamine and potassium ferricyanide. In all cases, the solid lines are spectra recorded under continuous illumination, while the dot-dashed and dotted lines are spectra recorded after 8 and 90 min of dark adaptation, respectively. Spectral condi-tions are given under ``Experimental Procedures.''



In the presence of hydroxylamine alone or in the presence of hydroxylamine and ferricyanide, EPR spectroscopy (Fig. 1, A and B, solid line) shows that a chlorophyll radical is present under illumination. The line width (7.5 G) and g value (2.0025) of this radical are both consistent with a chlorophyll origin (40, 48) . Spin quantitation of the spectra recorded under illumination (Fig. 1, A and B, solid line) indicates that the chlorophyll radical is present in approximately 10-20% of the centers. This is in contrast to earlier results, which reported no chlorophyll radical present when hydroxylamine was added as a reductant (26) . Note that the level of detection was not specified in this earlier study.

A difference in the decay of this chlorophyll signal is observed when samples containing hydroxylamine alone (Fig. 1 B) are compared to samples containing ferricyanide and hydroxylamine (Fig. 1 A). In the presence of ferricyanide, the chlorophyll radical is reduced by approximately 15% in 8 min (Fig. 1 A, compare dot-dashed and solid line). Approximately 30% of the radical has decayed after 90 min (Fig. 1 A, compare the dotted line and solid line). If ferricyanide is not present in the PSII membranes, only <10% reduction is seen after 90 min of dark adaptation (Fig. 1 B, dotted line), showing that, in the presence of hydroxylamine alone, the formation of the chlorophyll radical is relatively irreversible.

To summarize, our EPR results show that, when both hydroxylamine and ferricyanide are present, a chlorophyll radical is produced reversibly in approximately 5% of the centers. However, in the presence of hydroxylamine alone, less than 1% of the centers reversibly produce a chlorophyll radical. We would expect that Q

It should be noted that the EPR line shape of the radical varies from sample to sample, as does the amount of radical produced. Some samples exhibit a broader EPR signal that contains contributions from the tyrosine radical, D, in addition to chlorophyll contributions (data not shown). The presence of hydroxylamine in high concentrations leads to this variability, which is not observed in infrared studies of PSII in the absence of hydroxylamine (for example, see Refs. 11 and 12). Therefore, high concentrations of hydroxylamine are problematic in experiments conducted under steady state illumination. FTIR Spectra of PSII Membranes at -9 °C-Light-minus-dark difference FTIR spectra recorded on PSII membranes are shown in Fig. 2. In Fig. 2, A and B, both ferricyanide and hydroxylamine are present. In Fig. 2 A, there was a short dark adaptation time of 8 min between the light and dark spectra. In Fig. 2B, there was a long dark adaptation time of 90 min between the light and dark spectra. Both sets of data were obtained on the same sample, and both difference spectra exhibit strong vibrational modes. These are the conditions where our EPR experiment predicts that a chlorophyll radical will make the largest contribution to the spectrum. An increase in intensity is observed when Fig. 2 B is compared to Fig. 2 A. This increase in intensity roughly correlates with the results of our EPR experiment (Fig. 1 A) that showed a more extensive decay of the chlorophyll radical in 90 min than in 8 min. The use of hydroxylamine leads to some variability in the relative intensities of the difference infrared spectrum and in the amplitudes of the EPR signal, so an exact correlation of EPR and FTIR spectral intensities is not expected.


Figure 2: Light-minus-dark FTIR spectra recorded at -9 °C of manganese-depleted spinach PSII membranes. Samples were treated with either hydroxylamine and potassium ferricyanide ( A and B) or hydroxylamine alone ( C and D). The dark adaptation was 8 min in A and C and 90 min in B and D. The tick marks on the y axis correspond to 4 10absorbance units. Spectral conditions are given under ``Experimental Procedures.''



Fig. 2 , C and D, present difference spectra recorded on PSII membranes in the presence of hydroxylamine alone. In Fig. 2C, the dark adaptation was 8 min, and in Fig. 2 D, it was 90 min. Both sets of data were obtained on the same sample. These difference spectra exhibit only weak features (see Fig. 2 , C and D). These are conditions where our EPR control experiment showed only a small change in the concentration of chlorophyll radical even after prolonged (90 min) dark adaptation.

Our experiments provide evidence for a correlation between the appearance of a reversibly oxidized chlorophyll radical (EPR spectroscopy) and an increase in the intensity of the infrared spectrum. To demonstrate that an approximately 5% (moles of chl radicals/total moles of chl) contamination from a chlorophyll radical would be observable in the difference infrared spectrum, we present light-minus-dark difference infrared spectra (Fig. 3) on purified photosystem I from the cyanobacterium, Synechocystis sp. PCC 6803. The antenna size (35) of the PSI samples is approximately 90 chlorophyll per P. Illumination of photosystem I photooxidizes the primary chlorophyll donor, P


Figure 3: FTIR difference spectra recorded at -9 °C of purified PSI particles from the cyanobacterium, Synechocystis sp. PCC 6803. Samples contained chlorophyll in the amounts of 4.0 µg ( A), 2.0 µg ( B and D), and 0.4 µg ( C) plus 1.5 m M potassium ferricyanide. A, B, and C are light-minus-dark difference spectra in which the dark adaptation time was 8 min. D is a dark-minus-dark difference spectrum obtained after illumination. The tick marks on the y axis correspond to 4 10absorbance units.



In Fig. 3 A, the P

These results show that the oxidation of chlorophyll makes an intense contribution to the difference infrared spectra. Such a conclusion is consistent with the results of previous infrared studies on bacterial reaction centers (for example, see Ref. 32). These studies have shown that the vibrational spectrum associated with the oxidation of the bacteriochlorophyll donor dominates the difference spectrum. For example, vibrational modes of the primary donor are an order of magnitude more intense than those observed for the acceptor quinones (32) .

We also point out that because of the large sample sizes needed to obtain high signal-to-noise EPR data, the light source is not saturating under the EPR conditions. For FTIR experiments, a much smaller amount of sample is required. For this reason, we believe that the EPR results probably underestimate the number of chlorophyll radical-producing centers in the infrared experiment.

Our difference spectra resemble data obtained previously on hydroxylamine-treated PSII membranes (24) . In order to demonstrate this, we have reproduced the difference spectrum reported by Berthomieu et al. (24) (Fig. 4, solid line) and have compared it directly to one of our spectra (Fig. 4, dotted line), which was also recorded on hydroxylamine-treated PSII membranes. The dark adaptation time was 90 min (Fig. 4, dotted line). The superimposition shows that the spectra are similar. The difference observed in the 1670-1650 cmspectral region may be due to an increased contribution of D in Berthomieu et al.'s experiments, which involved much longer dark adaptations. Our previous work has assigned lines to redox active tyrosine D through the use of isotopomers of tyrosine (12) . In addition, the 1670-1650 cmspectral region may include contributions from the amide I band of the peptide backbone (31) . The spectra compared in Fig. 4 were obtained at a different temperatures, and this temperature difference could result in small differences in protein conformation and, thus, in amide I frequencies and intensities.


Figure 4: Light-minus-dark FTIR spectra of hydroxylamine-treated spinach PSII membranes either from this work ( dotted line) or as reproduced from Berthomieu et al. (24) ( solid line). The data reproduced from Berthomieu et al. (24) were obtained in the presence of DCMU at 15 °C.



The spectrum of Berthomieu et al., was attributed to Q

EPR and FTIR Spectra of PSII Complexes

The PSII membranes used in the experiments described above may contain small amounts of PSI. At room temperature, the primary chlorophyll donor of PSI, P, gives rise to an EPR signal. To distinguish whether the chlorophyll observed in Fig. 1is due to residual PSI or originates from PSII, EPR data were obtained on a PSII complex preparation that has no PSI contamination. PSI quantitation was performed on the same samples according to the method described in Noren et al. (35) ; PSI was not detectable in these samples by optical techniques.

EPR spectra recorded on PSII complexes in the presence of both hydroxylamine and ferricyanide are shown in the inset of Fig. 1; these spectra were recorded under illumination ( solid line), after 8 min of dark adaptation ( dot-dashed line), and after 90 min of dark adaptation ( dotted line). The EPR spectrum of a chlorophyll radical is observed under illumination, and this radical is reduced in the dark. Spin quantitation of the radical indicates approximately 10% of the PSII complexes exhibit the light-induced radical when hydroxylamine and ferricyanide are present. If only hydroxylamine is present, the chlorophyll radical is produced under illumination, but the radical does not decay appreciably in 90 min of dark adaptation (data not shown). This is a similar result to the one described above for PSII membranes. The broader line width and higher g value of the signal shown in the inset are due to an increased contribution from D.

Fig. 5 shows FTIR difference spectra recorded on manganese-depleted PSII complexes under the conditions used to record the EPR data. The signal-to-noise is increased upon comparison of Fig. 5 to Fig. 2, because PSII complexes have a smaller antenna size. The reduced antenna size will allow vibrational modes associated with structural changes in redox-active tyrosine, D, and other amino acids to be more easily observed. Thus, we expect there to be small differences in intensity and frequency between the two samples, even if chlorophyll and quinone vibrational lines contribute to both difference spectra. Spectra in Fig. 5, A and B, were recorded in the presence of hydroxylamine and ferricyanide with either an 8-min (Fig. 5 A) or a 90-min (Fig. 5 B) dark adaptation between the ``light'' and ``dark'' spectra. Intense difference spectra are observed (Fig. 5, A and B), and an increase in intensity is observed that correlates with the length of dark adaptation. Examination of Fig. 5, A and B, reveals positive lines with frequencies of 1456, 1478, 1506, 1718, and 1737 cmand negative modes at 1522, 1557, 1630, 1645, 1672, 1682, 1724, and 1743 cm. All of these frequencies are within 1-2 cmof corresponding lines in Fig. 2, A and B.


Figure 5: Light-minus-dark FTIR spectra recorded at -9 °C of manganese-depleted spinach PSII complexes. Samples were treated with either hydroxylamine and potassium ferricyanide ( A and B) or hydroxylamine alone ( C and D). The dark adaptation was 8 min in A and C and 90 min in B and D. The tick marks on the y axis correspond to 4 10absorbance units. Spectral conditions are given under ``Experimental Procedures.''



In contrast, in the presence of hydroxylamine, spectral features are weak, regardless of the length of dark adaptation (Fig. 5, C and D). However, when hydroxylamine is added in strict darkness and samples are then dark-adapted for 3 h or more, intense light-minus-dark difference spectra are obtained (data not shown). These spectra resemble those shown in Fig. 5, A and B. This strong infrared spectrum is again correlated with the production of the chlorophyll cation radical in an EPR control experiment (data not shown). This radical does not appreciably decay. In reference Berthomieu et al. (24) a very slow spectral decay behavior was also observed.

N-Labeling of Photosystem II

The work that we have presented thus far shows a correlation between the reversible production of a chlorophyll cation radical and an intense positive line at 1478 cmin the difference infrared spectrum (Figs. 1, 2, and 5). This line is observed either in spinach PSII membranes (Fig. 2) or in spinach PSII complexes (Fig. 5). There is also a correlation with the appearance of differential features in the 1750-1650 cmregion (Figs. 2 and 5). Although this chlorophyll cation radical is produced in only 5% of the centers, we have presented evidence that this a detectable amount of chlorophyll radical, as assessed by infrared spectroscopy (Fig. 3). We have also provided evidence that the frequency of the 1478 cmline is not responsive to the presence of DCMU (Fig. 4).

In light of these observations, we decided to investigate the origin of the intense 1478 cmline that is observed in the presence of hydroxylamine. Vibrational lines in complex biological spectra can be assigned through isotopic labeling. As the first step in our attempts to label chlorophyll and quinones specifically for infrared spectroscopy, we carried out global N-labeling of photosystem II. This can be performed by growing the cyanobacterium, Synechocystis sp. PCC 6803, in the presence of [N]nitrate and isolating photosystem II from the labeled cultures. This procedure should have the effect of replacing every N in the cell with N. To verify isotope incorporation, mass spectral analysis was performed on extracted chlorophyll from the same samples used to generate Fig. 6 B. Mass spectral analysis (37) shows that the total amount of N incorporation into chlorophyll is 97% (data not shown). 90% of the chlorophyll is labeled with N at all four ring positions (N), while the remaining 7% is either N, N, or N. This analysis is in agreement with previous N-labeling experiments in cyanobacteria (31, 36) .

In Fig. 6 B, we present light-minus-dark difference spectra obtained on control ( dashed line) and N-labeled ( solid line) PSII particles from Synechocystis. A 2 cmshift of the 1478 cmline is observed upon global N-labeling (Fig. 6 B, or see the expanded spectrum in Fig. 6 A). It should be noted that overlay of two control, N, difference spectra gives a reproducible frequency of 1478 cmfor this line (data not shown). This N shift was observed in PSII samples isolated from three different N-labeled cultures. Also, extensive HO exchange had no effect on the frequency of this line. Since plastoquinone contains no nitrogen atoms, a 2 cmN shift precludes assignment of the 1478 cmline to Q


Figure 6: Light-minus-dark FTIR difference spectra recorded at -9 °C of control (N) ( dashed line) or of N-labeled ( solid line) PSII particles from Synechocystis sp. PCC 6803. Panel A shows an expanded spectrum from 1500 to 1450 cm, while Panel B shows the 1900 to 1300 cmregion. Samples contained hydroxylamine and ferricyanide. The dark adaptation time was 8 min. The tick marks on the y axis correspond to 4 10absorbance units. Spectral conditions are given under ``Experimental Procedures.''



A difference infrared spectrum of N-labeled spinach photosystem II preparations that were treated with hydroxylamine has been described previously (26) . In this work, it was reported that the 1478 cmline did not shift as a result of isotopic labeling. The discrepancy with our result is probably the result of incomplete N-labeling of chlorophyll in the earlier study. No mass spectral analysis of chlorophyll was described in the previous study, and the extent of labeling was not reported.

On the basis of the N shift obtained, we favor the assignment of the 1478 cmline to a chlorophyll cation radical. This chlorophyll may be the primary donor or may be another chlorophyll oxidized by P

The assignment of the 1478 cmline to chlorophyll implies that the spectrum observed in the presence of hydroxylamine is actually dominated by the vibrational changes associated with chlorophyll oxidation, not by acceptor side changes. Assignment of this spectrum to a dominating chl- chl contribution accounts for the fact that a similar spectrum is observed in the absence of hydroxylamine in some PSII membrane preparations (27, 28) . Such a result could be obtained if contributions from a PSII chlorophyll oxidized in inactive centers dominated the spectrum (27, 28) .

Our FTIR difference spectra show spectral similarities with other infrared data recorded on bacteriochlorophyll and chlorophyll both in vitro and in vivo. For example, in all these cases, oxidation of chlorophyll or bacteriochlorophyll leads to differential features in the spectral region between 1750 and 1650 cm(51, 52) . These lines have been assigned to ester and carbonyl vibrations (Fig. 7) that are perturbed by oxidation. Note, however, that a recent study suggests that some lines in this spectral region may be more properly assigned to nonfundamental vibrations (53) . In in vitro studies of chlorophyll a oxidation, a differential feature at 1718/1693 cmwas assigned to the C=O vibration (Fig. 7), and a differential feature at 1751/1738 cmwas assigned primarily to the Cester carbonyl vibration (52) . By analogy, a strong differential feature at 1717/1700 cm, observed after photoxoidation of Pin photosystem I, was assigned to the Cketo group (52) . However, unambiguous assignment of the Cester carbonyl vibration was not possible, since two differential features, not one, were observed at higher frequency: 1753/1748 and 1741/1734 cm. The authors favored the assignment of both these spectral features to a Cester carbonyl vibration. They reasoned that the individual chlorophylls, making up a presumed dimeric cation radical, contributed independently to the difference spectrum. However, other possible explanations could not be excluded (52) .


Figure 7: Structure of chlorophyll a.



Based on these previous studies, we tentatively assign the differential feature at 1718/1701 cmin our spectrum (Fig. 2, A and B) to the Cketo C=O group of chlorophyll. Our difference spectra also exhibit two differential features in the 1725-1750 cmregion. If the cation radical is dimeric (see discussion below), these features could arise from the CC=O groups of the component monomers. These frequencies are slightly shifted in PSII complexes, as compared with PSII membranes. This may be an effect of the solubilization of the preparation in detergent, since the frequencies of these lines would be expected to be sensitive to changes in solvent. Increased contribution from amino acids could also have the observed effect of shifting observed vibrational lines of chlorophyll.

In addition to these characteristic lines in the 1750-1650 cmspectral region, the infrared difference spectrum associated with the oxidation of the primary bacteriochlorophyll donor, P- P, also exhibits an intense positive mode at 1474 cm(54) . This line may be analogous to the 1478 cmline observed in our spectra. However, the primary chlorophyll donor of PSI does not exhibit a strong line in the 1470 cmregion (see Fig. 3, for example), and this line is not observed either in bacteriochlorophyll (51) or in chlorophyll (52) upon oxidation in vitro. This spectral feature is also not very intense in Rb. capsulatus mutants that contain a bacteriochlorophyll-bacteriopheophytin heterodimer as the primary donor (55) . The significance of the appearance of this spectral feature is not understood, but we speculate that its appearance may be correlated with whether the chlorophyll/bacteriochlorophyll cation radical is a dimer.

It is of interest to compare our data, obtained in the presence of hydroxylamine, to the chlQ


Figure 8: Light-minus-dark FTIR difference spectra of spinach PSII complexes. In A, data were recorded at -9 °C on manganese-depleted preparations in the presence of hydroxylamine and potassium ferricyanide (same as Fig. 5 B). In B, data were recorded at 80 K on manganese-containing preparations. In both cases, cytochrome bis known to be in low potential (15).



Although superficially Fig. 8, A and B, are dissimilar, these spectra do have common vibrational lines. Observed spectral differences between Fig. 8, A and B, could have several possible causes. First, changes could be due to an alteration in the relative concentrations of the chlorophyll cation radical and the semiquinone anion radical. Second, differences could be due to an effect of hydroxylamine on the Q

In the data reported here, Z is not oxidized, because hydroxylamine is present in these samples (23, 43) . Under conditions where Z can be accumulated under illumination, isotopic labeling has shown that the tyrosine radical also makes a contribution to the 1478 cmspectral region (12) . This contribution can be distinguished from that of chlorophyll, since mass spectral analysis has shown that labeling of tyrosine with either C or H does not change the isotopic composition of chlorophyll (12) . When Z contributes to the spectrum, i.e. in the absence of hydroxylamine, a small N-induced change in line shape and intensity is observed in this region (31) . However, there is no significant alteration of the frequency of the line. Comparison of this result with Fig. 6 indicates that the 1478 cmmode, observed in the presence of hydroxylamine, contains fewer components. This is consistent with our conclusion that difference infrared spectrum, obtained in the presence of hydroxylamine, is dominated by the oxidation of chlorophyll.

Summary

We conclude that infrared difference spectra, recorded on hydroxylamine treated samples, reflect structural changes associated with both the oxidation of chlorophyll and the reduction of quinone. Therefore, this procedure cannot be used to isolate the acceptor side contribution to the difference infrared spectrum. Definitive identification of quinone contributions to the vibrational spectrum awaits isotopic labeling of plastoquinone.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM43273 and by a National Institutes of Health Training Grant Award GM08277 (to G. M. M.). Acknowledgement is also made of partial support from the donors of the Petroleum Research Fund, which is administered by the American Chemical Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 612-624-6732. Fax: 612-625-5780.

The abbreviations used are: PSII, photosystem II; PSI, photosystem I; chl, chlorophyll; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; EPR, electron paramagnetic resonance; FTIR, Fourier transform infrared; P, primary chlorophyll donor of PSII; P, primary chlorophyll donor of PSI; MES, 4-morpholineethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank the University of Minnesota Agricultural Experiment Station and T. Krick for assistance in obtaining the mass spectra. We are also grateful to Mary Bernard for assistance in sample preparation.


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