©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Difference Infrared Study of Hydrogen Bonding to the Z Tyrosyl Radical of Photosystem II (*)

(Received for publication, August 9, 1994; and in revised form, November 18, 1994)

Mary T. Bernard (1) Gina M. MacDonald (1) Anh P. Nguyen (2) Richard J. Debus (2) Bridgette A. Barry (1)(§)

From the  (1)Department of Biochemistry, University of Minnesota, St. Paul, Minnesota 55108 and the (2)Department of Biochemistry, University of California, Riverside, California 92521

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Photosystem II, the photosynthetic water oxidizing complex, contains two well characterized redox active tyrosines, D and Z. D forms a stable radical of unknown function. Z is an electron carrier between the primary chlorophyll donor and the manganese catalytic site. The vibrational difference spectra associated with the oxidation of tyrosines Z and D have been obtained through the use of infrared spectroscopy (MacDonald, G. M., Bixby, K. A., and Barry, B. A.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11024-11028). Here, we examine the effect of deuterium exchange on these vibrational difference spectra. While the putative C-O vibration of stable tyrosine radical D downshifts in ^2H(2)O, the putative C-O vibration of tyrosine radical Z does not. This result is consistent with the existence of a hydrogen bond to the phenol oxygen of the D radical; we conclude that a hydrogen bond is not formed to the Z radical. In an effort to identify the amino acid residue that is the proton acceptor for Z, we have performed global N labeling. While significant N shifts are observed in the vibrational difference spectrum, substitution of a glutamine for a histidine that is predicted to lie in the environment of tyrosine Z has little or no effect on the difference infrared spectrum. There is also no significant change in the yield or lineshape of the Z EPR signal under continuous illumination in this mutant. Our results are inconsistent with the possibility that this residue, histidine 190 of the D1 polypeptide, acts as the sole proton acceptor for tyrosine Z.


INTRODUCTION

PSII (^1)is one of two chlorophyll-containing reaction centers that cooperate to transfer electrons from water to NADP in oxygenic photosynthesis. PSII carries out the light-driven oxidation of water and reduction of plastoquinone; this enzyme is made up of at least eight subunits (for review, see (1) and (2) ). The hydrophobic D1 and D2 polypeptides bind most of the prosthetic groups that are involved in electron transfer (3) .

To initiate electron transfer in photosystem II, the primary chlorophyll donor, P, transfers an electron to a pheophytin molecule after photoexcitation. The pheophytin intermediate acceptor reduces a bound molecule of plastoquinone, the acceptor Q(A). Q(A) in turn reduces a second quinone, Q(B). Unlike Q(A), Q(B) can function as a two-electron acceptor. The chlorophyll cation radical, P, is reduced by a redox active tyrosine, Z, which in turn oxidizes the manganese cluster, the catalytic site of water oxidation. Photosystem II also contains a redox active tyrosine, D, which forms a stable radical, D, and has an undetermined function (for review, see (4) and (5) ). Recently, a new signal from a redox active tyrosine, M, has also been observed in three site-directed mutants of PSII(6, 7, 8) . The M tyrosine radical has an unusual structure(8) .

The current model for the location of redox active tyrosines D and Z is based on the assumption that they are located with approximate C(2) symmetry with respect to the primary chlorophyll donor (9, 10) . The crystal structure of the bacterial reaction center shows such C(2) symmetry in the placement of some of its prosthetic groups(11) . Site-directed mutagenesis experiments are consistent with this symmetric model for the location of the redox active tyrosines in PSII. These studies suggest that tyrosine D is located at the 160 position of the D2 subunit (12, 13) and that tyrosine Z is located at position 161 of the D1 subunit(6, 14, 15) .

In spite of this putative C(2) symmetry, D and Z have different functions and physical properties. For example, the midpoint potentials of these 2 tyrosines are predicted to differ by 240 mV. The rise and decay kinetics of the radicals are also dramatically different. In addition, Z is more accessible to reduction by exogenous donors than D (for review, see (16) ).

The EPR signal of D is observable for hours after illumination, and this signal was assigned to a neutral tyrosine radical by isotopic labeling(17) . The EPR signal of Z is observed under illumination in manganese-depleted preparations, and isotopic labeling has also been used to assign this signal to a neutral tyrosine radical (18) . One spin of D is obtained per reaction center; up to one Z spin per reaction center can be observed under illumination in manganese-depleted preparations (for review, see (16) ). The EPR lineshapes are broadened because of immobilization of the radicals (19) and are similar, but not identical, to each other(18) .

The protein environmental factors that influence the function of these redox active tyrosines are of interest. For example, a hydrogen bond to the phenol oxygen could influence the midpoint potential of a redox active tyrosine, since the residue deprotonates upon oxidation(20, 21) . D is known to be a hydrogen-bonded radical from magnetic resonance studies(22, 23, 24) . Due to difficulty in trapping Z, such a study has not been reported for tyrosine Z.

Site-directed mutagenesis has been used to explore the effect of changes in the environments of D and Z. In the structural models of PSII that have been developed, histidine 189 of D2 (Synechocystis numbering scheme) is located near tyrosine 160, which is the putative location of redox active tyrosine D. A second, C(2) symmetry related histidine (histidine 190 of D1) is located near tyrosine 161 of D1, which is the putative Z location (9, 10) . Recent site-directed mutagenesis studies have shown that some substitutions at histidine 189 of D2 lead to a loss of the characteristic 24 G D EPR signal and to the appearance of a narrow structureless signal(25, 26) . However, the EPR signal of tyrosine Z is not affected by substitutions at histidine 190 (27) .

The site-directed mutagenesis experiments described above provide evidence for a structural asymmetry that is imposed on the redox active tyrosines D and Z by their protein environment. Two lines of spectroscopic evidence also point to such a structural difference. First, isotopic labeling and EPR spectroscopy have shown that the EPR lineshapes of D and Z differ when the radicals are 3,5-deuterated(18) . Second, isotopic labeling and difference Fourier transform infrared spectroscopy have shown that the vibrational difference spectra associated with the oxidation of D and Z are also significantly different from each other(21) . Through isotopic labeling of tyrosine, vibrational modes in the difference (light minus dark) spectrum have been assigned to Z and D and to Z and D(21) . This procedure distinguishes contributions from chlorophyll and plastoquinone from tyrosine, since mass spectral analysis shows that these compounds are unlabeled(17, 21) . It was suggested that the observed spectral differences could be caused by a difference in the strength of a hydrogen bond. A difference in hydrogen bonding to D and Z has also been suggested in order to explain the results of the site-directed mutagenesis experiments described above(27) .

To test the hypothesis that Z and D differ in the strength of a hydrogen bond, we report here the results of D(2)O exchange and infrared spectroscopy. Difference infrared spectroscopy offers a direct way to determine if Z and D are hydrogen bonded, since the C-O stretching vibration of a hydrogen-bonded radical should downshift upon deuterium exchange. In addition, difference infrared spectroscopy provides a method with which the proton acceptor can be identified. If a residue is reversibly protonated and deprotonated upon charge separation and recombination, its vibrational signature should also appear in the light minus dark difference spectrum. In order to learn more about the environment of this redox active amino acid, we have measured the effect of D(2)O exchange, N substitution, and a mutation at histidine 190 D1 on the difference infrared spectrum associated with the oxidation of tyrosine Z.


EXPERIMENTAL PROCEDURES

Growth of Cyanobacteria, Isotopic Labeling, and Purification of PSII

Cultures of wild type and mutant Synechocystis sp. PCC 6803 were grown photoheterotrophically as described(28) . Global isotopic labeling with N was accomplished by replacing [^14N]nitrate in the BG-11 growth media with [N]nitrate, as described previously(29) . This treatment has the effect of substitution of all nitrogen atoms in the cell with N. For example, mass spectral analysis of chlorophyll shows that >90% was N-labeled at all four ring positions by this treatment (data not shown). Isotopically labeled sodium nitrate (99%) was from Cambridge Isotope Laboratories (Cambridge, MA). Construction of the HQ190D1 mutant was carried out by the methods previously described(30) , and further characterization will be given in a forthcoming manuscript. (^2)

Photosystem II particles were purified from Synechocystis cells by the procedure previously described (28) with the following modifications: a 400-ml gradient was employed for elution from the first fast flow Q (Pharmacia Biotech Inc.) column, and the sample was not precipitated after the second Mono Q (Pharmacia) column. The lauryl maltoside (dodecyl-beta-D-maltoside) was from Anatrace (Maumee, OH). Since the HQ190D1 mutant cells were light sensitive, all biochemical manipulations with the HQ190D1 mutant were performed in the dark. Manganese was removed from all PSII samples (except the HQ190D1 mutant, see below) as described(20) , except that incubation in NH(2)OH was only 40 min. The HQ19OD1 PSII samples were not manganese-depleted, since without this treatment the yield of Z under steady state illumination was similar to the yield obtained in control manganese-depleted PSII samples. This argues that this mutant cannot assemble a functional manganese cluster, in agreement with an earlier characterization of the HF190 and HY190 mutants in a eukaryotic green algae, Chlamydomonous reinhardtii(27) .

For most experiments, purified PSII particles were concentrated by binding the preparation to a Mono Q 5/5 column(31) . The column was then washed with 15 ml of buffer A (50 mM MES-NaOH, pH 6.5, 20 mM CaCl(2), 0.05% lauryl maltoside, 15 mM NaCl). The column was inverted(31) , and the sample eluted in a concentrated form with high salt buffer (buffer A with 275 mM NaCl). The sample was dialyzed overnight against 5 mM MES-NaOH, pH 6.0(20) , and was frozen at -80 °C before use.

For deuterium exchange, the concentration, dialysis, and freezing of the PSII sample were carried out in the same manner as described in the paragraph above except that D(2)O buffers were used (99.9%, Cambridge Isotopes). The fraction of amide groups exchanged by this procedure is approximately 20% based on the deuterium-induced change in ratio of the amide II and amide I bands in the infrared absorption spectrum(32) . This number is in good agreement with the extent of exchange achieved for bacteriorhodopsin by dehydration of the sample and rehydration with D(2)O(32) . The pD of the D(2)O buffers was the same as the pH values reported above. The pD is reported here as the uncorrected pH meter reading (33) and was adjusted using sodium deuteroxide (99.5%, Cambridge Isotopes). The D(2)O and H(2)O samples were different halves of the same pooled PSII preparation.

For some experiments, samples were concentrated in Centricon 100 concentrators (Amicon, Beverly, MA).

Oxygen Evolution Assays

Assays were performed as described (28) . 1 mM 2,6-dichlorobenzoquinone (recrystallized from methanol) and 1 mM potassium ferricyanide were used as acceptors. The HQ190D1 cells exhibited no detectable oxygen evolution (leq40 µmol O(2)/mg of chlorophyll-h). The range for oxygen evolution for all other cultures was 600-670 µmol O(2)/mg of chlorophyll-h and for all other PSII particles was 2200-3600 µmol O(2)/mg of chlorophyll-h.

Fourier Transform Infrared Spectroscopy at -9 °C

Data were obtained as described previously(20, 21) . PSII samples (30 µg of chlorophyll) were dehydrated 30-40 min/50 µl of sample on a germanium window with nitrogen at 0 °C(20) . The amide I band absorption at 1654 cm was always leq0.9 OD. 1000 scans were accumulated to construct each interferogram, and the spectral resolution was 4 cm. PSII samples of the HQ190D1 mutant were found to be light sensitive. Changes in the vibrational difference spectra over the course of data acquisition were observed in some mutant samples. Such progressive spectral changes are not observed in any other data sets and were also not observed in the HQ190D1 data reported here.

EPR Spectroscopy at -9 °C

Spectra were obtained and analyzed as described(20, 21) . Samples were dehydrated on a mylar substrate as described above.


RESULTS AND DISCUSSION

D(2)O exchange has predictable effects on the infrared absorption spectrum of proteins(34) . As expected for an extensively alpha helical protein, the PSII amide I band (1654 cm), which arises mainly from the C=O stretch of the peptide bond(34) , is not appreciably affected by exchange (data not shown). However, the amide II vibration (1549 cm) decreases in intensity after H-N deuteration due to the uncoupling of the C-N stretching and N-H bending modes(34) . New intensity, due to the C-N vibration, appears at 1456 cm (amide II`).

In Fig. 1A, we present the difference infrared spectrum associated with the oxidation of tyrosine Z in control (dashedline) and deuterium exchanged (solidline) PSII samples. The dark adaptation time between illumination and accumulation of the dark spectrum is 8 min. Such a short dark delay permits the decay of Z, but D will not significantly decay and will not contribute to these spectra. The data presented in Fig. 1A differ slightly from our earlier reported spectra because the 10% PSI contamination previously obtained (21) has been reduced to an undetectable level. As expected, only small frequency shifts are observed in the amide I region after exchange (compare solid and dashedlines). In contrast, a negative line at 1559 cm and a positive line at 1535 cm downshift approximately 90 cm. This is the approximate downshift of the amide II band in the infrared absorption spectrum after deuterium exchange, and this result provides evidence for the assignment of these positive and negative lines in the difference infrared spectrum to amide II. Deuterium exchange experiments on N-labeled samples confirm that there is a negative contribution from amide II` in the 1470 cm region after D(2)O treatment. The new shifted amide II` lines are broader than the corresponding amide II modes. This is possible since H-N deuteration changes the normal mode description(34) .


Figure 1: Difference (light minus dark) infrared spectra of purified PSII samples from the cyanobacterium, Synechocystis sp. PCC 6803. In each panel, the dashedline is the control spectrum. In A, PSII was D(2)O-exchanged (solidline); in B, PSII was globally labeled with N (solidline); in C, PSII was isolated from the HQ190D1 mutant (solidline). There was an 8-min dark adaptation time between illumination and accumulation of the ``dark'' spectrum. Samples were dehydrated and contained 1.5 mM potassium ferricyanide and 1.5 mM potassium ferrocyanide, which do not contribute to the spectrum in the region shown here(20, 21) . Spectra were recorded at -9 ± 1 °C. The data presented are the average of 5-23 difference spectra. The tickmarks on the yaxis correspond to 1 times 10 absorbance units. The data are normalized on the basis of amide I band intensity in the infrared absorption spectrum, that is, on the basis of the total amount of protein in the sample.



The effect of D(2)O exchange on the vibrational modes of Z will now be considered. To summarize some of the relevant previous assignments (Table 1), a component of the positive line at 1477 cm has been assigned to Z, and a component of the negative line at 1657 cm has been assigned to Z(21) . Negative lines at 1522 cm and in the spectral region from 1264 to 1231 cm were also assigned to Z in this earlier work. Furthermore, the component of the 1477 cm line previously assigned to Z was tentatively attributed to the C-O stretching vibration (7a), based on a comparison with vibrational studies of phenoxyl radicals (35) and with the tyrosyl radical in ribonucleotide reductase(36) . Our signal-to-noise was insufficient to identify any ring-stretching modes (for example, 8a) of Z. There is also a higher frequency component of the ``1477 cm'' feature that downshifts upon N labeling (see Fig. 1B) and is unaffected by ^2H or C labeling of tyrosine (refer to Fig. 3A in (21) ). Thus, there are at least two components of the spectral feature at 1477 cm in Fig. 1A. The component at higher frequency does not originate from tyrosine, and the component at slightly lower frequency is a Z vibrational mode.




Figure 3: Hydrogen bonding to the neutral tyrosine radicals, D and Z. Note that the protonation state and hydrogen bonding characteristics of the tyrosine residues, D and Z, are not as yet known.



The 1450 cm region of Fig. 1A is expanded in Fig. 2A. The difference spectra in H(2)O (dottedline) and D(2)O (solidline) have been scaled to give a 1477 cm line with the same amplitude in order to facilitate comparison. This accounts for the apparent base line displacement caused by the downshift of the amide II vibration in D(2)O (see ``Discussion''). It seems unlikely that both components of the 1477 cm feature would shift to the same extent in D(2)O. Therefore, any downshift of a 1477 cm component would cause a lineshape change and the appearance of a new shifted line. Fig. 2A shows that there is no lineshape change in the 1477 cm feature and no new shifted line in this spectral region. There is a small amplitude change at 1477 cm when control (Fig. 1A, dashedline) and deuterium exchanged (Fig. 1B, solidline) samples are compared. However, deuterium exchange experiments on N-labeled samples show a much smaller H/D-induced alteration in the amplitude of the 1477 cm feature when compared with a deuterium-exchanged ^14N sample (data not shown) since N-labeling has the effect of downshifting an amide II or II` line (Fig. 1B). Therefore, this experiment demonstrates that the H/D-induced difference in the amplitude of the 1477 cm feature in Fig. 1A is caused by the negative amide II` contribution. Our data demonstrate that there is no significant downshift of the C-O vibrational mode of Z upon deuterium exchange.


Figure 2: Comparison of the effects of D(2)O exchange on the vibrational difference spectrum associated with the oxidation of Z (A) and with the oxidation of Z and D (B). The tickmarks on both the yaxes correspond to 2 times 10 absorbance units. In both A and B, the dashedline is the control spectrum and was plotted on the scale shown on the left-handaxis, and the solidline is the spectrum obtained after deuterium exchange and was plotted on the scale shown on the right-handaxis. The small expansion of all the spectra obtained after deuterium exchange (solidlines) accounts for the base line displacement caused by downshift of the amide II band into this region (see text). The data shown in A are repeated from Fig. 1A and are the average of 23 (dashedline) and 11 (solidline) difference spectra. In B, the sample was dark adapted at 5 ± 2 °C for 5 h and contained no acceptors. The data in Fig. 2B are the average of 3 (dashedline) and 4 (solidline) difference spectra.



A hydrogen bonded radical would show a downshift of the C-O vibrational mode after deuterium exchange. Therefore, there are two possible explanations of our findings: first, deuterium exchange has not occurred in the vicinity of Z or, second, there is no hydrogen bond to Z.

First, we consider the possibility that deuterium exchange has not occurred in the vicinity of Z. Although an extensive amount of washing and dialysis of samples against D(2)O buffers was performed (see ``Experimental Procedures'') and although samples were frozen and thawed in D(2)O, the percentage of exchanged amide groups is approximately 20%. This low number of exchangeable H-N groups is typical of membrane proteins (for example, see (32) ). However, low amounts of global exchange in membrane proteins can be combined with an extensive amount of exchange in solvent-exposed regions(32) . As a control for exchange on the donor side of PSII in the samples used above, we consider the effect of exchange on the vibrational modes of tyrosine radical D in the same samples. D is known to be a hydrogen-bonded neutral radical (Fig. 3) from magnetic resonance results(17, 22, 23, 24) .

In Fig. 2B, we present the vibrational spectrum associated with the oxidation of both tyrosine Z and D. These data were obtained by allowing a 5-h dark incubation time so that both the D and Z radicals decay and contribute to the spectra. The same control (dashedline) and exchanged (solidline) samples were used to generate Fig. 2, A and B. In each panel, the spectra obtained after deuterium exchange are plotted on the same slightly expanded vertical scale to account for the ``base line displacement'' observed in Fig. 1A. We have previously assigned a line at approximately 1473 cm to D(21) (Table 1). Comparison of Fig. 2, A and B (control spectra, dashedlines) shows that, upon long dark adaptation, there is a change in lineshape in the 1477 cm region. This broadening on the low frequency side of the 1477 cm line is consistent with the appearance of a new vibrational mode at approximately 1473 cm.

In Fig. 2A, to which Z alone contributes, there is no significant spectral change at 1477 cm after deuterium exchange (solidline). However, Fig. 2B shows that, after D(2)O exchange, the 1473 cm line of stable radical D downshifts, probably to 1460 cm. An additional base line displacement occurs due to a larger contribution from the negative amide II` vibration. The data in Fig. 2B are consistent with the idea that the D radical is involved in a hydrogen bond and that the environment around D has exchanged with solvent (Fig. 3). Since Z is known to be more accessible to exogenous reductants than D(37) , this control experiment provides evidence that the lack of D(2)O effect on the vibrational lines of Z is not caused by lack of exchange. We conclude that Z is not hydrogen bonded (Fig. 3). This conclusion is based on the assignment of the 1477 cm line to the C-O stretching vibration. However, we see no other significant H/D shifts in the region between 1530 and 1477 cm; this is a reasonable spectral range for observation of the C-O stretch of Z(21) . Also, note that deuteration effects on the exchange-sensitive modes of the neutral residue, Z, are observed. Substantial intensity and frequency shifts are seen in the 1250 cm spectral region, as expected if tyrosine Z is protonated (Fig. 3) and if deuterium exchange has occurred (38) (data not shown). However, our detailed analysis of these spectral changes awaits a firm set of normal mode assignments.

Since Z is a neutral tyrosine radical(18) , and Z is likely to be protonated in the reduced form (Fig. 3), the tyrosine must deprotonate when it is oxidized. The nature of the proton acceptor is of importance for understanding the effect of the environment on the physical properties of redox active tyrosine, Z. The vibrational spectrum associated with the reversible protonation and deprotonation of this acceptor should appear in our difference infrared spectrum (Fig. 3). Since structural models of PSII predict that a histidine is in the environment of redox active tyrosine Z (9, 10) and since a histidine has been predicted to be the proton acceptor(12, 39) , we performed global N labeling to assess whether significant ^14N/N shifts are observed in the infrared spectrum. A comparison of control ^14N- (dashedline) and N-labeled (solidline) PSII particles is shown in Fig. 1B. A 15 cm shift is observed for the amide II vibration in the infrared absorption spectrum of N-labeled samples (data not shown). Therefore, we conclude that the negative line at 1559 cm and the positive line at 1535 cm, assigned above to amide II vibrations, shift to 1545 and 1519 cm, respectively, upon N labeling.

Aside from the changes in the amide II lines, there are other complex changes upon N labeling. For example, a positive line at 1549 cm downshifts, probably to give the positive shoulder at 1524 cm, and a negative mode at 1540 cm shifts to 1510 cm. We also observe a positive line at 1456 cm that downshifts upon N incorporation and intensity changes at approximately 1660 and 1630 cm. The difference infrared spectrum associated with the protonation of the imidazole group of histidine in vitro has been presented previously(20) . These in vitro spectra show positive lines at approximately 1650, 1630, 1540, 1390, and 1370 cm and negative lines at 1590, 1490, and 1410 cm. Also, the N induced shifts of an imidazole group range from 8 to 16 cm(40) . Therefore, the N shifts that we have observed in Fig. 1B may be consistent with a contribution to the spectrum from a histidine residue and, therefore, with a change in the structure or environment of a histidine upon charge separation. However, since substantial frequency changes can occur when in vitro data are compared with vibrational spectra of proteins, we cannot rule out a contribution from another nitrogen containing amino acid residue. Also, note that the structural change could be an alteration in the environment of the nitrogen containing amino acid and is not necessarily a protonation change. A shift in the high frequency component of the 1477 cm line is observed upon N labeling. Previously, the high frequency component of this line was tentatively attributed to Q(A)(21) on the basis of the work described in (41) . However, the N shift observed here appears to be inconsistent with this Q(A) assignment.

Since the result of our N labeling experiment is consistent with a histidine contribution to the difference spectrum, we generated a site-directed mutation at a histidine residue predicted to be in the environment of Z. Site-directed mutagenesis can distinguish changes on the donor and acceptor side, which isotopic labeling alone cannot do. Histidine 190 of the D1 polypeptide is predicted to lie close to tyrosine Z in structural models of PSII (9, 10) and has been predicted to be the proton acceptor for tyrosine Z(39) . If this hypothesis is correct, this residue should be reversibly protonated and deprotonated upon oxidation of Z by the primary chlorophyll donor. To test this hypothesis, a glutamine was substituted for this histidine, and the effects of this mutation were assessed. This mutant is inactive in oxygen evolution (see ``Experimental Procedures''). Also, it has been reported to have a low quantum yield for ZQ(A) formation (39) .^2

Fig. 4presents EPR spectra recorded on control (Fig. 4A) and HQ190D1 (Fig. 4B) PSII preparations. The EPR spectra were recorded under the conditions used for infrared spectroscopy. In each panel, the spectrum recorded under illumination (solidline) is the composite of the D and Z EPR spectra, while the spectrum recorded after 8 min in the dark (dottedline) is the D spectrum. Thus, the observed light-induced increase (compare solid and dottedlines) is due to the accumulation of Z in the absence of a functional manganese cluster. The data in Fig. 4show that, under steady state illumination in the presence of acceptors, HQ190D1 PSII preparations exhibit Z and D EPR lineshapes that are similar to the lineshapes observed in the control preparation. A similar result has been obtained by Roffey et al.(27) for the HF190 and HY190 mutants in the eukaroytic green algae, C. reinhardtii(27) . Fig. 4also shows that the yields of Z and D under continuous illumination are similar when the control (Fig. 4A) and mutant (Fig. 4B) preparations are compared. This is in contrast to the observations of Roffey et al.(27) who noted a low D yield in their mutants. Also, due to photosensitivity, Roffey et al.(27) could not measure a Z EPR spectrum under continuous illumination in the HF190D1 or HY190D1 mutants of C. reinhardtii. This observation is in contrast to our results on the cyanobacterial HQ190D1 mutant.


Figure 4: EPR spectra of PSII from control (A) and HQ190D1 (B) PSII samples from the cyanobacterium, Synechocystis sp. PCC 6803. The samples contained 90 µg (A) and 60 µg (B) of chlorophyll. In each panel, the solidline was recorded under illumination, the dottedline was recorded after an 8-min dark adaptation, and the dashedline was recorded after a 90-min dark adaptation. The conditions were as follows: gain, 2.0 times 10^4; microwave frequency, 9.1 GHz; modulation amplitude, 3.2 G; microwave power, 0.67 milliwatts; scan time, 4 min; time constant, 2 s. Samples were dehydrated and contained 1.5 mM potassium ferricyanide and 1.5 mM potassium ferrocyanide. Spectra were recorded at -9 ± 1 °C. The tickmarks on the xaxis correspond to 25 G. The g value of D in the control preparation is known to be 2.0046(44) .



The spectral shifts that result from N labeling (Fig. 1B) are quite dramatic, indicating that if protonation of a histidine is contributing to the difference infrared spectrum its impact on the difference spectrum is relatively large. Therefore, we would expect that if histidine 190 is the proton acceptor for Z, removal of this residue and substitution with a glutamine will have a similarly dramatic impact on the vibrational spectrum. In Fig. 1C, we present a comparison of difference infrared data recorded on purified PSII preparations from the HQ190D1 mutant (solidline) with infrared data recorded on a control preparation (dashedline). The HQ190D1 preparation contains a small amount of PSI contamination due to the decreased PSII content in this mutant (note the differential feature at 1750 cm). In spite of this, the control and mutant difference spectra are similar in frequency and relative intensity in every spectral region. Construction of a mutant-PSI double difference spectrum shows that most of the small differences in frequency that do exist arise from PSI (data not shown). If substitution of a glutamine for this histidine has no large influence on the vibrational difference spectrum, then it is unlikely that this mutation causes a significant alteration in the structural changes that occur upon charge separation. This result is inconsistent with the possibility that histidine 190 acts as the sole proton acceptor for tyrosine Z and is reversibly protonated and deprotonated upon charge separation. Therefore, the molecular basis of the photoheterotrophic phenotype in this mutant is still undetermined.

To summarize, our infrared results are consistent with a hydrogen bond to tyrosine radical D that is not formed to the Z radical (Fig. 3). This hydrogen bond downshifts the C-O frequency of D relative to Z and may account for some of the functional differences observed between the two redox active amino acids. This change in hydrogen bonding would be expected to increase spin density at the phenol oxygen in Z and to increase the g(x) component of the g tensor of Z with respect to D (see (42) and references therein). Other structural differences, such as a change in the geometry at the C1-Cbeta bond, may also exist. For example, reconciliation of the differences observed in the EPR spectra of 3,5-deuterated D and Z with the results reported here require such a change in geometry (18) in addition to the spin density redistribution predicted by our infrared studies. Although our data are consistent with the conclusion that Z is not hydrogen bonded, its C-O vibrational frequency is downshifted approximately 20 cm from the C-O vibration of the stable tyrosine radical in the enzyme, ribonucleotide reductase (36) . This spectral difference could be caused by an electrostatic perturbation, since the tyrosyl radical in ribonucleotide reductase is near an iron center(43) . We have also obtained evidence that structural changes occur in nitrogen-containing amino acid residues and/or prosthetic groups upon charge separation in PSII. These structural changes have a large impact on the difference infrared spectrum. Finally, a mutation at histidine 190 of D1 has little influence on the detailed structural changes that occur in PSII upon charge separation.


FOOTNOTES

*
Supported by National Institutes of Health Grants GM 43496 (to R. J. D.) and GM 43273 (to B. A. B.). 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.

(^1)
The abbreviations used are: PSII, photosystem II; P, the primary chlorophyll donor of photosystem II; MES, 4-morpholineethanesulfonic acid.

(^2)
H.-A. Chu, A. P. Nguyen, and R. J. Debus, submitted for publication.


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