High Field EPR Study of the Pheophytin Anion Radical in Wild Type and D1-E130 Mutants of Photosystem II in Chlamydomonas reinhardtii*

Pierre DorletDagger , Ling Xiong§, Richard T. Sayre§, and Sun UnDagger

Fom the Dagger  Département de Biologie Cellulaire et Moléculaire, Section de Bioénergétique, CNRS URA2096, Commissariat à l'Energie Atomique, Saclay, F-91191 Gif-sur-Yvette, France and the § Department of Plant Biology, Ohio State University, Columbus, Ohio 43210-1293

Received for publication, March 20, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The intermediate electron acceptor in photosystem II is a pheophytin molecule. The radical anion of this molecule was studied using high field electron paramagnetic resonance in a series of Chlamydomonas reinhardtii mutants. Glutamic acid 130 of the D1 polypeptide is thought to hydrogen bond the ring V carbonyl group of this radical. Mutations at this site, designed to weaken or remove this hydrogen bond, strongly affected the g tensor of the radical. The upward shift of the gx component followed the decreasing hydrogen bonding capacity of the amino acid introduced. This behavior is similar to that of tyrosyl and semiquinone radicals. It is also consistent with the optical spectra of the pheophytin in similar mutants. Density functional calculations were used to calculate the g tensors and rationalize the observed trend in the variation of the gx value for pheophytin and bacteriopheophytin radical. The theoretical results support the experimental observations and demonstrate the sensitivity of g values to the electrostatic protein environment for these types of radicals.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Photosystem II (PSII)1 is a large protein complex found in higher plants and green algae that catalyzes the oxidation of water. The structure of PSII has been very recently determined to 3.8 Å resolution (1). The primary electron acceptor in PSII is a pheophytin-a molecule. There are two symmetry-related pheophytins in PSII that differ with respect to the presence or absence of a hydrogen bonding interaction between the pheophytin ring V (Fig. 1 for ring designation) carbonyl group and the protein. Significantly, only the pheophytin (active branch) that is hydrogen-bonded to glutamic acid 130 of polypeptide D1 (D1-E130 residue) via the ring V carbonyl group is reduced following charge separation (2). Site-directed mutagenesis coupled with spectroscopic techniques have proven to be extremely useful tools to characterize pigment-protein interactions. The resolution of the x-ray structure is currently insufficient to accurately determine such interactions. In this paper, we report on HF-EPR measurements of the Pheo&cjs1138; radical in spinach as well as wild type and site-specific mutants of Chlamydomonas reinhardtii, which can be used to more precisely characterize such interactions between radicals and neighboring protein residues.


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Fig. 1.   Scheme and ring numbering of the pheophytin-a molecule. The position of a positive charge located at a distance R+ from the oxygen carbonyl group as used in the calculations is also shown.

The pheophytins in photosynthetic reaction centers have been studied using a variety of methods. Comparative measurements of the carbonyl vibrational frequencies of the bacterial and plant reaction centers using resonance Raman (3) and differential Fourier transform infrared spectroscopy (4) suggested that the ring V carbonyl group was likely to be hydrogen bonded in PSII. Such a hydrogen bond was also inferred from electron nuclear double resonance studies by using deuterium exchange experiments and in vitro pheophytin models (5). Modeling studies based on the sequence homology between the PSII D1 and D2 subunits and the bacterial reaction center L and M subunits have been carried out on C. reinhardtii, as well as on spinach, pea and Synechocystis (6, 7). Mutagenesis studies on Synechocystis have also been carried out at the predicted pheophytin hydrogen bonding site (2). These modeling and mutagenesis studies supported the conclusion of the earlier spectroscopic studies that the pheophytin ring V carbonyl group in all PSII complexes is hydrogen-bonded. The likely hydrogen bond donor was identified as the amino acid residue 130 of the D1 subunit, which is homologous to glutamic acid 104 of the L subunit (L-E104) of the bacterial reaction center from Rhodopseudomonas viridis. This residue is a glutamine in the case of cyanobacterial PSII and a glutamic acid in the chloroplastic PSII complex.

g values of organic radicals have been shown to be very sensitive probes of the molecular structure and local environment of the radicals (8-10). These g values are difficult to resolve using conventional 0.3 T/9 GHz EPR. However, by using much higher fields and frequencies, they can be measured more readily. Recently, we have reported the HF-EPR spectrum of the Pheo&cjs1138; radical from wild type spinach PSII as well as of the Bpheo&cjs1138; analog from R. viridis (11). We proposed that the differences between the g tensors of the two radicals reflect differences in intrinsic electronic structure and/or differences in local protein environment. In addition, the results of measurements performed on oriented samples allowed us to fix the three principle directions of the g tensor to the molecular frame. The gx direction was concluded to be along the ring V carbonyl bond, whereas the gz direction is perpendicular to the ring plane. The g tensor orientation is therefore similar to those of the tyrosyl (12) and semiquinone (10) radicals. In both cases, by using HF-EPR and theoretical calculations, it has been shown that the gx value is sensitive to the presence of hydrogen bonds (8, 9) and charged amino acid residues (13). This sensitivity is a direct consequence of the fact that both radicals possess C-O functional groups that carry a significant amount of unpaired spin density. Similarly, computational results (14) have shown that the Pheo&cjs1138; radical also carries appreciable spin density on the ring V carbonyl group. These observations suggest that the gx value should be sensitive to local electrostatic environment around the Pheo&cjs1138; ring V carbonyl group. In this paper, we have used the mutants of C. reinhardtii in conjunction with HF-EPR to demonstrate the sensitivity of the gx value of pheophytin radicals to the electrostatic environment and to directly confirm the presence of a hydrogen bond to the ring V carbonyl group.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The isolation of the C. reinhardtii wild type and mutants was done as described in Ling et al.2. The generation of the Pheo&cjs1138; radical in spinach PSII as well as the Bpheo&cjs1138; radical in R. viridis has been described elsewhere (11). The procedure to generate the Pheo&cjs1138; radical in C. reinhardtii wild type and mutants was similar except that the reduced samples were illuminated at 0 °C for 5 min.

The HF-EPR spectrometer as well as the procedure for simulating the spectra have been described in reference (11). The use of a Mn(II) g standard (g = 2.00101) (10) was required for calibration and to accurately measure g values. In this way, the g values of different samples could be compared with within 2 × 10-5.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fig. 2 shows the HF-EPR spectra recorded for the Bpheo&cjs1138; radical from R. viridis and the Pheo&cjs1138; radical from spinach and C. reinhardtii PSII particles (wild type and D1-E130H, D1-E130Q, and D1-E130L mutants). To accurately obtain the g values of the radical in each case, all six spectra were adequately simulated using a single g tensor and a line-broadening tensor, the principal axes of which were collinear with the g tensor. The latter tensor was needed to account for the observed anisotropic broadening that was presumably due to unresolved hyperfine couplings. Hyperfine interactions were not explicitly considered in the calculations. The simulation parameters are given in Table I. The spinach and wild type Pheo&cjs1138; g values were identical to within 4 × 10-5. The radical in spinach was slightly better resolved. When comparing the g values measured for the C. reinhardtii wild type and mutants, the relationship between hydrogen bonding and g values is clearly seen on the gx edge of the spectra. Like tyrosyl and semiquinone radicals, the gx value decreases with increasing hydrogen bonding. Replacement of the hydrogen bond donor by a leucine (D1-E130L) induces a shift of 20 × 10-5 in the gx value. A change of 3 × 10-5 in the gx values reported in Table I resulted in an increase of the root mean square difference between the experimental spectra and simulations of at least 10%. Hence, the differences in the reported gx values between the D1-E130H and D1-E130Q mutants and wild type were significant and indicated that the electrostatic influence of the leucine, histidine, glutamine, and glutamic acid side chains were not identical.


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Fig. 2.   HF-EPR spectra of the Pheo&cjs1138; in PSII from spinach, wild type (WT) and mutants (D1-E130H, -Q, -L) of C. reinhardtii and Bpheo&cjs1138; in R. viridis. Unbroken line, experiment; broken line, simulation. The vertical lines give the positions of the wild type gx and gz values. Experimental conditions: nominal microwave frequency 285 GHz; modulation amplitude 2.0 mT (PSII) and 1.0 mT (R. viridis); temperature 4.2 K except for D1-E130L taken at 3.0 K. See Table I for simulation parameters.

                              
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Table I
g values of Pheo&cjs1138; and Bpheo&cjs1138; from simulation of experimental data and ADF-ZORA calculations

To study the effect of the electrostatic environment on the Pheo&cjs1138; g tensor, g value calculations on prototypic Pheo&cjs1138; and Bpheo&cjs1138; were carried out using the relativistic zeroth order regular approximation (ZORA) method as implemented in the Amsterdam Density Functional (ADF) program package (16-21). The calculations utilized single zeta wavefunctions for hydrogens and carbons and double zeta wavefunctions for nitrogens and oxygens. For carbons, nitrogens, and oxygens, frozen core wavefunctions were used. The calculations were spin-restricted. The molecule used in the calculations was derived from the bacteriopheophytin structure (taken from the Protein Data Bank, accession number 1PRC). The structure was modified to a pheophytin, and standard protons were added. The structure of the entire Pheo&cjs1138; molecule was not used for the calculations for efficiency reasons. All side groups that were not conjugated with the ring conjugation system were replaced by hydrogens. The resulting molecule was geometry-optimized using the PM3 method as implemented in Gaussian 94 (22). Similar methodology has been used in previous density functional theory studies (14). The geometry-optimized structure was planar. g values using a saddle-shaped structure of the non-optimized structure did not yield significant differences. The geometry-optimized structure was used in all calculations including those involving external point charges. The structure was not reoptimized in the presence of the point charge as neither ADF nor Gaussian 94 packages permitted this. To simulate electrostatic effects, a positive point charge located 120° relative to the ring V carbonyl bond at varying distances (Fig. 1), was incorporated into the calculations. The results of the ZORA calculations are summarized in Table I along with the experimental measurements. The variations of the calculated gx values for the pheophytin and bacteriopheophytin model molecules as a function of the oxygen-point charge distance are plotted in Fig. 3.


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Fig. 3.   Dependence of the theoretical ZORA gx values on the charge-oxygen distance. Circles, calculation for bacteriopheophytin model; squares, calculation for pheophytin model; filled symbols, the limiting value in the absence of a charge; filled triangle, experimental value for R. viridis. The solid lines represent a simple 1/(R+ - 0.5)2 dependence.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The HF-EPR spectroscopy of the Pheo&cjs1138; radical in wild type and mutants of C. reinhardtii confirms the sensitivity of g values to the protein microenvironment for this type of radical as was predicted earlier (11). The shift observed in the gx value between the wild type and D1-E130L mutant is caused by a red shift of the n right-arrow pi * electronic excitation upon the loss of the hydrogen bond on the ring V carbonyl group. This can be compared with a hydrogen bond-induced gx shift of 200 × 10-5 for tyrosyl radicals (9) that carry ~25% of spin density on the phenolic oxygen. This result provides strong experimental evidence for the presence of non-negligible ring V oxygen spin density (14). The removal of the hydrogen bond interaction is supported by a blue shift of 2.5 nm in the Pheo&cjs1138; Qx (pi  right-arrow pi *) band observed in C. reinhardtii as well as in the corresponding mutant in Synechocystis relative to the wild type. The opposite shifts of the two types of electronic excitations are entirely consistent with generally accepted interpretation of hydrogen bonding effects on electronic absorption spectra (23). When the D1-Q130 residue in wild type Synechocystis is mutated to a glutamic acid, the Pheo&cjs1138; Qx (pi  right-arrow pi *) band red shifts to the same wavelength as that of plant (Pisum sativum) indicating a strengthening of the hydrogen bond (2). The HF-EPR results are entirely consistent with these optical results. The C. reinhardtii D1-E130Q mutant exhibits a gx shift of +10 × 10-5 indicating a weakening of the hydrogen bond when the glutamic acid is replaced by a glutamine. These shifts in electronic states point to the possibility that the redox potential of the pheophytin is also modified by the hydrogen bond.

In the context of hydrogen bonding, the similarity in g values between D1-E130H and D1-E130Q mutants and the wild type is not surprising. All three amino acids are capable of hydrogen bonding. In fact, the g values of the D1-E130H and D1-E130Q mutants and wild type differ only slightly. The ordering roughly follows that of increasing electronegativity: glutamic acid, histidine, glutamine, leucine. Because the dipole moment of an OH group is higher than that of an NH group one would expect that, for a given geometry, a histidine and a glutamine are likely to be weaker hydrogen bond donors than a glutamic acid. Without crystallographic data, we cannot rigorously exclude the possibility that the mutations have caused electrostatic changes in the pheophytin environment other than those intended. However, the observed trends in gx values are clearly consistent with the expected effects of hydrogen bonding and are very similar to those observed for tyrosyl and semiquinone radicals. To quantitatively analyze the magnitude of the hydrogen bonding effect, we carried out ab initio quantum mechanics calculation of the g values.

The Bpheo&cjs1138; ZORA g values were in good agreement with experiment. By contrast, the Pheo&cjs1138; ZORA g values only modestly agreed. For both Pheo&cjs1138; and Bpheo&cjs1138;, the calculated gx direction was within 10° of the carbonyl bond with the exact value depending on the charge-oxygen distance, and the gz direction was always perpendicular to the ring plane. This was independent of the dihedral angle of the side groups relative to ring plane. The near collinearity of the gx direction with the carbonyl bond is consistent with theoretical predictions (24-26) and our previous assignment based on HF-EPR measurements performed on oriented samples (11). The best agreement between the Bpheo&cjs1138; experimental data was found when the point charge was 2.0 Å away from the bacteriopheophytin oxygen atom. In the R. viridis crystallographic structure, the L-E104 carboxylic acid oxygen is 2.7 Å away with the proton presumably about 1 Å closer. Similar to tyrosyl and semiquinone radicals (15), the pheophytin gx values followed an r-2 dependence on the charge-oxygen distance (Fig. 3). For both radicals, the ZORA calculations predicted that a positive charge at 1.8 Å will contribute a shift of as much as 40 × 10-5 in the gx value. The difference between wild type and D1-E130L mutant was 20 × 10-5. Some of the differences between experiment and theory are likely due to the fact that a full positive charge has a much stronger effect than a hydrogen bond. Further computational studies are needed to better understand the discrepancies between the calculated and experimental g values. Nonetheless, the observed increase in the gx-values due to the weakening of the hydrogen bond in the mutants is predicted by the ZORA calculations.

The experimental and theoretical results reported in this paper suggest that the differences in g values between Bpheo&cjs1138; in R. viridis and Pheo&cjs1138; in spinach are largely due to structural differences. The specific gx values reflect hydrogen bonding to the ring V carbonyl group. These results taken together with previous work on tyrosyl, semiquinones, chlorophyll, and carotenoid radicals demonstrate the usefulness of HF-EPR in probing the structure of and protein environment around radicals.

    ACKNOWLEDGEMENTS

We thank Drs. A. W. Rutherford and T. Mattioli for fruitful discussions and G. Voyard for technical assistance.

    FOOTNOTES

* This work was supported by Grant RGO349 from the Human Frontiers Science Organization and by grants from the European Union through Human Capital and Mobility Grants FMRX-CT98-0214 and FMRX-CT96-0031 and from the Région Ile-de-France (contract Sesame).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 33-169082842; Fax: 33-169088717; E-mail: sun@ozias.saclay.cea.fr.

Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M102475200

2 X. Ling, M. Seibert, M. Wasielewski, and R. T. Sayre, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: PSII, photosystem II; HF-EPR: high field electron paramagnetic resonance, Pheo&cjs1138;: pheophytin anion; Bpheo&cjs1138;, bacteriopheophytin anion; ZORA, zeroth order regular approximation; ADF, Amsterdam density functional; mT, millitesla.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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