Fom the 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
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ABSTRACT |
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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.
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
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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
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EXPERIMENTAL PROCEDURES |
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The isolation of the C. reinhardtii wild type and
mutants was done as described in Ling et
al.2. The
generation of the Pheo
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 × 105.
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RESULTS |
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Fig. 2 shows the HF-EPR spectra
recorded for the Bpheo5. 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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To study the effect of the electrostatic environment on the
Pheo
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DISCUSSION |
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The HF-EPR spectroscopy of the Pheo
* 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
*) 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
*) 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 Bpheo2 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
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ACKNOWLEDGEMENTS |
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We thank Drs. A. W. Rutherford and T. Mattioli for fruitful discussions and G. Voyard for technical assistance.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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The abbreviations used are:
PSII, photosystem
II;
HF-EPR: high field electron paramagnetic resonance, Pheo
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REFERENCES |
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