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Article
Evidence for an Unprecedented Histidine Hydroxyl Modification on D2-His336 in Photosystem II of Thermosynechoccocus vulcanus and Thermosynechoccocus elongatus. Miwa Sugiura, Kazumi Koyama, Yasufumi Umena, Keisuke Kawakami, Jian-Ren Shen, Nobuo Kamiya, and Alain Boussac Biochemistry, Just Accepted Manuscript • Publication Date (Web): 09 Dec 2013 Downloaded from http://pubs.acs.org on December 10, 2013
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Evidence for an Unprecedented Histidine Hydroxyl Modification on D2-His336 in
2
Photosystem II of Thermosynechoccocus vulcanus and Thermosynechoccocus elongatus.
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Miwa Sugiura*1, 2, Kazumi Koyama1, Yasufumi Umena2, 3, Keisuke Kawakami3, Jian-Ren
5
Shen4, Nobuo Kamiya3, 5 and Alain Boussac6
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1
8
790-8577, Japan
9
2
Proteo-Science Research Center, Ehime University, Bunkyo-cho, Matsuyama, Ehime
PRESTO, Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawauchi, Saitama
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332-0012, Japan
11
3
12
Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585, Japan
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4
14
Technology/Faculty of Science, Okayama University, Tsushima-naka, Kita-ku, Okayama
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700-8530, Japan
16
5
17
558-8585, Japan
18
6
The OUC Advanced Research Institute for Natural Science and Technology (OCARNA),
Photosynthesis
Research
Center,
Graduate
School
of
Natural
Science
and
Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka
iBiTec-S, CNRS UMR 8221, CEA Saclay, 91191 Gif-sur-Yvette, France
19 20
Correspondence Author
21
Miwa Sugiura
22
E-mail:
[email protected] Phone: +81-89-927-9616
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Funding
25
This work has been supported by a grant of the JST-PRESTO program to M.S. (4018).
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Abbreviations
2
MALDI-TOF/MS,
3
spectroscopy; P680, chlorophyll dimer acting as the second electron donor; PSI, Photosystem I;
4
PSII, Photosystem II; QA, primary quinone acceptor; QB, secondary quinone acceptor; TyrD,
5
D2-Tyr160; TyrZ, D1-Tyr161; WT*1, WT*2, WT*3, cells containing only the psbA1, psbA2,
6
psbA3 gene in Thermosynecococcus elongatus, respectively.
matrix-assisted
laser
desorption/ionization-time
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Abstract
2
The electron density map of the 3D crystal of Photosystem II from Thermosynechococcus
3
vulcanus with a 1.9 Å resolution (PDB: 3ARC) exhibits, in the two monomers in the
4
asymmetric unit cell, an until now non-identified uninterpreted strong difference electron
5
density centered at a distance around 1.5 Å from the nitrogen Nδ of the imidazole ring of
6
D2-His336. By MALDI-TOF/MS upon tryptic digestion it is shown that in ≈ 20-30% of the
7
Photosystem II from both Thermosynechococcus vulcanus and Thermosynechococcus
8
elongatus the fragment containing the D2-His336 residue bears an extra mass of + 16 Da.
9
Such an extra mass likely corresponds to an unprecedented post-translational or chemical
10
hydroxyl modification of histidine.
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Elucidation of the process of photosynthesis at the molecular level is of paramount
2
importance to contribute to the answer of the problems arising from the global environmental
3
changes. Today, engineers and scientists face the challenge of fuels production using sunlight,
4
water and carbon dioxide. Yet, nature has been performing this reaction for more than 2
5
billion years using solar energy to remove protons and electrons from water, generate oxygen,
6
trap CO2 and store the energy in the chemical bonds of biomass (sugars, etc.). Thus
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photosynthesis is the ultimate energy input into the biosphere.. The light-driven oxidation of
8
water performed by Photosystem II (PSII) is the first step in the whole photosynthetic
9
process.
10
PSII from cyanobacteria is made up of 17 trans-membrane protein subunits and 3
11
extrinsic proteins [1] (the PsbY unit was missing in [1] but present in e.g. [2] and in
12
Sr-containing PSII [3]). Altogether these bear 35 chlorophylls, 2 pheophytins, 2 hemes, 1
13
non-heme iron, 2 plastoquinones (QA and QB), a Mn4CaO5 cluster, 2 Cl- ions, 11 carotenoids
14
and approximately 20 lipids [1]. The excitation resulting from the absorption of a photon is
15
transferred to the photochemical trap that undergoes charge separation. The positive charge is
16
then stabilized on P680, a weakly coupled chlorophyll dimer. Then, P680+• oxidizes TyrZ, the
17
Tyr161 residue of the D1 polypeptide, which in turn oxidizes the Mn4CaO5 cluster. On the
18
electron acceptor side the electron is transferred to the primary quinone electron acceptor, QA,
19
and then to QB, a two-electron and two-proton acceptor, e.g. [2-4]. The Mn4CaO5 cluster
20
accumulates oxidizing equivalents and acts as the catalytic site for water oxidation. The
21
enzyme cycles sequentially through five redox states, denoted Sn where n stands for the
22
number of stored oxidizing equivalents (n=0-4). Upon formation of the S4 state two molecules
23
of water are rapidly oxidized, resulting in the release of one O2 molecule and regeneration of
24
the S0 state [5, 6], see [7-10] for recent reviews. The main cofactors involved in PS II stable
25
oxygen evolution activity are borne by two proteins, D1 (PsbA) and D2 (PsbD)
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Thermosynechoccocus elongatus, a thermophilic cyanobacterium, became in the last 15
2
years one of the model organisms in the researches on photosynthesis for the following
3
reasons. First, the PSII of this organism turned out to be very robust allowing enzymological
4
studies with less risks of enzyme denaturation. Second, the first 3D crystallographic structures
5
of PSI and PSII were obtained with enzymes purified from this cyanobacterium [2, 11, 12].
6
Third, the genome was sequenced in 2004 [13]. Fourth, the molecular biology in this
7
organism is now well established, e.g. [14-24]. The structural resolution of three-dimensional
8
crystals obtained with PSII from T. elongatus increased up to 3.4 Å [12] and then to 2.9 Å [2]
9
and, more recently, the resolution increased up to 1.9 Å with a PSII enzyme purified from the
10
very similar cyanobacterium T. vulcanus [1].
11
Analyses of the electron density map calculated from the PSII structure from T. vulcanus
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[1] (PDB: 3ARC) revealed relatively strong difference electron density (>3 σ) found in the
13
vicinity of some residues. One example is the D1-Tyr246 residue for which an extra electron
14
density originating possibly from an HOO adduct has been suggested [25]. However, it
15
remains to be clarified if an interpretation of this different electron density is relevant to an
16
analysis of a crystal structure at 1.9 Å resolution. Here we have a question whether the
17
modification is due to functional reason or due to a consequence of radical production during
18
the X-ray exposure. Interestingly, another strong difference electron density concerns the
19
environment of D2-His336 (Figure 1).
20
The center of this difference electron density was detected at a distance around 1.5 Å
21
from the nitrogen Nδ of the imidazole ring of D2-His336 in both monomers (although it is
22
slightly larger in monomer B). Such an extra electron density could originate from either a
23
post-translational modification or a radical adduct resulting from PSII function or induced by
24
the X-ray beam. In the former case, we can also wonder if it is specific to T. vulcanus or if it
25
is also present in other cyanobacteria like T. elongatus. These two questions are addressed in
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the present report. The D2 protein was extracted from PSII purified from different T.
2
elongatus strains and from T. vulcanus. Then, the fragments containing the D2-His336
3
residue obtained by a trypic digestion were analyzed by MALDI-TOF spectrometry.
4 5 6
Experimental Procedures
7
PSII Samples.
8
PSII core complexes from the T. elongatus variants WT*1 [18], WT*2 [19] and
9
WT*3 [17], from the T. elongatus D2-His189Leu mutant [16] and from T. vulcanus were
10
purified as previously described [1, 16-19].
11 12
Mass Spectrometric Measurements.
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The isolated PSII were solubilised with 2% lithium lauryl sulfate and analyzed by
14
SDS-polyacrylamide gel electrophoresis with a 16–22% gradient gel containing 7.5 M urea as
15
described previously [14, 18]. All the buffers used were bubbled with N2 gas to remove O2
16
and therefore avoid artifactual amino-acid oxidations, e.g. (32). The CBB-visualized bands of
17
the protein extracts separated on the SDS gels were subjected to in-gel trypsin digestion as
18
previously reported [26]. Briefly, the gel bands were cut from the SDS gels along their band
19
edges, typically in a size of 1.5 mm wide and 1 mm high (1 mm thick), each gel piece was
20
equilibrated with a solution of 100 mM NH4HCO3 (50 mL) for 10 min, and 50 µL of
21
acetonitrile was further added in the tube. The tube was left for 10 min to remove CBB and
22
the gel piece was washed with water (50 µL) for 10 min and dehydrated with 20 µL of
23
acetonitrile for 15 min. The solution was discarded and the gel piece was dried in a vacuum
24
evaporator centrifuge for 20 min. The gel was rehydrated with a solution of 10 mM
25
dithiothreitol in 20 µL of 100 mM NH4HCO3 and kept at 56°C for 1 h. The tube was then
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cooled down to room temperature and the solution was discarded, 20 µL of 50 mM
2
iodoacetamide in 100 mM NH4HCO3 was added, and the alkylation of cysteine residues was
3
achieved by keeping the tube in the dark for 45 min with mild shaking (room temperature).
4
The gel piece was washed in 50 µL of 100 mM NH4HCO3 for 10 min, dehydrated with
5
acetonitrile for 15 min, dried in vacuum, rehydrated in a 10-µL aliquot of trypsin solution (5
6
µg mL-1 in 50 mM NH4HCO3), and trypsin digestion was done at 37°C for 13 h. The solution
7
was taken out to a new tube, the gel was further subjected to peptide extraction with 10 µL of
8
0.1% trifluoroacetic acid / 50% acetonitrile (v/v) for 20 min with sonication. The pooled
9
solution was subjected to vacuum centrifugation until the volume of the solution was reduced
10
to 2 µL. Then, the solution was diluted with 10 µL of water.
11
Mass spectra of tryptic peptides were acquired with a Voyager-DE PRO
12
MALDI-TOF mass spectrometer (Applied Biosystems) operated in the reflector mode at 20
13
kV accelerating voltage and 100 ns ion extraction delay with the nitrogen laser working at
14
337 nm and 3 Hz in the m/z range of 600 – 4000 as described earlier [20]. The tryptic peptides
15
were mixed with the same volume of a saturated matrix (sinapic acid, Fluka) solution that
16
consisted of 60% acetonitrile and 0.1% trifluoroacetic acid. Data Explorer software (Applied
17
Biosystems) was used for subsequent data processing and the generation of peak lists.
18
The fragments with a m/z value close to 2638 and 2654 and obtained upon tryptic
19
digestion of the D2 band in the SDS polyacrylamide gel electrophoresis isolated by
20
reverse-phase chromatography then further analyzed with LC-MS/MS (analysed by
21
Genomine, Inc.). Details are described in Supporting Information.
22 23
Results and Discussion
24
To assign the species responsible of the extra density seen close to D2-His336 the D2
25
protein band separated by an SDS-polyacrylamide gel was digested by trypsine. Then, the
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resulting fragments were first analyzed by MALDI-TOF mass spectroscopy. Figure 2 shows
2
MALDI-TOF spectra recorded in the m/z region from 2,600 to 2,700 where the peptide
3
fragment of D2 containing the His336 residue is detectable after trypic digestion, i.e. the
4
327-348 fragment (see supplementary material). The spectrum a corresponds to PSII purified
5
from T. elongatus (with PsbA1 as the D1 protein) and the spectrum b corresponds to
6
re-dissolved crystal sample of PSII from T. vulvanus.
7
The two peaks with m/z ≈ 2,638 and m/z ≈ 2,654 were observed in both the T.
8
vulcanus and T. elongatus samples. The former, which had the larger amplitude, was
9
observed at m/z = 2,638. This m/z value corresponds to the expected size for a non-modified
10
327-348 fragment when no amino acid modification had occurred (see also Table 1). The
11
second peak, at m/z ≈ 2,654, can match neither any fragments resulting from the tryptic
12
digestion of the D2 protein nor two adjacent fragments which would have not been separated
13
by the tryptic digestion (see supplementary material). It seems, therefore, very likely that the
14
two different peaks correspond to the same 327-348 fragment with the presence of an extra
15
mass in the second fragment resulting from certain proportion of PSII. The difference in the
16
m/z value between the two peaks is 16.11. This additional mass is too large for a CH3 adduct
17
(in this case the maximum additional mass would be 15.04) and too small for a SH adduct
18
(33.07). Therefore, an oxygen adduct seems to be the most likely possibility. An extra mass of
19
≈ 16 can correspond to a hydroxyl addition to the imidazole ring followed by a proton release.
20
The fragment exhibiting a m/z value of 2652.14, i.e. with an extra m/z value equal to +
21
16, was further purified by reversed chromatography (see supplementary material) and
22
analyzed with LC-MS/MS (Genomine, Inc.). Then, the 2,652 Da fragment was digested by
23
collisions in Ar gas. The new fragments were analysed by MALDI-TOF and the full sequence
24
of the original fragment was deduced as being "xWMAPxDxPHExFVFPEExLPx" with the
25
extra mass indeed born by the fragment containing the His336.
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To clarify whether the adduct described above depends on the source of PSII
2
preparations, the MALDI-TOF/MS analysis upon tryptic digestion was also done on i)
3
re-dissolved PSII crystal sample from T. vulcanus [1] that have not been subjected to X-ray
4
irradiation; and ii) PSII from T. elongatus in the presence of either dimethyl sulfoxide or
5
ethylene glycol, two chemicals potentially used in the post-crystallization process for
6
cryo-crystallography. As shown in the supplementary material, in all these cases the same
7
proportion of D2 was found to bear the same adduct.
8
To further probe the accuracy of the measurements above and a possible consequence
9
of the substitution of different psbA genes on the proportion of the PSII in which the adduct
10
was observed, different PSII variants that are composed of different D1 subunit such as
11
PsbA2 [19] and PsbA3 [17] instead of PsbA1, and a site-directed mutant, D2-His189Leu [16]
12
were analyzed. In all PSII complexes with the different D1 variant, a peak with m/z = 2,653
13
was observed in addition to the peak at m/z = 2,638 (data not shown). As for the peptide
14
fragment (181-233) containing the D2-His190Leu mutation it exhibited a m/z value of
15
5,714.91 instead of 5,738.80 in the wild type. This decrease in the m/z value by - 23.8956
16
corresponds to the expected decrease for a His to Leu mutation, which demonstrates the
17
accuracy of our measurement.
18
From the above results, it seems very likely that the extra mass observed from certain
19
proportion of the 327-348 fragment of D2 corresponds to the extra electron density detected
20
in the vicinity of His336 residue in the 1.9 Å resolution X-ray structural analysis, because no
21
other extra electron density was found around the 327-348 fragment of D2. Since the PSII
22
studied in this mass spectroscopic work have never been subjected to X-ray irradiation, this
23
adduct cannot originate from the binding of radicals generated by the X-ray beam. So, the
24
most likely explanation is that the extra mass of m/z = 16 corresponds either to a
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post-translational modification of D2-H336 or to a hydroxylation that would occur in the two
2
thermophilic cyanobacterial species.
3
Because no EPR signal corresponding to an oxidation of histidine with a OH• are detected
4
in PSII, the occurrence of such an oxidation can be reasonably ruled out. There are some
5
precedences for a peroxide adduct on the Cε atom of the imidazole side chain of histidine
6
such as 2-oxo-histidine found in oxidized PerR protein [27]. An hydroxyl adduct on
7
tryptophane has also been found in the lignin peroxidase [28]. However, this hydroxyl adduct
8
is observed either on the Cβ atom of the tryptophane or on a carbon atom, e.g. [29], in
9
hydroxyl radical-mediated modification of proteins.. Although adduct of oxygen to Cδ seems
10
possible, powerful oxidation reaction must be required for this adduct. So, PSII from T.
11
elongatus and T. vulcanus may have provided the first example for an unprecedented OH
12
adduct on the N atom of the imidazole. As a matter of fact, the addition of the OH group to
13
one of the C atoms of the imidazole side chain of D2-His336 would stabilize the hydrogen
14
bond network around this amino acid by also involving a water molecule.
15
As mentioned in the introduction, the extra electron density is detectable in both PSII
16
monomers of the dimer in the crystal structure. However, from the amplitude of the peaks at
17
2,637.88 and 2,653.99 of the mass-spectra shown in Figure 2, it is clear that the OH adduct
18
was not present in all of the PSII complexes, and we estimate that it is present in 20-30% of
19
the complexes. This contrasts, for example, with the formylation of PsbI in PSII from T.
20
elongatus which was observed in all of the reaction centers [20]. We also performed a short
21
refinement of the structure by restraining the B-factor of the O atom to the average value of
22
the nearby His and water molecule. The result showed that the occupancies of the two O
23
atoms are in the range of 0.5-0.6, which is higher than the 20-30% revealed from the
24
MALDI-TOF analysis. This discrepancy may be caused by several factors. For example, the
25
mass spectra reflects the whole fraction of PSII in solution, whereas the crystal structural
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analysis is performed with crystals, and it is possible that the modified His residue is
2
preferentially incorporated into the crystals. Alternatively, some of the fraction containing the
3
modified His residue may have escaped the mass detection probably due to de-hydroxylation
4
of the modified His residue by laser illumination or some difficulty in de-ionization of the
5
specific fragment, resulting in a lower occupancy. In any cases, the mass spectra provides a
6
good qualitative analysis in agreement with the assignment based on the electron density
7
obtained from the high resolution structural analysis, but it does not have a high accuracy in
8
quantification of each peak. Since the main purpose of this paper is on the identification of the
9
electron density seen near D2-His336, we consider that our results provided enough evidence
10
for the assignment of this extra density.
11
Further works will be required to identify the functional consequence of this
12
unprecedented histidine modification. It should be noted that the D2-His336 although very
13
well conserved is not present in Gloeobacter violaceus. However, the involvement of
14
D2-His336 in the structural stabilization of the hydrogen bond network in D2 can be proposed.
15
Figure 3A and B show distances and possible H-bonds between the modified His336 residue
16
and its environment. As shown in Figure 3B, the modified hydoxyl group of D2-His336 can
17
be hydrogen bonded to the N atom of its own main chain and linked to N atom of the main
18
chain of D2-Asn83 and O atom of main chain of D2-Thr60 through one of the water molecule.
19
Alternatively, two other functional roles could be suggested. In the first one, the D2-His336
20
residue would be the end of the proton pathway starting from TyrD-His189 in tunnel B as
21
shown in Figure 3C. In the second one, the D2-His336 would act as a hydroxyl radical
22
scavenger, and the hydroxylated residue observed in the present study could be a result of this
23
scavenging reaction. Some earlier literature has indeed looked at the hydroxylation of PSII
24
consequently to photo-damage, e.g. [33]. Side path electron transfers that could be the source
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of reactive oxygen species production could also potentially lead to the oxidative stress of the
2
PSII complexes, see for example [30].
3 4 5 6
Acknowledgements
7
Jérôme Lavergne and Pascal Arnoux are acknowledged to prompt us to do this work. We also
8
thank Professor Keiji Okada (Osaka City University) for helpful discussions.
9 10 11
Supporting Information
12
Details of Figure S1 - S2, and Table S1. This material is available free of charge via the
13
Internet at http://pubs.acs.org.
14 15 16
References
17 18
1. Umena, Y., Kawakami, K., Shen, J.-R. and Kamiya, N. (2011) Crystal structure of
19
oxygen-evolving Photosystem II at a resolution of 1.9 Å. Nature 473, 55–60.
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2. Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A. and Saenger, W. (2009)
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Cyanobacterial photosystem II at 2.9-Å resolution and the role of quinones, lipids, channels,
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and chloride. Nat. Struct. Mol. Biol. 16, 334–342.
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4. Diner, B. A. and Rappaport, F. (2002) Structure, dynamics, and energetic of the primary
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8. Dau, H., Zaharieva,I., and Haumann, M. (2012) Recent developments in research on water
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oxidation by photosystem II. Curr. Op. Chem. Biol. 16, 3-10.
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11. Jordan, P., Fromme, P.,Witt, H. T., Klukas, O., Saenger, W., Krauss, N. (2001)
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Three-dimensional structure of cyanobacterial photosystem I at 2.5 angstrom resolution.
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Nature 411, 909-917.
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12. Ferreira, K. N., Iverson, T. M., Maghlaoui, K., Barber, J. and Iwata, S. (2004)
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Architecture of the photosynthetic oxygen-evolving center. Science 303, 1831–1838.
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13. Nakamura, Y., Kaneko, T., Sato, S., Ikeuchi, M., Katoh, H., Sasamoto, S., Watanabe, A.,
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Iriguchi, M., Kawashima, K., Kimura, T., Kishida, Y., Kiyokawa, C., Kohara, M.,
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Matsumoto, M., Matsuno, A., Nakazaki, N., Shimpo, S., Sugimoto, M., Takeuchi, M.,
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Yamada, M. and Tabata, S. (2002) Complete genome structure of the thermophilic
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cyanobacterium Thermosynechococcus elongatus BP-1. DNA Res. 9, 123–130.
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14. Sugiura, M. and Inoue, Y. (1999) Highly purified thermo-stable oxygen evolving
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Photosystem II core complex from the thermophilic cyanobacterium Synechococcus
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elongatus having His-tagged CP43. Plant Cell Physiol. 40, 1219–123.
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15. Sugiura, M., Rappaport, F., Brettel, K., Noguchi, T., Rutherford, A. W. and Boussac, A.
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(2004) Site-directed mutagenesis of Thermosynechococcus elongatus photosystem II: the O2
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evolving enzyme lacking the redox active tyrosine D. Biochemistry 43, 13549-13563.
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16. Un, S., Boussac, A. and Sugiura, M. (2007) Characterization of the tyrosine-Z radical and
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its
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Thermosynechococcus elongatus. Biochemitry 46, 3138-3150.
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spin-coupled
S2TyrZ•
state
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Photosystem
II
from
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1 2
17. Sugiura, M., Boussac, A., Noguchi, T. and Rappaport, F. (2008) Influence of
3
histidine-198 of the D1 subunit on the properties of the primary electron donor, P680, of
4
Photosystem II in Thermosynechococcus elongatus. Biochim. Biophys. Acta 1777, 331–342.
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18. Ogami, S., Boussac, A. and Sugiura, M. (2012) Deactivation processes in
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PsbA1-Photosystem II and PsbA3-Photosystem II under photoinhibitory conditions in the
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cyanobacterium Thermosynechococcus elongatus. Biochim. Biophys. Acta 1817, 1322–1330.
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19. Sugiura, M., Ogami, S., Kusumi, M., Un, S., Rappaport, F. and Boussac, A. (2012)
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Environment of TyrZ in Photosystem II from Thermosynechococcus elongatus in which
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PsbA2 is the D1 protein. J. Biol. Chem. 287, 13336–13347.
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20. Sugiura, M., Iwai, E., Hayashi, H. and Boussac, A. (2010) Differences in the interactions
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between the subunits of Photosystem II dependant on D1 protein variants in the thermophilic
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cyanobacterium Thermosynechococcus elongatus. J. Biol. Chem. 285, 30008–30018.
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21. Kawakami, K., Umena, Y., Iwai, M., Kawabata, Y., Ikeuchi, M., Kamiya, N. and Shen,
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J.-R. (2011) Roles of PsbI and PsbM in photosystem II dimer formation and stability studied by
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deletion mutagenesis and X-ray crystallography. Biochim. Biophys. Acta 1807, 319-325.
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22. Takasaka, K., Iwai, M., Umena, Y., Kawakami, K., Ohmori, Y., Ikeuchi, M., Takahashi,
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Y., Kamiya, N. and Shen, J.-R. (2010) Structural and functional studies on Ycf12 (Psb30) and
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PsbZ-deletion mutants from a thermophilic cyanobacterium. Biochim. Biophys. Acta 1797,
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1 2
23. Nowaczyk, M. M., Krause, K., Mieseler, M., Sczibilanski, A., Ikeuchi, M. and Rogner, M.
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(2010). Deletion of psbJ leads to accumulation of Psb27-Psb28 photosystem II complexes in
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Thermosynechococcus elongatus. Biochim. Biophys. Acta 1817, 1339-1345.
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24. Iwai, M., Katoh, H., Katayama, M. and Ikeuchi, M. (2004) Improved genetic
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transformation of the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1.
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Plant Cell Physiol. 45, 171–175.
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25. Saito, K., Rutherford, A. W. and Ishikita, H. (2013) Mechanism of proton-coupled
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quinone reduction in Photosystem II. Proc. Natl. Acad. Sci. USA 110, 954-959.
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26. Manabe, T. and Jin, Y. (2007) Analysis of protein/polypeptide interactions in human
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plasma using non-denaturing micro-2-DE followed by 3-D SDS-PAGE and MS.
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Electrophoresis 28, 2065–2079.
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27. Traoré, D. A. K., El Ghazouani1, A., Jacquamet, L., Borel, F., Ferrer, J.-L., Lascoux, D.,
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Ravanat, J.-L., Jaquinod, M., Blondin, G., Caux-Thang, C., Duarte, V. and Latour, J.-M.
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(2009) Structural and functional characterization of 2-oxo-histidine in oxidized PerR protein.
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Nat. Chem. Biol. 5, 53-59.
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28. Choinowski, T., Blodig, W., Winterhalter, K. H. and Piontek, K. (1999) The crystal
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structure of lignin peroxidase at 1.70 Å resolution reveals a hydroxy group on the Cβ of
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tryptophan 171: A novel radicals formed during the redox cycle. J. Mol. Biol. 286, 809-827.
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29. Xu, G. and Chance, M. R. (2007) Hydroxyl Radical-Mediated Modification of Proteins as
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Probes for Structural Proteomics. Chem. Rev. 107, 3514-3543.
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30. Frankel, L. K., Sallans, L., Limbach, P. A. and Bricker, T. M. (2012) Identification of
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oxidized amino acid residues in the vicinity of the Mn4CaO5 cluster of Photosystem II:
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Implications for the identification of oxygen channels within the Photosystem. Biochemistry
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51, 6371-6377.
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31. Saito, K., Rutherford, A. W. and Ishikita, H. (2013) Mechanism of tyrosine D oxidation in
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Photosystem II. Proc. Natl. Acad. Sci. USA 110, 7690-7695.
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32. Sun, G., Anderson, V. E. (2004) Prevention of artifactual protein oxidation generated
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during sodium dodecyl sulfate-gel electrophoresis. Electrophoresis 25, 959–965.
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33. Sharma, J., Panico, M., Shipton, C. A., Nilsson, F., Morris, H. R. and Barber, J. (1997)
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Primary structure characterization of the Photosystem II D1 and D2 subunits. J. Biol. Chem.
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272, 33158–33166.
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1
Table 1. D2 Peptide Fragments Identified by MALDI-TOF Mass Spectroscopy upon Tryptic
2
Digestion in Different PsbA PSII Variants.
3 fragment
na
Calculated massb
Experimental massc 2653.99 (+17.7)
327 - 348
22
2636.2482 2637.88 (+1.63)
234 - 251
18
2028.9211
2029.23
305 - 317
13
1546.6828
1547.02
252 - 264
13
1543.7823
1545.45
13 - 23
11
1406.6868
1405.52
295 - 304
10
1256.6037
1258.35
4
a
5
theoretical mass; experimental mass by using the reflector mode.
number of residues in the fragments obtained by tryptic digestion of D2;
6
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b
average
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1
Figure Legends
2 3
Figure 1. Electron density distributions around D2-His336 of PSII from T. vulcanus at 1.9 Å
4
resolution (PDB: 3ARC). A, monomer A and B, monomer B. The distributions depicted in
5
gray are the 2Fo-Fc electron densities maps at 1 σ level, and those in green corresponds to
6
Fo-Fc difference Fourier maps at 3 σ level. These maps were calculated as an omit electron
7
density map corresponding to the unprecedented modification. Balls in orange show water
8
molecules.
9 10
Figure 2. MALDI-TOF mass spectra in reflector mode of isolated PsbA1-PSII complexes
11
from T. elongatus in red (spectrum a) and from re-dissolved crystals of T. vulcanus in black
12
(spectrum b). The m/z region shown corresponds to the 327-348 fragment containing the
13
His336 residue obtained upon tryptic digestion of D2. Adrenocorticotropic hormone fragment
14
(m/z 2,465.1989 in monoisotopic) and bovine insulin (m/z 5,730.6087 in monoisotopic) were
15
used to calibrate the m/z scale.
16 17
Figure 3. (A) Environment of the proposed hydroxylated D2-His336 based on the in the 1.9
18
Å resolution crystal structure. Dot meshes show amino acid residues in Van der Waals radii.
19
D2-His336 is surrounded by hydrophobic amino-acid residues. The modified hydroxyl group
20
on D2-His336 and the linked water molecule locate in the cavity surrounded with the
21
hydrophobic residues; (B) Hydrogen bond network. The crystal structure of native PSII
22
(PDB: 3ARC) was further refined including the hydroxylated D2-His336. Dotted lines and
23
numbers in parentheses show hydrogen bonds and average distance of two monomers,
24
respectively, in the 1.9 Å resolution crystal structure. Temperature factors corresponding to
25
water molecule, which hydrogen-bonded to the hydroxyl group in D2-His336, are both 29 Å2
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1
in monomer A and 34 Å2 in monomer B, respectively; these values are similar to those of
2
amino acid chains surrounding these molecules. The water molecule hydrogen-bonded to Ne
3
of D2-His336 is also stable with temperature factors of 29 Å2 in monomer A and 31 Å2 in
4
monomer B, respectively. (C) Structure of the tunnels connecting to lumen and involving
5
hydrogen bond network and water molecules from TyrD, D2-Tyr160, which were calculated
6
with CAVER2.1. Balls in orange show water molecules. The Tunnel A involves the proton
7
path from TyrD to D2-His61 suggested by Saito et al. [31]. Here we propose an additional
8
tunnel, Tunnel B, where proton might be transferred from D2-His189 to D2-His336 through
9
D2-Arg294, D2-Asn292, D2-Gln164 and water molecules. Since D2-His336 is blocked the
10
proton donation by hydrophobic residues, D2-Phe168 and D2-Pro335, from Tunnel A, the
11
Tunnel B is separated by the D2-His336 from Tunnel A. In the Tunnel B, D2-Arg357 and
12
D2-Asp276 form salt bridges with D2-Glu337 and D2-Arg272, respectively. These
13
electrostatic interactions might work to maintain stable the tunnel structure and the linkage
14
structure of the hydrogen-bond network. In contrast, D2-His336 might be the end of the
15
proton network, because D2-His336 might be the end of the proton network in the absence of
16
its ion pair partner.
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A!
Biochemistry
B!
Figure 1. Sugiura et al. ACS Paragon Plus Environment
Biochemistry
327 - 348! 2637.8797!
2653.9884!
Intensity / a.u.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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a!
b! 2600
2620
2640
2660
Mass (m/z)
Figure 2. Sugiura et al. ACS Paragon Plus Environment
2680
2700
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Biochemistry
A!
B! D2-‐Phe169 D2-‐Asn83
HOH
D2-‐Glu337
(2.9)
D2-‐Asn83 D2-‐Phe168 D2-‐His336
N
N
(3.0) N
D2-‐Ala82
O
(2.9) HOH (3.3) O
(3.0) D2-‐Pro335
N
D2-‐H336
E-‐Leu65
(3.0) HOH
D2-‐Thr60
C!
D2-‐H189
D2-‐R294
D2-‐N292
D2-‐Y160
D2-‐R272
D2-‐R180 D2-‐Q164
D2-‐D333
D2-‐D276 D2-‐H336 D2-‐F168
D2-‐H61
D2-‐E337 D2-‐R357
D2-‐P335 Tunnel A
Tunnel B
Figure 3. Sugiura et al. ACS Paragon Plus Environment
D2-‐Thr60
Biochemistry
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O
D2-‐His336
Table of contents Sugiura et. al.! ACS Paragon Plus Environment
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