Fragmentation of the Large Subunit of Ribulose-1,5-bisphosphate
Carboxylase by Reactive Oxygen Species Occurs near Gly-329*
Hiroyuki
Ishida,
Amane
Makino, and
Tadahiko
Mae
From the Department of Applied Biological Chemistry, Faculty of
Agriculture, Tohoku University, Sendai 981-8555, Japan
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ABSTRACT |
The large subunit (LSU) of
ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in the
illuminated lysates of wheat (Triticum aestivum L.)
chloroplasts is broken down by reactive oxygen radicals into 37- and
16-kDa polypeptides. Analysis of the terminal amino acid residues of
both fragments revealed that the C terminus of the 37-kDa fragment was
Ser-328 and the N terminus of the 16-kDa fragment was Thr-330. Gly-329,
which links the two fragments, was missing, suggesting that the
fragmentation of the LSU in the lysates driven by oxygen-free radicals
occurs at Gly-329. Purified rubisco, exposed to a hydroxyl
radical-generating system, was also cleaved at the same site of the
LSU. The cleavage site was positioned at the N-terminal end of the
flexible loop (loop 6) within the
/
-barrel domain, constituting
the catalytic site of rubisco. The binding of a reaction intermediate
analogue, 2-carboxyarabinitol 1,5-bisphosphate, to the active form of
rubisco completely protected the enzyme from the fragmentation. The
fragmentation was differentially affected by CO2,
Mg2+, ribulose 1,5-bisphosphate, or 2-carboxyarabinitol
1,5-bisphosphate. All these results indicate that the conformation of
the catalytic site of the enzyme is involved as an important factor
determining the breakdown of rubisco by reactive oxygen species.
Reactive oxygen species generated at its catalytic site by a
Fenton-type reaction may trigger the site-specific degradation of the
LSU in the lysates of chloroplasts in the light.
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INTRODUCTION |
Ribulose-1,5-bisphosphate carboxylase/oxygenase
(rubisco,1 EC 4.1.1.39)
catalyzes two competing reactions, photosynthetic CO2
fixation and photorespiratory carbon oxidation, in the stroma of the
chloroplasts. This enzyme is the most abundant protein, which accounts
for 30-35% of total leaf protein in C3 plants, and the
amount of rubisco can be a limiting factor for light-saturated photosynthesis in air (1-4). During senescence rubisco is one of the
early proteins that are broken down (5), therefore affecting photosynthesis and nitrogen economy in plants. Only limited information is available on the triggering mechanisms that cause rubisco
degradation in plants (6-8).
It is widely accepted that the initial step of the rubisco degradation
in leaves must occur within the chloroplast (6, 7). In illuminated
chloroplasts, in which light energy is harvested and converted to
chemical energy, production of reactive oxygen species occurs as an
unavoidable event, particularly under light or oxidative stress
conditions (9, 10) that cause degradation of rubisco (5, 11-15).
Reactive oxygen species are known to inactivate and modify enzymes in
diverse biological systems (16). Reactive oxygen species could be one
means by which rubisco degradation is triggered. For example, Mehta
et al. (5) showed that Cu2+-induced oxidative
stress caused intermolecular cross-linking of the large subunits of
rubisco via disulfide bounds within the holoenzyme, rapid and specific
translocation of the soluble enzyme complex to the chloroplast
membranes, and finally rubisco degradation. In addition, Desimone
et al. (15, 17) reported that light stress induces reactive
oxygen-mediated denaturation of rubisco followed by proteolytic
degradation of the large subunit (LSU) in chloroplasts (15) or its
lysates (17). These reports indicated that oxidative stress induces a
modification of rubisco protein, which then leads to its subsequent
degradation, perhaps by a protease. On the other hand, we recently
found that the LSU of rubisco can be directly fragmented into 37- and
16-kDa polypeptides by reactive oxygen species in chloroplast lysates
(18) and in intact chloroplasts (19) under illumination. This
fragmentation of rubisco was completely inhibited in the presence of
metal chelators (EDTA or 1,10-phenanthroline), catalase, or hydroxyl
radical scavenger (n-propyl gallate), but not in the
presence of protease inhibitors. A similar fragmentation was also
observed for the purified rubisco exposed to a hydroxyl radical-generating system.
We have now identified the cleavage site where LSU of rubisco in the
chloroplast lysates in the light and LSU of purified rubisco exposed to
hydroxyl radicals are fragmented. In addition, we found that effectors
potentially bound to its active site, such as CO2,
Mg2+, ribulose 1,5-bisphosphate (RuBP), and
2-carboxyarabinitol 1,5-bisphosphate (CABP), affect to different
extents the susceptibility of the enzyme to fragmentation mediated by
hydroxyl radicals. Of particular significance is the finding that CABP,
a reaction intermediate analogue of the carboxylation reaction of the
enzyme, which is known to tightly bind to the catalytic site of the
activated form of rubisco (20, 21), fully protected the enzyme from the
fragmentation. We suggest that one mechanism of the site-specific
fragmentation of rubisco in the illuminated lysates of chloroplasts may
involve the generation of reactive oxygen species, probably hydroxyl
radicals, at its catalytic site by a Fenton-type reaction.
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EXPERIMENTAL PROCEDURES |
Plant Material and Isolation of Chloroplasts--
Wheat
(Triticum aestivum L. cv. Aoba) seeds were planted on a
plastic net floating on tap water in a pot and grown in a phytotron with a day/night temperature of 20/18 °C and 70% relative humidity. The photoperiod was 12 h, with a quantum flux density of 300 µmol of quanta·m
2·s
1 at plant height.
Chloroplasts were isolated from the primary leaves of 12-day-old
seedlings by a mechanical method using continuous Percoll gradient
centrifugation as described previously (18).
Purification of Rubisco and Binding of CABP to Activated
Enzyme--
Rubisco was purified from wheat leaves as described
previously (18). The enzyme·CO2·Mg2+·CABP
complex was produced as follows. The purified enzyme (5 mg/ml) was
pretreated at 37 °C for 1 h and then activated with 10 mM NaHCO3 and 20 mM
MgCl2 at 25 °C for 20 min in 50 mM
Hepes-NaOH, pH 8.0, containing 1 mM dithiothreitol (DTT). A
2-fold excess (with respect to active sites) of CABP (a gift from Dr.
Tadaaki Yamashita) was added and allowed to stand for 20 min. After the reaction, the enzyme·CO2·Mg2+·CABP
complex was separated from unbound CABP by gel filtration through a
column of Sephadex G-25 (Amersham Pharmacia Biotech).
Gel Electrophoresis and Immunoblotting--
SDS-PAGE was
performed by the methods of Laemmli (22), except that SDS sample buffer
was changed to 100 mM Tris-HCl, pH 8.5, containing 1%
(w/v) SDS, 10% (v/v) glycerol, and 2.5% (v/v) 2-mercaptoethanol at
final concentrations. Native PAGE was performed (22) with a 5% (v/v)
polyacrylamide gel in which SDS was omitted. Two-dimensional
electrophoresis was carried out by the method of O'Farrell (23),
except that 2% (v/v) Ampholine (pH range, 3.5-10; Amersham Pharmacia
Biotech) was used in isoelectric focusing. Immunoblot analysis was done
as described previously (18).
Isolation of LSU Fragments from Chloroplast Lysates Exposed to
Light--
The chloroplast lysates were incubated at 4 °C for
1 h in the light at 2000 µmol of
quanta·m
2·s
1 in 50 mM
2-morpholinoethanesulfonic acid-NaOH, pH 5.7, containing 1 mM DTT and 10 µM E-64. After incubation, the
lysates were centrifuged at 39,800 × g for 30 min, and
the supernatant fraction was subjected to 33-55% (w/v)
(NH4)2SO4 saturation. The
precipitate was dissolved in 20 mM Tris-HCl, pH 7.6, containing 12.5% (v/v) glycerol, 1 mM DTT, and 1 mM EDTA, and passed through a column of Econo-Pac 10DG
(Bio-Rad) previously equilibrated with the same buffer. The eluted
protein fractions were applied to an ion exchange column of RESOURCE Q
(Amersham Pharmacia Biotech) previously equilibrated with the same
buffer using the FPLC system (Amersham Pharmacia Biotech). The proteins
were eluted with 40 ml of the same buffer containing a linear gradient
of 0-0.5 M NaCl at a flow rate of 1 ml/min. The fractions
containing rubisco and its fragments were loaded onto a gel filtration
column of Superdex 200 (Amersham Pharmacia Biotech) previously
equilibrated with 50 mM Na-phosphate, pH 7.5, containing
12.5% (v/v) glycerol, 1 mM DTT, and 1 mM EDTA, and eluted with the same buffer at a flow rate of 0.5 ml/min using the
FPLC system. The 37- and 16-kDa fragments co-eluted with intact rubisco
throughout the purification steps. For the isolation of 16-kDa
fragment, the final fractions containing rubisco and its fragments were
subjected to two-dimensional electrophoresis, and the separated
fragment was electroblotted onto a polyvinylidene difluoride membrane.
For the isolation of the relatively large quantity of 37-kDa fragment,
the eluted proteins from the gel filtration column were reduced in 0.1 M Tris-HCl, pH 8.5, containing 8 M urea and 40 mM DTT at 37 °C for 2 h under N2. Then,
the proteins were incubated with 0.6% (w/v) 4-vinylpyridine for 30 min. The pyridylethylated proteins were applied to a preparative
SDS-PAGE system (Mini Prep Cell; Bio-Rad). The acrylamide concentration in the separation gel was 11% (w/v), and the procedure was followed according to the manufacturer's instructions. The eluted fraction of
the 37-kDa fragment was precipitated and washed with acetone to remove
SDS. The precipitate was dissolved in 0.1 M Tris-HCl, pH
8.5, containing 8 M urea and loaded onto a reversed phase
column (ProRPC; Amersham Pharmacia Biotech) using HPLC (LC-10Ai series; Shimazu, Kyoto, Japan). The 37-kDa fragment was eluted with a 120-min
linear gradient of 5-80% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid at a flow rate of 0.3 ml/min.
Isolation of LSU Fragments from the Purified Rubisco Exposed to
Hydroxyl Radical-generating System--
Purified rubisco (1 mg/ml) was
exposed to the hydroxyl radical-generating system comprising 1 mM H2O2, 10 µM
FeSO4, and 20 mM ascorbic acid at 4 °C for
15 min in 50 mM Hepes-NaOH, pH 8.0, containing 5% (v/v)
glycerol and 1 mM DTT. The mixture was passed through a
column of Econo-Pac 10DG previously equilibrated with 0.1 M
Tris-HCl, pH 8.5, containing 1 mM EDTA and 1 mM
DTT. The 16-kDa fragment was isolated from this fraction by
two-dimensional electrophoresis as described above. The 37-kDa fragment
was also isolated by the same procedure using the preparative SDS-PAGE system and reversed phase HPLC as described above.
Proteolytic Digestion of the 37-kDa Fragment and Peptide
Characterization--
The 37-kDa fragment (~20 µg) was digested
with 0.4 µg of lysylendopeptidase (Wako Pure Chemical Industries,
Osaka, Japan) in 10 mM Tris-HCl, pH 9.0, containing 2 M urea at 37 °C for 15 h. The peptides were
separated by HPLC using a reversed phase column of µRPC (Amersham
Pharmacia Biotech). After the column was washed with 10% (v/v)
acetonitrile in 0.06% (v/v) trifluoroacetic acid, the peptides were
eluted with a 100-min linear gradient of 10-25% (v/v) acetonitrile in
0.06% (v/v) trifluoroacetic acid at a flow rate of 0.2 ml/min.
N-terminal amino acid sequencing was performed using a protein
sequencer (model 491; Applied Biosystems) equipped with an on-line
analyzer of the phenylthiohydantoin derivative of the amino acids.
Electrospray ionization-mass spectrometry analysis was performed using
a triple-quadruple mass spectrometer (API 365; Perkin-Elmer Sciex).
 |
RESULTS |
Identification of the Cleavage Site--
The chloroplast lysates
were incubated in the light, and proteins were electrophoretically
separated and then immunoblotted. The N-terminal 37-kDa and C-terminal
16-kDa fragments of the rubisco-LSU were the most dominant degradation
products. When a purified preparation of rubisco was exposed to a
hydroxyl radical-generating system, the cleavage of the LSU into 37- and 16-kDa fragments was also observed (18). To identify the cleavage
site, we isolated the 37- and 16-kDa fragments of the LSU from the
chloroplast lysates incubated in the light and those generated from the
purified rubisco after exposure to the hydroxyl radical-generating
system. The 37- and 16-kDa fragments always co-purified with the intact
rubisco throughout the purification steps,
(NH4)2SO4 precipitation,
ion-exchange chromatography (RESOURCE Q), and gel filtration (Superdex
200) (Fig. 1A). The purified
fraction from gel filtration showed only a single band on native PAGE
(Fig. 1B). These results indicate that both fragments
co-exist as parts of the holoenzyme form comprising hexaoctamer.

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Fig. 1.
Isolation of the rubisco containing the
fragmented LSU from the lysates of chloroplasts incubated in
light. The chloroplast lysates were incubated at 4 °C for 60 min in the light at 2000 µmol of
quanta·m 2·s 1 (lane 1). Then
these were subjected to centrifugation (lane 2), 33-55%
ammonium sulfate fractionation (lane 3), FPLC-RESOURCE Q
(lane 4), and FPLC-Superdex 200 (lane 5)
chromatography. Samples from each step were applied to SDS-PAGE
(A), and the fraction after FPLC-Superdex 200 was applied to
a native PAGE (B). The gels were stained with Coomassie
Blue. The arrowhead indicates the fragment of the LSU with
its molecular mass.
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Fig. 2 shows the polypeptide profiles on
two-dimensional electrophoresis of the purified fraction containing the
fragments of the LSU isolated from the chloroplast lysates (Fig.
2A) and those from the purified rubisco exposed to the
hydroxyl radical-generating system (Fig. 2B). The mobilities
on the gels and isoelectric points of the 37- and 16-kDa fragments were
the same in Fig. 2, A and B. Both the 16-kDa
fragments were electroblotted onto a polyvinylidene difluoride membrane
and applied to a protein sequencer. The N-terminal sequences of the
16-kDa fragment generated in the chloroplast lysates and from the
purified rubisco were TVVGKLEGEREMTLG and XVVXKLEGEXXMTLG, respectively. These
sequences correspond to the deduced residues 330-344 in wheat LSU
(24). When the 37-kDa fragment electroblotted onto the polyvinylidene
difluoride membrane was applied to a protein sequencer, no
phenylthiohydantoin derivatives of amino acids were released after
three cycles of Edman degradative sequencing. This was expected because
the N-terminal proline residue of wheat LSU is acetylated (25). We
therefore digested intact LSU and the 37-kDa fragments from the
chloroplast lysates in the light and the purified rubisco exposed to a
hydroxyl radical-generating system, with lysylendopeptidase, and the
released peptides were analyzed by reversed phase HPLC, respectively
(Fig. 3, A-C). The elution profiles of the peptides were quite similar in Fig. 3 A-C, except that peptide 1 found in Fig.
3A was missing in Fig. 3, B and C,
whereas peptide 2 or peptide 3 was found in Fig. 3, B or
C, respectively. Peptides 2 and 3 eluted at the same
retention time. The sequence of peptide 1 from intact LSU was
ALRMSGGDHIHSGTVVGK. This corresponds to deduced residues 317-334 in
wheat LSU. The sequences of peptide 2, from the 37-kDa fragment of the
chloroplast lysates, and peptide 3, from the 37-kDa fragment of the
purified rubisco exposed to hydroxyl radicals, were the same,
ALRMSGGDHIHS, which correspond to residues 317-328 in wheat LSU.
Because lysylendopeptidase cleaves the peptide bonds at the carboxylic
side of lysine, and the C-terminal amino acid of peptides 2 and 3 is
not lysine, both peptides are derived from the C-terminal region of the
37-kDa fragments. From electrospray ionization-mass spectrometry
analysis, masses of 1278.9 and 1295.0 were obtained for peptide 2. Because the theoretical mass of ALRMSGGDHIHS is 1279.6, these results indicated that peptide 2 was terminated at Ser-328 and did not contain
the Gly-329 residue. From these results, we concluded that the LSU is
cleaved at Gly-329 or at both ends of this residue into the 37- and
16-kDa fragments.

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Fig. 2.
Comparison of two-dimensional polypeptide
profiles of the partially fragmented rubisco isolated from the lysates
of chloroplasts incubated in the light (A) with those of
purified rubisco exposed to the hydroxyl radical-generating system
(B). The rubisco fraction containing the fragmented
LSU obtained from the chloroplast lysates after a series of
purification steps (Fig. 1) and the purified rubisco once exposed to
the hydroxyl radical-generating system were subjected to
two-dimensional gel electrophoresis, and the gels were stained with
Coomassie Blue. Arrowheads indicate the positions of the LSU
and the small subunit of rubisco and the fragments of the LSU with
their molecular masses.
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Fig. 3.
Elution profiles of the peptides after
lysylendopeptidase digestion of the intact LSU, the 37-kDa fragment of
the LSU from the chloroplast lysates, and the 37-kDa fragment from the
purified rubisco exposed to the hydroxyl radical-generating system by
reversed phase HPLC. A, intact LSU of rubisco; B,
the 37-kDa fragment of the LSU purified from the lysates of
chloroplasts that were incubated at 4 °C for 60 min in the light at
2000 µmol of quanta·m 2·s 1;
C, the 37-kDa fragment produced by the exposure of purified
rubisco to the hydroxyl radical-generating system comprising 1 mM H2O2, 10 µM
FeSO4, and 20 mM ascorbic acid. Each sample was
digested with lysylendopeptidase, and the products were separated on a
C18 reversed phase column (µRPC). After the column was
washed with 10% (v/v) acetonitrile in 0.06% (v/v) trifluoroacetic
acid, the peptides were eluted with a 20-ml linear gradient of 10-25%
(v/v) acetonitrile in 0.06% (v/v) trifluoroacetic acid at a flow rate
of 0.2 ml/min.
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CABP Binding Protects LSU from Fragmentation--
The cleavage
site of the LSU, Gly-329, is one of the residues that constitutes the
catalytic site of the enzyme (26). It is known that CABP, a reaction
intermediate analogue of the enzyme carboxylation reaction, tightly
binds to the active form (ternary complex of
enzyme·CO2·Mg2+) of rubisco and masks the
metal and the substrate binding sites (20, 21, 26). Thus, it was of
interest to examine the effect of the binding of CABP on the
fragmentation. The enzyme·CO2·Mg2+·CABP
complex (active form·CABP complex) added to the thylakoid fraction
was not fragmented even in the light, although the LSU of the purified
rubisco (inactive form) added to the thylakoid fraction was fragmented
in the light (Fig. 4). These results
clearly indicated that the binding of CABP to the active form of
rubisco protected the enzyme from the fragmentation.

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Fig. 4.
CABP protects the LSU of rubisco from
fragmentation. Purified rubisco (lanes 1-6)
and the rubisco·CO2·Mg2+·CABP complex
(lanes 7-12) were incubated at 4 °C with
(lanes 2-5 and 8-11) or
without (lanes 6 and 12) the thylakoid fraction
isolated from chloroplasts in the light at 2000 µmol of
quanta·m 2·s 1 (lanes
2-4, 6, 8-10, and 12) or in
darkness (lanes 5 and 11) for 5 min (lanes
2 and 8), 10 min (lanes 3 and 9),
or 20 min (lanes 4-6, and 10-12). Lanes
1 and 7 are untreated control samples. After
incubation, the samples were subjected to SDS-PAGE. Immunoblot analysis
was performed with antibodies against the LSU of rubisco.
Arrowheads indicate the fragments of the LSU with their
molecular masses.
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Effects of CO2, Mg2+, RuBP, and CABP on the
fragmentation of purified rubisco exposed to the hydroxyl
radical-generating system--
Purified rubisco was preincubated with
various combinations of CO2, Mg2+, RuBP, and
CABP. Then the mixtures were exposed to the hydroxyl radical-generating
system. The results are shown in Fig. 5.
The fragmentation of the rubisco LSU was most pronounced when the enzyme was incubated alone (Fig. 5, lane 2).
Preincubation of the enzyme with CO2 (Fig. 5, lane
5) or Mg2+ (Fig. 5, lane 9) did not cause
significant influence on the fragmentation. When the enzyme was,
however, incubated with CO2 and Mg2+ together,
and once the ternary complex
(enzyme·CO2·Mg2+, active form) was formed,
the fragmentation was significantly suppressed (Fig. 5, lane
6). The enzyme complex with RuBP (enzyme·RuBP) or CABP
(enzyme·CABP) was degraded less than the enzyme alone but more than
the enzyme·CO2·Mg2+ complex (Fig. 5,
lane 4 or 3). The complex of the active form with
CABP (enzyme·CO2·Mg2+·CABP) was not
fragmented at all (Fig. 5, lane 7), as in the case of the
complex of activated rubisco with the illuminated thylakoid (Fig. 4).
The active form complexed with RuBP
(enzyme·CO2·Mg2+·RuBP) was resistant to
fragmentation, as was the complex of the active form incubated with
CABP (Fig. 5, lane 8).

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Fig. 5.
Effects of CO2, Mg2+,
CABP, and RuBP on rubisco fragmentation by the hydroxyl
radical-generating system. Purified rubisco was first incubated
with 90 µM CABP (lane 3), 0.5 mM
RuBP (lane 4), 10 mM NaHCO3
(lane 5), 10 mM NaHCO3 and 20 mM MgCl2 (lanes 6-8), and
20 mM MgCl2 (lane 9) or without
effectors (lanes 1 and 2) at 25 °C for 10 min
in 50 mM Hepes-NaOH, pH 8.0, containing 5% glycerol and 1 mM DTT. The mixture for lane 7 was further
incubated with 90 µM CABP for 10 min. After the
incubation, the mixtures were held at 4 °C for 5 min and then
exposed to the hydroxyl radical-generating system comprising 1 mM H2O2, 10 µM
FeSO4, and 1 mM ascorbic acid at 4 °C,
except for the mixture for lane 1. RuBP at the final
concentration of 0.5 mM was added to the mixture for
lane 8 just before the exposure. After an exposure for 15 min, the mixtures were subjected to SDS-PAGE. Immunoblot analysis was
performed with antibodies against the LSU of rubisco.
Arrowheads indicate the fragments of the LSU with their
molecular masses.
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DISCUSSION |
The Cleavage Site of the LSU and Involvement of Hydroxyl
Radicals--
In the present study we determined the specific cleavage
site of the LSU fragmented in the lysates of chloroplasts incubated in
the light. The C-terminal amino acid residue of the 37-kDa fragment was
Ser-328, and the N-terminal amino acid residue of the 16-kDa fragment
was Thr-330, whereas the residue Gly-329 between them was missing. The
same terminal amino acids were determined for the LSU fragments derived
from the purified rubisco exposed to the hydroxyl radical-generating
system. These results confirm our previous finding that rubisco is
directly degraded into the N-terminal side fragment of 37 kDa and the
C-terminal side fragment of 16 kDa by reactive oxygen species, probably
hydroxyl radicals, in the lysates of chloroplasts (18). Jakob and Huber
(10) reported that hydroxyl radicals are produced in illuminated
thylakoids as a consequence of O2 reduction. They also
indicated that the light-dependent hydroxyl radical
formation might be brought about by a Fenton-type reaction.
Unfortunately, the mechanism of protein fragmentation by the
metal-catalyzed oxidation system is poorly understood. One of the
proposed mechanisms is the cleavage of the peptide bond by either the
diamide pathway or
-amidation pathway (16). After cleavage by either
pathway, the derived peptide fragments should have blocked N termini,
which are resistant to Edman degradation. However, in the present
study, the N-terminal amino acid sequence of the 16-kDa fragment of the
LSU could be identified by the standard sequencing procedure of the
Edman degradation. There are some reports that the cleavage of
polypeptide by reactive oxygen species produces fragments with free N
termini (27, 28), as found in the present study. However, it could not
be explained why the Gly-329 was missing. It is not likely that the
residue Gly-329 was removed by proteases in the lysates from the
terminal end of the 37- or 16-kDa fragment after the cleavage of the
LSU by reactive oxygen species, because Gly-329 was also missing in the
fragments derived from the purified rubisco exposed to the hydroxyl
radicals. Another, as yet unknown, mechanism of protein fragmentation
by reactive oxygen species needs to be considered.
Mechanisms of Site-specific Fragmentation of the LSU--
Rubisco
from higher plants is a hexadecamer of eight LSUs and eight small
subunits. The fragmentation of rubisco was specific to the LSU; no
fragmented product was found for the small subunits (data not shown).
In addition, the cleavage of the LSU was observed at a specific site.
As reviewed by Stadtman and Oliver (29), the metal-catalyzed oxidation
of proteins is a site-specific process involving the interaction of
oxygen or hydrogen peroxide and a redox-active metal at metal-binding
sites on the protein. Rubisco requires Mg2+ or
Mn2+ for the formation of a ternary complex of the
enzyme·CO2·Mg2+(Mn2+), which is
an active form of the enzyme. All the active sites of rubisco are on
the LSU. Fe2+ is potentially able to bind to the metal
binding site of the LSU, because Fe2+ can activate rubisco
instead of Mg2+ (30). Chloroplasts are especially rich in
iron and contain ~80% of the total iron of mesophyll cells (31).
Rubisco possesses oxygenase activity, and the binding site of
O2 is the same as that of CO2. Moreover,
H2O2 competitively interacts with the binding site for O2 and CO2 (32). Therefore, it can be
hypothesized that, under our experimental conditions, namely certain
light- or oxygen-stressed conditions, the production of hydroxyl
radicals via a Fenton-type reaction occurs at the catalytic site of
rubisco, which brings about the fragmentation of the LSU. The
fragmentation of the LSU was completely protected by CABP binding to
the catalytic sites of the enzyme (Figs. 4 and 5). The
enzyme·CO2·Mg2+·CABP complex is so stable
that neither activator CO2 nor Mg2+ can be
readily replaced by unbound ligand (20, 21). Thus, Fe2+ or
H2O2 (O2) is unable to bind to
their site at the active site, and the production of hydroxyl radical
is prevented. As a consequence of this, under these conditions, LSU
fragmentation does not occur even in the light. These results
further support the above hypothesis.
No cleavage of the LSU was observed in the presence of 0.1% SDS even
when the enzyme was exposed to the hydroxyl radical-generating system
(data not shown). In the presence of SDS, rubisco disrupts into
subunits and loses the enzyme activity (33). These results strongly
indicate that a correctly folded structure of the enzyme is required
for the fragmentation of rubisco by reactive oxygen species. This is
largely different from rubisco degradation mediated by a protease (34,
35), because proteolytic degradation is often stimulated by SDS.
The LSU consists of two separate domains, a smaller N-terminal domain
and a larger C-terminal domain. The N-terminal domain is built from a
five-stranded
-sheet and two
-helices on one side of the sheet,
and the C-terminal domain is from an eight-stranded parallel
/
-barrel, which has a flexible loop (loop 6), as commonly observed among
/
-barrel proteins. The catalytic site of rubisco is located at the interface between the C-terminal domain of one LSU
and the N-terminal domain of the other LSU. The cleavage site of the
LSU was between Ser-328 and Thr-330, possibly at Gly-329. This is
located at the N-terminal end of loop 6 within the
/
-barrel domain and composes one of the phosphate binding sites of RuBP (26).
The residue Gly-329 is highly conserved in plant species (36). It is
known that hydroxyl radicals are produced close to the site where the
metal is bound, and that they can only attack atoms or chemical bonds
located within a limited distance from their site of production.
However, Gly-329 is likely not to be the closest amino acid residue to
the O2 binding site (37). For example, Asp-203 and Glu-204,
constituting the metal binding site, or Gly-403 and Gly-404 are much
closer to the O2 binding site than Gly-329, respectively.
Therefore, preferential attack of Gly-329 or both of its sides by
reactive oxygen species could not be simply attributed to one specific
reason. Other factors, such as a primary structure (kinds and
properties) of amino acid residues juxtaposed to the Gly-329 or the
flexibility of loop 6, may come into play.
The site-specific cleavage of the LSU strongly depends on the
structural and conformational status of the catalytic site. Dissociation of the enzyme into its subunits did not cause any fragmentation. Binding of the activators (CO2 and
Mg2+), substrate (RuBP), and intermediate analogue (CABP)
to their binding sites within catalytic site changed the susceptibility of the enzyme to fragmentation. This is perhaps attributable to the
structural and conformational status of the catalytic site induced by
the effectors. The conformational nature of rubisco seems to be very
important in vivo, because under high light conditions rubisco is in the active form, and the level of RuBP in chloroplasts is
maintained at a higher level (38). Such conditions would make rubisco
more resistant to photo-oxidative stress.
Recently, Desimone et al. (17) reported that reactive oxygen
species first modify rubisco, which then makes it susceptible to
proteolysis. Although they did not show how rubisco is modified by
reactive oxygen species, they demonstrated that once rubisco is exposed
to reactive oxygen species, it is fragmented by a protease(s) in a
stromal fraction in an ATP-dependent manner (17).
Therefore, it seems that there are at least two triggering reactions
driven by reactive oxygen species for the fragmentation of the LSU of rubisco in the lysates of chloroplasts: direct fragmentation shown here
and modification followed by protease-dependent degradation reported by other authors.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Autar K. Mattoo for critical
reading of the manuscript and Drs. Martin Parry and Tomohisa Ogawa for
helping with computer analysis of the ternary structure of rubisco.
 |
FOOTNOTES |
*
This work was supported by Grant-in-Aid for Scientific
Research 09460036 and Grant-in-Aid for Scientific Research in Priority Areas 10170201 from the Ministry of Education, Science and Culture, Japan, by Grant JSPS-RFTF 96L00604 for Research for the Future from the
Japan Society for the Promotion of Science (to T. M.), and by research
fellowships from the Japan Society for the Promotion of Science for
Young Scientists (to H. I.).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: Dept. of Applied
Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Tel.:
81-22-717-8766; Fax: 81-22-717-8765; E-mail: hikomae{at}biochem.tohoku.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase;
LSU, large subunit;
RuBP, ribulose 1,5-bisphosphate;
CABP, 2-carboxyarabinitol
1,5-bisphosphate;
DTT, dithiothreitol;
PAGE, polyacrylamide gel
electrophoresis;
FPLC, fast protein liquid chromatography;
HPLC, high
performance liquid chromatography.
 |
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