(Received for publication, May 22, 1995; and in revised form, July 18, 1995)
From the
Illumination of the isolated reaction center of photosystem II
generates a protein of 41 kDa molecular mass. Using immunoblotting, it
is confirmed that the protein is an adduct of the D1 protein and the
-subunit of cytochrome b
. Its formation
seems to be photochemically induced, being independent of temperature
between 4 and 20 °C and unaffected by a mixture of protease
inhibitors. The maximum levels are detected when the pH is in the
region 6.5-8.5 and when illumination intensities are moderate.
Although higher light intensities induce a higher rate of formation,
the accumulation of elevated levels of the 41-kDa protein does not
occur due to light-induced degradation. This degradation is also
unaffected by the presence of protease inhibitors. Proteolytic mapping
and N-terminal sequencing indicates that the cross-linking process
involves the N-terminal serine of the
-subunit of cytochrome b
and D1 residues in the 239-244 FGQEEE
motif close to the Q
binding site. In conclusion, the
results indicate that the N terminus of the
-subunit is exposed on
the stromal side of photosystem II in such a way as to undergo
light-induced cross-linking in the Q
region of the D1
protein. They also suggest that the 41-kDa adduct may be an
intermediate before the light-induced cleavage of the D1 protein in the
FGQEEE region.
Photosystem II (PSII) ()is the oxygen-evolving system
of photosynthesis. It is a membrane-located multicomponent, pigment
protein complex that functions as a light-driven water/plastoquinone
oxidoreductase(1, 2) . The heart of the PSII complex
is the reaction center, in which the initial primary and secondary
charge separation occurs. In several respects, the PSII reaction center
shares features similar to those of the purple photosynthetic
bacteria(3) . In the bacterial reaction center, the cofactors
that catalyze charge separation are bound to a heterodimer consisting
of the L and M subunits. Determination of the structure of the reaction
center of Rhodopseudomonas viridis gave information at the
level of atomic resolution of the organization of the cofactors and the
polypeptide chains of the L and M subunits(4, 5) . The
amino acid sequences of the reaction center proteins, D1 and D2, share
sufficient homologies with the L and M subunits to allow a reasonable
model to be proposed, describing how these proteins fold and how the
redox-active cofactors are bound(6, 7, 8) .
From the comparative approach and from direct experimental evidence(9, 10) , it is quite clear that the D1 protein is analogous to the L-subunit of the bacterial system. Nevertheless, D1 protein shows the unusual characteristic of rapid turnover that is greater than in any other PSII protein and indeed faster than most other proteins in the photosynthetic membrane(11) . In contrast, the L-subunit does not turn over rapidly. The phenomenon of rapid turnover of D1 protein is even more amazing since, like the L-subunit, it forms the active branch of the PSII reaction center, being involved in both donor and acceptor side redox reactions.
There is good reason to believe that the rapid turnover of D1 protein is a consequence of the vulnerability of PSII to photodamage and that this effect is the basis of the physiological phenomenon of photoinhibition(12, 13) .
In studying
the rapid turnover of D1 protein in vivo, Greenberg et al.(14) concluded that the degradation involved a cleavage
that yielded a 23.5-kDa fragment containing the N terminus of the
protein. A similar photoinduced fragment has also been generated using in vitro systems(15, 16, 17) . In
the case of isolated PSII reaction centers, it was shown that a 23-kDa
N-terminal fragment of the D1 protein is observed when they are
illuminated under aerobic conditions in the absence of exogenous
electron acceptor(17) . Under such conditions, recombination
occurs between the radical pair state consisting of the oxidized
primary electron donor (P680) and reduced primary
electron acceptor pheophytin a (pheo
). This
recombination forms a triplet state of P680(18) , which is able
to generate singlet oxygen(19, 20) . It is likely,
therefore, that this highly reactive form of oxygen causes damage to
the D1 protein. The damage itself does not seem to be directly involved
in the cleavage process but triggers the D1 protein for degradation (21) possibly via a conformational change(22) . Because
this isolated complex consists only of the D1 and D2 proteins, the
- and
-subunits of cytochrome b
(Cyt b
), and the product of the psbI gene(9, 23) , it has been concluded that the
mechanism of cleavage is contained within the reaction center and that
no external proteases are involved. For this reason, we have undertaken
a study to elucidate the nature of the autocatalytic cleavage, which
generates the 23-kDa N-terminal fragments. Previous studies have
indicated that the cleavage occurs on the C-terminal side of residue
238 in the D1 sequence(14, 24) . As emphasized by
Greenberg et al.(14) , this cleavage site is located
in the loop joining putative transmembrane segments IV and V, which is
in the vicinity of the Q
binding region (6) .
The investigation that we report here was stimulated by the
observation that in addition to the appearance of the light-induced
23-kDa N-terminal fragment, a band was induced at about 41 kDa when
isolated reaction centers were exposed to strong illumination. This
band was reported by Shipton and Barber (25) and subsequently
shown, by immunological blotting, to consist of D1 protein and the
-subunit of Cyt b
(26) . Here, we
further characterize the properties of this D1 protein/
-subunit
Cyt b
adduct and consider how the formation of a
covalent linkage between these two reaction center subunits could be
involved in events leading to D1 protein cleavage and the generation of
the 23-kDa N-terminal fragment.
For N-terminal sequencing, proteins separated on gels were transferred onto polyvinylidene difluoride membranes (ProBlot, Applied Biosystems) according to Matsuidara(31) , using 10 mM CAPS, pH 11. Gel solutions were stirred overnight on Amberlite MB1, and 1 mM 2-dimethylamino-ethanethiol hydrochloride was added to all buffers. To locate bands, filters were stained with 0.4% Coomassie Brilliant Blue R-250 in 40% methanol and 1% acetic acid and destained in 40% methanol. N-terminal sequencing was performed by automated Edman degradation using an Applied Biosystems protein/peptide sequencer (model 477A).
Chlorophyll concentration was measured according to Arnon(33) . Densitometric analyses of immunostained gels were performed using a Hirchmann densitometer.
Figure 1:
Production of a 23-kDa D1 protein
fragment during illumination with no exogenous electron donors or
acceptors. Isolated reaction centers were diluted (50 µg of Chl
ml) into 50 mM MES, pH 6.0, 2 mMn-dodecyl
-D-maltoside and illuminated with
2000 µmol of quanta m
s
white
light at 23 °C. Samples were removed (1.5 µg Chl) for SDS-PAGE.
Western blots were immunodecorated with an N-terminal-specific D1
protein antibody,
-D1-N.
Figure 2:
Immunoblotting with antibodies anti-D1N (panel A), anti-D1C (panel B), and
anti-cyto (panel C) of undigested 41 kDa-band (lanes 1A, 1B, and 1C), undigested D1 (lanes 2A and 2B), undigested cyto
(lane 2C), Lys-C-digested 41-kDa band (lanes
3A, 3B, and 3C), Lys-C digested D1 (lanes 4A and 4B), and Lys-C-digested cyto
(lane 4C). Wheat RCII complexes were irradiated at a
chlorophyll concentration of 50 µg/ml for 15 min with 600 µmol
m
s
white light to induce the
formation of the 41-kDa adduct. After SDS-PAGE and acid-free Coomassie
staining, the corresponding band was cut out from the gel and loaded on
the stacker of a second identical gel. For proteolysis, acrylamide
bands were overlaid with 20 µl of Lys-C (10 units/ml), and
electrophoresis was started. When the tracking dye reached the bottom
of the stacking gel, power was switched off for 3 h to allow
proteolysis; gels were then run as usual. See Table 1for
N-terminal sequence of the C-terminal fragment of D1 induced by Lys-C
treatment (arrow on lane4B). Bands marked with a circle in lanes2C and 4C are aggregation products of cyto
. Arrows on lanes3B and 3C emphasize
the identity of the fragments detected by anti-D1C and
anti-cyto
from Lys-C proteolysis of the 41 kDa. Arrow on lane3A marks the N-terminal
fragment from Lys-C proteolysis of the 41-kDa adduct and D1, which
represents the 2-238 segment of the
protein.
PanelA of Fig. 2shows an immunoblot
with anti-D1N antiserum of the 41-kDa adduct (lanes1 and 3) and isolated D1 protein (lanes2 and 4), either undigested (lanes1 and 2) or digested (lanes3 and 4) with
Lys-C protease. As can be seen in lane4, a 19-kDa
fragment of the D1 protein is detected by anti-D1N, which corresponds
to the 2-238 segment of this protein(32, 36) .
The same 19-kDa fragment was observed when the 41-kDa band was
partially digested by Lys-C (arrow on lane3A). When an identical blot was probed with an antiserum
to the C-terminal region of the D1 protein (anti-D1C), the results
presented in panelB were obtained. In this case, a
10-kDa fragment of the D1 protein was detected (arrow on lane4B), which is the 239-344 C-terminal
segment. Indeed, N-terminal sequencing confirmed that the first 10
residues of this fragment corresponded to residues 239-248 (see Table 1). This 10-kDa C-terminal fragment, however, was not
detected as a consequence of partial digestion of the 41-kDa adduct;
instead, the anti-D1C detected a 21-kDa band (arrow on lane3B), which is thought to contain the 10-kDa
C-terminal portion of the D1 protein and the -subunit of Cyt b
. Indeed, this was confirmed by blotting with
anti-Cyt
antiserum (arrow on lane3 of panelC in Fig. 2).
As
the -subunit of Cyt b
does not contain any
lysine residues(35) , Lys-C proteolysis had no effect on this
isolated subunit (compare lanes2C and 4C in Fig. 2). Therefore, the above experiments show unequivocally
that the site for light-induced cross-linkage is located at an amino
acid residue lying on the C-terminal side of lysine-238 of the D1
protein.
Figure 3:
Limited proteolysis of cyto with V8 protease. A, Coomassie staining; B,
immunoblotting with anti-cyto
antiserum. Lane1, no protease was added. Lanes2 and 3 contain, respectively, 1.0 and 5.0 µg of protease.
N-terminal sequencing of cyto
and Sa-cyto
is reported in Table 1.
The effect of V8 proteolysis of the isolated D1 protein and the 41-kDa adduct was also investigated, as shown in Fig. 4. Immunoblotting with anti-D1C antiserum (panelA) showed that the digestion of D1 with V8 (lane2) resulted in the appearance of two or, more often, three bands in the range of 8-10 kDa. These bands probably correspond to Sa8 and Sa10 doublet described by Marder et al. (37) and Greenberg et al.(14) . As these bands were recognized by the C-terminal specific antibody (raised to the last 19 amino acid residues of the protein), they must contain the C terminus of D1. In particular, it was found by N-terminal sequencing (see Table 1) that the Sa8 fragment is derived from a cleavage at glutamate 244, as suggested previously by Marder et al.(37) . In the case of V8 proteolysis of the 41-kDa adduct (lane4 of panelA), the anti-D1C antibody recognizes three fragments in the range of 15-20 kDa and a 8-kDa fragment. As this last fragment has the same electrophoretic mobility and immunological reactivity as the Sa8 fragment from D1, it is reasonable to assume that they are the same proteolytic fragment (i.e. from 245 to the C terminus of the D1 protein (see Fig. 9)).
Figure 4:
Immunoblotting with antibodies anti-D1C (panel A), anti-cyto (panel B), and
anti-D1N (panel C) of undigested D1 (lanes 1A and 1C), undigested 41 kDa (lanes 3A and 3C),
undigested cyto
(lane 3B), digested D1 (lanes 2A and 3B), digested 41 kDa (lanes
1B), and digested cyto
(lane 2B). RCII
complexes were treated as described in the legend to Fig. 1.
Bands were isolated by SDS-PAGE and subjected to proteolysis for 1 h
using 1 µg of V8 protease.
Figure 9:
Representation of the proposed
cross-linkage site between the D1 protein and the cytochrome b
-subunit, resulting in the generation of
the 41-kDa light-induced band. The N-terminal serine residue of the
cytochrome b
-subunit seems to cross-link
with the FGQEEE motif on the D1 protein (shaded).
When anti-Cyt was
used to probe V8 proteolytic digestion products of the 41-kDa adduct (panelB, lane1), a number of
bands were detected in the 15-20-kDa range. Of note is that two
of them are the same as those detected by the anti-D1C antibody (marked
by circles in lane4 of panelA and lane1 of panelB). Since
the Sa8 fragment was detected after treatment of the 41-kDa adduct with
V8, it seems likely that cross-linking of the
-subunit and the D1
protein occurs on the N-terminal side of residue 244. This conclusion,
taken together with the result derived from the Lys-C experiments (Fig. 2), suggests that the linkage takes place between 239 and
244 of D1 protein, i.e. in the FGQEEE motif. Detection of the
V8-induced doublet of the 41-kDa band by both anti-D1C and
anti-Cyt
indicates that these contain the branched
peptide formed by the
-subunit (possibly minus a small 1-kDa
C-terminal region) of cytochrome b
and the C
terminus of the D1 protein. It is note worthy that, when the polyclonal
specific for the N terminus of D1 is employed to probe V8 digests (panelC of Fig. 4) of D1 (lane2) and the 41-kDa adduct (lane4),
essentially the same pattern is observed, confirming that the
-subunit is not linked with D1 on the N-terminal side of residue
238.
Figure 5:
Effect of different light intensity on the
relative level of the 41-kDa band. A, densitometric analysis
of immunoblots similar to that shown in B but containing
samples irradiated with different fluence rates. Squares, 2000
µmol quanta m s
; circles, 1000 µmol quanta m
s
; invertedtriangles, 400
µmol quanta m
s
; triangles, 100 µmol quanta m
s
. Each gel lane contained 0.2 µg of
chlorophyll. Immunoblot was probed with anti-cyto
antiserum. B, immunoblot of pea RCII complexes
irradiated with 600 µmol quanta m
s
for 0 (lane1), 2.5 (lane2),
5 (lane3), 7.5 (lane4), 10 (lane5), 15 (lane6), 20 (lane6), and 30 min (lane8).
When irradiation was performed in the presence of a mixture of protease inhibitors, no effect on the 41-kDa band was observed, neither on its appearance nor disappearance (data not shown). Similarly, as Fig. 6shows, formation of the 41-kDa adduct was independent of temperature over the range 4-20 °C.
Figure 6:
Effect of temperature on the relative
level of the 41-kDa RCII complexes was illuminated for the desired
period of time at 4 °C (squares), 10 °C (circles), and 20 °C (triangles). The level of
the 41-kDa band was evaluated by densitometry. Light intensity, 600
µmol quanta m s
.
In
further experiments, the effect on the presence/absence of oxygen and
pH were investigated. Very low levels of oxygen (less than 1
µM) were obtained by flushing the RCII suspension with
oxygen-free nitrogen and by including a chemical trap consisting of 10
mM glucose, 0.1 mg ml catalase, and 0.1 mg
ml
glucose oxidase. The results of this experiment
are shown in Fig. 7, where it can be seen that only those RCII
samples illuminated under aerobic conditions (lanes6-8) produced the 41-kDa band due to cross-linking
of D1 protein and the
-subunit of Cyt b
.
For studying the pH sensitivity, the RCII preparation was suspended in
buffers ranging in pH from 5.5 to 9.5. As Fig. 8shows,
irradiation at pH 5.5 does not result in the appearance of the 41-kDa
adduct. At pH 9.5, the formation of the adduct is also severely
inhibited. In contrast, the light-induced 41-kDa band is readily
detected at pH values 6.5, 7.5, and 8.5. When a sample that had been
irradiated at pH 7.5 (i.e. containing the 41-kDa band) was
incubated in the dark at pH 5.5 or 9.5, no effect on the level of the
adduct was noted, an observation that rules out the possibility that
the band is not observed in Fig. 7because of its instability
toward pH 5.5 or 9.5.
Figure 7:
Formation of the 41-kDa adduct depends on
the presence of oxygen. Isolated RCII complexes (50 µg of
chlorophyll/ml) were illuminated with 600 µmol quanta
m s
either under anaerobic (lanes3-5) or aerobic (lanes6-8) conditions. Anaerobic conditions were obtained
by using a chemical trap consisting of 10 mM glucose, 0.1
mg/ml catalase, and 0.1 mg/ml glucose oxidase. Buffer was also flushed
for 15 min with oxygen-free nitrogen. Samples were irradiated for 2.5 (lanes3 and 6), 5 (lanes4 and 7), and 10 min (lanes5 and 8). Lanes2 contain a dark control anaerobic
control. 0.2 µg of chlorophyll was loaded on each gel lane. Lane1 contains prestained molecular markers. Blot
was probed with anti-D1 N antiserum. HD, D1/D2
heterodimer.
Figure 8:
The effect of different pH levels on the
formation of the 41-kDa adduct. RCII complexes were diluted to 20
µg of chlorophyll/ml in 0.1 M Bis-Tris propane at the
indicated pH value. 2 mMn-dodecyl
-D-maltoside was also added. Irradiation was carried out
at 4 °C with 600 µmol quanta m
s
for the indicated period of time. Each gel
lane contained 0.2 µg of chlorophyll. LaneM contains prestained molecular markers. The blot was probed with
anti-cyto
antiserum.
The immunological blotting data presented above are
consistent with the previous conclusion that the 41-kDa band generated
by illuminating isolated PSII RCs is a cross-linked adduct of the D1
protein and the -subunit of Cyt b
.
Treatment of this adduct, isolated from illuminated RCIIs of wheat with
Lys-C, generated a 21-kDa fragment that consisted of the C-terminal
portion of the D1 protein and the
-subunit of Cyt b
. Thus, the cross-linking occurs on the
C-terminal side of residue 238 of the D1 protein. The C-terminal
portion of the D1 protein (from 239 to 344) was found to have an
apparent molecular mass of about 10 kDa, while the
-subunit of Cyt b
has about the same mass, thus accounting for
the observed 21-kDa value for the Lys-C-induced fragment of the adduct.
Experiments using S. aureus V8 protease further suggest
that the cross-linking site is on the N-terminal side of the tyrosine
residue 245 of the D1 protein. This conclusion is based on the fact
that the Sa8 fragment formed due to V8 cleavage at glutamate 244 on the
D1 protein is also generated when the 41-kDa adduct is digested with
this enzyme (e.g.Fig. 3, laneA4).
Thus, these results lead to the overall conclusion that the adduct is
formed by a cross-linking of the -subunit of Cyt b
with a residue in the 239-244 motif
FGQEEE (see Fig. 9).
The N terminus of the -subunit of
Cyt b
is not blocked and can be sequenced by
Edman degradation as indicated in Table 1. In contrast, the N
terminus of the D1 protein is blocked and cannot be readily sequenced
by the Edman procedure. We have also found that the 41-kDa adduct is
not amenable to N-terminal sequencing using Edman degradation. The
reason for this is unclear, but it could be that the N terminus of the
-subunit is not available in the adduct as it is in the free
protein. This therefore suggests that the cross-linking to the FGQEEE
motif involves the N-terminal residue of the
-subunit, which is a
serine. The action of V8 on the 41-kDa adduct is also consistent with
this since the products of this proteolysis did not indicate an
attachment of the C-terminal end of the
-subunit to the D1 protein
where the Cyt subunit has a V8 cleavage site (e.g.Fig. 4C)
The identified cross-linking site on
the D1 protein has the unusual feature of having three glutamic acid
residues adjacent to each other. Such a motif is not typical and occurs
in only a few proteins(24) . The pK value
for a single glutamic acid residue is 4.3 and thus at neutral pH would
be unprotonated. However, the existence of three adjacent carboxyl
groups of glutamic acid would be expected to significantly shift the
pK
to a higher value so that protonation would
occur at neutral pH. This shift of pK
could aid a
condensation reaction with the N-terminal serine of the
-subunit,
thus forming a peptide linkage. Such a reaction is endothermic and
would not be expected to happen spontaneously. Indeed, the
cross-linking is induced by illumination. The mechanism involved does
not seem to be enzymic (lack of effect of protease inhibitors and
temperature) but involves a physically induced reaction driven by the
absorption of light energy. Moreover, we have also shown that oxygen
needs to be present for the adduct to appear. It is difficult to
perceive how the proposed condensation reaction is driven. Under
anaerobic conditions the heme of Cyt b
becomes
photoreduced, but no such reaction occurs when oxygen is
present(38) . It is conceivable that the formation of the
adduct is dependent on the redox state of the heme, which affects the
conformation of the
-subunit. Alternatively, light-induced
oxidation processes could aid the cross-linking.
Although the
precise mechanism for the formation of this adduct is not clear, the
identification of the linkage site has at least two important
implications. The first is that the N terminus of the -subunit
must be exposed on the stromal side of PSII, as suggested by Tae et
al.(39) and Vallon et al.(40) .
Moreover, our data indicate that Cyt b
may be
more closely located at the D1 rather than the D2 protein and that the
N terminus of the
-subunit is positioned near to the Q
site. This conclusion would mean that the heme, which is probably
ligated to the histidine residues 22 and 17 on the
- and
-subunit, respectively(39) , is more intimate with Q
than Q
. No cross-linking product was observed with
the
-subunit, which, like the psbI gene product, is
blocked at its N terminus(23) .
The second important
implication is related to our finding that the formation of a D1
protein/Cyt b
-subunit adduct occurs via
cross-linking in the 239-244 motif of the D1 protein. Studies of
D1 protein degradation in both in vivo and in vitro systems identify the 239-244 region as the primary site of
cleavage when acceptor side photoinhibition occurs, generating a 23-kDa
N-terminal and a 10-kDa C-terminal fragment of the D1 protein. Studies
with isolated reaction centers have shown that the 41-kDa adduct is
formed prior to the appearance of the 23-kDa N-terminal D1 cleavage
product (e.g. see Fig. 1and (17) ). It is
possible, therefore, that the cross-linking of the
-subunit to the
D1 protein is the first step in the sequence of events leading to the
primary cleavage of this protein. The formation, for example, of a
branch peptide linkage could possibly aid a hydrolysis reaction,
resulting in cleavage of the D1 protein in the FGQEEE motif without
causing any degradation of the
-subunit itself in line with other
observations(41) . However, further experiments are required to
give credibility to this idea. Among the experiments to be carried out
will be those that identify whether the linkage and cleavage processes
involve the same specific residues.