(Received for publication, October 20, 1995; and in revised form, January 18, 1996)
From the
The PSI-C subunit of photosystem I shows similarity to soluble
2[4Fe-4S] ferredoxins. Alignment analysis clearly shows that
PSI-C contains an 8-residue internal loop and a 15-residue C-terminal
extension that are absent in the ferredoxins. The remaining residues in
PSI-C are likely to be folded in a way similar to the soluble
2[4Fe-4S] ferredoxins. Two modified PSI-C subunits lacking
either the 8-residue loop or 10 residues of the C terminus were
expressed in Escherichia coli and used to reconstitute a
barley P700-F core prepared to specifically lack PSI-C,
PSI-D, and PSI-E. As shown by EPR spectroscopy, the modified proteins
carry two [4Fe-4S] clusters with characteristics similar to
those of native PSI-C. Western blot analysis of the reconstituted
photosystem I complexes showed that the modified PSI-C proteins bind to
the P700-F
core. Flash photolysis revealed that in
photosystem I complexes reconstituted in the presence of PSI-D with the
C-terminally deleted PSI-C, the F
/F
back-reaction was less efficiently restored than with wild-type PSI-C.
The loop-deleted PSI-C was even less efficient. We attribute these
differences to altered binding properties of the modified proteins.
Comparison of reconstitutions performed in the presence and absence of
PSI-D shows that the loop-deleted PSI-C is unable to bind without
PSI-D, whereas the C-terminally deleted PSI-C binds only weakly with
PSI-D. These results imply that the internal loop of PSI-C interacts
with the PSI-A/B heterodimer and that the C terminus of PSI-C interacts
with PSI-D.
The photosystem I (PS I) ()reaction center complex
mediates electron transport from plastocyanin to ferredoxin in oxygenic
photosynthesis. PS I contains the primary electron donor P700 (a Chl a dimer) and the electron acceptors A
(Chl a), A
(phylloquinone), and three
[4Fe-4S] centers F
, F
, and
F
(1, 2, 3) . The terminal
acceptors F
and F
are bound to the PSI-C
subunit(4, 5) , while the remaining electron acceptors
are bound to the PSI-A
PSI-B heterodimer(6, 7) .
A major unresolved question is the precise pathway of electron flow
through the terminal acceptors F
and
F
/F
. F
has been shown to be
essential for electron transfer to ferredoxin but not for low
temperature photoreduction of F
, suggesting F
to be the final acceptor in the electron transfer chain (8, 9, 10) .
The amino acid sequence of
PSI-C is highly conserved among species and contains a repeated
CXXCXXCXXXCP motif that is characteristic of
2[4Fe-4S] proteins. Alignment analysis of these proteins
shows that, apart from an internal segment of 8 residues and 15
residues in the C terminus that are not present in the ferredoxins,
PSI-C is similar to the bacterial 2[4Fe-4S] ferredoxins (5, 11, 12) . The folding of PSI-C must be
very similar to that of the soluble ferredoxins, indicating that the 8
internal residues form an extra loop. Since the ferredoxins are soluble
and the PSI-C polypeptide is membrane-bound, the additional amino acids
in the loop and in the C terminus may be responsible for the binding of
PSI-C to the P700-F core(12) . EPR data obtained
with PS I crystals and comparison with the known structure of Peptococcus aerogenes ferredoxin allow the orientation of
PSI-C within the PS I complex to be limited to two possibilities by
positioning the two clusters and two highly conserved helices along a
fixed axis(13) . Ligands for F
are provided by
Cys
, Cys
, Cys
, and
Cys
, while Cys
, Cys
, Cys
and Cys
provide ligands for
F
(14) . The positions of two [4Fe-4S]
clusters within the complex are known from the crystal structure of PS
I(15) , but which cluster represents F
and which
represents F
cannot be distinguished at 6-Å
resolution. From alignment with P. aerogenes ferredoxin, the
internal loop and the C terminus are predicted to extend from opposite
sides of PSI-C. Therefore, determination of the orientation of the loop
and the C terminus relative to PS I would yield further information on
the positions of F
and F
.
We characterized
the interaction and binding of PSI-C within the PS I complex by
producing two PSI-C deletion mutants that lack either the 8-residue
internal loop or 10 residues of the C terminus. Using a modification of
the method of Parrett et al.(16) , we recently
established that urea treatment of barley PS I followed by a combined
detergent, salt, and urea wash leads to specific dissociation of the
PSI-C, PSI-D, and PSI-E subunits without affecting the remaining
polypeptides(17) . The resulting P700-F core can be
used for reconstitution by the addition of Escherichia coli expressed PSI-C and PSI-D in the presence of the reagents
Na
S, FeCl
, and 2-mercaptoethanol, which serve
to rebuild the FeS clusters of
PSI-C(17, 18, 19) . Using this reconstitution
system, we show that the PSI-C protein lacking the internal loop
interacts inefficiently with the P700-F
core, while the
protein lacking the C terminus interacts inefficiently with PSI-D.
Figure 1:
Amino acid sequences of mutant and
wild-type barley PSI-C(11) and Synechococcus sp. PCC 6301 PSI-C
(21) . The underlined sequences in the wild-type PSI-C proteins were deleted, resulting
in two modified proteins, PSI-C
L and
PSI-C
C. The residues differing between the
PSI-C
and the PSI-C
are shown in boldface type. The sequence of the 2[4Fe-4S]
ferredoxin from P. aerogenes(33) is shown for
comparison.
Figure 2:
Western blot analysis before and after
reconstitution of the P700-F core with the PSI-C
polypeptides. The PS I preparation before the urea treatment was
included. The samples were pelleted by ultracentrifugation, and equal
amounts of P700 were applied to each lane of the gel. The blots were
incubated with polyclonal antibodies raised against the PSI-C
subunit. Chl. indicates the position of pigments
released from PS I.
Figure 3:
EPR analysis of a P700-F core
rebuilt with PSI-C
C and PSI-C
. A, reconstituted with PSI-C
C and PSI-D; B, reconstituted with PSI-C
and PSI-D; C, mock reconstitution of the P700-F
core without
added proteins. The free radical signal at g = 2.0 was deleted
for clarity.
Similar experiments were performed with
complexes reconstituted with PSI-CL (Fig. 4).
When illuminated at 15 K, the low-field peaks of F
and
F
were clearly visible but small in amplitude, while the
midfield and high-field features were not well resolved. We next froze
the sample during illumination, conditions allowing two or more
electrons to accumulate in the electron acceptor system. The resulting
spectrum had peaks with much increased amplitude and was very similar
to that of the wild-type reconstitution, containing the features
characteristic of an F
/F
interaction spectrum.
The data show that PSI-C
L contains two functional
[4Fe-4S] clusters with properties similar to F
and F
.
Figure 4:
EPR analysis of a P700-F core
reconstituted with PSI-C
L and PSI-C
. A, reconstitution with PSI-C
L and PSI-D.
The sample was illuminated at 15 K, and the spectrum was acquired. B, subsequently the sample in panel A was thawed and
refrozen during illumination to allow photoaccumulation. C,
reconstitution with PSI-C
L and PSI-D,
photoaccumulated. D, mock reconstitution of the P700-F
core without added proteins, photoaccumulated. The free radical
signal at g = 2.0 was deleted for
clarity.
Figure 5:
Flash-induced absorption changes of P700
at 834 nm in PS I complexes reconstituted with varying amounts of
PSI-C. The rebinding was analyzed by the addition of different molar
ratios of the PSI-C (
), PSI-C
C
(
), and PSI-C
L (
). PSI-D was added in a
molar ratio of 20 PSI-D/1 P700 in all experiments. The samples (20
µg/ml Chl) were resuspended in 50 mM Tris-HCl, (pH 8.3), 2
mM sodium ascorbate, 60 µM DCPIP. The extent of
reconstitution was determined by fitting the traces to biphasic
exponential decay curves containing phases with time constants of
1 ms and >16 ms. The fraction constituting the slow decay phase
was calculated in percentage of the total absorbance
change.
The PS I complex was reconstituted using molar ratios of
20:20:1 (PSI-C:PSI-D:P700). The PS I complex reconstituted with PSI-D
and PSI-C, PSI-C
, or
PSI-C
C resulted in 88, 84, and 82% recovery of the
slow decay phase, respectively (Fig. 6A, Table 1).
Rebuilding with PSI-C
L and PSI-D resulted in an
incomplete reconstitution, with only 52% recovery of the PS I activity (Fig. 6A, Table 1). To assay stability, the
reconstituted complexes were washed with 0.1% Triton X-100 over an
ultrafiltration membrane. The PS I complex reconstituted with
PSI-C
and PSI-C
remained stable, showing 84
and 88% reconstitution, respectively, whereas the PS I complex
reconstituted with PSI-C
C was unstable as shown by a
reduction in the magnitude of the slow
A
decay from 82 to 53% (Fig. 6B, Table 1).
The PS I complex reconstituted with PSI-C
L was also
affected by the wash (52% before to 38% after). It should be noted that
mock reconstitution of the P700-F
core in the absence of
added PSI-C resulted in about 17% recovery of a slow absorbance decay.
This background value has not been subtracted from the data presented
in Table 1. Subtraction of the background value would obviously
accentuate the differences reported.
Figure 6: Flash-induced absorption changes of reconstituted PS I. The transients represent the primary data for one of the experiments summarized in Table 1. A, reconstitution in the presence of PSI-D. B, the samples analyzed in panel A after washing by ultrafiltration. C, reconstitution in the absence of PSI-D. D, the samples analyzed in panel C after washing by ultrafiltration.
Rebuilding the PS I complex
with the PSI-C and PSI-C
in the absence of
PSI-D resulted in retention of 64 and 66% of the slow decay phases,
respectively (Fig. 6C, Table 1), which is less
than when PSI-D was included for reconstitution (88 and 84%). The
PSI-C
C had an enhanced ability to bind to the
P700-F
core, retaining 85% of the slow phase.
PSI-C
L did not bind to the P700-F
core
in the absence of PSI-D. The
[F
/F
]
P700
back-reaction in all the PS I complexes
reconstituted without PSI-D was greatly reduced by washing (Fig. 6D, Table 1), indicating that PSI-C was
removed by this procedure.
Deletion of neither the internal 8-residue loop nor the 10
C-terminal residues of PSI-C affected the iron-sulfur clusters F and F
in the unbound proteins. Therefore, any
differences in reconstitution efficiency exhibited by these proteins
can be attributed to altered binding efficiency of the modified
proteins PSI-C
C and PSI-C
L to the
P700-F
core. PSI-C is a highly conserved subunit, and only
nine amino acid residues are different between PSI-C from barley and Synechococcus sp. PCC 6301 (Fig. 1). In all
experiments, the wild-type proteins PSI-C
and
PSI-C
have given identical results.
Western blot (Fig. 2) and EPR analysis ( Fig. 3and Fig. 4) show
that the P700-F core does not contain detectable amounts of
PSI-C. Nevertheless, the core exhibited 17% recovery of the slow phase
of the flash-induced absorbance change at 834 nm upon mock
reconstitution. This suggests that a small fraction of the slow
back-reaction is due to the presence of reconstitution reagents that
may function as electron acceptors. This would be in agreement with the
previous observation that electron transport to NADP
cannot be accomplished in the presence of the reagents employed
for F
/F
reconstitution(17) .
EPR
analysis at 15 K of the P700-F core reconstituted with the
PSI-C
L showed features of F
and
F
, but the electron transport was inefficient at 15 K (Fig. 4). However, when the sample was subjected to
photoaccumulation, the spectrum was nearly identical to that of PS I
reconstituted with PSI-C
and contained resonances typical
of an interaction spectrum of F
and F
. This is
in contrast to the PSI-C
C reconstitution, which
gives a spectrum similar to wild-type reconstitution when illuminated
at 15 K.
In order to measure NADP reduction, it is
essential to remove the reconstitution reagents, and this was generally
accomplished by ultrafiltration. However, in the case of
PSI-C
L significant loss of reconstitution efficiency
occurred as determined by flash photolysis, and no NADP
reduction could be detected. When a milder desalting procedure
was employed, there was no impairment of the reconstitution, and
NADP
reduction was detected. This shows that
PSI-C
L does bind to PS I in the presence of PSI-D,
but the binding is weak. The measured NADP
-reducing
rates are low compared with native PS I but correspond well to
previously observed values in reconstitution experiments(17) .
Washing in the presence of Triton X-100 causes a loss of activity
possibly due to loss of PSI-F and thereby an inefficient interaction
with plastocyanin(33, 34) . This effect of Triton
washing is also seen with native PS I(17) . In all cases, the
extent of NADP
reduction mirrored the extent of
reconstitution observed by flash analysis. Therefore, the data indicate
that PS I reconstituted with the mutant PSI-C polypeptides functions
identically to wild-type proteins in ferredoxin reduction. Further
flash experiments to determine the kinetic constants of ferredoxin
reduction will be needed to address this question in detail.
The
C-terminally truncated PSI-CC restored the
[F
/F
]
P700
back-reaction nearly as well as
PSI-C
and PSI-C
, but the complex was
unstable. When the PS I complex was washed intensively, the recovery of
reconstituted PS I decreased to 53%. Using PSI-C
and
PSI-C
the reconstituted PS I complexes were resistant to
the wash procedure. Thus the C terminus is not essential for correct
positioning of PSI-C on the P700-F
core, but it is
important for the stability of the resulting PS I complex. In reactions
with and without PSI-D, the reconstitution behavior of
PSI-C
C was similar, in contrast to the significant
effect of PSI-D observed with wild-type PSI-C. This suggests that the
C-terminal domain that was removed in PSI-C
C
includes the major site of interaction with PSI-D. PSI-C
,
PSI-C
, and PSI-C
C were almost
completely removed by washing of the PS I complexes rebuilt in the
absence of PSI-D. This is in agreement with previous results showing
PSI-D as essential for the stabilization of PSI-C in PS
I(17, 19) . However, since the activity of the PS I
complex rebuilt with PSI-C
C in the presence of PSI-D
was not entirely destroyed by the extensive wash, there must be some
additional sites of interaction with PSI-D that remain on
PSI-C
C.
Rebuilding the PS I complex with the loop
deletion mutant in the presence of PSI-D was achieved, but it was
incomplete. Furthermore, while the other proteins tested interacted
weakly with PS I in the absence of PSI-D, PSI-CL was
unable to interact functionally with PS I under these conditions. This
suggests that the PSI-D interaction provides the only functional link
between the PS I complex and PSI-C
L. The 8-residue
loop, which was deleted in this mutant, must therefore contain sites of
interaction between PSI-C and the P700-F
core. The sites of
interaction between PSI-C and the PS I polypeptides have been shown to
be limited to PSI-A, PSI-B, PSI-D, and PSI-E (35, 36) (
)in the native PS I complex. The
loop was shown not to be involved in binding of PSI-D, and
reconstitution can be performed completely in the absence of PSI-E
(this paper and (17) and (19) ); therefore, the
binding activity of the loop must involve interaction with PSI-A and
PSI-B. Studies of Rodday et al.(36, 37) suggest that arginine residues in the
conserved domain in PSI-A and PSI-B that binds F
are
important for the interaction with PSI-C. The 8-residue internal loop
contains two negative charges that may be responsible for the specific
interaction with the arginines. To interact with PSI-A and PSI-B and
most likely with residues very close to the ligands of F
,
we propose that the loop of PSI-C must face the thylakoid membrane,
whereas the C terminus providing the binding site for PSI-D is likely
to be oriented toward the stroma.