(Received for publication, April 6, 1995; and in revised form, August 7, 1995)
From the
A Photosystem I (PS I) complex reconstituted with PsaC-C51D
(aspartate in lieu of cysteine in position 51) shows light-induced EPR
signals with g values, line widths, and photoreduction behavior
characteristic of F. Contrary to an earlier report, a
[3Fe-4S] cluster was not located in the reconstituted PS I
complex. Instead, a second set of resonances with g values of 2.044,
1.942, and 1.853 becomes EPR-visible when the C51D-PS I complex is
measured at 4.2 K. This fast relaxing center, termed F
` is
likely to represent a [4Fe-4S] cluster in the mixed ligand
(3Cys
1Asp) site. Redox studies show that the E
of F
` and F
are -630 mV and
-575 mV, respectively. Room temperature optical studies support
the presence of two functioning electron acceptors subsequent to
F
, and NADP
photoreduction rates mediated
by ferredoxin and flavodoxin are nearly equivalent to the wild type. In
addition to [3Fe-4S] clusters and S = ground
state [4Fe-4S] clusters, the free PsaC-C51D protein shows
resonances near g = 5.5, which may represent a population of
high spin (S = ) [4Fe-4S] clusters in the
mixed ligand F
` site. Similar to the C14D-PS I mutant
complex, it is proposed that the P700-F
core selectively
rebinds those free PsaC-C51D proteins that contain two
[4Fe-4S] clusters. These studies show that primary
photochemistry and electron transfer rates in PS I are relatively
unaffected by the presence of a highly reducing, mixed ligand cluster
in the F
` site.
Photosystem I (PS I) ()is a multisubunit
pigment-protein complex located in the thylakoids of chloroplasts and
inner membranes of cyanobacteria (reviewed recently in (1) and (2) ). This enzyme functions primarily as a light-driven
cytochrome c
:ferredoxin oxidoreductase,
transforming a photon into stable chemical free energy. An
indispensible feature of PS I is a series of bound electron acceptors
that function to suppress the back reaction with P700
by providing a kinetically favorable pathway to ferredoxin. One
strategy for studying the function of the bound electron carriers is to
modify their structure and redox potentials, with the expectation that
this will lead to changes in function. This approach is currently being
applied to individual components of the PS I reaction center, where it
is anticipated that subtle structural alterations will influence
electron transfer rates and photosynthetic efficiency.
The majority
of the bound electron carriers in PS I are located on the PsaA and PsaB
polypeptides, including P700, A, A
, and
F
. The terminal electron carriers of PS I, designated
F
and F
, are two [4Fe-4S] clusters
located on the stromal, extrinsic PsaC polypeptide. This 9-kDa protein
is potentially more amenable to chemical and genetic modification than
are the larger subunits of the complex because it can be expressed as
the apoprotein in Escherichia coli(3, 4) .
Further, this polypeptide can be removed from the PS I complex by
chemical treatment, and either wild-type PsaC or site-directed mutants
of PsaC can be rebound onto a purified P700-F
core
(reviewed in (5) ). Since there is no three-dimensional
structure for PsaC (a crystal structure of PS I is published at a
resolution of 6 Å(6) ), one challenge is to identify
changes in amino acids that will lead to insights concerning the
function of these two iron-sulfur clusters.
One obvious target for
study is the ligands to the F, F
, and F
iron-sulfur clusters. Site-directed mutagenesis has been used
recently to alter the cysteine residues in PsaC with the expectation
that the substitute amino acid will affect the spectral, thermodynamic,
and kinetic properties of the F
and F
clusters.
Based on precedent with naturally occurring ferredoxins, the second
cysteine in each CXXCXXCXXXCP binding motif
was replaced with aspartic acid(7) . The free PsaC-C14D and
PsaC-C51D proteins were found to contain [3Fe-4S] and
[4Fe-4S] clusters(8) . When PsaC-C51D and PsaC-C14D
were rebound to a P700-F
core in the presence of PsaD, the
[4Fe-4S] cluster in the PsaC-C51D-PS I complex assumed g
values and photoreduction behavior that identified that cluster as
F
. The [4Fe-4S] cluster in the rebound C14D
mutant assumed g values and photoreduction behavior that identified
that cluster as F
(7) .
One complication was
that, although a [3Fe-4S] cluster was found in the
PsaC-C51D-PS I complex, it was not reduced by light(7) . This
was troubling, especially since the midpoint potential of the
[3Fe-4S] cluster in the free PsaC-C51D protein (E of -98 mV) should have allowed it to
function as an electron acceptor (8) . A further complication
was that a [3Fe-4S] cluster was not found in the mirror image
PsaC-C14D-PS I complex; instead, an unusual set of resonances around g
= 2.0 were found that were also unresponsive to light. In this
paper, the results of a comprehensive search for the [3Fe-4S]
clusters in a PS I complex reconstituted with PsaC-C51D are reported.
The ``missing'' [3Fe-4S] clusters were not located,
and it is suspected that in the earlier work they represented a
population of unbound PsaC-C51D that was incompletely removed from the
complex. Instead, a spin system was found that showed characteristics
of a [4Fe-4S] cluster in the mixed ligand F
site.
This new center, termed F
`, showed photochemical behavior,
spin relaxation properties, and a midpoint potential different from
F
in the wild-type PS I complex. The consequence of this
change on the low temperature photoreduction of F
` and
F
and on the room temperature photoreduction of
NADP
are reported.
The iron-sulfur clusters were reinserted into
the unbound C51D mutant according to a modification of published
procedures(10) . Briefly, the purified apoproteins were added
to a final concentration of 0.5 mg ml to a solution
of 50 mM Tris-HCl (pH 8.3) containing 1% (v/v)
-mercaptoethanol. After 5 min of stirring at room temperature, a
solution of 30 mM FeCl
was added slowly to a final
concentration of 0.15 mM. After an additional 5 min of
stirring, a solution of 30 mM Na
S was added slowly
to a final concentration of 0.15 mM. The reaction mixture was
incubated at 4 °C for 12 h. The reconstituted iron-sulfur protein
was purified free of inorganic reagents by gel filtration over Sephadex
G-25 in an anaerobic chamber (Coy Laboratory Products Inc.) and
concentrated under anaerobic conditions to a final protein
concentration of 20 mg ml
.
Reconstitution of the
PsaC-C51D mutant holoprotein with P700-F cores from Synechococcus sp. PCC 6301 was performed as
described(4, 10, 12) . Unbound PsaC and PsaD
were removed by gel exclusion chromatography over Sephadex G-75.
Figure 1:
EPR spectra of the PsaC-C51D-PS I
complex under different conditions of illumination. Spectra on the left (A, C, and E) were recorded
with the EPR resonator tuned to perpendicular mode; spectra on the
right (B, D, and F) were recorded with the
resonator tuned to parallel mode. The conditions were freezing in
darkness (A, B), illumination at 15 K minus freezing
in darkness (C, D), and freezing during continuous
illumination minus freezing in darkness (E, F). The
large resonance near 3450 G in panels C and E is the
photochemically generated P700 radical at g =
2.002. The g values of 2.067, 1.934, and 1.882 are diagnostic of the
F
cluster; the g values of 2.044, 1.942, and 1.853 are
diagnostic of the F
cluster. The 250-µl sample
contained 1 mg ml
chlorophyll, 300 µM
DCPIP, and 1 mM sodium ascorbate in 50 mM Tris
buffer, pH 8.3, containing 0.04% Triton X-100. Spectrometer conditions
were as follows: temperature 15 K; microwave power, 20 mW; microwave
frequency, 9.648 GHz (perpendicular mode), 9.340 (parallel mode);
modulation amplitude, 10 G at 100 kHz. The parallel mode signal was
amplified 2-fold in software.
To eliminate the possibility that [3Fe-4S]
clusters are present in the F site but are reduced under
these conditions, the PsaC-C51D-PS I complex was reexamined in the
presence of 5 mM potasssium ferricyanide and after
electrochemical poising to a solution potential of +400 mV. No
resonances were found at or near g = 2.02 that would indicate an
oxidized [3Fe-4S] cluster (data not shown). A reduced
[3Fe-4S] cluster is paramagnetic with an integer spin state
of S = 2 and can usually be observed as a broad,
asymmetric resonance at g values of 10-12 (the zero-field
splitting parameter must be smaller than the X-band microwave quantum).
The complex was examined after reduction with sodium dithionite at pH
10.0 and after electrochemical poising at a solution potential of
-650 mV. No resonances were found near g = 10-12
that would indicate the presence of a reduced [3Fe-4S]
cluster (data not shown). The complex was also examined using parallel
mode EPR spectroscopy, for which the transition probability of an S = 2 spin system is maximum when the alternating magnetic
field is polarized parallel to the static magnetic field. Provided this
``
M
= 2'' transition is
allowed, this mode provides an enhancement in the sensitivity of
integer spin systems as characterized by reduced [3Fe-4S]
clusters. As shown in Fig. 1B, a dark-frozen sample
showed no significant resonances at temperatures of 4.2-30 K in
any field position (the transition at 140 mT is due to contaminating
ferric iron). Likewise, there was no evidence for a reduced
[3Fe-4S] cluster around g = 10-12 when the
sample was illuminated at 15 K (Fig. 1D) or when the
sample was thawed and photoaccumulated by freezing during illumination (Fig. 1F; the resonance at g = 2.002 is due to
the strong P700
radical, which is observed as a weak
resonance due to imperfections in the dual mode cavity). The absence of
a signal under both oxidizing and reducing conditions demonstrates that
a [3Fe-4S] cluster is not present in the F
site
of the PsaC-C51D-PS I complex.
Figure 2:
Redox titration of the PS I complex
reconstituted with PsaC-C51D at 15 K. The spectra in the top part of the figure show the intensity of the g = 2.067, 1.934,
and 1.882 resonances of F when the potential was poised at
-530 mV (A), -580 mV (B), and -640
mV (C). The amplitude of the resonances as a function of
imposed potential shown in the bottom part of the figure for the g = 2.067, 1.934, and 1.882 resonances. The E
and n values are calculated by a least
squares algorithm to a linearized form of the data. The 400-µl
sample contained 1.2 mg/ml chlorophyll, 0.5 mM
4,4`-dimethyl-N,N`-trimethylene-2,2`-dipyridinium
dibromide, 0.5 mMN,N`-trimethylene-2,2`dipyridinium dibromide, and 0.5
mM 1,1`-dimethyl-4,4`-bipyridinium dichloride in 250 mM glycine buffer, pH 10.5. Spectrometer conditions were as follows:
microwave power, 20 mW; microwave frequency, 9.448 GHz; modulation
amplitude, 10 G at 100 kHz. The sample was kept in total darkness
throughout the entire procedure.
Figure 3: Temperature dependence of the EPR resonances in a PsaC-C51D-PS I complex when illuminated at 15 K. The 250-µl sample contained 1 mg/ml chlorophyll, 300 µM DCPIP, and 1 mM sodium ascorbate in 50 mM Tris buffer, pH 8.3. The sample was frozen in darkness and illuminated at 15 K. Spectrometer conditions: microwave power. 40 mW; microwave frequency, 9.448 GHz (standard resonator); modulation amplitude, 32 G at 100 kHz.
Fig. 4shows the EPR
spectrum of the PsaC-C51D-PS I complex when the sample was frozen
during illumination to photoaccumulate the electron acceptor system.
The amplitude of the F resonances at g = 2.067,
1.934, and 1.882 increase as the temperature is lowered from 32 to 18
K. This is followed by a decrease in amplitude as the temperature is
lowered further from 15 to 6 K due to the onset of microwave power
saturation. The F
cluster, seen most clearly in the high
field resonance at g = 1.769, achieves a maximum intensity at a
temperature of 6-8 K. A closer examination of the EPR spectrum of
the PsaC-C51D-PS I complex at very low temperatures revealed several
new features. A shallow peak at g = 2.089 and a broad highfield
trough at g = 1.847 begins to develop at a temperatures of 12 K,
becoming clearly visible only at a temperatures around 6 K. In the
midfield region, the g = 1.934 resonance from F
decreases in amplitude between 12 and 6 K, and a new
derivative-shaped resonance at g = 1.922 begins to appear as a
slight bend on this signal. These new features are most evident at 6 K,
where the contribution of the F
cluster is diminished.
Figure 4: Temperature dependence of the EPR resonances in a PsaC-C51D-PS I complex when illuminated at 298 K. The 250-µl sample contained 1 mg/ml chlorophyll, 300 µM DCPIP, and 1 mM sodium ascorbate in 50 mM Tris buffer, pH 8.3. The sample was illuminated during freezing to 6 K. Spectrometer conditions were as follows: microwave power, 40 mW; microwave frequency, 9.448 GHz (standard resonator); modulation amplitude, 32 G at 100 kHz.
A
plot of signal intensity versus temperature for the g =
1.934 and 1.882 resonances of F and for the g =
1.847 and 1.922 resonances of the new spin system indicates that the
relaxation behavior of F
, inferred from the temperature
dependence and half-saturation parameter P
(data
not shown), are similar to that of F
in the wild-type PS I
complex. The amplitude of the g = 1.769 resonance of F
is maximum at 6-8 K, similar to its behavior in the
wild-type complex. In contrast, the amplitude of the signals derived
from the new spin system increases as the temperature is lowered from
12 to 4.2 K. It is surprising that the F
cluster has not
changed under these conditions, i.e. that it does not
contribute to an interaction spectrum as seen in the wild-type complex
when F
and F
are reduced together in the same
reaction center. Instead, the pattern of reduction implies that two
spin systems are represented, one by the F
cluster, with a
temperature optimum of 18 K and the second by the presumed spectrum of
F
`.
Fig. 5A shows the midfield spectral region between 340
and 356 mT, where the intensity of the g = 1.922 resonance can
be measured in the absence of reduced F. The most
prominent feature is the relatively shallow slope of the derivative
resonance, denoting a broader line width, and the lack of the prominent
subtraction artifact. As depicted in Fig. 5A, the
intensity of the midfield F
` resonance increases when the
potential is lowered from -595 to -645 and to -690
mV. The end point of the titration was determined by thawing the most
reducing sample and photoaccumulating the acceptors by illuminating the
sample during freezing. The highfield resonance of F
` at g
= 1.847 is in an undisturbed spectral region, and its intensity
was determined directly (the low field resonance is in a region too
cluttered to allow an accurate determination). As shown in Fig. 5B the midfield feature at g = 1.922 and
the highfield trough at g =1.847 titrate at a midpoint potential
of -630 mV (n = 1). This value is considerably
more reducing than the midpoint potential of the F
cluster
in a wild-type spinach PS I complex (E
of
-520 mV).
Figure 5:
Redox titration of the PS I complex
reconstituted with PsaC-C51D at 4.2 K. The spectra in panel A show the intensity of the g = 1.922 resonances of
F` when the potential was poised at -595, -645,
and -690 mV. The fit of the intensity of the resonances as a
function of imposed potential is shown in panel B for the g
= 1.847 highfield and the g = 1.922 midfield resonances.
The E
and n values are calculated by the
best fit to the data. The 400-µl sample contained 1.2 mg/ml
chlorophyll, 0.5 mM
4,4-dimethyl-N,N`-trimethylene-2.2`-dipyridinium
dibromide, 0.5 mMN,N`-trimethylene-2,2`-dipyridinium dibromide, and
0.5 mM 1,1`-dimethyl-4,4`-bipyridinium dichloride in 250
mM glycine buffer, pH 10.5. Spectrometer conditions were as
follows: microwave power, 20 mW; microwave frequency, 9.452 GHz
(standard resonator); modulation amplitude, 10 G at 100 kHz. The sample
was kept in total darkness throughout the entire
procedure.
Figure 6: Time course of flash-induced absorbance changes in the near-IR. Panels A and B show absorption changes at 77 K, and panels C and D depict multiple flash experiments at room temperature. The complexes in panels A and B are suspended in media containing 25 mM Tris-HCl buffer, pH 8.3, and 60% glycerol, 10 mM sodium ascorbate, and 4 µM DCPIP. The flashes were separated by 10-s intervals. The absorbance changes are depicted for the first through eighth flashes, respectively. The data depict the result of a single measurement. The complexes in panels C and D were incubated with 100 µM PMS and approximately 3 mM sodium dithionite for 7 s in the dark prior to the onset of the flash train. The flashes were separated by 100 ms. The absorbance changes are depicted for the first through fourth flashes, respectively. The data depict the result of a single measurement.
Optical absorption studies were also carried
out at room temperature to determine whether F` and F
function as efficient electron acceptors during single turnover
flashes. The number of acceptors functioning subsequent to F
can be determined by the number of electrons that can be
stabilized for more than 1 ms on the acceptor side of PS I during a
series of saturating flashes(18) . The pattern of the
flash-induced absorbance changes in response to the first and the
second flashes arises from the donation of reduced PMS to
P700
(Fig. 6C). In the wild-type PS I
complexes, about 80-90% of the total amplitude is derived from a
component with a lifetime of 4-5 ms. In the PsaC-C51D-PS I
complex the lifetime is longer, perhaps because of deleterious effects
on the donor side due to the urea treatment. In response to the third
and fourth flashes, the decay of the absorption change becomes
considerably faster. In the wild-type, the components with lifetimes of
230 µs (43% on the third and 52% on the fourth flash) and
2-2.5 ms (54% on the third and 43% on the fourth flash) become
dominant. These components are most likely ascribed to back reactions
from A
and/or
F
. The remaining slower part of the decay
is approximated by a base line that probably corresponds to electron
donation from PMS to P700
. In the PsaC-C51D-PS I
complex (Fig. 6D), the components with lifetimes of 200
µs (29%) and 750 µs (41%) are dominant on the third flash, and
components with lifetimes of 65 µs (22%) and 410 µs (46%) are
dominant on the fourth flash. The two fast components are assumed to
result from back reactions of A
and/or
F
, although the F
back reaction is somewhat faster in the PsaC-C51D-PS I complex
than in the wild-type PS I complex. The remainder of the decay is
represented by a 5-ms component (
20% for both the third and fourth
flashes) and a base line. Were the F
` cluster
photochemically inactive at room temperature, the back reaction of
F
would override the PMS forward reaction
on the second flash. Instead, comparison of wild-type and PsaC-C51D
sets of kinetic data shows that both F
` and F
function as electron acceptors at room temperature.
Figure 7:
EPR spectrum of the iron-sulfur clusters
in the free PsaC-C51D protein. The [3Fe-4S] cluster and the [4Fe-4S]
cluster are reduced by the addition of sodium dithionite at pH
10. The wide field scan shows the S =
,
[4Fe-4S]
cluster near g = 2 and the S = , [4Fe-4S]
cluster at g
= 5.5; the S = 2, [3Fe-4S]
cluster is not present at g
= 12.
Spectrometer conditions were as follows: dual mode resonator, microwave
power, 80 mW; microwave frequency, 9.644 GHz; modulation amplitude, 10
G at 100 kHz, temperature, 4.2 K.
In an earlier paper(8) , it was shown that
substitution of aspartic acid in position 51 leads to the incorporation
of a high potential [3Fe-4S] cluster (E = -98 mV) in the modified site and a low potential
[4Fe-4S] cluster (E
= -580
mV) in the unmodified site of PsaC-C51D. On rebinding PsaC-C51D to a
P700-F
core, the g values of 2.063, 1.934, and 1.880, the
relatively narrow line widths, and the differential response to light
at 15 K and at 298 K allowed us to identify the [4Fe-4S]
cluster in the PsaC-C51D-PS I complex as F
(7) . In
the present work, the [3Fe-4S] cluster could not be located
in the reconstituted PsaC-C51D-PS I complex. Instead, a set of
resonances characteristic of F
were found that were
EPR-visible at 6 K when the sample was frozen in darkness and
illuminated at low temperatures. Although the highfield resonance at g
= 1.847, is similar to that of the reduced F
cluster
in the wild-type PS I complex (under conditions where there is no
magnetic interaction with F
), the line widths are somewhat
broader. Also, the temperature and power dependences imply a spin
relaxation rate more rapid than F
alone in the wild-type PS
I complex (and even more rapid than the F
cluster). It is
therefore likely that the [3Fe-4S] cluster observed in Zhao et al.(7) was due to a population of unbound
PsaC-C51D that was incompletely removed from the complex. The most
significant difference in protocol is that in this study, the
PsaC-C51D-PS I complex was purified by anaerobic gel filtration
chromatography to remove free PsaC-C51D; in the earlier study, the
sample was purified by repeated ultrafiltration over a 100-kDa cut-off
membrane.
The tacit assumption is that F` is a fast
relaxing, S =
, [4Fe-4S] cluster in
the aspartate-modified site. This is reasonable since there the
insertion of a rubredoxin-like single iron center or a ferredoxin-like
[2Fe-2S] cluster into a [4Fe-4S] or a
2[4Fe-4S] protein is probably unlikely. Also, the spin state
of a rubredoxin-like [Fe] center would likely be S = 5/2, and the temperature dependence of a ferredoxin-like
[2Fe-2S] cluster should allow EPR detection at temperatures
approaching 77 K. There is precedent for the insertion of an iron into
a [3Fe-4S] cluster; for example, the [3Fe-4S] and
[4Fe-4S] forms of Azotobacter vinelandii and Azotobacter chroococcum ferredoxins are interconvertible (21) . The reduced [3Fe-4S] cluster in the double
cluster ferredoxin III from Desulfovibrio africanus and in the
single cluster ferredoxin from Pyrococcus furiosus contains an
aspartic acid in the second cysteine position of the
CXXDXXCXXXCP motif and can incorporate an
iron to form an S = , [4Fe-4S]
cluster(22) . Evidence for the presence of a S = , [4Fe-4S] cluster that may likewise have been
derived from the insertion of iron into the [3Fe-4S] cluster
resident in the modified site in the free PsaC-C14D protein has been
obtained (see also (11) and (19) ). Our hypothesis is
that the [3Fe-4S] and S = ,
[4Fe-4S] clusters in the modified site of PsaC-C51D co-exist
in solution but that the P700-F
core selectively binds only
those proteins that contain two cubane clusters (see (11) ).
Further, the spin state of the majority population of the cluster must
change from S = to S =
on
binding and/or on reduction of F
, since the resonances of
F
and F
are visible in the g = 2 region.
In summary, the EPR and optical data show that electron transfer can
occur to a mixed ligand [4Fe-4S] cluster at both cryogenic
and room temperatures. The EPR resonances of the F` cluster
are visible in the g = 2 region when the measurement is made at
temperatures less than 6 K. The optical absorption measurements made at
room temperature indicate that there are two electron acceptors
functioning after F
, and the measurements made at low
temperature show that the same amount of P700
is
generated irreversibly as in the wild-type PS I complex under these
conditions. The room temperature optical data indicate that the
efficiency of electron transfer is largely unaffected by the change in
the reduction potential of F
` (E
= -630 mV) to a value 55 mV more reducing than
F
(E
= -575 mV). Rates of
NADP
photoreduction mediated by ferredoxin or
flavodoxin are also relatively unaffected by an F
that is
considerably more reducing than in wild-type spinach PS I (E
= -520 mV). The overall large
Gibbs free energy change between F
(E
-710 mV) and ferredoxin (E
-420 mV) is most likely be responsible for driving the reaction
against the changed reduction potential of F
`.