(Received for publication, April 6, 1995; and in revised form, August 3, 1995)
From the
Cysteines 14, 21, 34, 51, or 58 in PsaC of photosystem I (PS I)
were replaced with aspartic acid (C21D and C58D), serine (C14S, C34S,
and C51S), and alanine (C14A, C34A, and C51A). When free in solution,
the C34S and C34A holoproteins contained two S= ground
state [4Fe-4S] clusters; all other mutant proteins contained
[3Fe-4S] clusters and [4Fe-4S] clusters; in
addition, there was evidence in C14S, C51S, C14A, and C51A for high
spin (S = ) [4Fe-4S] clusters, presumably in
the modified site. These findings are consistent with the assignment of
C14, C21, C51, and C58, but not C34, as ligands to F and
F
. The [4Fe-4S] clusters in the unmodified sites
in C14S, C51S, C14A, and C51A remained highly electronegative, with E
values ranging from -495 to -575 mV.
The [3Fe-4S] clusters in the modified sites were driven 400
to 450 mV more oxidizing than the native [4Fe-4S] clusters,
with E
values ranging from -98 mV to
-171 mV. A C14D/C51D double mutant contains [3Fe-4S]
and S= [4Fe-4S] clusters, showing that the
3Cys
1Asp motif is also able to accommodate a low spin cubane.
When C34S, C34A, C14S, C51S, C14A, and C51A were rebound to
P700-F
cores, electron transfer to F
/F
was regained, but functional reconstitution has not yet been
achieved for C21D, C58D, or C14D/C51D. These data imply that PsaC
requires two iron-sulfur clusters to refold, one of which must be a
cubane. Since two [4Fe-4S] clusters are found in all
reconstituted PS I complexes, the presence of two cubanes in free PsaC
may be a necessary precondition for binding to P700-F
cores.
PsaC in photosystem I (PS I) ()shares similarities
with a number of soluble 2[4Fe-4S] ferredoxins in terms of
molecular mass, amino acid sequence, and the presence of two
CXXCXXCXXXCP iron-sulfur cluster binding
motifs. The protein has greatest similarity to the amino acid sequence
of Chromatium vinosum ferredoxin, for which no
three-dimensional structure is available. However, there is enough
sequence homology with the 54-amino acid 2[4Fe-4S] ferredoxin
from Peptococcus aerogenes(1) and the first 58
residues of the 106-amino acid
[3Fe-4S]
[4Fe-4S] ferredoxin from Azotobacter vinelandii(2) to make predictions of
tertiary structure based on the known three-dimensional structure of
these proteins (Fig. 1). Assuming that the overall folding of
the PsaC polypeptide backbone is analogous to these known structures,
cysteines 11, 14, 17, and 58 should provide the ligands to one
[4Fe-4S] cluster, and cysteines 21, 48, 51, and 54 should
provide the ligands to the second [4Fe-4S] cluster. The two
clusters in PsaC should also be related by a pseudo-2-fold axis of
symmetry, so that cysteines 11, 14, 17, and 58 correlate with cysteines
48, 51, 54, and 21, respectively.
Figure 1:
Backbone
structure of PsaC based on modeling studies with P. aerogenes (adapted from (19) and (20) ) with the proposed
assignment of cysteines based on experimental findings in (5) .
The similarity between the two proteins is highlighted by the x-ray
crystal structure of the cyanobacterial PS I reaction center, which
shows that the distance between the iron-sulfur clusters is 12 Å,
identical to the intercluster distance in P. aerogene ferredoxin. An extra 10 amino acids are required between the two
iron-sulfur binding motifs, and an extra 14 amino acids are required on
the C terminus for accurate alignment. The cysteines chosen for
site-directed mutagenesis (C14D, C14S, C14A, C21D, C51D, C51S, C51A,
and C58D) are identified; in addition, a double and triple mutant
(C14D/C51D and C14D/C51D/C34S) were generated. Cysteine 34 lies in the
loop region between cysteines 21 and 48. Iron-sulfur clusters F and F
were identified (5) by their g values
and response to illumination in C14D- and C51D-rebound PS I
complexes.
The sequences of naturally
occurring ferredoxins suggest modifications that may be tolerated in
PsaC. For example, several naturally occurring ferredoxins contain
aspartic acid at the position analogous to cysteine 14 in PsaC. These
proteins are capable of supporting a mixed spin (S =
and ) [4Fe-4S
3Cys1Asp] cluster in the modified
site(3, 4) . Using site-directed mutagenesis, mutant
PsaC proteins have been constructed with similar mutations (PsaC-C14D
and PsaC-C51D), and the free proteins were found to contain
[3Fe-4S] clusters at the modified sites and
[4Fe-4S] clusters at the unmodified
sites(5, 6) . When rebound to PS I cores, the g values
and reduction behavior of the [4Fe-4S] clusters in the
unmodified sites indicated that F
is ligated by residues
11, 14, 17, and 58 and that F
is ligated by residues 21,
48, 51, and 54(5) . A ninth cysteine is located at position 34
in all PsaC proteins sequenced thus far, and based on the structural
analogy with P. aerogenes, this residue is not predicted to be
involved in ligation of either cluster.
The goal of this study is to
determine the effect of cysteine replacements on the refolding of PsaC
and on the rebinding of modified PsaC proteins to P700-F cores. Mutant PsaC proteins were constructed in which one of the
cysteine ligands to an iron-sulfur cluster was changed to the charged
amino acid aspartate (PsaC-C14D, previously reported in (6) ,
and PsaC-C51D, in the accompanying paper(36) ), the polar amino
acid serine (PsaC-C14S and PsaC-C51S), or the neutral amino acid
alanine (PsaC-C14A and PsaC-C51A). Modifications not previously known
in ferredoxins (PsaC-C21D and PsaC-C58D) were also made to determine
whether a [3Fe-4S] cluster could be accommodated when the
proline-proximal cysteine in either cluster-binding motif is altered.
In an effort to determine whether cysteine 34 is a ligand to a cubane
iron, serine (PsaC-C34S) and alanine (PsaC-C34A) mutations were
constructed and characterized at this site. Finally, novel double and
triple mutants containing one aspartate in both iron-sulfur cluster
binding sites (PsaC-C14D/C51D and (PsaC-C14D/C51D/C34S) were
constructed. The mutant proteins were studied for their ability to
ligate a [3Fe-4S] or a [4Fe-4S] cluster, to rebind
to P700-F
cores, and to reestablish electron transfer to
F
and F
.
Purified PsaC was refolded with iron-sulfur clusters as
described (7) and purified by ultrafiltration in an Amicon cell
over a YM-5 membrane (Amicon, Beverly, MA) with 50 mM Tris-HCl, pH 8.3, and 0.1% -mercaptoethanol. For
electrochemistry, the PsaC holoproteins were desalted, and the
-mercaptoethanol was removed by gel filtration chromatography over
a Sephadex G-25 (Pharmacia Biotech Inc.) column under anaerobic
conditions in a Coy controlled environment chamber (Grass Lake, MI).
Figure 2:
EPR spectra of the oxidized
[3Fe-4S] (dotted line) and reduced
[4Fe-4S] (solid line) clusters in free PsaC-C14S (A), PsaC-C51S (B), PsaC-C14A (C) and
PsaC-C51A (D) mutant proteins. The proteins were oxidized by
brief exposure to air at pH 8.3 and reduced in the presence of sodium
dithionite at pH 10.5. Signal intensity was measured in a matrix of
temperature and microwave power to determine the temperature optimum
and half-saturation parameter, P, for each
cluster. Spin quantitation was performed at the temperature optimum and
at power settings an order of magnitude below the P
value. The vertical axis shows signal intensity with the
reduced [4Fe-4S] cluster scaled arbitrarily to unity spin
concentration; scaling factors of 0.25 (A), 2.0 (B),
1.0 (C), and 0.25 (D) were applied to the
[3Fe-4S] clusters. Spectrometer conditions were as follows:
standard mode resonator, microwave power, 10 mW for the
[4Fe-4S] clusters, 1 mW for the [3Fe-4S] clusters;
microwave frequency, 9.456 GHz; modulation amplitude, 10 G at 100 kHz;
temperature, 15 K for the [4Fe-4S] clusters and 35 K for the
[3Fe-4S] clusters.
The temperature optimum of the [3Fe-4S] clusters in
PsaC-C14S, PsaC-C14A, PsaC-C51S, and PsaC-C51A is 30 K (determined at 5
mW of microwave power). The temperature optima of the
[4Fe-4S] clusters in PsaC-C14S and PsaC-C51S are 12 and 9 K,
and in PsaC-C14A and PsaC-C51A they are 12 and 15 K (determined at 20
mW of microwave power). The overall pattern is that the presence of a
charged, polar, or hydrophobic amino acid result in small differences
in the spin relaxation properties, as inferred from the temperature
optimum and half-saturation parameter P, of the
[3Fe-4S] clusters in the modified sites or in large changes
in the relaxation properties of the [4Fe-4S] clusters in the
unmodified sites (data not shown).
To demonstrate that the
[3Fe-4S] clusters are indeed present in these
proteins (i.e. that chemical reduction does not result in
their destruction), the four mutant proteins were analyzed by
perpendicular and parallel mode EPR. A reduced [3Fe-4S] is
detectable because it is paramagnetic with a ground state spin S= 2, and can be observed in normal mode EPR as a
single, asymmetric resonance at g = 10-12, which extends
into zero field. Under mildly oxidizing conditions, we were unable to
detect any significant resonances in PsaC-C14A, PsaC-C51A, PsaC-C14S,
or PsaC-C51S between 0 and 100 mT (data not shown). When the samples
were treated with sodium dithionite at pH 10.5 to reduce both the
[3Fe-4S] and [4Fe-4S] clusters, the reduced
[3Fe-4S]
clusters were easily observed around g
= 10-12 at high microwave powers and at very low
temperatures (Fig. 3, solid lines). The reduced
PsaC-C14D (34) and PsaC-C51D (15) mutant proteins also
show a single asymmetric resonance around g = 10-12, which
tails toward the low field, with broadening into zero field. When
analyzed by parallel mode EPR (Fig. 3, dotted lines),
the resonances around g = 10-12 from the integer S= 2 [3Fe-4S]
clusters are
enhanced and sharpened, and the resonances around g = 2 due to
the half-integer [4Fe-4S]
clusters have
disappeared (data not shown).
Figure 3:
Perpendicular (solid line) and
parallel mode (dotted line) EPR studies of reduced
[3Fe-4S] clusters in free PsaC-C14S (A), PsaC-C51S (B), PsaC-C14A (C), and PsaC-C51A (D) mutant
proteins. The samples were reduced with sodium dithionite in 330 mM glycine buffer, pH 10.5, containing 0.67% -mercaptoethanol.
The resonance at 160.5 mT is probably due to a small amount of
octahedrally coordinated iron that has remained in the oxidized state.
The vertical axis shows signal intensity, with the reduced
[3Fe-4S] cluster scaled arbitrarily to unity spin
concentration; the comparison depicts relative intensity of the
reduced, high spin [4Fe-4S] cluster. Spectrometer conditions
were as follows: dual mode resonator, microwave power, 80 mW; microwave
frequency, 9.647 GHz (perpendicular mode), 9.349 GHz (parallel mode);
modulation amplitude, 10 G at 100 kHz; temperature, 4.2
K.
This signal is also present, albeit at a
reduced intensity, when the altered amino acid contains a hydrophobic
side group such as alanine in PsaC-C14A and PsaC-C51A (Fig. 3, C and D). The intensity of the g 5.5 resonances
are considerably weaker in the alanine mutants, but the g value, line
shape, and temperature dependence are similar to the aspartate and
serine mutants. The implication is that a ligand has been recruited
from other than the replacement amino acid to occupy the fourth
coordination site of the iron, a good candidate being water, hydroxide,
or the free thiolate from
-mercaptoethanol present in the
reconstitution mixture. The occurrence of high spin [4Fe-4S]
clusters in the free PsaC proteins is relevant to the finding (see
below) that mutant proteins that are rebound to the P700-F
core contain two [4Fe-4S] clusters.
Figure 5: EPR spectrum of reduced [4Fe-4S] clusters in free PsaC-C34S, wild-type PsaC, and PsaC-C34A. The clusters were reduced by the addition of a minimal amount of sodium dithionite to a solution of free protein at 2.5 mg/ml in 250 mM glycine, pH 10.5. The g values are not identified due to the broad line widths of the resonances. The magnetic fields differ in the two spectra due to the use of the standard and dual mode resonators; the g values of the principal features of the resonances are nearly equal. Spectrometer conditions were as follows: standard mode resonator; microwave power, 10 mW; microwave frequency, 9.456 GHz; modulation amplitude, 10 G at 100 kHz (A); dual mode resonator; microwave power, 10 mW; microwave frequency, 9.647 GHz; modulation amplitude, 10 G at 100 kHz (B); dual mode resonator; microwave power, 10 mW; microwave frequency, 9.646 GHz; modulation amplitude, 10 G at 100 kHz (C).
The
redox titration of the mutant proteins does not have this complication,
since the [3Fe-4S] cluster is EPR-visible in the g = 2
region only when oxidized, and the [4Fe-4S] cluster is
EPR-visible in the g = 2 region only when reduced. As shown in Fig. 4, A and C, the [4Fe-4S]
clusters in the unmodified sites of PsaC-C51S and PsaC-C14S titrate
according to a well-behaved Nernstian response with midpoint potentials
of -495 mV (n = 1.0) and -520 mV (n = 0.98), respectively. The clusters in the unmodified sites
have been driven alternately more oxidizing and more reducing than the
comparable clusters in PsaC-C51D (E =
-580 mV; (6) ) and PsaC-C14D (E
= -515 mV; (6) ). As shown in Fig. 4, B and D, the [3Fe-4S] clusters in the
modified sites of PsaC-C51S and PsaC-C14S titrate with midpoint
potentials of -145 mV (n = 1.0) and -132 mV (n = 1.0), respectively. The replacement serine drives
the potential of the [3Fe-4S] clusters in the modified sites
more reducing than the comparable clusters in the PsaC-C51D and
PsaC-C14D proteins (E
= -98 mV; (6) ). Thus, in the serine series of mutants, the relative
potentials of the [4Fe-4S] clusters are inverted, making the
serine substitutions of particular interest for functional studies
should this inversion of potential be maintained when the mutant
proteins are rebound to P700-F
cores.
Figure 4:
Redox
titration of the [4Fe-4S] (panels A and C)
and [3Fe-4S] clusters (panels B and D) in
free PsaC-C51A (A and B, open squares),
PsaC-C51S (A and B, filled circles),
PsaC-C14A (C and D, open squares), and
PsaC-C14S (C and D, closed circles) mutant
proteins. The titrations were carried out by electrochemical poising at
pH 9.0. The end points of the titration were determined in the presence
of excess potassium ferricyanide and sodium dithionite at pH 10.5 (not
shown). The precision of the measurement is shown in the depiction of
C51S, which was performed on three independent samples reconstituted at
three different occasions. The individually determined values are E = -493, n = 1.01 (squares); E
= -493 mV, n = 0.97 (circles); E
= -491 mV, n = 1.02 (triangles). Spectrometer conditions were as follows: standard
mode resonator, microwave power, 10 mW for the [4Fe-4S]
clusters, 1 mW for the [3Fe-4S] clusters; microwave
frequency, 9.455 GHz; modulation amplitude, 10 G at 100 kHz;
temperature, 15 K for the [4Fe-4S] clusters and 35 K for the
[3Fe-4S] clusters.
The substitution
of alanine for cysteine was also studied, with the expectation that a
hydrophobic amino acid would also affect midpoint potential of the
iron-sulfur clusters. As shown in Fig. 4, A and C, the [4Fe-4S] clusters in PsaC-C14A and PsaC-C51A
titrate with midpoint potentials of -551 mV (n =
0.98) and -575 mV (n = 0.94), respectively. As
shown in Fig. 4, B and D, [3Fe-4S]
clusters in PsaC-C51A and PsaC-C14A titrate with midpoint potentials of
-167 mV (n = 0.96) and -171 mV (n = 1.04). Thus, in the alanine series of mutants, the
midpoint potentials of the [3Fe-4S] clusters are more
reducing than in the aspartate and serine mutants. The midpoint
potentials of the [4Fe-4S] clusters in the unmodified sites
of PsaC-C14D (E = -515 mV) and
PsaC-C51D (E
= -580 mV) proteins were
previously shown to be similar to F
and F
in
wild-type PsaC (Table 1).
When the iron-sulfur
clusters were inserted into the purified PsaC-C34S apoprotein, the EPR
spectrum showed no detectable [3Fe-4S] clusters under mildly
oxidizing conditions (not shown). When the PsaC-C34S protein was
reduced with sodium dithionite at pH 10.5 (Fig. 5A), the EPR
spectrum showed a rhombic signal with broad line widths and with the
characteristic splitting observed in proteins that contain two
[4Fe-4S] clusters(26) . Indeed, the g values and line
widths are nearly identical to those of the [4Fe-4S] clusters
representing F and F
in free PsaC (Fig. 5B). The clusters in PsaC-C34S and in PsaC have
similar but not quite identical relaxation properties, as depicted by
the a slight change in line shape as the temperature is lowered from 30
to 5 K (data not shown). Cysteine 34 was also replaced with alanine
(PsaC-C34A), an amino acid incapable of providing a ligand to an iron.
The EPR spectrum of the holoprotein showed no [3Fe-4S]
clusters in the oxidized state, but a rhombic spectrum in the reduced
state. As with PsaC-C34S, the g values and line widths are nearly
identical to those of the [4Fe-4S] clusters representing
F
and F
in free PsaC (Fig. 5C).
The EPR spectroscopic properties of the free PsaC-C34S and the
PsaC-C34A proteins are consistent with the proposal that cysteine 34
does not ligate an iron. This assessment is supported by the wild-type
EPR spectral properties of F
and F
in
Photosystem I complexes reconstituted with PsaC-C34S and PsaC-C34A (see
below).
Fig. 6A shows the EPR spectrum of
PsaC-C14D/C51D in the presence of sodium ascorbate. The presence of a
peak at g = 2.02 and a trough at g = 1.98 indicates the
presence of oxidized [3Fe-4S] clusters. When the sample is
reduced with dithionite at pH 10.5, a rhombic EPR signal is observed
with broad line widths in the g = 2 region that is virtually
identical to the low spin [4Fe-4S] cluster seen in the single
mutants. This could indicate the presence of one type of cluster
located in the F site and another type of cluster
located in the F
site, or alternatively there may be no
site differentiation. Independent of the issue of site distribution of
the two types of clusters, a mixed ligand 3Cys
1Asp site in PsaC
is clearly capable of supporting a cubane. The only distinction is that
the spin state of the [4Fe-4S] cluster in the double mutant
is S=
, while the spin state of the mixed
ligand [4Fe-4S] clusters in the single mutants is S= . The likelihood is that the double mutant cannot
support two [3Fe-4S] clusters; the implication is that the
minimum requirement for a stable protein is the presence in PsaC of at
least one cubane cluster.
Figure 6: EPR spectra of the oxidized [3Fe-4S] (dotted line) and reduced [4Fe-4S] (solid line) clusters in free PsaC-C14D/C51D (A), PsaC-C14D/C51D/C34S (B), PsaC-C21D (C), and PsaC-C58D (D) mutant proteins. The proteins were oxidized by brief exposure to air at pH 8.3 and reduced in the presence of sodium dithionite at pH 10.5. The vertical axis shows signal intensity, with the reduced [4Fe-4S] cluster scaled arbitrarily to unity spin concentration; no attempt was made to scale the spin concentration of the [3Fe-4S] clusters relative to the [4Fe-4S] clusters. Spectrometer conditions were as follows: standard mode resonator, microwave power, 10 mW for the [4Fe-4S] clusters, 1 mW for the [3Fe-4S] clusters; microwave frequency, 9.458 GHz; modulation amplitude, 10 G at 100 kHz; temperature, 15 K for the [4Fe-4S] clusters and 35 K for the [3Fe-4S] clusters.
One potential complication is that the
nonligating cysteine in position 34 may have been recruited to function
as the ligand to one of the two iron-sulfur clusters in the
PsaC-C14D/C51D double mutant. A ligand rearrangement has been reported
to occur in A. vinelandii ferredoxin I (AvFdI), where C20A is
rearranged in the region of the [4Fe-4S] cluster to allow it
to use the free cysteine 24 as a replacement ligand(28) . A
triple mutant of PsaC was therefore constructed to additionally
substitute cysteine 34 with serine: PsaC-C14D/C51D/C34S. By invoking
reasoning similar to that for the PsaC-C34S single mutant (see above),
were this serine to be recruited to provide a ligand to an iron, the g
values and line widths of the [4Fe-4S] cluster should be
sufficiently different to distinguish it from the wild type. As shown
in Fig. 6B, the EPR spectrum of the triple mutant in
the presence of sodium ascorbate shows the presence of a
[3Fe-4S] cluster and, after the addition of sodium dithionite
at pH 10.5, the presence of an S= ,
[4Fe-4S] cluster. There are no significant differences in the
g values, line shapes, temperature optima, or half-saturation
parameters between the double and triple mutants (data not shown).
These data verify that both high and low spin [4Fe-4S]
clusters can be supported in a mixed ligand 3Cys
1Asp environment
in PsaC.
The EPR
spectra of the PsaC-C21D and PsaC-C58D mutant proteins are shown in Fig. 6, C and D. In the presence of sodium
ascorbate, both proteins show a peak at g = 2.02 and a trough at
g = 1.998, characteristic of an oxidized [3Fe-4S]
cluster. When PsaC-C21D and PsaC-C58D were reduced with sodium
dithionite at pH 10.5, the [3Fe-4S] clusters disappeared and
a rhombic EPR signal with broad line widths appeared, which is
characteristic of a reduced [4Fe-4S] cluster. The maximum
signal intensity of the [4Fe-4S] clusters in PsaC-C21D and
PsaC-C58D was achieved at 15 K when measured at 10 mW of power, and the
half-saturation parameter (P) was also similar
to [4Fe-4S] clusters in the other mutant proteins. These
results indicate that it is possible to create a ferredoxin containing
a [3Fe-4S] cluster by replacing the fourth as well as the
second cysteine in each of the two
CXXCXXCXXXCP motifs. A corollary to this
finding is that in addition to cysteines 14 and 51, cysteines 21 and 58
also provide ligands to the cubane clusters in PsaC. Since neither
PsaC-C21D nor PsaC-C58D rebind to the P700-F
core under
conditions appropriate for PsaC (shown in Fig. 7), the spectral
and redox properties of these proteins were not studied further.
Figure 7:
Spectroscopic evidence for the rebinding
of PsaC-C34S and PsaC-C34A to P700-F cores. The mutant PsaC
proteins were rebound to P700-F
cores in the presence of
excess PsaD for 12 h and purified as described under ``Materials
and Methods.'' For optical studies (A), the solution
contained 15 µg/ml chlorophyll, 30 µM dichlorophenol-indophenol, and 0.1 mM sodium ascorbate in
50 mM Tris-HCl, pH 8.3. The onset of the flash is depicted by
the arrow; this is followed by a 10-µs instrumental dead
time due to the lifetime of the xenon flash and then by the decay of
the P700
radical. The measurements were made at 698 nm
as described under ``Materials and Methods.'' For EPR
studies, the samples were concentrated to 500 µg/ml chlorophyll,
100 µM dichlorophenol-indophenol, and 1 mM sodium
ascorbate in 50 mM Tris-HCl, pH 8.3. The spectrum was recorded
after freezing the reconstituted PsaC-C34S-PS I complex (B)
and the reconstituted PsaC-C34A-PS I complex (C) during
illumination. Spectrometer conditions were as follows: standard
resonator in panel B at microwave frequency of 9.444 GHz; dual
mode resonator in panel C at microwave frequency of 9.645 GHz;
microwave power, 20 mW; modulation amplitude, 10 G at 100 kHz,
temperature, 15 K. The resonances occur at different field positions in panels B and C due to the resonant frequencies of the
standard mode and dual mode cavities.
When
PsaC-C14A, PsaC-C14S, PsaC-C51A, and PsaC-C51S were combined with
P700-F cores and analyzed by room temperature optical
kinetic spectroscopy, all four mutant reaction centers supported long
lived charge separation to F
/F
. Similarly, all
reconstituted PS I complexes showed electron transfer to (the
equivalent of) F
and F
when analyzed by low
temperature EPR spectroscopy. The detailed electron transfer properties
of these mutant reaction center complexes will be reported elsewhere. (
)
The EPR spectroscopic properties of the iron-sulfur clusters
in free and PS I-rebound PsaC proteins were determined after the
introduction of charged (aspartate), polar (serine), and hydrophobic
(alanine) amino acids in the second position and after the introduction
of a charged (aspartate) amino acid in the fourth position, of each
CXXCXXCXXXCP motif (c.f.Fig. 1). The following generalizations can be made when the
second cysteine of the motif is changed. First, a [3Fe-4S]
cluster can be found in the oxidized protein and is assumed to be
resident in the modified site. The midpoint potentials of the
[3Fe-4S] clusters are 400 mV more oxidizing than the
wild-type [4Fe-4S] clusters, and the spin relaxation
properties, inferred from the temperature dependence and
half-saturation parameter, show differences among the three classes of
amino acids. Second, a low spin [4Fe-4S] cluster is found in
the reduced protein and is assumed resident in the unmodified site. The
replacement amino acids, which include those with charged side groups,
polar groups, and a hydrophobic side group, show no consistent pattern
in affecting the reduction potential of the cluster in the unmodified
site (Table 1). Third, high spin [4Fe-4S] clusters are
tentatively identified in the PsaC-C14S, PsaC-C51S, PsaC-C14A, and
PsaC-C51A reduced proteins and are assumed resident in the modified
site. High spin [4Fe-4S] clusters have also been tentatively
identified in free C14D-PsaC (15) and free C51D-PsaC (see
accompanying paper(36) ).
One likely origin of the high spin (S = ) [4Fe-4S] cluster is the conversion of
a reduced [3Fe-4S] cluster as described previously in Peptococcus furiosus(3) and Desulfovibrio
africanus ferredoxins(4) , which contain similar
CXXDXXCXXXCP motifs. Alternately, the
[3Fe-4S] clusters may be derived from the loss of an iron
from an oxidized [4Fe-4S] cluster, which may have been
inserted in vitro as an intact cubane into the mixed-ligand
site of the free PsaC protein. These are the limiting cases; there may
well exist a dynamic equilibrium between the [3Fe-4S] and
[4Fe-4S] clusters through insertion and loss of an iron
controlled by redox potential. While the presence of a high spin
[4Fe-4S] cluster was not surprising in the aspartate (or
serine) mutants, it was not expected in the alanine mutants. The issue
is whether the aspartate and/or serine oxygens provide ligands to the
cubane iron as does aspartate in P.
furiosus(29, 30) , or whether water, hydroxide,
or the thiolate from -mercaptoethanol present in the
reconstitution mixture, has contributed the fourth ligand. In this
respect, it is interesting that only those mutant proteins that show
evidence for a high spin [4Fe-4S] cluster in the mutant site
are capable of reconstituting onto P700-F
cores.
An
additional new finding is that the PsaC-C14D/C51D double mutant does
not contain two [3Fe-4S] clusters; rather it contains
[3Fe-4S] and low spin [4Fe-4S] clusters in a nearly
stoichiometric ratio (data not shown). In light of the above data on
the single mutants, it is intriguing that two [4Fe-4S]
clusters are not present in the PsaC-C14D/C51D double mutant. The
presence of both types of clusters in the PsaC-C14D/C51D double mutant
is in agreement with the premise that PsaC must contain at least one
cubane cluster for stability. It is also interesting that an earlier
attempt to introduce two [3Fe-4S] clusters into A.
vinelandii Fd I also failed(28) . This protein normally
contains one [3Fe-4S] and one [4Fe-4S] cluster.
When a cysteine ligand of the [4Fe-4S] cluster was altered, a
nearby cysteine was recruited as a replacement ligand, resulting in a
protein with structural modifications but still containing one
[3Fe-4S] and one [4Fe-4S] cluster. In an attempt to
minimize the chance that cysteine 34 provides a ligand to one of the
clusters in PsaC-C14D/C51D, the triple mutant PsaC-C14D/C51D/C34S was
constructed. This mutant does not contain a recruitable cysteine
residue; hence, the [4Fe-4S] cluster must reside in a mixed
ligand site. Nevertheless, serine cannot be dismissed as a potential
ligand since it supports a [2Fe-2S] cluster with modified
optical and EPR properties in a site-directed mutant of ferredoxin (31, 32, 33) and NADH-quinone oxidoreductase (34) and a low spin [4Fe-4S] cluster with modified
EPR properties in a site-directed mutant of the interpolypeptide
F cluster in PS I(23, 24) . However, the g
values, line widths, temperature optimum, and P
values of the PsaC-C14D/C51D/C34S mutant were identical to those
of the PsaC-C14D/C51D double mutant, in agreement with the proposal
that cysteine 34 is not a ligand to a cubane iron in this instance.
The detection of a high spin [4Fe-4S] cluster in the C14X and C51X (where X represents A, D, or S) single mutants provides a precedent for the presence of a [4Fe-4S] cluster in the C14D/C51D double mutant. The only significant difference is the S = spin state of the [4Fe-4S] cluster in the single mutants and the S = spin state of the [4Fe-4S] cluster in the double mutant. It should be noted that P. furiosus ferredoxin contains a single, mixed ligand [4Fe-4S] cluster that exists in both S = (20%) and S = (80%) ground states(3) . The cross-over energy between the high and low spin states is likely to be small; indeed, ethylene glycol and urea change the spin state in the A. vinelandii ferredoxin(35) . One factor appears to be related to the degree of exposure to solvent, a consideration that may be relevant in the spin state changes that have been postulated to occur between the free and PS I-rebound PsaC-C51D(15) .
In summary, these studies indicate that PsaC refolds only in the presence of two iron-sulfur clusters when cysteine is replaced in positions 14 and 51 by aspartate, serine, and alanine, and in positions 21 and 58 by aspartate. Since there was no occurrence of a mutant PsaC that contained only one [3Fe-4S] cluster, only one [4Fe-4S] cluster, or two [3Fe-4S] clusters, a corollary to the above premise is that a stable three-dimensional structure may require that two conditions be met: 1) two iron-sulfur clusters are present, and 2) one of the two clusters is a cubane. The inability to observe a [3Fe-4S] cluster in the C14D/C51D-PS I complex implies that it may be difficult to introduce this type of motif into PsaC in vivo. In light of photosynthesis, two points can be made. First, the important feature for electron transfer is that the mixed ligand, [4Fe-4S] clusters in reconstituted PsaC-C14D-PS I(34) , PsaC-C14S-PS I, PsaC-C14A-PS I, PsaC-C51D-PS I(15) , PsaC-C51S-PS I, and PsaC-C51A-PS I complexes are capable of accepting electrons at both cryogenic and room temperatures. Second, a checkpoint for the assembly of PsaC onto PS I cores may be that the free PsaC protein must contain two [4Fe-4S] clusters.