(Received for publication, July 3, 1996, and in revised form, November 20, 1996)
From the Department of Biochemistry and Center for
Biological Chemistry, University of Nebraska, Lincoln, Nebraska
68588-0664 and the Department of Energy Plant Research
Laboratory and ** Department of Biochemistry, Michigan State University,
East Lansing, Michigan 48824-1312
A psaC deletion mutant of the unicellular cyanobacterium Synechocystis sp. PCC 6803 was utilized to incorporate site-specific amino acid substitutions in the cysteine residues that ligate the FA and FB iron-sulfur clusters in Photosystem I (PS I). Cysteines 14 and 51 of PsaC were changed to aspartic acid (C14DPsaC, C51DPsaC, C14D/C51DPsaC), serine (C14SPsaC, C51SPsaC), and alanine (C14APsaC, C51APsaC), and the properties of FA and FB were characterized by electron paramagnetic resonance spectroscopy and time-resolved optical spectroscopy. The C14DPsaC-PS I and C14SPsaC-PS I complexes showed high levels of photoreduction of FA with g values of 2.045, 1.944, and 1.852 after illumination at 15 K, but there was no evidence of reduced FB in the g = 2 region. The C51DPsaC-PS I and C51SPsaC-PS I complexes showed low levels of photoreduction of FB with g values of 2.067, 1.931, and 1.881 after illumination at 15 K, but there was no evidence of reduced FA in the g = 2 region. The presence of FB was inferred in C14DPsaC-PS I and C14SPsaC-PS I, and the presence of FA was inferred in C51DPsaC-PS I and C51SPsaC-PS I by magnetic interaction in the photoaccumulated spectra and by the equal spin concentration of the irreversible P700+ cation generated by illumination at 77 K. Flash-induced optical absorbance changes at 298 K in the presence of a fast electron donor indicate that two electron acceptors function after FX in the four mutant PS I complexes at room temperature. These data suggest that a mixed-ligand [4Fe-4S] cluster is present in the mutant sites of C14X-PS I and C51X-PS I (where X = D or S), but that the proposed spin state of S = 3/2 renders the resonances undetectable in the g = 2 region. The C14APsaC-PS I, C51APsaC-PS I and C14D/C51DPsaC-PS I complexes show only the photoreduction of FX, consistent with the absence of PsaC. These results show that only those PsaC proteins that contain two [4Fe-4S] clusters are capable of assembling onto PS I cores in vivo.
There are several exceptions to the nearly universal occurrence of cysteine thiolate ligands to iron-sulfur clusters in the ferredoxin class of electron transfer proteins (1). Examples include the N-ligands from histidine residues in the Rieske subclass of iron-sulfur proteins (2-4), O-ligands from (most likely) aspartate in Fd III from Desulfovibrio africanus (5), and a proposed O-ligand from serine to the pentacoordinated iron in the P-cluster of nitrogenase (6). Recently, oxygen ligands have been introduced in lieu of cysteine thiolates by site-directed mutagenesis in proteins that contain [2Fe-2S], [3Fe-4S], and/or [4Fe-4S] clusters. Examples include the introduction of serine for cysteine in the interpolypeptide FX cluster of Photosystem I (PS I)1 (7, 8) and the [4Fe-4S] cluster of Escherichia coli fumarate reductase (9). The consequence of these changes include a change in the EPR spectrum and a decreased electron transfer efficiency in the case of serine-ligated FX and a change in the cluster midpoint potential and intercluster spin interaction in the case of E. coli fumarate reductase.
One instance of a functional [4Fe-4S] cluster derived from an aspartate-for-cysteine change is the modified PsaC subunit of PS I. In a previous study, E. coli expressed mutant PsaC proteins were reconstituted onto P700-FX cores, and the assignment of the ligands for the two terminal electron acceptors was determined by EPR spectroscopy (10). The substitution of aspartate for cysteine in position 14 of PsaC led to the retention of a S = 1/2, [4Fe-4S] cluster at the unmodified site with g values characteristic of FA. Similarly, the substitution of aspartate for cysteine in position 51 of PsaC led to the retention of a S = 1/2, [4Fe-4S] cluster at the unmodified site with g values characteristic of FB. Since the pattern of cysteine ligation in PsaC is expected to be identical to that of ferredoxins with two [4Fe-4S] clusters whose structures have been determined, it follows that FB is ligated by cysteines 11, 14, 17, and 58 and FA is ligated by cysteines 21, 48, 51, and 54 (10).
The in vitro reconstitution experiments led to several hypotheses regarding alternative ligands to iron-sulfur clusters: 1) unbound PsaC refolds only in the presence of one [3Fe-4S] and one [4Fe-4S] or two [4Fe-4S] clusters when cysteine is replaced in positions 14 and 51 by aspartate, serine, and alanine (11, 12). According to these results, a stable three-dimensional structure requires the presence of two iron-sulfur clusters, one of which must be a cubane. 2) The failure to observe a [3Fe-4S] cluster in the in vitro reconstituted C14XPsaC-PS I or C51XPsaC-PS I complexes (where X = D, A, or S) indicates that the binding of PsaC onto P700-FX cores requires the presence of two [4Fe-4S] clusters. If rigorously true, it may be difficult to introduce a [3Fe-4S] cluster motif into PsaC in vivo. 3) An oxygen ligand to the [4Fe-4S] cluster in the mutant site of the C14DPsaC-PS I and the C51DPasC-PS I complexes leads to a high-spin state (likely S = 3/2).
In this paper, these hypotheses were tested through the generation of a series of in vivo mutations in FA and FB. The choices to replace cysteine residues with aspartic acid, serine, and alanine were made because aspartate is known to support [3Fe-4S] and [4Fe-4S] clusters in naturally occurring ferredoxins (13, 14), serine can support [3Fe-4S] and [4Fe-4S] clusters in the FX binding site of PS I (7) and in the Azotobacter vinelandii hydrogenase small subunit (15), and alanine, with the absence of a suitable ligand, should support only [3Fe-4S] clusters. This paper summarizes the EPR spectral characteristics and electron transfer properties of aspartate (C14DPsaC, C51DPsaC, C14D/C51DPsaC), alanine (C14APsaC, C51APsaC), and serine (C14SPasC, C51SPsaC) mutations in PsaC in Synechocystis sp. PCC 6803. The premise tested is that [4Fe-4S] clusters will only be assembled in those mutants where oxygen ligands are available from the side chains of the replacement amino acids. A companion paper (16) describes the genetics, physiology, and electron transfer efficiency on the acceptor side in these in vivo PsaC mutants.
The protocols for the isolation of the cyanobacterial
thylakoid membranes and the purification of the n-dodecyl
-D-maltoside PS I complexes are described in Ref.
16.
Electron
paramagnetic resonance (EPR) studies were performed using a Bruker
ECS-106 X-band spectrometer equipped with either a standard resonator
(ER4102 ST) or a dual-mode resonator (DM/4116). Samples contained
either 0.5 mg ml1 (wild type) or 1 mg ml
1
(mutants) chlorophyll, 1 mM sodium ascorbate, 30 µM DCPIP in 50 mM Tris-HCl, pH 8.3. For
chemical reduction of the FA and FB clusters,
samples were suspended at a chlorophyll concentration of either 0.5 mg
ml
1 (wild type) or 1 mg ml
1 (mutants) in
250 mM glycine, pH 10, with 50 mM sodium
dithionite. For analysis of the P700+ cation, samples were
suspended at a chlorophyll concentration of 0.47 mg ml
1
in 50 mM Tris, pH 8.3, 30 µM DCPIP, and 200 µM sodium ascorbate. The spectra were taken at 77 K and
at microwave power levels that were in the square-law region and well
below saturation. The charge separation between P700 and
FA/FB is irreversible at this temperature during the time scale of the measurement (17). All data manipulations and graphics were performed using IGOR Pro (WaveMetrics, Lake Oswego,
OR). Simulations of the S = 1/2 EPR spectra were performed using
the "EPRSim" XOP for IGOR Pro 3.0 (recompiled by John Boswell, Oregon Graduate Institute), an adaptation of the FORTRAN program "QPOW" (18).
The number of
electron acceptors functioning after A1 was determined at
room temperature using a train of appropriately spaced sequential
single turnover flashes similar to that described in (19). In this
protocol, reduction of P700+ by reduced phenazine
methosulfate (PMS) overrides the faster reaction(s) involving
FX and/or A1
on the
first and second flashes, when the electrons are consequently stabilized on both FA and FB. On the third and
fourth flashes, the faster back reactions from
FX
and A1
with
lifetimes shorter than 1 ms become dominant. Hence, when two acceptors
function after FX, the kinetics for the second flash is
similar to that for the first, and the kinetics for a fourth flash is
similar to that for the third. In practice, the kinetics on the second
flash may contain some contribution from FX
due to double hits on the 10-µs duration xenon flash. This method, originally introduced by Sauer et al. (20) to determine the number of electrons acceptors in PS I, has been used to characterize PS
I complexes with FB destroyed by HgCl2
treatment (21, 22) in vitro mutants in the ligands to
FA and FB in PsaC (23, 24) and in
vivo mutants in the ligands to FA and FB
in PsaC (25).
The measurements were performed using the same spectrometer as in Ref.
26 except for a replacement of the measuring beam source with a 832-nm
laser diode (PMT25, Power Technology Inc., Mabelvale, AR). The output
power was 25 mW, and due to a higher stability of the output, the
reference channel was not used. Repetitive flashes at 15-ms intervals
were provided by a model 6100E-72 xenon flash lamp (Photochemical
Research Associates, London, Ontario, Canada) at full-width
half-maximum of 10 µs and flash energy of ~5 mJ. A fiber optic
light guide was used to direct the actinic beam to the cuvette in
direction prependicular to that of the measuring beam. The saturating
intensity of the flash was confirmed by using the neutral density
filters and by monitoring the A832 signal.
The samples were suspended in 20 mM MES buffer, pH 6.3, at
a chlorophyll concentration of 25 µg ml
1 with 0.05%
-DM, and PMS and sodium dithionite from freshly prepared solutions
were added to concentrations of 10 µM and 2 mM, respectively. The concentration of PMS was chosen to
assure re-reduction of P700+ prior to recombination with
FA
and/or FB
.
Sodium dithionite was used to completely reduce the PMS to prevent the
oxidized form from functioning as an electron acceptor. The optical
path length for the measuring light was 1 cm. The samples were
preincubated in the dark in the spectrometer for 4 min prior to flash
excitation.
For numeric analysis of the data we have made a simultaneous global fit
of all four kinetics to the sum of two exponentials with common
lifetimes and common initial amplitude (assuming that the amount of
P700+ formed on each flash should be the same upon almost
complete relaxation of the absorbance change produced by the preceding flash). For each set of four kinetics we have derived the initial amplitude of the absorbance change, the lifetime of the slow component accounting for P700+ reduction by reduced PMS, the lifetime
of the fast component accounting for P700+ reduction by
FX and A1
, and a
set of four amplitudes of the slow component. In general, this model
gave a high quality fit in the millisecond time domain (where the
kinetics is mostly governed by the slower PMS component), but in some
cases the deviations in the sub-millisecond time domain could be higher
due to the presence of two exponentially decaying components in this
time domain.
When PS I
complexes from a mutant expressing C14DPsaC were frozen in
darkness and illuminated at 15 K, the EPR spectrum showed a S = 1/2 ground state iron-sulfur cluster with g values of 2.047, 1.945, and
1.851 (Fig. 1, left column). The resonances, which are
diagnostic of FA, do not diminish in intensity in a
subsequent period of darkness. The temperature dependence of the
resonances at 18 K (not shown), 15 K (Fig. 1A), 12 K (Fig.
1B), 9 K (Fig. 1C) to 6 K (Fig. 1D)
indicates a maximum signal intensity at 15 K, similar to the wild type,
although the resonances are present over a much broader and lower range
of temperatures. (The trough between 365 and 370 mT may represent the
midfield resonance of FX; the low-field trough around 390 mT is barely visible). There is no indication of a S = 1/2 ground
state iron-sulfur cluster characteristic of reduced FB in
the g = 2 spectral region.
Resonances characteristic of FA were also present in the g = 2 spectral region when PS I complexes from the a mutant expressing C14DPsaC were frozen during illumination (Fig. 1, right column). Under these conditions, more than one electron is promoted to the acceptor system. The intensity of the FA resonances increase from 15 K (Fig. 1E), to 12 K (Fig. 1F), to 9 K (Fig. 1G), and become maximum at <6 K (Fig. 1H). This is a temperature behavior different from conditions where only one electron is promoted (compare Fig. 1, B and D with F and H), indicating that the FA cluster is relieved of power saturation, most likely by the presence of a nearby spin system. One candidate is reduced FX, which is observed at temperatures <9 K (see Fig. 1, G and H) as a broad, rhombic spectrum with midfield and high-field resonances at g = 1.852 and 1.755 (the low-field peak is obscured by the g = 2.044 resonance of FA). However, it has been shown in a PS I complex which lacks FB that reduced FX does not influence the microwave power or temperature dependence of FA (27). Assuming that no changes are introduced other than creating an empty site in FB, these data indicate that FX does not influence the spin relaxation properties of FA. The implication is that the enhanced relaxation rate of FA is due to the presence of reduced FB, which we propose is present as a high-spin (likely S = 3/2) [4Fe-4S] cluster in the mutant site. At this time, it is only possible to infer the presence of FB, since we have not located a low-field resonance around g = 5.5 which could be attributed to a highly rhombic S = 3/2 spin system. High-spin iron-sulfur clusters are difficult to observe when the g anisotropy is distributed over a large spectral range. Note also that there is also an apparent broadening of the g = 1.852 resonance at 6 K (Fig. 1H), but this is probably due to overlap from the midfield resonance of FX.
CysteineThe PS I
complexes from the C14SPsaC mutant have EPR spectral
properties that are similar, although not identical, to those for the
C14DPsaC-PS I complex. When the PS I complexes from a mutant expressing the C14SPsaC mutant were frozen in
darkness and illuminated at 15 K, the EPR spectrum was characteristic
of the FA cluster with g values of 2.045, 1.944, and 1.854 (Fig. 2, left column). There is no indication
of reduced FB in the g = 2 spectral region. The
FA resonances are present over a narrower range of
temperatures than in C14DPsaC-PS I, increasing in intensity from 15 K (Fig. 2A), becoming maximum at 12 K (Fig.
2B) and decreasing from 9 K (Fig. 2C) through 6 K
(Fig. 2D). This temperature behavior is similar than that of
FA in the wild type cyanobacterial PS I complex, indicative
of an enhanced spin relaxation mechanism at this site.
Resonances characteristic of FA are found with the same temperature behavior when PS I complexes from the mutant C14SPsaC are frozen during illumination (Fig. 2, right column). The FX cluster is visible in the 9 K (Fig. 2G) and 6 K spectra (Fig. 2H), with a high-field resonance at g = 1.761 and a midfield feature around 365 mT. The latter is likely to be responsible for some, but not all, of the features around 360 mT in the 6 K spectrum. There is evidence at 12 K (Fig. 2F, arrows) and 9 K (Fig. 2G, arrows) for additional resonances at g = 2.102 and 1.883, and for a broader line width of the high-field resonance at g = 1.846. Except for the temperature dependence, these resonances are strikingly similar to those observed in the PS I complexes containing C51SPsaC (see below).
CysteineWhen PS
I complexes from a mutant expressing C51DPsaC were frozen
in darkness and illuminated at 15 K, the EPR spectrum showed a set of
resonances characteristic of FB, with g values of 2.067, 1.931, and 1.880 (Fig. 3, left column). The
intensity of the resonances are weak, but are formed irreversibly. The
maximum signal intensity occurs at 15 K (Fig. 3A), with
lower intensities being observed at 12 K (Fig. 3B), 9 K
(Fig. 3C), and 6 K (Fig. 3D). This temperature optimum is similar to that of FB in the wild type complex.
However, a large amount of irreversible P700+ is generated;
this rules out charge separation with FX or A1 (discussed below).
The spectrum is considerably more complex when the
C51DPsaC-PS I complex is frozen during illumination (Fig.
3, right column). As the temperature is lowered from 15 K
(Fig. 3E) to 12 K (Fig. 3F), the FB
resonances remain dominant with a temperature optimum identical to that
of a sample illuminated at 15 K. At temperatures of 9 K (Fig.
3G) and 6 K (Fig. 3H), FB diminishes
due to microwave power saturation, but at 6 K a broad peak at g = 2.096, a change in the slope of the midfield feature at g = 1.925, and a high-field trough at g = 1.843 become visible (Fig.
3H, arrows). These features are assumed to be derived from a
mixed-ligand cluster, termed FA, in the modified site. The
maximum signal intensity of the modified FA
cluster occurs
at temperatures between 4.2 and 6 K, a value which is lower than the 15 K optimum of FA in a wild type complex. The FX
cluster is also seen at 6 K, with its high-field trough at g = 1.76 and a midfield resonance between 350 and 360 mT, which is obscured
by the presence of resonances from FA and/or FB.
When PS I
complexes from a mutant expressing C51SPsaC were frozen in
darkness and illuminated at 15 K, the EPR spectrum was characteristic
of FB, with g values of 2.067, 1.930, and 1.879 (Fig.
4, left column). There is no indication of
reduced FA in the S = 1/2 spectral region. The
intensity of the FB resonances are also weak, but again,
are formed irreversibly. The maximum signal intensity occurs at 15 K
(Fig. 4A), becoming progressively weaker at 12 K (Fig.
4B), 9 K (Fig. 4C), and 6 K (Fig. 4D).
The maximum at 15 K is similar to that of FB in the wild
type complex. Because of the large amount of P700+ radical
generated, we again surmise that the FA cluster is present and functional, but that it is not visible in the g = 2 region when FB is oxidized in the same reaction center. In terms
of g values, temperature dependence and spectral lineshape, the
FB cluster in the C51SPsaC-PS I mutant is
identical to that in the C51DPsaC-PS I mutant.
A stronger set of resonances characteristic FB are found
when the C51SPsaC-PS I complex is frozen during
illumination (Fig. 4, right column). The signal attains
maximum intensity at 15 K (Fig. 4E) and decreases at 12 K
(Fig. 4F) due to the onset of microwave power saturation;
below 9 K (Fig. 4, G and H), the resonances are
within the noise. At 9 K (Fig. 4G) and 6 K (Fig.
4H) high-field resonance of the FX cluster can
be observed at g = 1.756, but the low-field and mid-field
resonances are obscured by other peaks. At temperatures of 6 K and
lower (Fig. 4H), a new set of features at g = 2.117, 1.916, and 1.837 become visible, these are attributed to a mixed-ligand
FA cluster in the modified site. Their maximum signal
intensity occurs at
4.2 K, a temperature lower than that of
FA in the wild type PS I complex. The spectral g values and lineshapes are similar to those the in vivo
C51DPsaC-PS I complex; a set of common features includes
the temperature dependence of the FB resonances, the
appearance of new resonances at very low temperatures, and the apparent
lack of magnetic interaction between the FB cluster
observed at 15 K and the new spin system(s) observed at lower
temperatures.
If FB in the
C14DPsaC-PS I and C14SPsaC-PS I complexes and
if FA in the C51DPsaC-PS I and
C51SPsaC-PS I complexes are present as a high spin
iron-sulfur clusters, then the relative spin concentrations of
FA and FB in the non-mutant sites should be
equivalent to that in the wild type. One obvious consequence is that
the digital summation of the C14XPsaC-PS I
spectrum and the C51XPsaC-PS I spectrum (where
X = D or S) should yield a wild type spectrum. This is
shown in Fig. 5A for C14DPsaC-PS
I (data from Fig. 1A) and C51DPsaC-PS I (data
from Fig. 3A), where, under conditions where there is no
magnetic interaction between FA and FB, the resulting admixture of FA and FB is identical
to a wild type PS I complex (Fig. 5B). The relative spin
concentrations of FA and FB in the
C14DPsaC-PS I and C51DPsaC-PS I complexes were
determined by double integration of the simulated spectra. As shown in
Fig. 5C, FA (dotted line) can be
accurately simulated assuming g values of 2.047, 1.945, and 1.851 and
line widths of 33, 21, and 17 MHz and FB (dashed
line) can be simulated assuming g values of 2.067, 1.931, and
1.880 and line widths of 50, 32, and 33 MHz. Using these parameters,
the composite spectrum of FA/FB in the wild type and in the digitally summed mutant spectra can be reproduced assuming a 3.17 ratio of FA to FB (solid
line).
A second consequence is that the P700+ cation radical
should also be equivalent in spin concentration in the wild type,
C14XPsaC-PS I, and
C51XPsaC-PS I complexes (where X = D or S), regardless of the spin concentration of FA or
FB seen in the g = 2 region. Fig. 6
shows the P700+ radical in a sample after freezing in
darkness and illumination at 77 K (dotted line) and in a
subsequent period of darkness (solid line). The former
corresponds to the charge separated state of P700+
(FB/FA
plus
FX
), and the latter represents the permanent
charge separated state of P700+
(FB
/FA
). In wild
type PS I complexes, charge separation is not quantitative, but is
progressively induced in a series of laser flashes up to a maximum of
two-thirds of the reaction centers (28). Hence, the amount of
irreversibly formed P700+ radical paired with
FA and FB in the wild type PS I complex is less
than the amount seen on continuous illumination. As shown in Fig. 6,
the amount of irreversible P700+ generated in the
C14XPsaC-PS I and
C51XPsaC-PS I complexes is roughly equivalent,
yet the spin concentration of FB is only 24% that of
FA (Fig. 5C). This strongly implies that the
majority of irreversible P700+ is paired with a missing
spin system in the C51DPsaC-PS I complex, presumably the
high-spin FA cluster. Similarly, there is a smaller amount
of irreversible P700+ paired with a missing spin system in
the C14DPsaC-PS I complex, presumably the high-spin
FB cluster. One final detail is that there is a greater
amount of irreversible P700+ formed in the
C14SPsaC-PS I and C51SPsaC-PS I complexes than in the wild type and aspartate mutants, but this could be due to a
greater efficiency of forward electron transfer from FX to FA/FB at cryogenic temperatures in the serine
mutants.
Cysteine
Western blots showed the absence of
PsaC (as well as PsaD and PsaE) on the C14APsaC-PS I and
C51APsaC-PS I complexes (16). From this result, it was
anticipated that when these complexes were frozen in darkness and
illuminated at 9 K, the EPR spectrum would be devoid of any resonances
characteristic of FA or FB. As expected, a set
of broad resonances characteristic of FX and not
FA or FB were found with g values of 2.070, 1.877, and 1.773 and at a temperature optimum of 9 K (data not shown).
When the C14APsaC-PS I and C51APsaC-PS I
complexes were frozen under illumination, the FX resonances
are present at a ~2-fold higher spin concentration (Fig.
7, A and B).
Cysteines 14 and 51 to Aspartic Acid: C14D/C51DPsaC-PS I
A double replacement, with aspartic acid in positions 14 and 51, was generated in an attempt to localize two mixed-ligand [4Fe-4S] clusters in PsaC, one in the FA site and the other in the FB site. The prediction, based on the in vitro reconstitutions of PsaC onto P700-FX cores (12), was that the C14D/C51DPsaC-PS I double mutant would not contain two [3Fe-4S] clusters; rather it would contain one [3Fe-4S] cluster and one low-spin [4Fe-4S] cluster in a nearly stoichiometric ratio (data not shown). Hence, the absence of two [4Fe-4S] clusters should preclude binding of PsaC in the in vivo mutant. Indeed, immunoblots showed that no PsaC was assembled in the in vivo C14D/C51DPsaC-PS I mutant complex (16). The light-induced EPR spectrum showed reduced FX, with g values of 2.054, 1.873, and 1.779 when a dark-frozen C14D/C51DPsaC-PS I complex was illuminated at 9 K (not shown). When the double mutant was frozen under illumination, the FX resonances are present at a higher spin concentration (Fig. 7C).
Number of Acceptors Functioning in the C14XPsaC-PS I and C51XPsaC-PS I ComplexesEPR studies provide indirect evidence for the existence of two acceptors, FA and FB, functioning at low temperature in the C14XPsaC-PS I and C51XPsaC-PS I complexes (where X = D or S). To investigate the number of electron acceptors functioning at room temperature, the kinetics of P700+ reduction were monitored at 832 nm upon excitation with trains of four consecutive flashes in the presence of a fast donor, PMS. To establish the validity of the experiment, a wild type and a HgCl2-treated PS I complex were first analyzed. The HgCl2-treated PS I complex (isolated with Triton X-100) has been studied by low-temperature EPR spectroscopy and NADP+ reduction protocols and shown to be 95% depleted in FB while retaining FX and FA. As a control for the mutants we have studied the DM-PS I complex from Synechocystis sp. 6803, which gave virtually the same results with respect to the derived fit parameters and their dependencies on the flash number as the Triton X-100-PS I from Synechococcus sp. 6301 (26). A matrix of conditions showed that a flash interval of 15 ms is optimal for all samples; for the control sample a 10-s interval between flash trains is sufficient for complete recovery of the four-flash kinetic pattern, but in the case of HgCl2-treated sample even a longer interval is insufficient, leading to a less distinct difference between the first and second kinetics.
When this protocol is applied to the wild type PS I complex (Fig.
8A and Table I), a slow
component with a lifetime of 3.3 ms was found which constitutes almost
100% of initial amplitude on the first flash and about 71% on the
second flash, whereas on the third and the fourth flashes its
contribution is less than 40%. When applied to the
HgCl2-treated sample, the contribution of the slow
component on the first flash is only 36%, which is consistent with a
lower efficiency of forward electron transfer from FX in
the absence of FB (see Ref. 26). The amplitudes of the slow
component in the HgCl2-treated sample on the second, third,
and fourth flashes are remarkably similar, contributing ~23% to the
total amplitude of the absorbance change. In qualitative compliance
with previous findings (21), the equality of the second and third
kinetics of P700+ recovery upon repetitive flash excitation
is considered an important criterion (along with the EPR data) for the
absence of one cluster on PsaC.
Kinetics of absorbance changes at 832 nm
(A). A, PS I complex from wild type
Synechocystis sp. PCC 6803, an average of eight four-flash
trains applied to the same sample at 10-s intervals; B,
HgCl2treated PS I complex from wild type Synechocystis sp.
PCC 6803, an average of two four-flash trains applied to different samples; C, C14DPsaC-PS I complex; D,
C51DPsaC-PS I complex; E, C14SPsaC-PS I complex; F,
C51SPsaC-PS I complex. Samples C-F involve an
average of two four-flash trains applied to different samples. The
experimental protocol involved excitation of PS I complexes with trains
of four consecutive flashes in the presence of 10 µM PMS
and 2 mM sodium dithionite and two-exponential fits of the
kinetics. The experimental data are depicted as the dotted line; the multiexponential fit is overlaid as a solid
line. The parameters of the fit are summarized in Table I.
|
The kinetics on a single flash in the presence of the slow donor,
reduced DCPIP, showed that three of the PsaC complexes
(C14DPsaC-PS I, C51DPsaC-PS I, and
C14SPsaC-PS I) have a distinct backreaction from reduced
[FA/FB] in the
tens-of-milliseconds time scale, whereas C51SPsaC-PS I has
a very low amplitude of the
[FA/FB]
backreaction, and
C14APsaC-PS I, C51APsaC-PS I, and
C14D/C51DPsaC-PS I are devoid of this component (16).
Hence, we performed the multiple flash experiments only with the former
four mutant PS I complexes. As seen from Fig. 8, C-F and
Table I, all four PS I mutant complexes show a significant difference
between the amplitudes of the slow component appearing on the second
and the third flash. Namely, the percentage of the third amplitude
relative to the second makes up 75, 58, 70, and 80% for
C14DPsaC-PS I, C51DPsaC-PS I,
C14SPsaC-PS I, and C51SPsaC-PS I, respectively.
The same parameter makes up 52% for the wild type and 97% for the
HgCl2-treated sample. This makes the mutant PS I complexes
distinctly different from the FB-less HgCl2
sample and demonstrates the availability of two functional electron
acceptors in these complexes.
The following generalizations can be made about the C14XPsaC-PS I and C51XPsaC-PS I mutants (where X = D, S, and A). First, only [4Fe-4S] clusters are found in the in vivo mutant PS I complexes, even though the unbound PsaC mutants contain [3Fe-4S] and mixed-ligand [4Fe-4S] clusters in the altered site (11, 12). The PsaC protein does not assemble in the in vivo mutants for which cysteines 14 and 51 are substituted with alanine, C14APsaC, and C51APsaC, most likely because a [4Fe-4S] cluster cannot assemble in the absence of a suitable ligand. The absence of an oxygen or sulfur ligand from the replacement amino acid, alanine, and the resulting destabilizing effect of a [3Fe-4S] cluster on PsaC binding to the P700-FX heterodimer are apparently the reasons for the inability of the C14APsaC and C51APsaC mutants to bind to PS I. Second, all mutant PS I complexes that contain mixed-ligand [4Fe-4S] clusters are capable of electron transfer to FA and FB at 15 K. These mutant complexes are also capable of supporting electron throughput to NADP+ at room temperature. This is also true for PS I complexes reconstituted in vitro with C14DPsaC (23) and C51DPsaC (24). Third, the results of the in vivo experiments agree with the cysteine ligand assignments to FA and FB made using in vitro reconstitution of E. coli expressed proteins onto P700-FX cores (10).
Although the optical results provide a qualitative, rather than
quantitative, estimate of the efficiency of the FA and
FB photoreduction, it is clear that both acceptors are
photochemically active in C14DPsaC-PS I,
C51DPsaC-PS I, C14SPsaC-PS I, and
C51SPsaC-PS I complexes and that they may operate with
lower quantum efficiencies than in the wild type. However,
heterogeneity due to incomplete binding of PsaC would give rise to the
same kinetics in single turnover experiments as does forward electron
transfer inefficiency. To this end, we have taken care to isolate
near-homogeneous PS I complexes from the thylakoids based on the
different densities of PS I reaction centers with and without PsaC,
PsaD, PsaE, and PsaL. Nevertheless, low-temperature optical kinetic
measurements, single-turnover EPR experiments, and quantitative
enzyme-linked immunosorbent assays are being pursued to unambiguously
distinguish between inefficient electron transfer and sample
heterogeneity. The lower amount of FA/FB
photoreduction is also manifest in lower contribution of the
P700+ reduction from reduced PMS to the overall
A832 relative to the back-reaction(s) from
FX
and A1
, even on
the first flash. We note, however, that the C51SPsaC mutant
shows both a decreased contribution of the
FA/FB backreaction on a single flash in the
presence of a slow electron donor (16) and a less distinct difference
between the kinetics upon multiple flash excitation in the presence of
a fast donor (Table I).
The similarities
and the differences between the in vivo engineered and
in vitro reconstituted C14DPsaC-PSI and
C51DPsaC-PS I complexes depend on whether the mutation is
in the FA or the FB site. Under conditions of
photoaccumulation, the in vivo C51DPsaC-PS I
complex is similar in spectral appearance to E. coli-expressed C51DPsaC-PsaC reconstituted in
vitro onto P700-FX cores (23). These similarities
indicate that oxygen from aspartate (alternatively, water or
OH) provide the ligands to FA in the modified
site. The slight differences in the g values between the in
vivo and in vitro PS I complexes may be related to
species differences (29), since the in vivo C51DPsaC-PS I mutant PS I complexes were derived from
Synechocystis sp. PCC 6803, whereas the in vitro
mutant PS I complexes were hybrids, composed of a
Synechococcus sp. PCC 6301 P700-FX core, PsaC
and PsaE derived from Synechococcus sp. PCC 7002, and a PsaD protein derived from Nostoc sp. PCC 8009 (30). Under
conditions where only one electron was promoted to the acceptor side,
only a low spin concentration of the FB cluster was
observed in the in vivo and in vitro mutants; yet
the size of the P700+ radical indicates that the majority
of the electrons were promoted to the proposed high-spin FA
cluster. However, a very low spin concentration of FA was
also observed in the in vitro mutant, which may have been
derived from a minority population of a S = 1/2 cluster. One
candidate is a sulfur thiolate ligand derived from carryover of the
-mercapatoethanol used in the reconstitution protocol as suggested
in Ref. 31.
In contrast, the in vivo C14DPsaC-PS I complex
differs substantially from the in vitro reconstituted
C14DPsaC-PS I complex. In the in vivo mutant,
the FB cluster is not observed in the g = 2 region,
but is inferred from the size of the P700+ radical and the
presence of new EPR resonances at very low temperatures which may be
derived from intercluster spin interaction between FA and
FB. The proposal is that the FB cluster is
present as a high-spin system (likely S = 3/2). A search for the
expected g = 5 to 6 low-field resonance of a S = 3/2 cluster
failed; however, because of the broad anisotropy of a highly rhombic
spin system, along with its detection as a first derivative, the
resonances may be too weak to be detected at these sample
concentrations. In the in vitro mutant, the FB
cluster was observed as a ground state S = 1/2 spin system with g
values of 2.118, 1.911, and 1.883. The difference in the in
vivo engineered and in vitro assembled C14DPsaC-PS I complexes is that only oxygen ligands are
available in the former, whereas sulfur thiolate ligands may also be
available in the latter. The -mercaptoethanol used in the in
vitro iron-sulfur reinsertion protocol may have been recruited as
a ligand at the mutated site of the in vitro complex (31).
It is likely that sulfur provides a better ligand to an iron-sulfur
cluster, replacing some or all of the oxygen-ligated cluster in the
in vitro experiments. In this mutant, FB may be
present as a mixed population of sulfur-ligated S = 1/2 clusters
visible in the g = 2 region, and oxygen-ligated S = 3/2
clusters invisible in the g = 2 region. The fraction of sulfur-
and oxygen-ligated clusters may have more to do with steric hindrance
or accessibility to solvent than with inherent differences in the
C14DPsaC and C51DPsaC sites of PsaC.
When the psaC gene is interrupted in C. reinhardtii, neither the PS I reaction center subunits nor the small polypeptides accumulate in the thylakoid membranes of the transformants (32). In this eukaryotic organism, PsaC appears to be an essential component for the stable assembly of PS I.
When the psaC gene is interrupted in A. variabilis, the PS I reaction center accumulates (33), but PsaC,
PsaD, and PsaE are missing (34). Using site-directed mutagenesis,
cysteine 13 (equivalent to Cys-14 in Synechococystis sp. PCC
6803) and cysteine 50 (equivalent to Cys-51 in
Synechococystis sp. PCC 6803) were changed in
vivo to aspartic acid in A. variabilis (25). The
authors of this work argue that since the former mutant lacks an EPR
spectrum characteristic of FB, yet the organism grows at wild type rates and reduces NADP+, this cluster is
dispensable. A comparison of the respective EPR spectra, however, shows
striking similarities between the mutants in A. variabilis
and Synechocystis sp. PCC 6803. In both organisms, under
conditions of photoaccumulation, the FA cluster in the
C14DPsaC mutants appears identical to the wild type, while the FB cluster is not observed. Under similar conditions,
the FB cluster in the C51DPsaC mutant also
appears identical to the wild type; however, the FA cluster
can be observed at low temperatures. The FA cluster is
strikingly similar in the two organisms, showing a large midfield
derivative resonance around g = 1.94 consisting of two closely
spaced resonances (best visible in Synechocystis sp. PCC
6803) and possibly derived from magnetic coupling between FB and FA. We argue from the spin relaxation
data and from the spin concentration of P700+ generated
irreversibly at low temperatures that the FB cluster in the
C14DPsaC mutant is present, but that a spin state of S = 3/2 renders it undetectable in the g = 2 region.
Whereas the optical results in Synechocystis sp. PCC 6803 concerning the availability of two electron acceptors in the
C51DPsaC mutant (C50D in A. variabilis) agree
with (25), results on the C14DPsaC mutant (C13D in A. variabilis) do not. In the A. variabilis mutant, the
contribution of the slow (PMS-driven) component to the absorbance
change on the third flash was considered to be relatively close to that
on the second flash; the difference between the second and third traces
was attributed to a variable amount of FA
oxidation during the relatively long intervals between the flashes (50 ms). However, this interval is quite comparable with the intrinsic lifetime of P700+
[FA/FB]
, making it difficult to
determine whether this small difference is solely the result of
FA
oxidation or the result of a second
photochemically active cluster on PsaC. It is obvious that the C13D
mutant has a very high contribution of a phase decaying faster that 500 µs, which may be related to either an inefficiency in the electron
transfer from FX to the PsaC-bound cluster(s) or
heterogeniety of the sample. Our C14DPsaC-PS I complex (16)
shows a similar inefficiency (or a heterogeneous population), making it
necessary to optimize the PMS concentration and flash and dark
intervals to differentiate the kinetics between the successive
flashes.
The inability to engineer a PsaC protein with either a missing cluster or a [3Fe-4S] cluster in vitro in PsaC from Synechocystis sp. PCC 7002 (12) or in vivo in Synechocystis sp. PCC 6803 (this work) is further evidence that two [4Fe-4S] clusters must be present for PsaC to bind to the PS I core. This result is consistent with in vitro reconstitutions of E. coli-expressed mutant PsaC proteins, where only those proteins with two intact [4Fe-4S] clusters could be rebound onto P700-FX cores (31). There could, of course, be species-dependent differences in the behaviors of the filamentous A. variabilis and the unicellular Synechocystis sp. PCC 6803, but the similarity in the amino acid sequence of PsaC in the two organisms argues against this interpretation.
Magnetic Interaction between Mixed-ligand and All-cysteine FA and FB Iron-Sulfur ClustersThe
photoaccumulated spectra of the mutant PsaC complexes contain
additional features which raise several interesting issues. In the
C14DPsaC-PS I complex, the temperature behavior implies the
presence of an independent spin system which is not visible in the
g = 2 region. In the C14SPsaC-PS I complex, additional spectral features appear at very low temperatures, which imply that
this second missing spin system may have crossed over from the proposed
S = 3/2 state to the S = 1/2 state. Yet, this new spin system
does not appear to be magnetically coupled to the FA
resonances which are seen at higher temperatures. In the
C51DPsaC-PS I complex, only the FB resonances
are seen at temperatures of 15 K and higher, but additional spectral
features become apparent (Fig. 3H, arrows) at very low
temperatures, which do not appear to be spin-coupled to FB.
Yet, the spectrum is complex, including the presence of two resonances
in the mid-field region around 350 mT, suggesting that this may
represent spin-spin interaction between FA
and FB
. Another possibility is that altered g
values of the modified cluster might lead to a spectrum that appears
similar to an interaction spectrum. Nevertheless, the presence of new
spectral features at very low temperatures in the
C51SPsaC-PS I complex indicates the presence of a second
spin system in addition to FA. The presence of the missing
spin system under conditions where only one electron is promoted (Figs.
1, 2, 3, 4, left column) becomes manifest under conditions where
more than one electron is promoted (Figs. 1, 2, 3, 4, right
column). The salient issue is that there are more experimental
features than can be accounted for than by a straightforward magnetic
interaction of FA and FB.
The studies presented here demonstrate that mixed-ligand [4Fe-4S] clusters can assemble in the PsaC protein of PS I in vivo and that PS I complexes containing such mixed-ligand clusters can bind to PS I core complexes and function in electron transfer reactions from P700 to ferredoxin or flavodoxin. The efficiency of iron-sulfur center insertion into the mutant proteins, or the stability of mutant proteins after cluster insertion, varies depending upon the chemical nature of the side group on the replacement amino acid. Differences observed between the spectroscopic properties of PS I complexes containing mutant PsaC proteins formed by in vitro or in vivo methods are most likely due to the chemical nature of the ligands to the [4Fe-4S] clusters. The common denominator is that only those PsaC proteins which contain two [4Fe-4S] clusters are capable of assembling onto P700-FX cores either in vivo or in vitro.