From the Department of Biochemistry and Molecular
Biology, Pennsylvania State University, University Park, Pennsylvania
16802, the ¶ Department of Biochemistry, University of Nebraska,
Lincoln, Nebraska 68588, and the § Department of Energy
Plant Research Laboratory, Michigan State University,
East Lansing, Michigan 48824-1312
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ABSTRACT |
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The FX electron acceptor in
Photosystem I (PS I) is a highly electronegative
(Em = Iron-sulfur clusters are widely represented in various proteins
and multiprotein complexes and, in the majority of instances, are
involved in electron transfer reactions. Cubane ([4Fe-4S]) clusters
are 1-electron carriers that function between two of the three
oxidation states, 2+/1+ or 3+/2+ (for review, see Ref. 1). The midpoint
potentials of the near-geometrically identical [4Fe-4S] clusters are
modulated by different protein environments and can range from +350 to
Unlike the non-heme iron that bridges the two subunits of the
quinone-type reaction centers (represented by Photosystem II and the
reaction centers of purple bacteria) and that does not participate in
the electron transfer, FX serves as an indispensable component of the electron transfer chain of PS I (6, 7). According to
our current understanding, light-induced electron transport between the
PS I-bound cofactors follows a linear path: P700 (Chl
a dimer) A comparison of the FX domain among eukaryotes and
prokaryotes shows that the cysteine ligands on PsaB are located in a
nearly invariant region of 100 amino acids that surround and include helices VIII and IX (8) (helices j (or j') and
k (or k') in current nomenclature (9)). An
additional interesting feature of FX is that an aspartate
residue is adjacent to each conserved cysteine on PsaB
(PCDGPGRGGTCD) and adjacent to the
proline-proximal cysteine on PsaA (PCDGPGRGGTCQ). According
to the 4.5-Å electron density map, the FX cluster is
located just inside the membrane region of the stromal side of two
membrane-spanning The effects of ligand substitution on the properties of a variety of
iron-sulfur clusters were recently reviewed (10). The ability to
substitute oxygen for sulfur ligands has been demonstrated in PS I
reaction centers by substitution of serines for the cysteine ligands to
FX with serines in Synechocystis sp. PCC 6803. This change did not prevent the assembly of PS I or the low temperature photoreduction of FA or FB (11). The
mixed-ligand [4Fe-4S] clusters in the FX site of
C565SPsaB and C556SPsaB (12) were found to be
relatively inefficient in transferring electrons from A1 to FA/FB (13). These studies showed that although
serine could serve as a potential ligand, the stability of the cluster
and the efficiency of electron transfer were decreased relative to the
wild type. The FX region has also been altered in
Chlamydomonas reinhardtii using site-directed mutagenesis.
Among the findings were that the proline-cysteine motif on PsaB in this
organism is not essential for assembly of the PS I reaction center
(14); the reaction center polypeptides PsaA and PsaB do not accumulate in the D562NPsaB and R566EPsaB mutants (15);
and the D576LPsaA mutant fails to grow photoautotrophically
(16).
To test the hypothesis that the conserved cysteine-adjacent aspartate
acid residues play a role in modulating electron transfer through
FX, we used site-directed mutagenesis to change each
cysteine-proximal aspartic acid residue on PsaB of
Synechocystis sp. PCC 6803 to the positively charged amino
acid lysine (D566KPsaB and D557KPsaB) and to
the neutral amino acid alanine (D566APsaB and
D557APsaB). Because D566APsaB showed a large
change in the EPR spectrum of FX, we combined two
single-site mutants to create the double-site mutants alanine/alanine
(D557APsaB/D566APsaB) and lysine/alanine (D557KPsaB/D566APsaB). We measured
photoautotrophic growth, chlorophyll content, electron transfer
throughput from cytochrome c6 to ferredoxin and
flavodoxin, efficiency of electron transfer to
FA/FB on a single-turnover flash, and EPR
spectral properties of FX in the single and double
aspartate mutants.
Site-directed Mutagenesis--
Oligonucleotides were designed to
effect the desired change in the coding sequence of psaB
while also destroying or adding a restriction site. Site-directed
mutations were made with an Amersham Pharmacia Biotech in
vitro mutagenesis system. Incorporation of mutations was verified
by restriction mapping and by DNA sequencing, and the mutated
variations of pLS3531 (11) were then used to transform strain
Growth of Cells, Oxygen Evolution Rates, and Chlorophyll Content
in Whole Cells--
Cell growth rates, oxygen evolution rates, and
chlorophyll content in the whole cells were determined as described
(18). For large-scale culture, wild-type and mutant cells were grown under light-activated heterotropic growth conditions in carboys with 15 liters of BG-11 medium supplemented with 5 mM glucose. In
the case of mutants, the medium was supplemented with spectinomycin (20 mg ml Isolation of Thylakoid Membranes and PS I
Complexes--
Thylakoid membranes were isolated from
Synechocystis sp. PCC 6803 according to the procedure
described (19). The cells were broken in buffer containing 20 mM MES, pH 7.2, 800 mM sucrose, and a protease
inhibitor (phenylmethylsulfonyl fluoride) using a prechilled Bead
Beater (Biospec Products, Inc., Bartlesville, OK) in 10 cycles, where
one cycle consists of a 45-s on-period and a 10-min off-period. After
removing the unbroken cells by low speed centrifugation, the membranes
were pelleted by ultracentrifugation. The membranes were resuspended in
50 mM Tris, pH 8.3; frozen in liquid nitrogen with 10%
(v/v) glycerol; and stored at Electron Paramagnetic Resonance Spectroscopy--
EPR studies
were performed using a Bruker ECS-106 X-band spectrometer with a
standard-mode resonator (ST 8615) that had either a grid or a
quarter-wave stub for light entry. Cryogenic temperatures were
maintained with a liquid helium cryostat and an ITC-4 temperature controller (Oxford Instruments, Oxford, United Kingdom). The microwave frequency was measured with a Hewlett-Packard 5340A frequency counter,
and the magnetic field was calibrated using DPPH
(
Differential EPR spectra of FX were obtained by subtracting
the spectrum of a sample treated in darkness with sodium hydrosulfite at pH 10.5 (to reduce FA and FB) from a sample
that was treated identically, but frozen during illumination to
additionally reduce FX. The resonances observed between 340 and 360 mT are derived from FA and FB, most
likely due to less than quantitative dark reduction with hydrosulfite
(the narrow line widths of FA and FB tend to
exaggerate their apparent spin concentration relative to the broader
line widths of FX). Computer simulations of EPR spectra
were performed using WINEPR SimFonia Version 1.25 (Bruker EPR), where
the g-values were obtained by visual best-fit comparison with the experimental data.
Room Temperature Transient Absorbance
Spectroscopy--
Transient absorbance changes of P700 at
826 nm ( Determination of Efficiency of Forward Electron
Transfer--
Forward electron transfer between FX,
FB, and FA is difficult to measure by optical
spectroscopy because the broad S Steady-state Rates of Electron Transfer--
Rates of flavodoxin
photoreduction were measured in DM-PS I complexes resuspended at 5 µg
ml Growth Rates and Physiological Characteristics of Wild-type and
Mutant Strains--
Mutant cells of Synechocystis sp. PCC
6803 were tested for their ability to grow on BG-11 plates with and
without supplemental glucose. Doubling times and chlorophyll/cell
ratios were determined using mid-log phase liquid cultures grown
photoautotrophically upon bubbling with air (Table
I). All single aspartate mutant strains
had the ability to grow mixotrophically with supplemental glucose at
rates close to the wild-type strain. Strains D566APsaB, D566KPsaB, and D557APsaB grew
photoautotrophically at wild-type rates at light intensities ranging
from 2.5 to 60 µE m
Double aspartate mutant strains grew mixotrophically with supplemental
glucose at rates close to the wild-type strain except for
D557KPsaB/D566APsaB, which grew at all but the
highest tested light intensity of 60 µE m Steady-state Rates of Electron Transfer in PS I
Complexes--
Steady-state rates of electron transfer were measured
in the wild-type and mutant DM-PS I complexes by reduction of
flavodoxin, by reduction of NADP+ mediated by flavodoxin,
and by reduction of NADP+ mediated by ferredoxin. All
assays employed cytochrome c6, the physiological
electron donor to P700+, and the ionic strength
of the media was optimized to support the highest rates of electron
transfer. The rates of flavodoxin photoreduction in wild-type DM-PS I
complexes were 580 µmol mg Low Temperature EPR Spectra of FA and
FB--
FA and FB were
irreversibly photoreduced when dark-frozen membranes or DM-PS I
complexes from the single mutants D566APsaB, D566KPsaB, D557APsaB, and
D557KPsaB, and the double mutants
D557APsaB/D566APsaB and
D557KPsaB/D566APsaB were illuminated at 15 K. The principal g-values of FA at 2.048, 1.944, and 1.851 and FB at 2.069, 1.930, and 1.881 and the ratio
of photoreduced FA to FB of ~2.5:1 are identical to those in wild-type membranes and DM-PS I complexes (data
not shown). FA and FB were completely
photoreduced and showed the characteristic interaction spectrum of
FA Low Temperature EPR Spectra of FX--
The EPR
spectrum of FX in the wild-type DM-PS I complex is shown in
Fig. 1A. The numerical
simulation yields an acceptable match to the FX spectrum in
the non-occluded region of the spectrum upfield from FA and
FB, with g-values of 2.096, 1.853, and 1.757 and
line widths of 9.2, 8.8, and 84 mT, correspondingly. The resonance with
g = 2.002 corresponds to the
P700+ cation radical (not fully shown on the
vertical scale used in the figures). The g-values and line
widths of the EPR spectra of the membranes isolated from the single
lysine mutants D557KPsaB (Fig. 1B,
inset) and D566KPsaB (Fig. 1C,
inset) and the single alanine mutant D557APsaB
(Fig. 1D, inset) are similar to those of
FX in wild-type membranes. The high-field resonance occurs in the g = 1.744-1.764 region in these mutants;
however, the midfield and low-field regions are not depicted in
membranes due to spectral overlap with FA/FB.
However, the EPR spectra of DM-PS I complexes isolated from
D557KPsaB, D566KPsaB, and D557APsaB
(Fig. 1, B-D) are broadened, and they cannot be numerically
simulated with any degree of accuracy. These spectra may result from a
superposition of two or more conformers, which may result from
increased conformational flexibility in the detergent-isolated PS I
complexes.
The g-values of the EPR spectra of membranes isolated from
the single alanine mutant D566APsaB (Fig. 1E,
inset) and the double alanine mutants
D557APsaB/D566APsaB (Fig. 1F,
inset) and D557KPsaB/D566APsaB (Fig.
1G, inset) are shifted downfield relative to
FX in wild-type membranes. In particular, the high-field
resonance occurs near g = 1.795 compared with
g = 1.755 in the wild type. The g-values and
line widths of the spectra of FX in the isolated DM-PS I
complexes from the three D566APsaB mutants (Fig. 1,
E-G) appear similar to those of FX in the
membranes. The relaxation behavior is not greatly affected as judged by
the low temperature optima (9 K) at 100 mW ( Room Temperature Transient Absorbance Spectroscopy--
Fig.
2 (A-G) shows
The
In D557KPsaB, the component with a lifetime of ~15-20
µs still had a relatively high contribution of 26.5% to the total
absorbance change at flash intensities as low as 0.2 mJ, energies that
produce a much lower (<10%) contribution of tens-of-microseconds
components in the wild type (21). As shown in Fig.
3, at a flash intensity of 0.2 mJ, the
amplitude of the absorbance change in D557KPsaB calculated
on the basis of components with lifetimes slower than 7 ms was similar
to that of the other single mutants. This finding further elaborates an
equally efficient electron transfer from P700 to
FA/FB in this mutant. At the same time, the
decrease in the overall absorbance change upon reduction of the
excitation flash energy occurs mainly in the fast 15-20-µs
components (Fig. 3). This finding implicates that 3Chl
decay is a significant component in this time range. Although some
contribution of A1
The Relationship between Growth and Electron Transfer--
Our studies
of D557PsaB and D566PsaB show that the ability
of Synechocystis sp. PCC 6803 to grow photoautotrophically
depends on the position and identity of the substituting residue. There was no phenotypic difference from the wild type when aspartate 566PsaB or 557PsaB was substituted with
alanine. However, when aspartates 566PsaB and
557PsaB were both changed to alanine, the organism grew
slower than the wild type. This shows that a single change to a neutral
amino acid has no detectable consequence, but the cumulative effect of
a double change to two neutral amino acids leads to a noticeable change
in photoautotrophic growth characteristics. There was also no
phenotypic difference from the wild type when aspartate
566PsaB was changed to lysine, but when aspartate
557PsaB was changed to lysine, the organism grew slowly and
failed to grow at low light intensity. When aspartate 566PsaB was changed to alanine and aspartate
557PsaB was changed to lysine, the organism failed to grow
at any light intensity. Hence, position 557PsaB is more
sensitive to the identity of the substituted amino acid than is
position 566PsaB. These results in Synechocystis
sp. PCC 6803 are different from those obtained with the D562N mutant of
C. reinhardtii (comparable to D557 in Synechocystis sp. PCC 6803), in which no PS I complexes
accumulated in the mutant cells (15).
Yet, despite the effect of the studied mutations on growth
characteristics, there is little or no effect on the rate or efficiency of electron transfer through FX. The findings on the
functional properties of the aspartate mutants can be summarized as
follows. First, the reduction of FA and FB in
the single and double aspartate mutants occurs quantitatively at
cryogenic temperatures. The ratio of reduced FA to
FB after dark-freezing and illumination at 15 K is the same
as in the wild type, and the interaction spectrum of
FA Differences in Chlorophyll Content in Aspartate Mutants--
Given
the efficient electron transfer through FX, the proximal
cause behind the early bleaching of the double mutants and the
incapability of the double mutant
D557KPsaB/D566APsaB to grow photoautotrophically is not due to inefficiency in electron transfer through PS I. However, it was found that D557KPsaB and
D557APsaB/D566APsaB, the two slow growing
strains, had about two-thirds chlorophyll content compared with the
other strains in actively dividing photoautotrophic cells (Table I).
This suggests that reduced chlorophyll levels in these two strains
correlate with the slow growth. The lower chlorophyll levels may
reflect lower PS I levels in these cells since most of the chlorophyll
in cyanobacteria is bound to PS I. The implication is that these two
mutations, although having no detectable effects on electron transfer
efficiency, make PS I complexes less stable. It may be further
speculated that the stability of PS I is even lower in the double
mutant D557KPsaB/D566APsaB, which could not
grow photoautotrophically. Lower levels of PS I in these three mutants
could be largely responsible for the observed growth phenotypes.
Differences in EPR Spectra between Membranes and
Detergent-isolated PS I Complexes--
When the isolated membranes are
examined, the g-values and line widths of FX in
the D557APsaB, D566KPsaB, and
D557KPsaB mutants appear identical to those in the wild
type. When PS I complexes are solubilized with n-dodecyl
Functional Role of Cysteine-adjacent Aspartates in
FX-binding Site--
The question behind this study was
whether elimination of the electrostatic contribution by nearby charged
aspartate residues may induce a sufficiently large change in the
thermodynamic properties of FX to influence the efficiency
of electron transfer from A1
Since the single-turnover flash efficiency for photoreduction of
FA/FB in certain aspartate mutants is, at best,
only slightly reduced compared with that in the wild type, the
equilibrium constant between FX and FA will
remain no less than 10. This translates to a midpoint potential of
FX in the aspartate mutants that is more electronegative
than the midpoint potential of FA by at least 60 mV. Hence,
there is a certain dynamic range available for a change in the
reduction potential of FX from the wild-type value of -705
mV (2) without major consequence on electron distribution between the
terminal iron-sulfur clusters. Direct measurement of the FX
reduction potential in the FX niche mutants may be needed to resolve the basis for major differences in the electron transfer efficiencies between individual electron acceptors that were found especially in the cysteine-to-serine mutants (13), but also for the
minor differences that were seen in some of the aspartate mutants (this
work). Nevertheless, this work shows that the efficiency of electron
transfer through FX remains relatively unaffected by the
presence or absence of cysteine-proximal aspartate residues on PsaB.
705 mV) interpolypeptide [4Fe-4S] cluster
ligated by cysteines 556 and 565 on PsaB and cysteines 574 and 583 on
PsaA in Synechocystis sp. PCC 6803. An aspartic acid is
adjacent to each of these cysteines on PsaB and adjacent to the
proline-proximal cysteine on PsaA. We investigated the effect of
D566PsaB and D557PsaB on electron transfer
through FX by changing each aspartate to the neutral
alanine or to the positively charged lysine either singly
(D566APsaB, D557APsaB, D566KPsaB, and D557KPsaB) or in pairs
(D557APsaB/D566APsaB and
D557KPsaB/D566APsaB). All mutants except for
D557KPsaB/D566APsaB grew photoautotrophically, but the growth of D557KPsaB and
D557APsaB/D566APsaB was impaired under low
light. The doubling time was increased, and the chlorophyll content per
cell was lower in D557KPsaB and
D557APsaB/D566APsaB relative to the wild type
and the other mutants. Nevertheless, the rates of NADP+
photoreduction in PS I complexes from all mutants were no less than
75% of that of the wild type. The kinetics of back-reaction of the
electron acceptors on a single-turnover flash showed efficient electron
transfer to the terminal acceptors FA and FB in
PS I complexes from all mutants. The EPR spectrum of FX was
identical to that in the wild type in all but the single and double
D566APsaB mutants, where the high-field resonance was
shifted downfield. We conclude that the impaired growth of some of the
mutants is related to a reduced accumulation of PS I rather than to
photosynthetic efficiency. The chemical nature and the charge of the
amino acids adjacent to the cysteine ligands on PsaB do not appear to
be significant factors in the efficiency of electron transfer through
FX.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
705 mV (versus H2). One of the lowest potential redox-active iron-sulfur clusters is
FX 1
(Em =
705 mV) (2), which functions as an
intermediate electron acceptor in PS I. FX is a relatively
rare form of an interpolypeptide [4Fe-4S] cluster, where two cysteine
ligands are provided by PsaA and two cysteine ligands are provided by PsaB (3, 4). The EPR spectral properties of FX are unusual in that the average of the g-values 2.096, 1.853, and 1.757 is lower than in typical low potential cubane clusters, and its line widths are broad relative to the other two [4Fe-4S] clusters
(FA and FB), which serve as the terminal
electron acceptors in PS I. The EPR spectrum of FX shows a
low temperature optimum (~8 K) and a relatively high microwave
half-saturation power (P1/2 > 200 mW at 8 K),
which indicates an efficient spin relaxation mechanism (5). It is not
known which features of the protein are responsible for conferring the
unusual redox and spectroscopic properties to FX.
A0 (Chl a monomer)
A1
FX ([4Fe-4S] cluster)
FA ([4Fe-4S] cluster)
FB ([4Fe-4S]
cluster). P700, A0, A1, and
FX are located on the PsaA/PsaB reaction center
heterodimer, and FA and FB are located on an
extrinsic, ferredoxin-like protein termed PsaC (for review, see Ref.
8).
-helices. Even though the low potential
FX cluster operates between the +2 and +1 oxidation states,
a more comprehensive view incorporates the charge of the four cysteine
thiolate ligands,
[[4Fe-4S](S-Cys)4]
3/
2, whereupon the
overall charge on the FX cluster can be considered to
increase from
2 to
3 on reduction. There is a high electrostatic price paid for stabilizing a charge in the normally low protein dielectric, and such highly charged sites are usually located on the
surface of proteins. Since FX is located just within the membrane phase, one explanation of its low reduction potential is that
the charge is destabilized due to the low dielectric medium. A second
explanation for its low reduction potential is that the cysteine-proximal aspartic acid residues may be deprotonated in the
membrane (although there are no experimental data to support this
point), and they may be positioned so that they interact electrostatically with the negatively charged iron-sulfur clusters. It
follows that the additional work to move an electron into this environment would translate to a lower reduction potential of FX. Substitutions of these aspartates with amino acids
differing in charge may alter the reduction potential and hence the
efficiency of electron transfer through FX.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B-RCPT. Spectinomycin-resistant colonies were selected under
light-activated heterotropic growth conditions as described (17).
1; Sigma). After harvesting with a Sorvall
continuous flow rotor (DuPont), the cells were stored in BG-11 medium
with 15% (v/v) glycerol at
95 °C. For oxygen evolution
measurements, the light from a slide projector was passed through a
copper sulfate solution and filtered with a red cutoff filter (
> 640 nm), providing saturating photon flux density of 480 µmol
m
2 s
1. PS I-mediated oxygen uptake rates
were determined using the isolated thylakoid membranes as described
(11).
95 °C until use. PS I complexes were
prepared using a protocol described (20) with a minor modification.
Membranes were solubilized in 1% n-dodecyl
b-D-maltoside (DM) (Calbiochem) at 4 °C for 1 h at a Chl concentration of 1 mg ml
1. Trimeric PS I complexes
were isolated from the lower green band after sucrose density (0.1-1.0
M sucrose) ultracentrifugation of the solubilized membrane
suspension for 24 h at 4 °C. The isolated PS I complexes were
dialyzed overnight in 50 mM Tris-HCl, pH 8.3; resuspended
in the same buffer containing 15% (v/v) glycerol and 0.03% DM; frozen
as small aliquots in liquid nitrogen; and stored at
95 °C.
,
'-diphenyl-
-picryl hydrazil) as the standard. Sample
temperatures were monitored by a calibrated thermocouple located 3 mm
beneath the bottom of the quartz sample tube and referenced to liquid
nitrogen. Samples were illuminated with a 150-watt xenon arc source
(Model 66057, Oriel Corp., Stratford, CT) passed through 5 cm of water
and a heat-absorbing color filter to remove the near-IR light. Samples used for EPR measurements contained 1 mg ml
1 Chl, 1 mM sodium ascorbate, and 30 µM DCPIP in 50 mM Tris, pH 8.3. Samples requiring reduced FA
and FB were suspended to 1 mg ml
1 Chl in 250 mM glycine, pH 10.5, prior to the addition of 50 mM sodium hydrosulfite.
A826) were measured in the
microseconds to tens-of-seconds time domain with a laboratory-built
double-beam spectrophotometer as described previously (21). Samples for
optical experiments were suspended under anaerobic conditions at 50 µg ml
1 Chl in 25 mM Tris buffer, pH 8.3, with 4 µM DCPIP, 10 mM sodium ascorbate, and
0.03% DM. The photocurrents generated by UDT PIN-10D photodiodes were
converted to photovoltages using 1-kOhm resistors and amplified using
an EG&G Model 113A differential amplifier with a high frequency
roll-off of 300 kHz and a gain of 10. The signal was fed to a Model
11A33 plug-in and digitized using a Tektronix DSA601 digital signal
analyzer. Excitation was provided by 10-ns flashes from a
frequency-doubled, Q-switched Nd-YAG laser (DCR-11, Spectra-Physics,
Mountain View, CA) at 50-s intervals at flash energies of 16 mJ or 200 µJ. Each trace was recorded as 16-20 averages. The
A826 decay curves were fitted to the sum of
several exponentials with a base line using the Marquardt algorithm in
Igor Pro Version 3.1 (Wavemetrics, Lake Oswego, OR). Following deconvolution, each individual component was plotted with a vertical offset relative to the next component (with a longer lifetime) or the
base line, the offset being equal to the amplitude of the latter component.
Fe charge transfer bands are
highly similar for all three iron-sulfur clusters. However, the
efficiency of forward electron transfer from P700 to
FA/FB (i.e. the number of electrons
leaving P700 that arrive at FA and
FB) can be determined indirectly by measuring the relative
contributions of the back-reactions of A1
,
FX
, and
[FA/FB]
with
P700+. In a previous study (21), we measured
the lifetimes of these back-reactions in PS I complexes devoid of
particular acceptors using two independent techniques, near-IR
absorbance difference spectroscopy of P700+
(
A) and photovoltage (
) generated by PS I complexes
incorporated into liposomes. In wild-type PS I complexes suspended in
medium with a relatively slow electron donor (reduced DCPIP),
P700+ should be reduced by
FA- or DCPIP, whereas faster kinetics with
lifetimes shorter than several milliseconds represent those centers
that have lost the terminal iron-sulfur clusters (FA and
FB) during the isolation. In chemically prepared
P700-FX cores, the major contribution to
A and the
decay shows lifetimes of ~400 µs and
1.5 ms (21). In PS I complexes isolated from a PsaC-less in
vivo mutant of Synechocystis sp. PCC 6803, the major
contribution to
A700 decay also shows
biphasic kinetics with similar lifetimes of ~300 µs and 1.05 ms; a
point-by-point difference spectrum of the PsaC deletion mutant in the
blue shows that the two kinetic phases are identical, with a bleaching
from 400 to 500 nm and a broad peak at 430 nm characteristic of the
weak S
Fe charge transfer bands of an iron-sulfur protein (19). In
a P700-A1 core devoid of FX,
FB, and FA, the major contribution to
A in the near-IR decays bi-exponentially with life times
of 10 and 110 µs (21, 27). A point-by-point difference spectrum of
the P700-A1 core shows that the two kinetic
phases are identical, with an absorbance increase from 340 to 410 nm
characteristic of the 1-electron reduction of a quinone (27). Faster
kinetics should also be observed in the case of a decreased efficiency
of electron transfer to the terminal iron-sulfur clusters. At the same
time, the decay of an antenna chlorophyll triplet (3Chl)
generated at high excitation energies may also contribute to kinetics
in the tens-of-microseconds time domain. Therefore, the efficiency of
electron transfer from P700 to
FA/FB can be compared between different
preparations at identical chlorophyll concentration by calculating the
contribution of kinetic components with life times longer than 5-10 ms
to the overall absorbance change of P700 measured in the
near-IR upon a saturating flash. The resulting values are depicted by
horizontal arrows pointing to the absorbance change
axis in Figs. 2 and 3. The Chl a/P700 ratio
was calculated based on this absorbance change. The differential extinction coefficient of P700 at 826 nm equal to 5800 cm
1 (28) was used.
1 chlorophyll in a 1.3-ml volume of 50 mM
Tricine, pH 8.0, in the presence of 15 µM flavodoxin, 50 mM MgCl2, 15 µM cytochrome
c6, 6 mM sodium ascorbate, and
0.05% DM (22). The measurement was made by monitoring the rate of
change in the absorption of flavodoxin at 467 nm. Rates of
flavodoxin-mediated NADP+ photoreduction were measured in
the same medium, except for the addition of 0.5 mM
NADP+, 0.1% b-mercaptoethanol, and 0.8 M
spinach ferredoxin:NADP+ oxidoreductase (Sigma) and
lowering the MgCl2 concentration to 10 mM.
Rates of ferredoxin-mediated NADP+ photoreduction were
measured under the same conditions, except for the substitution of 5 µM spinach ferredoxin (Sigma) for 15 µM
flavodoxin. Flavodoxin was purified from a strain of Escherichia coli containing the Synechococcus sp. PCC 7002 isiB gene (23) using DEAE-Sepharose CL-6B and Sephadex G-25.
Cytochrome c6 was isolated from Spirulina
maxima as described (24). Rates of NADP+ reduction
were measured by monitoring the rate of change in the absorption of
NADPH at 340 nm. The measurements were performed using a modified Cary
219 spectrophotometer with appropriate narrow band slit and
interference filters to protect the photomultiplier from the actinic
light. The sample was illuminated from two opposite sides using two
banks of high intensity, red (~670 nm) light-emitting diodes (LS1,
Hansatech Ltd.). The light intensity incident on the sample was
saturating at the chlorophyll concentration used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 s
1. Strain
D557KPsaB either did not grow or grew extremely slowly at
2.5 µE m
2 s- 1 (and the colonies were much
smaller), but it grew at wild-type rates at 5.5 and 11 µE
m
2 s
1. At the higher light intensities of
20 and 60 µE m
2 s
1, however,
D557KPsaB again grew more slowly than the wild type. The
chlorophyll content of the whole cells of the single aspartate mutants
was similar to that of the wild type, except for D557KPsaB, which had about two-thirds of the wild-type chlorophyll content per
cell (Table I). Whole chain oxygen evolution rates of light-activated heterotrophic grown cells of the single aspartate mutants were within
20% of those of the wild type, except for D557KPsaB, for which the rates were somewhat lower. The rates of methyl
viologen-mediated O2 uptake by thylakoid membranes of the
single mutants were likewise similar to or slightly reduced compared
with the wild-type rates. The amounts of PsaA, PsaB, PsaC, PsaD, and
PsaE polypeptides assayed by Western blotting were about the same in
the wild type and the single aspartate mutants on a chlorophyll basis
(25). The ability to grow photoautotrophically, to accumulate
chlorophyll, and to evolve O2 at rates comparable to the
wild type indicates accumulation of high levels of PS I and PS II in
the single aspartate mutants.
Characteristics of whole cells, thylakoid membranes, and PS I complexes
of wild-type and mutant strains.
2
s
1. We noted that both double mutant strains bleached
early when grown mixotrophically between light intensities of 7.5 and
22 µE m
2 s
1. Strain
D557APsaB/D566APsaB grew slowly
photoautotrophically, but D557KPsaB/D566APsaB
failed to grow photoautotrophically at all light intensities ranging
from 2.5 to 60 µE m
2 s
1. The chlorophyll
content of the photoautotrophically growing cells of
D557APsaB/D566APsaB was about two-thirds of
that of the wild type (Table I). Whole chain oxygen evolution rates of
light-activated heterotrophic grown cells of the double aspartate
mutants were ~20-25% greater than those of wild type. This finding
implies that PS I is functional in the non-photoautotrophic
D557KPsaB/D566APsaB double mutant. As shown in
Table I, the chlorophyll levels in mid-log phase photoautotrophic cells
were significantly lower in D557KPsaB and
D557APsaB/D566APsaB, the two mutant strains
that had significantly longer doubling times compared with the other strains. This suggests that reduced chlorophyll levels in these two
strains may be responsible for the slow growth.
1 Chl h
1, and
the rates of ferredoxin- or flavodoxin-mediated NADP+
photoreduction were ~300 and 400 µmol mg
1 Chl
h
1, respectively (Table I). The rates of flavodoxin
photoreduction in DM-PS I complexes from the four single aspartate
mutants and the two double aspartate mutants were in the range of
500-600 µmol mg
1 Chl h
1, similar to the
wild-type values. When flavodoxin-mediated NADP+
photoreduction was measured, all aspartate mutants showed rates better
than 240 µmol mg
1 Chl h
1, and when
ferredoxin-mediated NADP+ photoreduction was measured, the
rates ranged from 340 to 410 µmol mg
1 Chl
h
1. Thus, impairment of photoautotrophic growth in
certain mutants is not accompanied by a decline in whole chain electron
transfer rates in either the single or double aspartate mutant strains.
/FB
with
g-values of 2.048, 1.940, 1.921, and 1.885 when membranes or
DM-PS I complexes of the single and double mutants were frozen during
illumination. The EPR spectral properties of
FA
and FB
are
therefore unaffected by substitutions of the cysteine-proximal aspartic
acid residues on PsaB even though extramembrane loops in the
FX region of PsaA and PsaB appear to be in close contact with PsaC (26).
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Fig. 1.
EPR spectra of wild-type (A)
and mutant strains D557KPsaB
(B), D566KPsaB (C),
D557APsaB (D), D566APsaB
(E),
D557APsaB/D566APsaB
(F), and
D557KPsaB/D566APsaB (G).
DM-PS I complexes are depicted in the main spectra, and thylakoid
membranes are depicted in the insets. The spectra were
obtained with FA and FB pre-reduced with
hydrosulfite at pH 10.5 and illuminated during freezing to 9 K. Background spectra were recorded in chemically reduced dark-adapted
samples frozen to 9 K and subtracted from the light-induced spectra.
Spectrometer settings were as follows: temperature, 9 K; microwave
power, 40 mW; microwave frequency, 9.4521 (A), 9.4476 (B), 9.4499 (C), 9.4496 (D), 9.4487 (E), 9.3591 (F), and 9.3593 (G) GHz;
receiver gain, 2 × 104; modulation amplitude, 32 G at
100 kHz; magnetic field, 3600 G with scan width of 1000 G; and four
scans averaged. The magnetic field positions of the resonances in the
double mutants (F and G) are shifted in resonant
frequency compared with the single mutants (B-E) as a
consequence of adding a quarter-wave optical stub to the cavity window.
The wild-type spectrum of FX was simulated (dashed
lines) using g-values of 2.096, 1.853, and 1.755.
3 dB) of microwave power
and by the inability to microwave power-saturate the signal at 9 K
(data not shown). The similarity of the EPR spectra in all three mutant
strains indicates that the amino acid in the position
566PsaB has a major influence on the electronic properties
of FX.
A826 kinetics of the DM-PS I complexes from
the wild type and the aspartate mutants measured in the presence of the
artificial electron donor, reduced DCPIP. As detailed previously (21,
27), the slowest component in the wild-type DM-PS I complex (Fig.
2A) has a lifetime of 2.2 s, and it arises from
P700+ reduction by the external electron donor,
ascorbate-reduced DCPIP. The major pathway of
P700+ reduction following a single-turnover
flash is charge recombination with
[FA/FB]
, and the kinetics of
this back-reaction in wild-type PS I complexes are approximated by two
components with lifetimes of ~25 and 112 ms (Fig. 2A). In
most cases, another component with a lifetime of ~250-300 ms (221 ms
in the preparation used in this work) can usually be resolved. At
present, we cannot unambiguously ascertain whether this component
represents P700+ reduction from an external
donor or a back-reaction with endogenous acceptor. It should be noted,
however, that kinetic components with lifetimes longer than 100 ms were
not observed in absorbance changes in the near-IR (
A) and
photovoltage (
) decay measurements performed in the absence of
exogenous mediators (21). Faster components (Fig. 1A) are
typically present in all PS I preparations, they usually make up
10-20% of the overall
A amplitude and may be derived
from the back-reactions of FX
and
A1
in a population of damaged PS I complexes.
The lifetimes of 2.6 ms and 543 µs are characteristic of the
back-reactions from FX
when FA
and FB are absent, and the 89-µs component is
characteristic of A1
back-reaction when
FX, FA, and FB are absent (21,
28).
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Fig. 2.
Kinetics of absorbance change at 826 nm
( A) measured in wild-type
(A), D557KPsaB (B),
D566KPsaB (C), D557APsaB
(D), D566APsaB (E),
D557APsaB/D566APsaB (F), and
D557KPsaB/D566APsaB (G) PS I
complexes in the presence of 1 mM sodium ascorbate and
4 µM DCPIP in 50 mM
Tris-HCl, pH 8.3, and illuminated by saturating laser flashes (16 mJ)
at 50-s intervals. Horizontal arrows point to the
absorbance change values calculated as the sum of the initial
amplitudes (Ampl.) of the components with lifetimes longer
than 7 ms.
A826 kinetics of D566KPsaB
(Fig. 2C), D557APsaB (Fig. 2D), and
D566APsaB (Fig. 2E) are similar to those of the
wild-type PS I complex. The overall
A826
absorbance change in D557KPsaB (Fig. 2B) shows a
much higher initial amplitude than in the wild type (5.9 versus 3.75 mOD) or the other single aspartate mutants. However, the contribution of the ~20-µs component to the overall P700+ decay kinetics in D557KPsaB
is much greater than the contribution of kinetically similar components
in D566APsaB, D566KPsaB, or D557APsaB. Unlike the milliseconds-to-seconds components,
the 20-µs component did not saturate when the excitation flash
intensity was increased to 150 mJ (data not shown), indicating that the decay of an antenna chlorophyll triplet (3Chl) most likely
contributes to the
A826 in this time range. If only those components with lifetimes longer than 7 ms are taken into
account when calculating the overall absorbance change, then the
resulting amplitude of 2.4 mOD (Fig. 2B) lies in the same range as the corresponding value calculated in the wild type and other
mutants at equal chlorophyll concentrations (this value is indicated by
horizontal arrows pointing to the absorbance change axis in Fig. 2). Therefore, the efficiency of electron transfer to
FB appears to be either unaffected by the mutation in
D557KPsaB or affected to the same extent as in other
mutants. The 2.4-mOD absorbance change in D557KPsaB
corresponds to a reasonable Chl a/P700 value of
~121 calculated based on a differential extinction coefficient of
P700 at 826 nm (29). This ratio is close to the Chl
a/P700 value of 91 that is obtained in the wild
type (Fig. 2A) or in the other mutants where Chl
a/P700 falls within the range of 97-122 (Fig.
2, C-G). These values are compatible with the high whole
chain electron transfer rates in D557KPsaB calculated on an
equal chlorophyll basis (Table I). The implication of this result is
that the absorbance change that decays with a lifetime of ~20 µs in
D557KPsaB is not related to P700
photochemistry, but is rather due to the decay of 3Chl
generated at flash intensities that saturate charge separation in the
reaction center. In this respect, the kinetic properties of
D557KPsaB are qualitatively different from those of the
C565SPsaB mutant, which was shown to have a decreased
quantum efficiency of electron transfer via FX (13). The
decrease in quantum efficiency was manifest in C565SPsaB by
a considerably lower amplitude of the sum of components with lifetimes
slower than 7 ms relative to the wild type at equivalent chlorophyll
concentrations (30).
back-reaction cannot be
rigorously excluded in D557KPsaB, it is most likely that
the fastest components represent uncoupled antenna chlorophyll
molecules, which are responsible for generation of 3Chl
states even at low flash energies.
View larger version (23K):
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Fig. 3.
Kinetics of absorbance change at 826 nm
( A) measured in D557KPsaB
(A), D566KPsaB (B),
D557APsaB (C), and D566APsaB
(D) PS I complexes in the presence of 1 mM
sodium ascorbate and 4 µM DCPIP in 50 mM Tris-HCl, pH 8.3, and illuminated by undersaturating
laser flashes (200 µJ) at 50-s intervals.
Horizontal arrows point to the absorbance change values
calculated as the sum of the initial amplitudes (Ampl.) of
the components with lifetimes longer than 7 ms.
A826 absorbance change in
D557APsaB/D566APsaB (Fig. 2F) and
D557KPsaB/D566APsaB (Fig. 2G) also
shows a higher initial amplitude than the wild type (4.9 and 4.1 versus 3.75 mOD). Similar to D557KPsaB, both
double aspartate mutants have a higher contribution of 3Chl
to the decay kinetics than the wild type. Applying a lower flash energy
to the double aspartate mutants led to a preferential decrease of the
fastest (10-15 µs) component, without completely saturating the
amplitudes of the FA/FB-related components
(data not shown). When the microsecond kinetic phases are neglected, the remaining amplitudes of 3.0 and 3.2 mOD, along with a high contribution of the components related to the
[FA/FB]
back-reaction, indicate
relatively efficient electron transfer from P700 to
FA/FB in the double aspartate mutants.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and FB
after
photoaccumulation is identical to that in the wild type. Second,
light-driven rates of NADP+ photoreduction in the single
and double aspartate mutants are comparable to those in the wild type,
provided that a physiologically relevant donor (cytochrome
c6) and acceptor (ferredoxin or flavodoxin) are
used. The most adversely affected mutant, D566KPsaB, shows rates of flavodoxin-mediated and ferredoxin-mediated NADP+
reduction that are within 75 and 80%, respectively, of the wild type,
yet this mutant grew at wild-type rates. Third, transient optical
spectroscopy showed efficient photoreduction of FA and FB in D566KPsaB, D566APsaB, and
D557APsaB at room temperature on a single-turnover flash.
Electron transfer between A1 and FX may be
slightly less efficient in D557KPsaB,
D557APsaB/D566APsaB, and
D557KPsaB/D566APsaB, where a small contribution
to the back-reaction may occur from A1
, but
the effect, if present, is largely masked by a large contribution of
the decay of 3Chl in the same time range.
-D-maltoside, the high-field g-value (the
only reliable resonance, since the midfield resonance is often obscured
by FA/FB) remains centered around
g = 1.75-1.76, but the line width broadens
considerably. We suggest that conformational flexibility accounts for
the heterogeneity of FX in the detergent-isolated PS I
complexes. The spectrum likely represents an envelope in which the
g-tensor is distributed due to the presence of a relatively large number of conformers. We speculate that the presence of a salt
bridge involving D557PsaB and D566PsaB with an
unidentified basic residue on a nearby helix may bring conformational
stability to the extramembrane region that binds FX. The
breaking of these bonds, by substituting either a positively charged
amino acid such as lysine or a neutral amino acid such as alanine,
would lead to a greater flexibility of the structure. A larger number of conformations, which would reflect a high degree of flexibility of
the detergent-solubilized reaction centers, would become available and
would be frozen-in at low temperature, leading to an inhomogeneously broadened set of lines. Notable differences from the wild type are
found in the single and double D566APsaB aspartate mutants. Although the line widths are not affected, the g-values have
moved downfield, with gx shifting from 1.758 to
1.795. This change derives from an alteration in the electronic
properties of the FX cluster. The alanine/alanine
(D557APsaB/D566APsaB) and lysine/alanine
(D557KPsaB/D566APsaB) double mutants are nearly identical to the D566APsaB single mutant, indicating that
D566PsaB is a major EPR spectral determinant of the
g-tensor for FX in the wild type. The change
from aspartate to positively charged lysine appears to have a minimal
effect on the spectral properties of FX, except for the
linewidth broadening that occurs when PS I complexes are removed from
the membrane using a detergent. It is interesting that substitution of
aspartate with alanine, a neutral amino acid with a hydrophobic side
group, rather than lysine, a positively charged amino acid, results in
the shifts in the g-values of FX.
to
FA/FB. The results of this study indicate that
the chemical nature and the charge of the amino acids adjacent to the
cysteine ligands on PsaB are not significant factors in the ability of FX to accept an electron from A1
or to donate an electron to FA/FB. It would be
reasonable to conclude that either the aspartate residues are not
charged or, if they are charged, their influence on those factors that
control the electron transfer efficiency through FX is
negligible. Although we have not undertaken the direct measurement of
the midpoint potential of FX in these mutants, relevant
data indicate that, in certain instances, the midpoint potential of an
intermediate electron carrier is not the major factor determining the
efficiency of forward electron transfer in a chain of electron
carriers. The presence of a thermodynamically uphill electron transfer
step from FA to FB in PS I (27, 31) gives an
example that an unfavorable Gibbs free energy change between cofactors
does not preclude efficient forward electron transfer from the PS I
complex to ferredoxin or flavodoxin. This step, of course, occurs in
the context of an overall favorable change in Gibbs free energy from
A0
to the terminal acceptor, ferredoxin or
flavodoxin. The same consideration likely applies to electron transfer
among the tetraheme cytochromes in the Rhodopseudomonas
viridis reaction center (32) and among the three iron-sulfur
clusters in the nickel-iron hydrogenase of Desulfovibrio
gigas (33). We suggest that apart from direct redox titration,
single-turnover flash efficiency monitored by P700+ re-reduction kinetics is the single best
parameter for detecting changes in the thermodynamic properties of the
electron acceptors.
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FOOTNOTES |
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* This work was supported by Grant 96-37306-2632 from the United States Department of Agriculture, National Research Initiative Competitive Grants Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Molecular Biology and Biochemistry,
University of California, Irvine, CA 92697.
** Present address: FB Biology/Botany, Philipps University, Karl-von-Frisch-Strasse, D-35032 Marburg, Germany.
Supported by National Science Foundation Research Training
Grant DBI-960223.
§§ To whom correspondence should be addressed. Tel.: 814-865-1163; Fax: 814-863-7024; E-mail: jhg5{at}psu.edu.
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ABBREVIATIONS |
---|
The abbreviations used are:
FX, FA, and FB, [4Fe-4S]-type electron acceptors
bound to Photosystem I chlorophyll-protein complex;
A1, the
secondary electron acceptor in Photosystem I, a phylloquinone;
PS, PS
I, Photosystem I;
Chl, chlorophyll;
MES, 4-morpholineethanesulfonic
acid;
DM, n-dodecyl -D-maltoside;
DM-PS I, Photosystem I complex isolated using n-dodecyl
-D-maltoside (containing FX, FA,
and FB iron-sulfur clusters);
DCPIP, 2,6-dichlorophenolindophenol;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
OD, optical density.
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REFERENCES |
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