From the Department of Molecular Biology and Plant
Biology, University of Geneva, 30 Quai Ernest Ansermet,
CH-1211 Geneva, Switzerland and the ¶ Biologisches Institut II,
University of Freiburg, Schänzlestraße 1, D-79104 Freiburg, Germany
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ABSTRACT |
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A strain of the cyanobacterium
Synechococcus elongatus was generated that expresses a
hybrid version of the photosystem I subunit PsaF consisting of the
first 83 amino acids of PsaF from the green alga Chlamydomonas
reinhardtii fused to the C-terminal portion of PsaF from S. elongatus. The corresponding modified gene was introduced into
the genome of the psaF-deletion strain FK2 by cointegration
with an antibiotic resistance gene. The transformants express a new
PsaF subunit similar in size to PsaF from C. reinhardtii that is assembled into photosystem I (PSI). Hybrid PSI complexes isolated from these strains show an increase by 2 or 3 orders of
magnitude in the rate of P700+ reduction by C. reinhardtii cytochrome c6 or plastocyanin
in 30% of the complexes as compared with wild type cyanobacterial PSI.
The corresponding optimum second-order rate constants
(k2 = 4.0 and 1.7 × 107
M1 s1 for cytochrome
c6 and plastocyanin) are similar to those of
PSI from C. reinhardtii. The remaining complexes are
reduced at a slow rate similar to that observed with wild type PSI from
S. elongatus and the algal donors. At high concentrations
of C. reinhardtii cytochrome c6, a
fast first-order kinetic component (t1/2 = 4 µs)
is revealed, indicative of intramolecular electron transfer within a
complex between the hybrid PSI and cytochrome
c6. This first-order phase is characteristic
for P700+ reduction by cytochrome
c6 or plastocyanin in algae and higher plants.
However, a similar fast phase is not detected for plastocyanin. Cross-linking studies show that, in contrast to PSI from wild type
S. elongatus, the chimeric PsaF of PSI from the transformed strain cross-links to cytochrome c6 or
plastocyanin with a similar efficiency as PsaF from C. reinhardtii PSI. Our data indicate that development of a
eukaryotic type of reaction mechanism for binding and electron transfer
between PSI and its electron donors required structural changes in both
PSI and cytochrome c6 or plastocyanin.
One of several minor differences found in the otherwise remarkably
conserved electron transfer chains of oxygenic photosynthesis of
cyanobacteria, algae, and land plants concerns the type of electron
carrier proteins used to transfer electrons from the cytochrome
b6/f complex to photosystem I and the
way they interact with
PSI1 (1, 2). All
cyanobacteria investigated utilize a cytochrome c6 as a soluble periplasmic electron carrier. In
several cases, e.g. Synechococcus elongatus,
cytochrome c6 is the only electron carrier in
the periplasma and is constitutively expressed (1-3). Other
cyanobacteria like Anabaena sp. PCC 7119 and
Synechocystis sp. PCC 6803 and most algae examined, however,
use both cytochrome c6 and the copper-containing
plastocyanin as alternative periplasmic electron carrier proteins
(4-8). In these organisms, they are differentially expressed depending
mostly on the relative availability of copper and iron in the culture
medium (5, 8). In contrast, plastocyanin is expressed constitutively in
photosynthetic land plants that lack cytochrome
c6. Thus, in the evolution of oxygen-evolving organisms a tendency to replace the originally used c-type
cytochrome by plastocyanin is clearly discernible.
The PSI complex functions as a light-driven oxidoreductase that
transfers electrons from cytochrome c6 or
plastocyanin to ferredoxin or flavodoxin (see Refs. 9 and 10 for a
review). According to the established atomic structure of PSI from
S. elongatus, the primary donor of PSI, P700, is located
within the highly conserved reaction center core close to the
periplasmic surface of the photosynthetic membrane (9, 11, 12). Two
horizontal helixes l and l', attributed to the PSI core subunits PsaA
and PsaB, are thought to form a recognition site for binding of the
periplasmic electron carriers (11, 12). However, despite the high
degree of structural conservation of the PSI core subunits in all
oxygen-evolving organisms, the mechanism of interaction between
plastocyanin or cytochrome and PSI varies in different species.
In higher plants, electron transfer from plastocyanin to
P700+ is a biphasic process that includes a first-order
kinetic component with a half-life of about 12 µs which is attributed
to electron transfer from plastocyanin to P700+ within a
stable complex between plastocyanin and PSI formed prior to the
photooxidation of P700 (13-15). Biochemical studies indicate that the
PsaF subunit of PSI is involved in the formation of this complex
(16-18). Similar first-order kinetic components with half-lives of
about 3 µs are observed for the reduction of P700+ by
both plastocyanin and cytochrome c6 in the green
alga Chlamydomonas reinhardtii (19). Electron transfer from
both donors to PSI from a psaF-deficient mutant of C. reinhardtii was shown to be drastically slower indicating that
PsaF is essential for efficient electron transfer from both
plastocyanin and cytochrome c6 to PSI (19, 20).
Similar first-order kinetics have also been observed for PSI reduction
by plastocyanin or cytochrome c in the green algae
Chlorella and Monoraphidium braunii (21, 22). This suggests that the formation of complexes between PSI and these
electron donors which involve the PsaF subunit is likely to occur
generally in algae and plants. In contrast, in the cyanobacterium Synechocystis sp. PCC 6803, the reduction of
P700+ by cytochrome c6 or
plastocyanin is a second-order process and fast phases which can be
attributed to electron transfer within a stable preformed complex could
not be detected (3, 23-25). In addition, spectroscopic investigations
of PSI complexes from S. elongatus and
Synechocystis sp. PCC 6803 lacking the PsaF subunit show
that the absence of PsaF does not affect the rates of P700+
reduction by cytochrome c6 (3, 25).
It has been shown that the PsaF subunit of PSI from spinach cross-links
at one of its N-terminal lysines between residues 10-23 or 24-51 to
the conserved acidic amino acids 42-44 and 59-61 of plastocyanin,
respectively (26). This region close to the N-terminal end of PsaF
could form an amphipathic In Vitro Site-directed Mutagenesis, Molecular Cloning Strategies,
and Vectors--
DNA manipulations were performed in Escherichia
coli strain XL1 blue according to standard protocols (28).
Integrative cartridge vectors were constructed by modifications of a
genomic 2.45-kb BamHI/XhoI fragment from S. elongatus carrying the psaF/psaJ operon subcloned into
pBSC M13+ (29) (Fig. 1). A DNA construct, pCRSEF/2, was
constructed in which the region of the psaF gene from
S. elongatus encoding the mature PsaF subunit was
substituted completely by the psaF region from C. reinhardtii (30). First, an EcoRV site was introduced at codons 24 and 25 of psaF (i.e. codons 1 and 2 of the mature PsaF from S. elongatus), and the termination
codon was modified to create an XbaI site. These
modifications were introduced by inverse PCR in the presence of
oligonucleotides SEF1 (5'-TGCGATATCAGCGGAGGCTAGG-3') and SEF2
(5'-TCTCTAGATTTGCTGTTTGTTG-3') to create the vector pSEF1. Second, a
psaF cDNA clone from C. reinhardtii (30) was
modified by PCR in the presence of oligonucleotides CRF1
(5'-CCGATATCGCGGGCCTGACC-3') and CRF2 (5'-CAGCTAGCGGGGAGACACGG-3') to
create an EcoRV site at codons 63 and 64 (i.e.
codons 1 and 2 of the mature PsaF) and an NheI site at the
termination codon. This fragment was inserted into the EcoRV
and XbaI sites of pSEF1 to produce the plasmid pCRSEF/2
which carries the psaF/psaJ operon from S. elongatus in which the part of the C. reinhardtii psaF
gene encoding the entire mature PsaF is fused in frame to the
presequence of psaF from S. elongatus.
In addition, a second plasmid was constructed in which the codons for
amino acids Ile64 to His145 of PsaF from C. reinhardtii were
inserted in frame between codon Asp24 and codon Ala83 of psaF from S. elongatus. First, the genomic
ClaI/XhoI fragment from S. elongatus
was modified to create a BalI site at codons 81 and 82 by
PCR in the presence of oligonucleotide FBal
(5'-CTTGGCCATGCCGGTGATTTTC-3') and the M13 reverse primer. This PCR
fragment was inserted into the BalI and XhoI
sites of vector pCRSEF/2 to create vector FBalCRF/2. The fusion part of
the DNA construct was sequenced. Finally, the SmaI fragment
of pHP45 Culturing Conditions, Transformation, and Selection of S. elongatus--
Wild type and mutant strains from S. elongatus were cultivated as described (32). Genetic manipulations
of the psaF/psaJ locus from S. elongatus were
carried out using the
psaF Isolation of Protein Components from S. elongatus and C. reinhardtii--
Photosystem I complexes were extracted from
PSII-depleted membranes by 0.6% w/v Analytical Methods, Cross-linking Procedures, and Immunoblot
Analysis--
For the fast immunoblot analysis of photosynthetic
membranes, 3.5-ml cultures of S. elongatus were grown to
OD750 = 1, harvested by centrifugation, washed once in 1 ml
of HMCM buffer (20 mM Hepes, pH 7.8, 10 mM
CaCl2, 5 mM MgCl2, 0.5 M mannitol), resuspended in 50 µl of HMCM buffer, and
frozen. The thawed suspension was incubated with 2 mg/ml lysozyme for
30 min at 48 °C and frozen. Cells were then lysed by the addition of
10 volumes of MCM (MMCM minus mannitol), and the photosynthetic
membranes were recovered by centrifugation in a microcentrifuge at
4 °C for 10 min at maximum speed. Membranes were washed once in 500 µl of MCM supplemented with 0.1% sulfobetain 10, pelleted by
centrifugation, and resuspended in SDS loading buffer at a
concentration of approximately 0.25 mg of chlorophyll/ml.
Cytochrome c6 and plastocyanin were chemically
cross-linked to photosystem I essentially as described (19); PSI
particles at a concentration of 0.1 mg of chlorophyll/ml in 30 mM Hepes, pH 7.5, 3 mM MgCl2, and 1 mM ascorbate were incubated in the presence of 20 µM plastocyanin or cytochrome c6
with 5 mM
N-ethyl-3-(3-diaminopropyl)carbodiimide and 10 mM N-hydroxysulfosuccinimide for 45 min in
darkness. The reactions were terminated by addition of ammonium acetate
to a final concentration of 0.2 M and diluted 4-fold. PSI
complexes were sedimented by centrifugation at 200,000 × g for 45 min and resuspended in 20 mM Hepes, pH
7.5, 0.05% Triton X-100. Analytical SDS-polyacrylamide gel
electrophoresis was carried out using 15 or 16.5% (w/v) polyacrylamide
gels (36). For immunoblot analysis, PSI complexes equivalent to 4 µg
of chlorophyll and membrane preparations equivalent to 10 µg of
chlorophyll were analyzed. Western blots and antibody incubations were
carried out essentially as described (36). Immunodetection reactions
were performed using anti-rabbit IgG antibodies linked to horseradish
peroxidase followed by enhanced chemiluminescence detection (ECL,
Amersham Pharmacia Biotech).
Flash-absorption Spectroscopy--
Flash-induced absorption
changes at 817 nm were measured at 296 K on a single beam
spectrophotometer essentially as described (15). Flash excitation was
performed using a frequency-doubled Nd:YAG laser (5 ns full width at
half maximum). The measuring light was provided by a luminescence diode
(Hitachi HE8404SG, 40 milliwatts, 30 nm full width at half maximum),
filtered through an 817-nm interference filter (9 nm full width at half
maximum), and passed through a cuvette with an optical path length of 1 cm that contained 200 µl of sample. For flash-absorption experiments, PSI reaction centers were suspended in the presence or absence of
cytochrome c6 or plastocyanin at a standard
concentration of 50 µM chlorophyll in 30 mM
Mops, pH 7.0, supplemented with 0.05% Vector Construction--
To generate an S. elongatus
strain carrying the psaF gene from C. reinhardtii, integrative vectors were introduced into the S. elongatus strain FK2 that carries a psaF gene
interrupted by a kanamycin resistance cassette and thus served as a
psaF-free background ((32) see Fig.
1, middle part). Vector
constructions were performed using the genomic fragment from S. elongatus carrying the psaF/psaJ locus ((29) see
top of Fig. 1) as follows. First, we took into account that
the PsaF subunit is expressed as a precursor protein carrying an
N-terminal export sequence required for the translocation of the N
terminus of the protein into the periplasma. In order to be efficiently
recognized by the cyanobacterial export apparatus, the hybrid
psaF gene should therefore contain the entire cyanobacterial
export sequence and the ASA-D cleavage site of the export protease.
Second, in cyanobacteria, psaF and psaJ are arranged in an operon. In order to avoid any interference with the
expression of the PsaJ subunit which is likely to be involved in the
binding of PsaF to PSI, the organization of the psaF/psaJ operon should be maintained (25) (see Fig. 1).
As shown in Fig. 1 (bottom) and Fig.
2, the integrative vector FBalCRF/3
carries a gene encoding a hybrid PsaF protein that contains the
cyanobacterial signal sequence (up to codon Asp-24 of S. elongatus psaF), the N-terminal domain of PsaF from C. reinhardtii (30) (i.e. between codons Asp-63/Ile-64 to
codon His-145), and the hydrophobic C-terminal part of the
cyanobacterial subunit (starting with codon Ala-60 of S. elongatus psaF) which is assumed to anchor the protein to the
hydrophobic core of PSI. A streptomycin/spectinomycin resistance gene
cassette was inserted at the HpaI site located 165 base
pairs downstream of psaJ, well downstream of the transcribed region of the psaF/psaJ operon (see "Experimental
Procedures" for details of the construction). In addition to
FBalCRF/3, a vector, pCRSEF/3, was constructed in which the complete
region of the psaF gene from S. elongatus
encoding the entire mature PsaF subunit was replaced by the
corresponding part of psaF from C. reinhardtii
(see "Experimental Procedures"). However, following the
introduction of this vector into S. elongatus, C. reinhardtii PsaF was not recovered in PSI although it was verified
by Southern blot analysis that the gene was cointegrated together with
the antibiotic resistance marker and inserted correctly into the genome (not shown).
Characterization of the Mutant H53 Expressing a Chimeric PsaF
Subunit--
Following the electroporation of the
psaF-deletion strain FK2 from S. elongatus in the
presence of the plasmid pFBalCRF/3, streptomycin-resistant
transformants with the desired Kms/Smr
phenotype were selected by replica plating on solid media, and those
expressing the chimeric C. reinhardtii-S. elongatus PsaF protein were screened by immunoblot analysis using anti-C.
reinhardtii-PsaF antibodies (not shown, see "Experimental
Procedures"). The organization of the psaF/psaJ locus of
one of these strains, H53, was investigated by Southern blot analysis
shown in Fig. 3. First, upon
hybridization with a 500-base pair fragment from a C. reinhardtii
psaF cDNA clone carrying part of the gene encoding the mature
PsaF, a single 6.3-kb genomic EcoRV restriction fragment is
detected that is not present in S. elongatus wild type nor
in strain FK2 (Fig. 3A). The same DNA fragment is detected
in H53 DNA after hybridization with a probe carrying the
Smr/Spr genes inserted into
pFBalCRF/3 (Fig. 3B). Taken together, these blots indicate
that the region of the psaF gene of C. reinhardtii has been inserted with the antibiotic marker gene into
the genome of S. elongatus. Furthermore, when the blots were
probed with the ClaI/XhoI fragment carrying the
psaF, psaJ, and rpl9 genes, only
single EcoRV fragments of ~13 and 14.5 kb are observed in DNA from wild type and strain FK2. The size difference is due to the
presence of the Kmr marker gene in FK2 (Fig.
3C, see Fig. 1). For strain H53, however, two
EcoRV fragments of ~8.0 and 6.3 kb are detected,
indicating that a new EcoRV site has been introduced into
the psaF/psaJ locus of H53. Since the
Smr/Spr marker genes do not
contain an EcoRV site, the existence of a new
EcoRV restriction site together with the
Smr/Spr genes at the
psaF/psaJ gene locus indicates that the entire part of the
psaF gene that originates from C. reinhardtii has
been introduced into strain H53, because C. reinhardtii psaF
is flanked by a new EcoRV site and the
Smr/Spr genes in the integrative
vector pFBalCRF/3 (Fig. 1). Finally, when the blots were probed with a
fragment carrying the Kmr marker present in strain
FK2, no hybridization signal was observed in DNA from strain H53 (Fig.
3D). This result indicates that H53 represents a fully
segregated mutant in which the original psaF/psaJ gene locus
of strain FK2 that carried a kanamycin resistance gene has been
completely replaced by the new psaF gene at the
psaF/psaJ gene locus in all copies of the polyploid genome.
Essentially the same conclusion was obtained by a similar Southern blot
analysis that was carried out using EcoRI-restricted DNA
(not shown).
The expression pattern of the psaF genes in S. elongatus wild type and strains FK2 and H53 was monitored by
Western blot analysis of photosynthetic membranes and isolated
photosystem I complexes using anti-C. reinhardtii-PsaF
antibodies (Fig. 4). This antibody recognizes the cyanobacterial PsaF subunit in membranes and photosystem I preparations in of wild type S. elongatus. As expected
from the deduced sequences, the apparent mass of the cyanobacterial PsaF subunit is 15 kDa, approximately 3 kDa smaller than its algal counterpart (Fig. 4, lanes 1, 4, and 5). In
strain FK2, the corresponding subunit is absent (Fig. 4, lanes
2 and 6). However, in membranes and PSI from strain H53
a new version of the PsaF protein is detected which exceeds the
original cyanobacterial PsaF subunit by 2.5 kDa in mass and is only
slightly smaller than PsaF from C. reinhardtii (Fig. 4,
lanes 3 and 7). Thus, the modified
psaF gene of strain H53 is expressed, and the hybrid PsaF
protein is assembled into the cyanobacterial photosystem I complex. In
addition, the fact that the protein is very similar in size to the
mature algal PsaF subunit indicates that the protein is exported across
the photosynthetic membrane and processed correctly by the transit
peptidase. Essentially the same results were obtained by Western blot
analysis using anti-S. elongatus-PsaF antibodies, with the
exception that these antibodies do not detect C. reinhardtii
PsaF (not shown). However, the visualization of this new PsaF subunit
in PSI particles by a general staining technique was not unambiguously
possible since the protein comigrates with the abundant allophycocyanin
and phycocyanin subunits of phycobilisomes in SDS-polyacrylamide gels.
Upon removal of these contaminations by ion-exchange chromatography,
PSI preparations were obtained that no longer show an increased rate of
P700+ reduction in the presence of cytochrome
c6 described below and are thus no longer suited
for further investigations (not shown). In this respect it has been
reported for PSI from plants and algae that the fast electron transfer
from plastocyanin to P700+ is impaired in preparations
purified by ion-exchange chromatography, an effect that is correlated
with the loss of the PsaF subunit from the reaction centers (18, 19).
Apparently, the hybrid PSI complexes from S. elongatus
strain H53 show a similar behavior.
Cytochrome c6 from C. reinhardtii Forms a Complex with
the Hybrid PSI from S. elongatus H53--
In order to study the
properties of the hybrid PsaF subunit within PSI from the H53 strain,
electron transfer from cytochrome c6 and
plastocyanin to PSI was monitored by flash absorption spectroscopy. Fig. 5 shows the absorbance transients at
820 nm of PSI particles from wild type S. elongatus and the
mutant strains FK2 and H53 induced by a laser flash in the presence of
10 µM cytochrome c6 from S. elongatus (A), and 10 µM cytochrome
c6 (B) or 10 µM
plastocyanin from C. reinhardtii (C). In the
presence of S. elongatus cytochrome c6 electron transfer is monophasic for
photosystem I from wild type S. elongatus with a half-life
of ~20 ms (Fig. 5A), and at first glance, the introduction
of the hybrid PsaF in PSI from H53 causes only a small increase of the
apparent rate of P700+ reduction (see below for a more
detailed analysis). Cytochrome c6 and
plastocyanin from C. reinhardtii, however, react much slower with PSI isolated from wild type S. elongatus or the
psaF deletion strain FK2, and the kinetic traces can be
fitted to monophasic decays (Fig. 5, B and C).
For C. reinhardtii cytochrome c6,
electron transfer to the cyanobacterial PSI is about four times slower (t1/2 ~ 80 ms) than with the cyanobacterial cytochrome, and the electron transfer from plastocyanin shows a
half-life of ~1 s, which is ~50 times slower than for cytochrome c6 from S. elongatus. In contrast,
the electron transfer to the chimeric PSI from S. elongatus
strain H53 by cytochrome c6 and plastocyanin
from C. reinhardtii shows a faster and a slower component, accounting for about 30 and 70% of the total amplitude, respectively (Fig. 5, B and C). The rates of both phases
depend on the donor concentrations, indicating second-order processes.
The fast component of these biphasic decays has a half-life of 2.1 and
6.5 ms for cytochrome c6 and plastocyanin (at 10 µM each), respectively, whereas the slow kinetic
component with both electron transfer donors is about 2 orders of
magnitude slower than the fast phase and similar to those observed for
PSI from wild type S. elongatus. A similar ratio of 30-70%
between the fast and slow phase is also observed for P700+
reduction in photosynthetic membranes of the mutant strain H53 (not
shown). This suggests that only one-third of the PSI reaction centers
have incorporated a functional chimeric PsaF, due perhaps to a
decelerated expression of the modified gene in S. elongatus, as a likely result of the high G/C codon bias of C. reinhardtii or due to an imperfect translocation, assembly, or
stability of the hybrid PsaF subunit in the cyanobacteria.
Fast first-order kinetic components that are indicative of electron
transfer reactions within a preformed complex between cytochrome
c6 and PSI are directly observable only at high
concentrations of cytochrome c6 and plastocyanin
for PSI from C. reinhardtii (19). For PSI from strain H53,
the kinetic traces observed at high cytochrome
c6 concentrations can be deconvoluted into three components (Fig. 6C) as
follows: first, a fast component with a constant half-life of 4 µs is
resolved, similar to those found for intramolecular electron transfer
between cytochrome c6 and PSI from C. reinhardtii within a stable, preformed complex (19); second, an
intermediate component (t1/2 = 154 µs) with a
half-time that decreases with increasing concentration of reduced donor
protein typically for second-order reactions between soluble reactants
(see also Fig. 5). The amplitudes of the first- and second-order phases
contribute to about 15 and 25% of the entire signal, respectively. The
third, very slow component (t1/2 = 70 ms) with an
amplitude of ~65% of the total signal is attributed to PSI lacking a
chimeric PsaF. In contrast, the absorbance transients of
P700+ reduction in H53 PSI show only two kinetic components
at plastocyanin concentrations up to 500 µM that are both
concentration-dependent (Fig. 6B). The slower
component (t1/2 = 49 ms at 300 µM
plastocyanin) most likely reflects the reduction of PSI complexes
without a functional hybrid PsaF and plastocyanin, whereas the faster
component (t1/2 = 96 µs at 300 µM
plastocyanin) is attributed to the reaction of plastocyanin with PSI
containing the chimeric PsaF. No fast first-order component can be
detected with plastocyanin (Fig. 6B). Thus, the presence of
the hybrid PsaF subunit in the cyanobacterial PSI is not sufficient to
generate a tight complex between plastocyanin and PSI although its
presence is sufficient for complex formation with cytochrome
c6 from C. reinhardtii.
Whereas the introduction of the hybrid PsaF into the cyanobacterial PSI
has a strong effect on the binding of the algal donors, the effects
observed with the cyanobacterial cytochrome c6
are minor (see Fig. 5). However, upon analyzing the transient signals in more detail, it appears that although the kinetics of
P700+ reduction by S. elongatus cytochrome
c6 are monophasic for PSI from wild type
S. elongatus (see lower panel of residuals in
Fig. 5A) and the psaF deletion mutant (not
shown), they are biphasic in the presence of the hybrid PsaF
(upper panel of residuals in Fig. 5A). Therefore,
in order to analyze a possible interaction between the hybrid PsaF and
the cyanobacterial cyt c6 in more detail,
P700+ reduction was monitored under a wide range of salt
concentrations. Fig. 7 shows the effect
of the ionic strength on the rate of the kinetic components for
cytochrome c6 from S. elongatus
(open symbols) and C. reinhardtii (closed
symbols). At low ionic strength (<1 mM
MgCl2) the kinetics of P700+ reduction by
S. elongatus cytochrome c6 are
monophasic for PSI from wild type S. elongatus (open
rectangles; t1/2 ~50 ms) and the
psaF deletion mutant (t1/2 ~50 ms, not
shown) and biphasic in the presence of the hybrid PsaF, displaying two
kinetic components with half-lives of ~17 and ~70 ms (open
circles and open triangles in Fig. 7). The amplitude ratio of these two components is similar to the one found for PSI from
S. elongatus H53 in the presence of the algal donors. At
~30 mM MgCl2 the kinetics become almost
monophasic, and at higher salt concentrations deviations from
monophasic behavior are again detected. These observations are best
rationalized when different salt dependences for the rates of the two
kinetic components are assumed. For the determination of individual
rate constants, the amplitude ratio for the two components was
therefore kept fixed at a ratio of 30:70 during the final curve-fitting
analysis of the data (Fig. 7). The component with a relative amplitude of 30% (open circles) shows optimum rates (1.5 × 107 M PSI from S. elongatus H53 Cross-links to Plastocyanin and
Cytochrome c6--
The interactions of isolated PSI
particles from C. reinhardtii, S. elongatus wild
type, and strain H53 with plastocyanin and cytochrome
c6 from C. reinhardtii and S. elongatus were examined by immunoblot analysis of cross-linked
complexes using C. reinhardtii PsaF antibodies (Fig.
8). Cross-linking products between
plastocyanin or the two cytochromes and PsaF from PSI from C. reinhardtii with molecular masses of 29 and 28.5 kDa were
detected, respectively (Fig. 8, left part). Essentially the
same cross-linking pattern was obtained with PSI from S. elongatus H53 (Fig. 8, center). The cross-linking
products observed for the hybrid PsaF from strain H53 were very similar
with respect to molecular masses and intensity to the corresponding
products in C. reinhardtii. However, no cross-linking was
obtained when the electron donors were incubated with PSI particles
from wild type S. elongatus (Fig. 8, right
panel). Thus, the N-terminal part of C. reinhardtii
PsaF is necessary and sufficient for cross-linking plastocyanin and
cytochrome c6 from C. reinhardtii and
S. elongatus to photosystem I. However, for S. elongatus cytochrome c6, the efficiency of
cross-linking to PSI from C. reinhardtii or S. elongatus H53 is low in comparison with plastocyanin or cytochrome
c6 from C. reinhardtii.
Comparison of the PsaF subunits of PSI from eukaryotic and
prokaryotic photosynthetic organisms has revealed that the former contain a basic region near their N-terminal end which is absent in
cyanobacteria. This region has been postulated to form an amphipathic helix whose positively charged face interacts electrostatically with
acidic patches of plastocyanin (26). Studies by site-directed mutagenesis of the psaF gene from C. reinhardtii
have confirmed this hypothesis and have further shown that this region
forms a recognition site for the binding of plastocyanin and cytochrome c6. In particular, Lys-16 to Lys-23 of PsaF
appears to play a crucial role in the electrostatic interaction with
both electron donor proteins (37).
In this study we have used a complementary approach to study the
function of the N-terminal region of the eukaryotic PsaF by inserting
this sequence into a cyanobacterial PsaF protein. We have thereby shown
that it is possible to express a chimeric algal-cyanobacterial PsaF
protein in a psaF-deficient strain of S. elongatus and to incorporate it into its PSI complex. This report
thus demonstrates that site-directed mutagenesis of non-essential photosynthetic genes can be performed in S. elongatus, the
cyanobacterium from which the crystallographic structure of PSI was
determined (11, 12). The organization of the psaF/psaJ
operon of the S. elongatus strain analyzed in this work
corresponds to a combination of a eukaryotic psaF with a
cyanobacterial psaJ gene that is very similar to the
psaF/psaJ operon of Cyanophora paradoxa and
P. purpurea (38, 39) indicating that the basic N-terminal
domain of PsaF was already present very early in the evolution of algae and land plants (1) (see Fig. 2).
The introduction of the N-terminal basic patch of PsaF from C. reinhardtii into the cyanobacterial subunit clearly improves the
binding of plastocyanin or cytochrome c6 from
C. reinhardtii to cyanobacterial PSI. The electron transfer
rates are increased by 2 and 3 orders of magnitude, and the
second-order rate constants (k2 = 4.0 and
1.7 × 107 M The new N-terminal domain of PsaF in the hybrid photosystem I also
influences the interaction with cytochrome c6
from S. elongatus, but the effects are apparently more
subtle. In contrast to its algal counterpart, this protein reacts very
efficiently with S. elongatus PSI regardless of the presence
of a cyanobacterial type PsaF (Fig. 5). However, the salt dependence of
the faster of the two second-order kinetic components observed for
P700+ reduction of the hybrid PSI by cytochrome
c6 from S. elongatus qualitatively
resembles the one observed for cytochrome c6
from C. reinhardtii (Fig. 7), and the cyanobacterial protein
cross-links to the eukaryotic PsaF subunit, although less efficiently
than its algal counterpart (Fig. 8). In addition, the presence of the eukaryotic PsaF subunit results in a 4-fold rate increase in the rate
of P700+ reduction by S. elongatus cytochrome
c6 at optimal salt concentrations. This suggests
that the introduction of the new basic domain on PsaF during evolution
resulted in a small immediate improvement of the reaction between
cyanobacterial cytochrome c6 and PSI. However,
the rates observed for cytochrome c6 from
C. reinhardtii are still 4-5 times faster than those of the
cyanobacterial cytochrome, a clear indication that during the evolution
from cyanobacteria to algae further structural changes must have been
introduced into cytochrome c6 in order to
develop an efficient interaction with the basic domain of PsaF. In this
context, the slow P700+ reduction rates that are observed
with cytochrome c6 in the PsaF-less complex from
C. reinhardtii but not from cyanobacteria also show that
during evolution the PsaF subunit has become an essential component in
the binding of cytochrome to PSI.
This essential role of the N-terminal domain of eukaryotic PsaF is even
more pronounced in the case of the binding of plastocyanin to PSI in
C. reinhardtii (19). The extremely poor rates of electron transfer between plastocyanin and the cyanobacterial PSI (10 times slower than with algal cyt c6) indicates that,
during evolution, the eukaryotic plastocyanin most likely lost
essential structural elements required for the recognition of
cyanobacterial PSI. These are still present to some extent in algal
cytochromes. With regard to its binding mechanism to PSI, cytochrome
c6 from C. reinhardtii thus appears
to represent an evolutionary intermediate between cytochrome
c6 from S. elongatus and plastocyanin
from C. reinhardtii, since it interacts efficiently with the
positively charged patch of a eukaryotic PsaF and still partially
retains the ability to interact with the recognition site for the
periplasmatic electron donors of a cyanobacterial PSI. In this context,
it is noteworthy that cyanobacterial cytochromes contain a single
arginine residue at the otherwise hydrophobic surface that contacts
PSI. This residue is conserved in algal cytochromes and in
cyanobacterial plastocyanin but is absent in eukaryotic plastocyanin
and may thus play an important role in the interaction between PSI and
the periplasmic electron donors in cyanobacteria.
Taken together, our data indicate that during the evolution from
cyanobacteria to algae and land plants, the reaction pathway from the
periplasmic electron donor proteins to photosystem I changed and
required structural changes in both PSI and cytochrome c6 or plastocyanin. For PSI, these were (i) the
introduction of the positively charged N-terminal recognition site of
PsaF to bind plastocyanin and cytochrome efficiently and to locate them in the vicinity to P700, and (ii) structural change(s) that most likely
occurred on the PSI subunits PsaA or PsaB. In addition, changes were
required on cytochrome c6 and plastocyanin in
order to adapt to the new mechanism. For cytochrome
c6 from C. reinhardtii, the presence
of an algal type PsaF subunit suffices to establish tight complex
formation with PSI, whereas for plastocyanin both changes are required.
In this respect, the algal cytochrome c6 appears
to be an evolutionary intermediate between a cyanobacterial cytochrome
c6 and an algal/land plant plastocyanin.
INTRODUCTION
Top
Abstract
Introduction
References
-helix, whose positively charged face may
interact with plastocyanin (26). Amino acid sequence comparison,
however, shows that a 27-amino acid domain of this well conserved N
terminus of PsaF from plants and algae is missing in cyanobacteria (9,
27). It was therefore suggested that the introduction of this new
N-terminal domain into algal and plant-type PsaF may be responsible for
the formation of the complex between PSI and cytochrome
c6 or plastocyanin that is characteristic for
PSI from algae and plants (26). In order to test this hypothesis we
have introduced a chimeric psaF gene containing the
N-terminal coding region from the green alga C. reinhardtii
into the genome of a psaF-deletion strain of S. elongatus and report the functional characterization of a
cyanobacterial photosystem I complex carrying an algal type PsaF subunit.
EXPERIMENTAL PROCEDURES
(31) carrying the streptomycin/spectinomycin resistance
genes was inserted into the HpaI site of pCRSEF/2 and FBalCRF/2, generating plasmids pCRSEF/3 and FBalCRF/3 which served as
integrative vectors for the genetic manipulation of S. elongatus.
/Kmr strain FK2, which
carries a psaF gene disrupted by a kanamycin resistance
marker (32). Prior to transformation, FK2 was grown in medium D
supplemented with 40 µg/ml kanamycin. Cells were transformed by
electroporation in the presence of plasmids pCRSEF/3 or FBalCRF/3 essentially as described previously, selected for streptomycin resistance in liquid cultures, and colony-purified on solid media (32).
From these initial clones, strains with the desired
Kms/Smr phenotype were selected by replica
plating on solid media containing either 2 µg/ml streptomycin or 25 µg/ml kanamycin. Cells carrying the C. reinhardtii psaF
gene were identified by either immunoblot analysis of photosynthetic
membranes isolated from small scale cultures using anti-C.
reinhardtii (20) or anti-S. elongatus PsaF
antibodies2 or by Southern
blot analysis of genomic DNA as described (32).
-dodecyl maltoside and purified
by centrifugation in sucrose gradients as described (33, 34). PSI from
C. reinhardtii was isolated as described (19), and
plastocyanin and cytochrome c6 were isolated
from C. reinhardtii following the protocol of Ref. 6 with
modifications described in Ref. 19. Cytochrome c6 from S. elongatus was isolated
essentially according to Ref. 35. Plastocyanin and cytochrome
c6 concentrations were determined spectroscopically using absorption coefficients of
597 nm = 4.9 mM1
cm1 and
552 nm= 20 mM1 cm1, respectively (19).
-dodecyl maltoside, 0.2 mM methyl viologen, 0.1 mM diaminodurene, 1 mM sodium ascorbate, and MgCl2 as indicated in
the figure legends. For measurements at high donor concentrations (Fig.
6), a cuvette with 3-mm optical path length was used that contained 30 µl of sample. Four individual signals were averaged, and the
resulting kinetic traces were fitted to a sum of one or two exponential components and a constant offset with the program GNUPLOT (Unix version
3.5, Williams), performing nonlinear least squares fitting using the
Marquart-Levenberg algorithm.
RESULTS
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Fig. 1.
Construction of an integrative vector for the
complementation of the psaF/psaJ-deletion strain FK2
from S. elongatus with the psaF gene
from C. reinhardtii. The physical maps of the
psaF/psaJ operon in wild type and the FK2 strain are shown
in the upper and central part. The structure of
the integrative vector FBalCRF/3 used to generate the S. elongatus strain H53 that contains a hybrid psaF gene
is displayed in the lower part. Vector FBalCRF/3 contains a
psaF gene consisting of the first 83 codons of the mature
PsaF subunit of C. reinhardtii inserted between the region
encoding the presequence and the C terminus of psaF from
S. elongatus (S.e.). A gene conferring resistance
to streptomycin and spectinomycin has been included for phenotypic
selection. Portions of the psaF gene originating from
C. reinhardtii (C.r.) are displayed in
white, and the cleavage site of the signal peptidase is
indicated by an arrow. For DNA sequences see Refs. 29 and
30. The Smr/Spr gene cassette was
obtained from pHP45 (31); the Kmr gene originated
from pRL161 (32). Restriction sites in brackets were lost
during construction. For details of the cloning procedures see
"Experimental Procedures."
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Fig. 2.
Sequence analysis of psaF
from the S. elongatus strain H53.
A, nucleotide and deduced amino acid sequence of the hybrid
psaF gene from vector FBalCRF/3. The part of the amino acid
sequence originating from C. reinhardtii is
underlined. EcoRV and BalI restriction
sites were introduced during construction of the vector and are not
present in wild type S. elongatus psaF. B, amino acid
sequence alignment of the mature PsaF from C. reinhardtii
(30), S. elongatus wild type (29), and strain H53, and
Porphyra purpurea (39). Conserved amino acids are indicated
by asterisks, and conserved lysine residues at the
N-terminal part of the algal PsaF which are functionally involved in
the binding of cytochrome c6 and plastocyanin
(37) are underlined.
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Fig. 3.
Southern analysis of genomic DNA from wild
type and mutant strains of S. elongatus. Lane 1, wild type DNA; lane 2, DNA from strain FK2; lane
3, DNA from strain H53. DNA samples were restricted by
EcoRV separated on a 0.8% agarose gel and transferred to
nylon membranes. The blots were probed with the following biotinylated
DNA probes: A, PCR fragment of a psaF cDNA
clone from C. reinhardtii encoding the part for the mature
PsaF; B, the SmaI fragment of plasmid pHP45
containing the Smr/Spr genes; C,
ClaI/XhoI fragment containing
psaF/psaJ from S. elongatus; D, HincII
fragment of plasmid pRL161 carrying Kmr gene.
Bars to the left indicate the positions of the
-HindIII restriction fragments.
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Fig. 4.
Immunoblot analysis of membranes and
photosystem I preparations from wild type S. elongatus
and strains FK2 and H53 probed with antibodies against PsaF from
C. reinhardtii. Lane 1, S. elongatus wild type membranes; lane 2, membranes from
strain FK2; lane 3, membranes from strain H53; lane
4, C. reinhardtii PSI; lane 5, S. elongatus wild type PSI; lane 6, PSI from strain FK2;
lane 7, PSI from strain H53. Membrane samples equivalent to
10 µg of chlorophyll and PSI preparations equivalent to 4 µg of
chlorophyll were separated by SDS-polyacrylamide gel electrophoresis
and transferred to nitrocellulose membrane. The blot was probed with an
anti-C. reinhardtii-PsaF antiserum and developed by luminol
detection. Bars to the left indicate the position
of recombinant molecular mass marker proteins (Sigma) whose molecular
masses (in kDa) are indicated.
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Fig. 5.
Flash-induced absorption changes measured at
817 nm with isolated photosystem I complexes from wild type S. elongatus and from the mutant strains FK2 and H53.
Kinetic traces were recorded in the presence of cytochrome
c6 from S. elongatus
(S.e.) (A), cytochrome c6
from C. reinhardtii (C.r.) (B), and
plastocyanin from C. reinhardtii (C). The
concentrations of reduced donor protein were 10 µM.
MgCl2 was present at 1 mM (A), or 10 mM (B and C). Transients obtained
with PSI from wild type S. elongatus (WT) are
displayed on dotted lines, and those obtained with PSI from
the mutant strains FK2 and H53 are shown on continuous
lines. The vertical dashed lines separate regions
recorded on different time scales. In order to be directly comparable,
the kinetic traces of the mutant PSI were normalized to the same
initial amplitude as for wild type PSI. (The differences in amplitude
was <10%.) Residuals for fits of the traces of P700+
reduction of PSI from wild type S. elongatus and strain H53
by cytochrome c6 from S. elongatus
with one (dotted trace) or two exponential components
(solid trace) are shown in the panels directly
below A. Results of fits: S. elongatus H53 PSI,
two components, t1/2 = 7.2 ms (30%) and
t1/2 = 27 ms (70%) (solid trace); wild
type PSI, one component, t1/2 = 20 ms (for details
see text.)
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Fig. 6.
Flash-induced absorption changes measured at
817 nm with isolated photosystem I complexes from the S. elongatus strain H53 in the presence of 300 µM plastocyanin (trace
b) or cytochrome c6
(trace c). Trace a (dotted
line) shows the transient signal of a control experiment recorded
with PSI from S. elongatus H53 in the absence of soluble
donors. The kinetic traces are the average of four measurements in the
presence 100 µM PSI and 3 mM
MgCl2. The vertical dashed lines separate
regions recorded on different time scales.
1 s
1) at
~3-10 mM divalent ions, i.e. at an ionic
strength that is close to the optimal salt concentrations found for the
faster phase of P700+ reduction of the hybrid PSI by
C. reinhardtii cytochrome c6 (4 × 107 M
1 s
1 at
3-10 mM MgCl2). In addition, the general shape
of the salt dependence of this component is very similar for the algal
and the cyanobacterial cytochrome and reminiscent of the one observed for C. reinhardtii photosystem I (19). On the other hand,
the salt dependence of the slower phase of P700+ reduction
of PSI from S. elongatus H53 by S. elongatus cyt
c6 (open triangles, 70% relative
amplitude) is very similar to the one observed for PSI from the wild
type (open rectangles) and the psaF-deletion
strain FK2 (not shown), and those observed for the slow phase in the
presence of C. reinhardtii cytochrome
c6 (closed triangles) is similar to
the one observed for PSI from the C. reinhardtii
psaF-deletion strain b3f (19). Thus, the salt dependences of
the slow phases (70% relative amplitude) corroborate the attribution
of these phases to the reduction of those PSI complexes from strain H53
that lack a functional hybrid PsaF subunit. The existence of a new,
faster kinetic component for P700+ reduction of the hybrid
PSI by cytochrome c6 from S. elongatus with a salt dependence reminiscent of the one observed
for C. reinhardtii cyt c6 shows that
the cyanobacterial cytochrome is affected by the introduction of a
eukaryotic PsaF. However, the difference between the rate of the faster
and slower components is rather small (factor <4) in comparison to
those observed between the corresponding two phases for cytochrome
c6 from C. reinhardtii (factor
~200) which is fully adapted to use the eukaryotic PsaF subunit
efficiently.
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Fig. 7.
Salt dependence of the rate of the
bimolecular reaction between cytochrome c6
and PSI. Open symbols, second-order rate constants for
cyt c6 from S. elongatus;
closed symbols, second-order rate constants for cyt
c6 from C. reinhardtii. For
conditions and kinetic deconvolution see Fig. 5 and text. For PSI
carrying the hybrid PsaF (strain H53), the two kinetic components with
relative amplitudes of 30 and 70% are indicated by circles
and triangles, respectively. For PSI from wild type S. elongatus the rate of the monophasic decay is given
(squares).The ionic strength was adjusted by varying the
concentration of MgCl2.
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Fig. 8.
Immunoblot analysis of cross-linked
photosystem I complexes from C. reinhardtii
(Cr, left panel), S. elongatus strain H53 (central panel), and
S. elongatus wild type (S sp,
right panel) probed with anti-C.
reinhardtii-PsaF antibodies. For each type of PSI,
cross-linked complexes generated in the absence of donor protein
(lanes 1) and in the presence of either plastocyanin
(Pc, lanes 2) or cytochrome
c6 from C. reinhardtii (Cyt.
Cr, lane 3) or cytochrome c6
from S. elongatus (Cyt. S sp, lane 4)
were analyzed. Samples corresponding to 2 µg of chlorophyll were
loaded on each lane, fractionated by SDS- polyacrylamide gel
electrophoresis, electrotransferred to nitrocellulose membrane, and
probed with antibodies against C. reinhardtii
DISCUSSION
1
s
1 for cytochrome c6 and
plastocyanin, respectively) are similar to the values found for
electron transfer to PSI from wild type C. reinhardtii (19).
In addition, a fast first-order electron transfer occurs between
cytochrome c6 from C. reinhardtii and the hybrid PSI indicating that the N-terminal domain of the eukaryotic PsaF subunit is required and sufficient for complex formation between
cytochrome c6 and PSI. Finally, the electron
donors from C. reinhardtii can be cross-linked almost
equally well to PSI complexes from S. elongatus H53 and
C. reinhardtii, demonstrating that the basic N-terminal part
of PsaF which is unique to eukaryotes is important for binding of the
electron donors from C. reinhardtii and even from
cyanobacteria. Surprisingly, however, no fast first-order kinetic
component is detected for P700+ reduction by C. reinhardtii plastocyanin, indicating that although the electron
transfer to the cyanobacterial PSI is drastically improved by the
N-terminal domain of PsaF, its interaction cannot be stabilized and
thus cannot lead to the formation of an intermolecular electron
transfer complex. This indicates that, besides the N-terminal region of
PsaF, additional structural differences must exist between PSI from
S. elongatus and C. reinhardtii which affect the
docking of plastocyanin to PSI. Site-directed mutagenesis of
plastocyanin (26, 40-42) suggests that the binding of plastocyanin to
PSI involves long range electrostatic interactions between PsaF and plastocyanin and a docking mechanism which brings the flat hydrophobic surface of plastocyanin in close contact to the PSI core proteins. The
absence of complex formation with plastocyanin could indicate that the
electrostatic interaction with PsaF is incompletely restored in the
chimeric PSI due to a slight misalignment of the hybrid PsaF within
PSI. Alternatively, a second recognition site is different in PSI from
cyanobacteria, most likely the hydrophobic contact surface on the PsaA
or PsaB subunits. However, gross structural alterations of the hybrid
PsaF are not likely since the intermolecular electron transfer with a
half-life of 4 µs observed in the complex with algal cytochrome
c6 is the same as within the eukaryotic complex.
This is indicative of a similar orientation within both electron
transfer complexes, since the half-life of electron transfer is very
sensitive to changes in distance between two redox partners (43).
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ACKNOWLEDGEMENTS |
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We thank Dr. Wolfgang Haehnel for supporting this work and Christa Reichenbach for the isolation of S. elongatus cytochrome c6.
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FOOTNOTES |
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* This work was supported by a Grant from the Swiss National Foundation (to J.-D. R.).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.
§ Recipient of a long term fellowship from the Human Frontier Science Program.
Supported by Deutsche Forschungsgemeinschaft Grants UM988/3-1
and SFB 388/A1.
** To whom correspondence should be addressed. Tel.: (49) 761-203-2697; Fax: (49) 761-203-2601; E-mail: muehlenh{at}uni-freiburg.de.
The abbreviations used are: PSI, photosystem I; cyt c6, cytochrome c6; Pc, plastocyanin; PsaF, subunit III of photosystem I; psaF, gene of the PsaF subunit; P700, primary donor of photosystem I; PCR, polymerase chain reaction; Mops, 4-morpholinepropanesulfonic acid; kb, kilobase pairs.
2 U. Mühlenhoff, unpublished.
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REFERENCES |
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