(Received for publication, July 3, 1996, and in revised form, November 20, 1996)
From the DOE Plant Research Laboratory and ** Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824-1312 and the § Department of Biochemistry, The George W. Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664
Two [4Fe-4S] clusters,
FA and FB, function as terminal electron
carriers in Photosystem I (PS I), a thylakoid membrane-bound protein-pigment complex. To probe the function of these two clusters in
photosynthetic electron transport, site-directed mutants were created
in the transformable cyanobacterium Synechocystis sp. PCC
6803. Cysteine ligands in positions 14 or 51 to FB and
FA, respectively, were replaced with aspartate, serine, or
alanine, and the effect on the genetic, physiological, and biochemical characteristics of PS I complexes from the mutant strains were studied.
All mutant strains were unable to grow photoautotrophically, and
compared with wild type, mixotrophic growth was inhibited under normal
light intensity. The mutant cells supported lower rates of whole-chain
photosynthetic electron transport. Thylakoids isolated from the
aspartate and serine mutants have lower levels of PS I subunits PsaC,
PsaD, and PsaE and lower rates of PS I-mediated substrate
photoreduction compared with the wild type. The alanine and double
aspartate mutants have no detectable levels PsaC, PsaD, and PsaE.
Electron transfer rates, measured by cytochrome
c6-mediated NADP+ photoreduction,
were lower in purified PS I complexes from the aspartate and serine
mutants. By measuring the P700+ kinetics after a single
turnover flash, a large percentage of the backreaction in the aspartate
and serine mutants was found to be derived from A1 and
FX, indicating an inefficiency at the FX FA/FB electron transfer step. The alanine and
double aspartate mutants failed to show any backreaction from
[FA/FB]
. These results indicate
that the various mutations of the cysteine 14 and 51 ligands to
FB and FA affect biogenesis and electron transfer differently depending on the type of substitution, and that
the effects of mutations on biogenesis and function can be biochemically separated and analyzed.
Photosystem I (PS I)1 functions as a
plastocyanin:ferredoxin oxidoreductase in the thylakoid membranes of
chloroplasts. In cyanobacteria, cytochrome c6
and flavodoxin serve as alternate donors and acceptors under conditions
of low copper and low iron. The PS I complex contains the
photosynthetic pigments, the primary donor P700, and five electron
transfer centers (A0, A1, FX,
FA, and FB) that are bound to the PsaA, PsaB,
and PsaC proteins. In cyanobacteria, the PS I complex contains at least
eight other polypeptides that are ancillary to electron transfer in
their function (1). The cofactors of PS I participate in electron transport across the membrane, oxidizing plastocyanin, and reducing ferredoxin according to the following sequence: plastocyanin (copper) or cytochrome c6 (heme) P700 (Chl
a dimer)
A0 (Chl a)
A1 (phylloquinone)
FX (a [4Fe-4S]
cluster)
FA or FB ([4Fe-4S] clusters)
ferredoxin ([2Fe-2S] cluster) or flavodoxin (flavin).
The PsaC subunit, encoded by the psaC gene, provides the ligands for two [4Fe-4S] clusters, FA and FB. Previous studies showed that the introduction of aspartic acid in position 14 (C14DPsaC) and in position 51 (C51DPsaC) led to the introduction of [3Fe-4S] and mixed-ligand [4Fe-4S] clusters in the modified FB and FA sites, respectively, of Escherichia coli-expressed PsaC proteins (2). However, when the mutant PsaC proteins were rebound to P700-FX cores, only mixed-ligand [4Fe-4S] clusters were found in the modified sites of the reconstituted C14DPsaC-PS I (3) and C51DPsaC-PS I (4) complexes. In both mutant PS I complexes, electrons could be transferred to the mixed-ligand iron-sulfur cluster at 15 K, and room temperature NADP+ photoreduction was supported at rates similar to the wild type.
The formation of mixed-ligand [4Fe-4S] clusters with altered spectral and redox properties in vitro provided a rationale for probing the functions of FA and FB in vivo. The ability to support [4Fe-4S] clusters that are able to transfer electrons at both 15 and 298 K suggests that the putative oxygen-ligated iron-sulfur clusters should be functional in living organisms. The step taken in this work is to move mutations in these ligands into a genetic system such as Synechocystis sp. PCC 6803 and to study the in vivo consequences on growth and electron transfer. Unlike other cyanobacteria, two different psaC genes have been reported in Synechocystis sp. PCC 6803. One (psaC1) (5) has a deduced amino acid sequence identical to that of tobacco, while the other (psaC2) (6) has a deduced amino acid sequence similar to those reported for other cyanobacteria. The psaC1 gene is not involved in PS I, nor can it substitute for psaC2 when the latter is insertionally inactivated. The amino acid sequence of Synechocystis sp. PCC 6803 PsaC matched that predicted from psaC2. Insertional inactivation of psaC2 prevented the formation of PsaC, thus demonstrating that this gene encodes the PS I-bound polypeptide. Further work showed that the PsaC polypeptide is necessary for stable assembly of PsaD and PsaE into PS I complexes in vivo and that PsaC, PsaD, and PsaE are not needed for assembly of PsaA/PsaB dimer and electron transport from P700 to FX (7). In this paper the term psaC refers to the gene psaC2.
Site-directed mutagenesis and transformation of Synechocystis sp. PCC 6803 has been successfully used in the study of PS I biogenesis and function (10, 11). In the work presented here, a set of strains with mutations in PsaC has been created in which a cysteine ligand to FA and/or FB is substituted by aspartate, serine, or alanine in positions 14 and/or 51. The genetic, physiological, and biochemical characterization of the PS I mutants will be presented. A companion paper (12) describes the EPR and optical kinetic properties of the mixed-ligand FA and FB clusters in site-modified PS I complexes.
Experiments were performed
using a glucose-tolerant strain of Synechocystis sp. PCC
6803, which was acclimated for growth on solid medium in the dark.
Except for tests for photoautotrophic and mixotrophic growth, cells
were grown at 30 °C under light-activated heterotrophic growth
(LAHG) conditions, as described previously (13). Antibiotics were added
in the following concentrations: kanamycin (Km), 5 mg/liter; gentamicin
(Gm), 1 mg/liter. Transformations were performed essentially as
described (15), except in the case of strain C-RCPT, which was
carefully maintained in dim light (5 µmol m
2
s
1 during 2-h incubation) throughout the procedure, since
it is light-sensitive (its mixotrophic growth with added glucose is inhibited at light intensities greater than 10 µmol m
2
s
1). Tests for photoautotrophic and mixotrophic growth
were performed using solid media with or without supplemental glucose
in a chamber providing continuous light. The light intensity was varied
by covering plates with layers of cheese cloth and were monitored using
a L1-185A photometer (LICOR, Lincoln, NE). Large cultures were grown
in carboys (15 liters) under LAHG conditions and were bubbled with
air.
Nucleic acids were manipulated using standard methodology (16), unless otherwise stated. Site-directed mutagenesis was performed using an oligonucleotide-directed in vitro mutagenesis kit as directed by the manufacturer (Amersham Corp.). For amplification of psaC from cyanobacterial strains, cells picked from a medium sized colony or equivalent amount of cells collected from liquid culture were washed once with water and used as template. Amplification products were purified using a PCR purification kit (Promega Corp., Madison, WI). Procedure for preparation of cyanobacterial DNA was adapted from Ref. 17, with two "loopfuls" of cells scraped from plates or cells from 10 ml of liquid culture being used to extract DNA.
Transformation of Synechocystis 6803 and Selection ConditionsSynechocystis sp. PCC 6803 that had been maintained under LAHG conditions for at least two subcultures (cells were subcultured once a week) was transformed with plasmids containing resistance genes to kanamycin or gentamicin. Selection for antibiotic-resistant colonies was performed under LAHG conditions. Resistant colonies were re-streaked to single colonies with at least five serial transfers to obtain full segregation of the mutation, as verified by restriction enzyme analysis of PCR products, direct sequencing of PCR products, Southern hybridizations, and growth tests.
Western Blot Analysis of Thylakoid Membrane ProteinsThylakoid membranes were isolated and SDS-polyacrylamide gel electrophoresis and immunoblotting were performed as described (18). To resolve the PsaA/PsaB proteins, 10% SDS-polyacrylamide gel electrophoresis gels were used; 17% gels were used to resolve PsaC, PsaD, PsaE, and PsaF. D2 protein in Photosystem II (PS II) was resolved with a 17% gel to serve as a control. Protein assays were performed using the method of (19). Equal amounts of chlorophyll (4 µg for PS I complexes) were loaded in each lane. Equal amounts of protein (150 µg) was loaded in each lane for thylakoids preparations. Rabbit antiserum to PsaC or PsaD were raised using protein purified from strains of E. coli expressing, respectively, the psaC gene from Synechococcus sp. PCC 7002 or the psaD gene from Nostoc sp. PCC 8009 (20). Antibodies against PsaA/B proteins from Synechococcus were raised in rabbits as described previously (21). Antibodies against PsaE and PsaF (gifts from Dr. Parag Chitnis, Kansas State University) were raised in rabbits immunized with PsaE and PsaF purified from Synechocystis sp. PCC 6803. Rabbit antibodies to the D2 polypeptide from spinach (gifts from Dr. Wim Vermaas, Arizona State University) were raised as described (22). Chlorophyll was extracted with methanol and quantified using published extinction coefficients (23).
Oxygen EvolutionLAHG-grown cells were washed once in 40 mM HEPES buffer, pH 7.0 and cells containing 10 µg Chl were resuspended in 1 ml of the same buffer and illuminated by saturating light at 25 °C. Rates of oxygen evolution were determined with a Rank-type oxygen electrode unit. Whole-chain electron transport was measured in the presence of 10 mM NaHCO3. PS II electron transport was measured in the presence of 1 mM 2,6-dichloro-p-benzoquinone.
Membrane Isolation and Purification of PS I ComplexThylakoid membranes from Synechocystis sp. PCC
6803 were isolated using a modification of the procedure described in
Ref. 7. The cells were suspended in 0.8 M sucrose, 50 mM HEPES buffer, pH 7.8, and pelleted by centrifugation.
The cells were washed twice and suspended in the same buffer containing
0.8 M sucrose and protease inhibitors. The cells were
broken in 10 cycles of a prechilled bead-beater (Biospec Products,
Bartelsville, OK); one cycle consisted of a 45-s "on phase" and a
10-min "off phase." The cell solution was removed from the beads by
vacuum suction and centrifuged at rav of
5,000 × g to remove unbroken cells. The supernatant
was pelleted by high speed ultracentrifugation at 100,000 × g with repetitive washing. The thylakoid membranes were
suspended in 50 mM Tris, pH 8.3, frozen in liquid nitrogen, and stored at 95 °C. n-Dodecyl
-D-maltoside-PS I (DM-PS I) complexes were isolated
using the protocol described in Ref. 24 with minor modifications. The
membranes were solubilized in 1% n-dodecyl
-D-maltoside (DM, Calbiochem) at 4 °C for 1 h at
the Chl concentration of 0.5 mg ml
1. DM-PS I complexes
were isolated from the lower green zone of PS I trimers which appeared
after centrifugation of the solubilized membrane suspension loaded in a
sucrose density gradient (0.1-1.0 M sucrose) for 24 h
at 4 °C. In the alanine and double aspartate mutants, a mixture of
PS I monomers and trimers was used, because it was difficult to
completely separate the lower and the upper bands. The problem is that
PsaL is easily lost in the presence of 1% DM at 0.4 mg Chl/ml due to
the absence of PsaD (9). Even though the lower part of the broad band
containing mostly PS I trimers was used (containing <10% of the total
Chl), there was nonetheless some contamination by PS I monomers.
Isolated PS I complexes were dialyzed in 50 mM Tris, pH
8.3, resuspended with the same buffer containing 15% glycerol and
0.03% DM, frozen as small aliquots in the liquid nitrogen, and stored
at
95 °C.
The steady state reductase activities of PS I complexes were measured according to Ref. 25. The absorbance kinetics were measured using a Cary 219 spectrophotometer with the photomultiplier shielded by appropriate narrow band and interference filters. The sample was illuminated from both sides using two banks of high intensity, LEDs emitting at ~660 nm (LS1, Hansatech Ltd.). The light intensity was saturating at the chlorophyll concentration used.
Rates of flavodoxin photoreduction were measured in a 1.3-ml volume
using 15 µM flavodoxin and DM-PS I at 5 µg
ml1 of Chl in 50 mM Tricine, pH 8.0, 50 mM MgCl2, 15 µM cytochrome c6 from Spirulina maxima, 6 mM sodium ascorbate, 0.05% DM. 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 a 1.3-ml volume using 15 µM flavodoxin and
0.8 µM spinach ferredoxin:NADP+
oxidoreductase (Sigma), and DM-PS I at 5 µg
ml1 of chlorophyll in 50 mM Tricine, pH 8.0, 10 mM MgCl2, 15 µM cytochrome c6 from S. maxima, 6 mM
sodium ascorbate, 0.05% DM, 0.5 mM NADP+,
0.1%
-mercaptoethanol. Rates of ferredoxin-mediated
NADP+ photoreduction were measured in a 1.3-ml volume using
5 µM spinach ferredoxin and 0.8 µM spinach
ferredoxin:NADP+ oxidoreductase (Sigma),
and DM-PS I at 5 µg ml
1 of chlorophyll in 50 mM Tricine, pH 8.0, 10 mM MgCl2, 15 µM cytochrome c6 from S. maxima, 6 mM sodium ascorbate, 0.05% DM, 0.5 mM NADP+, 0.1%
-mercaptoethanol. The
flavodoxin was purified from the a strain of E. coli
containing the gene by DEAE-Sepharose CL-6B and Sephadex G-75 (both
from Sigma) (26, 27). NADP+ reduction were
measured by monitoring the rate of change in the absorption of NADPH at
340 nm.
Transient
absorbance changes of P700 at 820 nm (A820)
were measured from the microseconds to tens-of-seconds time domain with
a laboratory-built double-beam spectrometer as described in Ref. 28
upon excitation with a frequency-doubled Nd-YAG laser with a flash
energy of 250 mJ. The decay transients were fitted to "sum of several
exponentials with base line" using the Marquardt algorithm in Igor
Pro. The user-defined fit function enabled a fit up to 7 exponentials
with all amplitudes and rate constants set free during the fit. In most
cases the fit comprised a base-line component accounting for long term
phases and/or possible drift of signal zero during long time scale
acquisition. The quality of the fit was estimated using standard
techniques, including analyses of the residuals plots and comparison of
the X2 values and standard errors of the fit parameters
between different fits.
Genetic Characteristics of the PS I Mutants
Construction and Characterization ofTo allow rapid
segregation of mutations in the psaC gene, a recipient
strain of Synechocystis sp. PCC 6803 was engineered with the
psaC coding region deleted and replaced by a
kanamycin-resistant (KmR) cassette (Fig.
1A). PCR-amplified psaC upstream
(454 bp) and downstream (208 bp) flanking regions, with linker
sequences for BamHI and EcoRI at either end, were
cloned into pUC118 (29). A KmR cassette excised from pUC4K
(30) using EcoRI was inserted into the EcoRI site
that separated the upstream and downstream flanking regions to form the
plasmid pUC118-C. Glucose-tolerant but otherwise wild type
Synechocystis sp. PCC 6803 was transformed with pUC118-
C, and KmR colonies were selected under LAHG conditions and
were genetically verified by Southern hybridization using a 1.5-kb
EcoRI-NcoI fragment containing psaC as
probe and by PCR. Complete segregation of the deletion mutation was
confirmed (data not shown).
PsaC Mutagenesis and Genetic Characterization of the Mutants
Plasmids for site-directed mutagenesis were constructed
in vitro and manipulated in E. coli. A 1.5-kb
EcoRI-NcoI fragment containing psaC
was cut out of the plasmid p6.1S3.5I (6) and cloned into pUC119 (29) at
the SmaI site to create plasmid pC. A 2.0-kb gentamicin
resistance (GmR) cassette, cut with BamHI from
pUC119-gen (31), was inserted at BbsI site downstream of
psaC gene on pC to create plasmid pCG. Single-stranded DNA
of pC was used as template for site-directed mutagenesis.
Oligonucleotides were designed to effect the desired changes in the
coding sequence, while also destroying an endonuclease restriction site
(RsaI for the Cys-14 site and BbvI for the Cys-51 site). The resulting change in digestion pattern serves as an effective
and simple means of screening for the desired mutation. After
verification of the mutations on pC using restriction mapping and DNA
sequencing, a 953-bp BglII-PstI fragment
containing the psaC mutation was cut out of pC and ligated
with a 5.7-kb partial digestion product from pCG devoid of the
corresponding fragment to form pCG with mutated psaC gene.
The mutated pCG plasmids were checked by restriction mapping and DNA
sequencing to verify the presence of the desired mutations and the
proper sequences. Taking advantage of a XbaI site in between
Cys-14 site and Cys-51 site, pCG with a double mutation
C14D/C51DPsaC was created by ligating a 758-bp
XbaI-XbaI fragment from pCG with a single
mutation C14DPsaC and a 5.9-kb
XbaI-XbaI fragment from pCG with a single
mutation C51DPsaC. Plasmid pCG with wild type
psaC and its mutated variants (shown with asterisk on
psaC) were then used to transform the strain C-RCPT (Fig.
1B). GmR colonies were selected under LAHG
conditions and were genetically verified by three different methods:
(i) Southern hybridization of genomic DNA using a 1.5-kb
EcoRI-NcoI fragment containing psaC as
probe; (ii) restriction mapping and direct DNA sequencing of PCR
product amplified from single colonies; and (iii) growth in the
presence of kanamycin. Gene replacement by double crossover and
complete segregation of all the mutations was confirmed (data not
shown).
Physiological Characterization of the PS I Mutants
Growth Analysis of the MutantsAs shown in Table
I, all of the PsaC mutants were unable to grow
autotrophically under light intensities ranging from 2.2 to 22 µmol
m2 s
1. They were able to grow
mixotrophically under 2.2 µmol m
2 s
1 but
not under 22 µmol m
2 s
1. In addition,
C51D and C14S were not able to grow photoautotrophically or
mixotrophically under 60 µmol m
2 s
1. All
PsaC mutants showed similar growth rates to wild type when growing
under LAHG or mixotrophically under 2.2 µmol m
2
s
1. These results show that the mutants are deficient in
photosynthesis, that they are light-sensitive, and that their
mixotrophic growth is inhibited by moderate light intensity of 22 µmol m
2 s
1. The likely source of light
inhibition is the overreduction of the quinone pool by PSII activity.
This interpretation arises from results (data now shown) where
C51DPsaC was found to grow mixotrophically under 22 µmol
m
2 s
1 light in the presence of 5 mM DCMU, a PS II inhibitor. Transformation of
C14SPsaC and C51DPsaC with wild type
psaC DNA restored photoautotrophic growth and relieved light
inhibition under mixotrophic conditions, demonstrating that the only
lesion in these mutants is on psaC (data not shown).
|
Whole-chain electron
transport (H2O CO2) and PS II electron
transport (H2O
2,6-dichloro-p-benzoquinone)
were measured using LAHG-grown cells. As shown in Table I, there was
essentially no O2 evolution in
C-RCPT, indicating that
PsaC is required for whole-chain photosynthetic electron transport.
This is consistent with previous observations (7). Compared with wild
type control, all the PsaC mutants had lowered whole-chain
O2 evolution rates to varying degrees, with Asp and Ser
mutants showing the highest rates, and Ala mutants and the double Asp
mutant showing the lowest rates. However, all the mutants, including
C-RCPT, showed near-wild type levels of PS II O2
evolution rates, indicating electron transport in PS II was not
affected in the short term in the mutants. Variation of these rates
most probably represents physiological variations of the whole cells of
several independent cultures of each mutant tested over a period of
several months.
Table I shows rates
of flavodoxin reduction and ferredoxin- and flavodoxin-mediated
NADP+ photoreduction for DM-PS I complexes isolated from
the wild type and mutant strains. The wild type PS I complex supports
high rates of reductase activity in the range of 800-900 µmol/mg of
Chl/h. The C-RCPT mutant (which lacks PsaC, PsaD, and PsaE) shows
the rate of flavodoxin photoreduction of 40 µmol/mg of Chl/h and the rates of flavodoxin and ferredoxin-mediated NADP+
photoreduction of 70 and 40 µmol/mg Chl/h. The
C14DPsaC-PS I and C51DPsaC-PS I complexes show
similar levels of reductase activity, but at a reduced level compared
with the wild type PS I complex: 460-510 µmol/mg of Chl/h for
flavodoxin reduction, 700 µmol/mg of Chl/h for flavodoxin-mediated
NADP+ photoreduction, and 480-540 µmol/mg of Chl/h for
ferredoxin-mediated NADP+ photoreduction. Both the
C14SPsaC-PS I and C51SPsaC-PS I complexes show
nearly the same level of reductase activity as the aspartate mutants.
However, the C14APsaC-PS I, C51APsaC-PS I, and
C14D/C51DPsaC-PS I complexes supported minimal rates of PS
I reductase activities similar to those for the
C-RCPT complex.
Biochemical Characterization of the PS I Mutants
PS I Subunit Composition in the MutantsImmunoblotting of
thylakoid membrane proteins was performed on an equal protein basis,
and PS I complexes on an equal chlorophyll basis, using antibodies
against the proteins PsaA/B, PsaC, PsaD, PsaE, and PsaF. Compared with
wild type thylakoid membranes, the levels of PsaC, PsaD, and PsaE were
lower to varying degrees in all mutants (Fig. 2). The
Asp and Ser mutants demonstrated moderately low levels of these
proteins, while Ala mutants and the double Asp mutant did not contain
detectable amounts of these three subunits. All mutants contained
near-wild type levels of PsaF and PsaA/PsaB in all samples (Fig. 2). It
is difficult to quantitate PsaA/PsaB with immunoblots (10) or by
enzyme-linked immunosorbent assay2;
however, it is assumed that the levels of PsaA/PsaB in the PsaC mutant
thylakoids are similar to that in wild type, since it has been
demonstrated that assembly of the PsaA/PsaB dimer does not require
PsaC, PsaD, or PsaE (7, 9).
In all mutants the levels of PsaC, PsaD, and PsaE appeared to be closely related, an observation that is consistent with the finding that the stable binding of PsaD and PsaE to PS I complex is dependent on the presence of PsaC (7, 8). The levels of PsaA/B and PsaF are representative of the amounts of PS I core in thylakoids (32). The levels of PsaC, PsaD, and PsaE represent the amounts of these three subunits bound on PS I core to form a complete PS I complex. These results show that the amount of complete PS I complexes is lower to varying degrees in all the mutants compared with the wild type. In thylakoids from the Asp and Ser mutants, a minor portion of PS I contains PsaC, PsaD, and PsaE bound to form a complete PS I complex, while the majority of PS I cores are lacking these subunits (it is difficult to specify the precise amount from the Western blots and efforts to do this by enzyme-linked immunosorbent assay techniques were unsuccessful). In thylakoids from Ala mutants and the double Asp mutant, no complete PS I complexes are found. To our knowledge this is the first report that the population of PS I complexes is found to be heterogeneous in vivo as a result of mutagenesis.
Heterogeneous PS I populations, assayed by immunoblot analysis, were
also observed for purified PS I complexes from the Asp and Ser mutants
after solubilizing the membranes with 1% DM at 1 mg/ml Chl for 20 min
followed by a single sucrose density centrifugation step (data not
shown). Heterogeneity complicates functional analysis of the mutant PS
I complexes, making it difficult to distinguish between (i) inefficient
electron transfer from A1 to FX to
FA/FB and (ii) a mixed population of
P700-FX cores and P700-FA/FB
complexes. To solve this problem, a purification procedure with sucrose
density gradient centrifugation was adopted to separate the PS I core subpopulation (i.e. those devoid of PsaC, PsaD, and PsaE
proteins) from the integral PS I complex. The separation principle is
based on the finding that at high ratios of DM to Chl, PsaL is readily lost in PS I complexes that lack PsaC, PsaD, and PsaE (9). On the other
hand, the PsaL-less P700-FX core was shown to be present in
the upper, monomeric band, while the intact PS I complex is present in
the lower, trimeric band (33). As the result nearly homogeneous PS I
holocomplexes, as analyzed by immunobloting, were isolated from the Asp
and Ser mutants. Compared with wild type PS I complexes, all the
mutants had similar levels of PsaB and PsaF; and the single Asp and Ser
mutants had similar levels of PsaC, while the Ala mutants and the
double Asp mutant had no detectable levels of PsaC (Fig.
3).
Single Turnover Flash Studies of Mutant PS I Complexes
The
effects of different ligand substitutions on P700+
re-reduction kinetics were determined in homogeneous DM-PS I complexes after a single turnover, saturating flash. In wild type PS I complexes, the vast majority of the backreaction is derived from
[FA/FB]. As shown in Fig.
3A, 60% of the recombination kinetics are derived from the
25- and 112-ms components attributed to P700+
[FA/FB]
recombination. An
additional 26% are derived from slower phases with lifetimes of
221 ms and 2.2 s, leading to an 86% efficient electron transfer
to FA/FB. The slowest kinetic phases are due to
exogenous donors undergoing redox reactions with
P700+ and come about in reaction centers where
[FA/FB]
has become oxidized by
exogenous electron acceptors in the medium. The sum contribution of
earlier acceptors, including FX
and
A1
, is 14% of the total absorption change.
The major contribution to the absorbance change in the
Synechocystis sp. PCC 6803 PS I complex with chemically
reduced terminal iron-sulfur clusters and in the P700-FX PS
I core isolated by urea treatment of the PS I complex is brought about
by the components with lifetimes of ~400 and 1.5 ms, which result
from back reaction of P700+ and
FX (34). The faster components with lifetimes
of ~10 and 100 µs appearing in these preparations may result from
recombination of P700+ and A1
(35) in a fraction of centers which either have a lower quantum efficiency of electron transfer between A1 and FX due to
some changes of FX microenvironment or have the
FX cluster missing or chemically reduced. There is also an
evidence that decay of 3Chl formed upon laser flash
excitation in the antenna may contribute to
A decay in
the tens-of-microseconds time domain.
The kinetics of the C14D/C51DPsaC-PS I (Fig.
4B), C14APsaC-PS I (Fig.
4C), and C51APsaC-PS I (Fig. 4D)
complexes are similar to those for the P700-FX PS I core
isolated by urea treatment of the PS I complex (data not shown; see
Ref. 34). The majority of the backreaction kinetics for all three
mutant complexes is derived from tens-of-microseconds to milliseconds
decay phases, with little or no measurable backreaction in the
tens-to-hundreds of milliseconds time scale. Since the PsaC protein is
missing in these mutants (Figs. 2 and 3), and therefore the
FA and FB clusters, which govern the
tens-of-milliseconds decay kinetics are lacking, this is the expected
result. The larger A in these three mutants, which is due
to a significant microsecond component, may be derived from additional
absorption changes from the decay of 3Chl in the antenna
bed. The optical kinetic data are in agreement with the steady state
rate data (Table I) in which no significant NADP+ reduction
occurs in the absence of FA and FB.
Kinetics of A820
absorbance changes measured at room temperature in wild type PS I
complex (A), C14D/C51DPsaC-PS I complex (B), C14APsaC-PS I complex (C), and
C51APsaC-PS I complex (D). The wild type
and mutant PS I complexes were isolated from membranes with
dodecyl-
-D-maltoside. The reaction media is 25 mM Tris buffer, pH 8.3, with
0.03% DM, 10 mM sodium ascorbate, and 4 µM
DCPIP; the chlorophyll concentration is 50 µg ml
1. Each
trace (dots) is an average of 16 measurements taken at 50-s
intervals. The multiexponential fit is overlaid as a solid line; residuals of the fit shown at the top of the
graph. The major individual exponential components are shown as
dashed lines, with the percent on the right
ordinate and with offsets equivalent to the relative contribution
of the component. The vertical dotted bars separate the time
domains in which most of the backreactions of A1
(left), FX (middle), and
FA/FB (right) correspondingly
occur.
The kinetics of the C14DPsaC-PS I, C14SPsaC-PS
I, C51DPsaC-PS I, and C51SPsaC-PS I complexes
are mixed "core"- and "complex-type," with each mutant showing
a slightly different fractions of the backreaction derived from
tens-of-microseconds to milliseconds decay phases (Fig.
5). The C14DPsaC-PS I complex (Fig.
5A) has the largest, and the C51SPsaC-PS I
mutant (Fig. 5D) has the smallest, percentage of electrons
which arrive at FA/FB. The
[FA/FB] backreaction in
C14DPsaC-PS I (Fig. 5A) has lifetime components of 33 and 335 ms which contribute 26% to the total absorption change;
an additional 15% is contributed by the slower donation to
P700+ by external donors, leading to a 41% efficient
transfer to FA/FB. The remainder of the
backreaction occurs from components with lifetimes of 712 µs (19%)
and 2.4 ms (20%) derived from FX, and 19% occurs from a
component with a lifetime of 45 µs. The
[FA/FB]
backreaction in
C51DPsaC-PS I (Fig. 5B) has lifetimes of 9 and 106 ms which contribute 15% to the total absorption change; an additional 13% is contributed by the slower donation to
P700+ by external donors, leading to a 28% efficient
electron transfer to FA/FB. The remainder of
the backreaction occurs from components with lifetimes of 206 µs
(20%) and 1.0 ms (12%) derived from FX, and over 39%
occurs from a component with a lifetime of 34 µs.
Kinetics of A820
absorbance changes measured at room temperature in
C14DPsaC-PS I complex (A),
C51DPsaC-PS I complex (B), C14SPsaC-PS I complex (C), and
C51SPsaC-PS I complex (D). The wild type
and mutant PS I complexes were isolated from membranes with
-D-dodecyl maltoside. The reaction medium is 25 mM Tris buffer, pH 8.3, with 0.03% DM, 10 mM sodium
ascorbate, and 4 µM DCPIP; the chlorophyll concentration
is 50 µg ml
1. Each trace (dots) is an
average of 16 measurements taken at 50-s intervals. The
multiexponential fit is overlaid as a solid line; residuals
of the fit shown at the top of the graph. The major
individual exponential components are shown as dashed lines, with the percent on the right ordinate and with offsets
equivalent to the relative contribution of the component. The
vertical dotted bars separate the time domains in which most
of the backreactions of A1 (left),
FX (middle), and FA/FB
(right) correspondingly occur.
The C14SPsaC-PS I complex shows relatively similar kinetic
behavior to the above two mutants. The
[FA/FB] backreaction in
C14SPsaC-PS I (Fig. 5C) has lifetime components of 51 and 148 ms which contribute 26% to the total absorption change;
an additional 6% is contributed by the slower donation to
P700+ by external donors, leading to a 32% efficient
transfer to FA/FB. The remainder of the
backreaction occurs from components with lifetimes of 1.13 ms (25%)
and 2.81 ms (13%) derived from FX, and over 31% occurs
from components with lifetimes of 32 and 198 µs. The
[FA/FB]
backreaction in
C51SPsaC-PS I (Fig. 5D) has lifetime components of 28 and 89 ms which contribute only 9% to the total absorption change; an additional 12% is contributed by the slower donation to
P700+ by external donors, leading to a 21% efficient
electron transfer to FA/FB. The remainder of
the backreaction occurs from components with lifetimes of 580 µs
(41%) and 2.3 ms (22%) derived from FX, and only 16%
occurs from a component with a lifetime of 36 µs.
Multiple site-specific mutations to individual cysteine ligands for each of the two PS I [4Fe-4S] centers FA and FB were used to produce seven mutant strains of PsaC in Synechocystis sp. PCC 6803. Cysteines 14 and 51 were changed to alanine (C14APsaC, C51APsaC), aspartic acid (C14DPsaC, C51DPsaC), and serine (C14SPsaC, C51SPsaC), and the results were compared with in vitro reconstitution studies (3, 4, 36). In each instance, the PsaC mutant strains could not grow under standard photoautotrophic growth conditions, but could grow mixotrophically under weak light, indicating a light-sensitive lesion in PS I that had become limiting for growth. The characteristics of these separate lines were dependent on the specific ligand substitution for the cysteines in two separable ways. First, some of the mutations resulted in PS I reaction centers where stable incorporation of PsaC was precluded by the specific mutation. These strains included the substitutions C51APsaC, C14APsaC, and the double aspartate substitution C14D/C51DPsaC. Second, some of the mutations resulted in PS I reaction centers incorporating lower-than-wild type levels of PsaC. These strains included the substitutions C51DPsaC, C51SPsaC, C14DPsaC, and C14SPsaC. In spite of the heterogeneity of PS I found in the membranes, near-homogeneous PS I complexes with bound PsaC, PsaD, and PsaE as judged by protein blotting were isolated from these four mutant strains by detergent fractionation.
These results vary from those recently published for two mutant strains constructed in Anabaena variabilis ATCC 29413 (37). In A. variabilis, the C13D and C50D mutants (the same functional cysteines in mutants C14DPsaC and C51DPsaC in Synechocystis sp. PCC 6803), grew photoautotrophically, and electron transport rates, measured using ascorbate/DCPIP as a donor, were similar to the wild type. Species-specific differences may be invoked to account for these differences. For example, A. variabilis 29413 is a filamentous organism and a natural heterotroph and does not require special light-pulse treatment as does Synechocystis sp. PCC 6803 (5) to grow heterotrophically.
PsaC Mutations and PS I BiogenesisThe biogenesis and redox properties of membrane complexes containing bound iron-sulfur clusters is dependent upon the associated protein structure; however, protein structure can also be modified by incorporation of mutations to ligands of iron-sulfur clusters (38). The in vivo experiments with Synechocystis sp. PCC 6803 demonstrate that some mutations give rise to altered forms of PsaC that are not stable in the PS I complex: C51APsaC, C14APsaC, and the double mutant C14D/C51DPsaC. In these cases little PsaC is seen in thylakoid membranes, and none is detected in purified PS I complexes (Figs. 2 and 3).
The Ala mutants, which contain an aliphatic side group, are not capable of providing a ligand to an iron in the modified site of the cluster. These substitutions are only capable of supporting [3Fe-4S] clusters (39). The implication is that PsaC proteins containing [3Fe-4S] clusters are unable to bind to P700-FX cores. We suspect that PsaC hosting a [4Fe-4S] cluster and a [3Fe-4S] cluster, as has been found in the unbound PsaC mutants C14DPsaC, C51DPsaC, C14SPsaC, and C51SPsaC (2, 39) has a structure sufficiently different from that of wild type PsaC to preclude its incorporation into PS I. The single substitutions with either aspartic acid (C14DPsaC, C51DPsaC) or serine (C14SPsaC, C51SPsaC) lead to lower-than-wild type levels of PsaC incorporation into PS I complexes in thylakoids (Fig. 2A). The Asp and Ser mutants, which contain carboxylate and hydroxy side groups respectively, are potentially capable of providing an oxygen ligand to an iron in the modified site of the cluster. These substitutions are capable of supporting mixed-ligand [4Fe-4S] clusters. The implication is that two [4Fe-4S] clusters must be present in PsaC to be incorporated into PS I complexes. It is likely that the C51DPsaC, C51SPsaC, C14DPsaC, and C14SPsaC proteins also have altered structures, resulting in less-than-perfect interactions with PsaA/PsaB. Yet, size and charge considerations are only one possibility. If the iron-sulfur center insertion requires an efficient ligand exchange reaction at the Cys-14 or Cys-51 positions (this is not provided by the Ala mutations and may be altered to some extent in the Ser and Asp changes), then PsaC biogenesis could be suppressed with the resulting phenotype. Consequently, the mutant PsaC proteins may not bind tightly, or they may dissociate easily, or they may be degraded in the cell, leading to a decreased amount of fully assembled PS I complex.
PsaC Mutations and Electron TransportThe
C51DPsaC, C51SPsaC, C14DPsaC, and
C14SPsaC mutant strains demonstrate whole chain oxygen
evolution of modest but significant levels along with at least 50% or
better wild type PS I electron transport capacity. Characterization of
isolated PS I complexes (Fig. 3) demonstrates that electron transport
is less efficient when incorporating a modified PsaC subunit. Assuming
that the levels of PsaC revealed by Western blots in the isolated PS I complexes of the above four mutants are similar to the wild type, the
A820 kinetics results imply that the lower
rates of electron transport are due to a qualitative functional
alteration of PS I. The single turnover flash data are in an agreement
with the NADP+ reduction data and show that the inefficient
electron transfer step occurs between FX and
FA/FB regardless of whether the mutation is in
cysteine 14, which is associated with the FB site, or in cysteine 51, which is associated with the FA site.
Particular values of lifetimes and contributions of the
A820 decay phases vary slightly from
preparation to preparation even in the wild type samples. However,
analysis of decay kinetics over several orders of time scale provides a
rationale to distinguish between the "integral-complex" and
"core-type" kinetics signature and detect a decrease in
efficiency of electron transport to FA and FB (34). We have tabulated the overall amplitude of the
A820 kinetic components with lifetimes longer
than 7 ms as an indicator of efficient photoreduction of FA
and FB. As shown in Table I, electron transfer to
FA/FB in the serine and aspartate mutants roughly correlates with the rates of ferredoxin-mediated
NADP+ photoreduction.
Comparison of some of these mutants (C14SPsaC,
C51SPsaC) with analogous in vitro PS I mutants
(36) indicates that FA/FB photoreduction in the
in vivo mutants samples occurs with a lower apparent quantum
efficiency. One possible explanation is that the external thiolate of
-mercaptoethanol, used in the iron-sulfur insertion protocol,
provides the ligand to the [4Fe-4S] cluster. The PsaC conformation
would be rendered closer to that of the wild type, which would then
confer higher efficiency of forward electron transfer to the PsaC-bound
clusters. On the other hand, the higher contribution of the fast
kinetic phases (in the microseconds to milliseconds time domain) in the
in vivo PS I mutants may occur due to decrease of the amount
of PsaC per reaction center, which would not be resolved in the Western
blots of the PsaC protein. In any event, one important question
regarding the functional properties of PsaC in the in vivo
mutants is whether only one or both of the PsaC-bound [4Fe-4S]
clusters are functioning as the electron acceptors. The goal of the
companion paper (12) is to probe this question using low-temperature
EPR spectroscopy and absorbance difference kinetics measurements using
repetitive flash excitation.
Given the proposed redox equilibrium between A1 and
FX (40), a reasonable rationale for the function of
FA and FB is to draw the equilibrium completely
away from A1, thereby ensuring a high quantum yield in PS
I. The single substitutions with either aspartic acid
(C14DPsaC, C51DPsaC) or serine
(C14SPsaC, C51SPsaC) are sufficiently similar
in structure and charge for the mutant PsaC subunit to function in this
capacity. For the Ala substitutions, C14APsaC and
C51APsaC, and the double Asp mutant,
C14D/C51DPsaC, the modified PsaC did not stably
incorporate into PS I thylakoids in vivo. These
mutants also demonstrated greater impairment in whole chain oxygen
evolution than the other mutants, with rates similar to the C-RCPT
recipient strain entirely lacking a functional PsaC. PS I electron
transport measured in three different assays also displayed base-line
activity, indicating that no functional PS I was present.
Decreased amounts of complete PS I complexes and reduced electron
transport efficiencies in complete complexes could both lead to total
reduced PS I function, making PS I activity the rate-limiting step in
photosynthetic electron transport. The reduced PS I activity apparently
causes dramatically different phenotypes in growth of the PsaC mutants
by two different mechanisms. Under low light the reduced PS I activity
limits photosynthetic conversion of light energy to the extent that
cells cannot grow photoautotrophically; under high light (22 µmol
m2 s
1) reduced PS I activity causes light
inhibition on growth by a yet unknown mechanism. The light sensitivity
issue has already been addressed in cyanobacterial PS I-less mutants
(41); the major problem is that the electron transfer chain stays
reduced, leading to the sensitivity to light.
Mutations in PsaC may therefore alter the structure of the protein, resulting in altered interaction with PS I core and thus decreased amount of PS I holocomplex in the thylakoids. Suppressor mutations on PsaA or PsaB should alter the structure of the PS I core sufficiently to restore this interaction and have the potential to partially restore PS I function. Spontaneous pseudorevertants have recently been isolated from the site-directed mutants based on the restoration of photoautotrophic growth. Characterization of the suppressor mutations is likely to provide new insights into protein-protein interactions in PS I and into the function of PS I in energy metabolism.