(Received for publication, December 22, 1995; and in revised form, March 6, 1996)
From the
The ADC4 mutant of the cyanobacterium Synechocystis sp.
PCC 6803 was studied to determine the structural and functional
consequences of the absence of PsaD in photosystem I. Isolated ADC4
membranes were shown to be deficient in ferredoxin-mediated
NADP reduction, even though charge separation between
P700 and F
/F
occurred with high efficiency.
Unlike the wild type, F
became preferentially photoreduced
when ADC4 membranes were illuminated at 15 K, and the EPR line shapes
were relatively broad. Membrane fragments oriented in two dimensions on
thin mylar films showed that the g tensor axes of
F
and F
were identical in the ADC4 and wild type strains, implying that
PsaC is oriented similarly on the reaction center. PsaC and the
F
/F
iron-sulfur clusters are lost more readily
from the ADC4 membranes after treatment with Triton X-100 or chaotropic
agents, implying a stabilizing role for PsaD. The specific role of
Lys
of PsaD, which can be cross-linked to Glu
of ferredoxin (Lelong et al.(1994) J. Biol. Chem. 269, 10034-10039), was probed by site-directed mutagenesis.
Chemical cross-linking and protease treatment experiments did not
reveal any drastic alterations in the conformation of the mutant PsaD
proteins. The EPR spectra of F
and F
in
membranes of the Lys
mutants were similar to those of the
wild type. Membranes of all Lys
mutants showed wild type
rates of flavodoxin reduction and flavodoxin-mediated
NADP
reduction, but had 10-54% decrease in the
ferredoxin-mediated NADP
reduction rates. This implies
that Lys
is a dispensable component of the docking site
on the reducing side of photosystem I and an ionic interaction between
Lys
of PsaD and Glu
of ferredoxin is not
essential for electron transfer to ferredoxin. These results
demonstrate that PsaD serves distinct roles in modulating the EPR
spectral characteristics of F
and F
, in
stabilizing PsaC on the reaction center, and in facilitating
ferredoxin-mediated NADP
photoreduction on the
reducing side of photosystem I.
Photosystem I (PS I) ()in cyanobacteria and
chloroplasts is a multisubunit membrane-protein complex that catalyzes
electron transfer from reduced plastocyanin (or cytochrome c
) to oxidized ferredoxin (or flavodoxin) (see (1) and (2) for recent reviews). The PsaA and PsaB
subunits form a heterodimeric core that harbors
100 antenna Chl a molecules, the primary electron donor, P700, and a chain of
electron acceptors A
, A
, and F
.
PsaC, PsaD, and PsaE are peripheral subunits that are located on the n-side (stromal in chloroplasts and cytoplasmic in
cyanobacteria) of the photosynthetic membrane. PsaC binds the terminal
electron acceptors, F
and F
, each a
[4Fe-4S] cluster. PsaE may be involved in ferredoxin
reduction(3, 4, 5, 6) and cyclic
electron flow around PS I(7) . The nature of interactions
between PsaE and ferredoxin are not well understood. The remaining
proteins of cyanobacterial PS I are integral membrane proteins. Among
them, PsaL is required for the formation of PS I
trimers(8, 9) . PsaF can be chemically cross-linked in vitro to plastocyanin(10, 11) . PsaJ and
PsaI may be involved in proper assembly of PsaF and PsaL,
respectively(12, 13) . PsaM has been implicated in
cyclic electron flow around PS I. (
)Role of PsaK has not
been identified.
The extrinsic protein, PsaD, has two reported
functions in the PS I complex of cyanobacteria, algae and higher
plants. The first function, deduced from in vitro reconstitution experiments, is to stabilize PsaC on the PS I
reaction center(14) . When PsaC is rebound to a P700-F core in the absence of PsaD, F
rather than F
becomes photoreduced at 15 K, and the EPR line widths of
F
and F
are
significantly broader than the wild type. These experiments did not
investigate the ability of PsaE to bind independently of PsaD. The
second function, inferred from cross-linking studies, is to serve as a
``docking'' protein to facilitate interaction of soluble
ferredoxin with the PS I
complex(15, 16, 17) . Recent cross-linking
experiments have shown that Lys
of PsaD from Synechocystis sp. PCC 6803 can be cross-linked to Glu
in ferredoxin(18) . Therefore these two residues come in
physical proximity with each other during at least one stage of
electron transfer from PS I to ferredoxin. These results indicate a
ferredoxin-docking function of PsaD, but do not illustrate a functional
requirement of PsaD for NADP
photoreduction. The
latter issue was addressed with membranes of a PsaD-less cyanobacterial
mutant, where it was shown that ferredoxin-mediated NADP
photoreduction was severely inhibited(3) . This defect
may be due to either the lack of ferredoxin-docking site in the
PsaD-less membranes or the absence of PsaD may cause alterations in
PsaC and its F
/F
redox centers. In this paper,
we further characterized ADC4, a PsaD-less cyanobacterial mutant
strain, to investigate proposed roles of PsaD in ferredoxin-mediated
NADP
photoreduction and in stabilizing PsaC on the
reaction center. We also used site-directed mutagenesis to alter
Lys
in PsaD to Arg, Glu, Asp, Asn, Ser, and Gly and
examined functional significance of the physical proximity between
Lys
of PsaD and Glu
of ferredoxin during
electron transfer from PS I.
Figure 1:
Mutagenesis of the PsaD subunit of PS
I. A, the restriction map of DNA region around psaD in the wild type (WT) strain is shown on the top
line. The restriction map of insert in the pCD40 plasmid that was
used to generate the ADC4 strain (middle line) and of insert
in the pKD1 (bottom line) is also shown. The downward and upward arrowheads represent positions of
oligonucleotides complimentary to the lower and upper strands,
respectively. Arrows show location, size, and direction of
open reading frames for psaD, Km (gene for
kanamycin resistance), and Cm
(gene for chloramphenicol
resistance). B, Western blot of the membrane proteins from the
wild type (WT), ADC4, and KD1 (K106K) strains using anti-PsaD
antibody. Thylakoid membranes containing 10 µg of Chl were used for
electrophoresis and electrotransfer.
The codon for Lys in PsaD was changed using PCR-mediated mutagenesis(20) .
The sequences of mutagenic oligonucleotides are given in Table 1.
The DNA fragments containing mutations were cloned into pKD1 by
replacing its EcoRI-SacI fragment. The amplified
fragment was completely sequenced to confirm the presence of desired
mutation and to ensure the fidelity of Taq polymerase. The
ADC4 strain was transformed with the recombinant DNAs containing
mutations, and mutant strains were obtained according to the procedure
described earlier for the KD1 strain. The presence of mutant genes in
the resulting strains was confirmed by amplification and sequencing of psaD.
The
P700-F/F
complex and the P700-F
core were prepared as described
previously(26, 27) . Reconstitution of the
F
/F
iron sulfur clusters and rebinding of PsaC,
PsaD, and PsaE to the PS I core were performed at ratios of 15 PsaC:20
PsaE:1 P700-F
core and 7 PsaC:5 PsaD:10 PsaE:1 P700-F
core according to (14) . Dissolved oxygen in the cuvette,
as measured in an oxygen electrode, decreased to near-zero levels
shortly after addition of
-mercaptoethanol.
Rates of flavodoxin photoreduction by photosynthetic membranes were
measured by monitoring the change in the absorption of flavodoxin at
467 nm under saturating actinic light(3, 27) . Rates
of flavodoxin- or ferredoxin-mediated NADP photoreduction were measured using ferredoxin:NADP
oxidoreductase (Sigma) and by monitoring the change in the
absorption of NADPH at 340 nm(3, 27) . Spinach
ferredoxin was purchased from Sigma. A strain of E. coli with
a plasmid containing the flavodoxin gene of Synechococcus sp.
PCC 7002 was provided by Dr. D. Bryant (Pennsylvania State University).
The expressed flavodoxin was purified by DEAE-Sepharose and gel
filtration chromatography.
Targeted mutagenesis was used to generate the ADC4 mutant
strain of Synechocystis sp. PCC 6803. This strain lacks PsaD
in its membranes, but contains all other PS I subunits, including PsaE
and PsaC(3) . The wild type thylakoids reduce NADP at the rate of 658 µmol
h
mg
Chl
when ferredoxin is used as the electron acceptor (Table 2). The ADC4 thylakoids are severely deficient in reducing
NADP
using ferredoxin as electron acceptor, showing
less than 10% of wild type rates. When overexpressed Synechocystis sp. PCC 6803 PsaD was reconstituted with the PsaD-less thylakoids,
72% the ferredoxin-mediated NADP
reductase activity
was restored. These results confirm our previous observation that PsaD
is essential for electron transfer using ferredoxin as
mediator(3) . In vitro reconstitution experiments have
shown that PsaD influences the binding of PsaC to the reaction center
and the EPR spectral characteristics of the F
/F
clusters(14) . The defect in electron transfer to
ferredoxin may result from the changes in PsaC and its redox centers
that are caused by the absence of PsaD. We therefore examined PsaC in
the PsaD-less mutant strain using biochemical and spectroscopic
techniques.
Figure 2: Relative PsaC contents of the wild type and PsaD-less thylakoids after treatment with Triton X-100 or chaotropic agents. Thylakoid membranes from the wild type (WT) and PsaD-less mutant (ADC4) were treated with detergent and different chaotropes as described under ``Experimental Procedures.'' The relative amounts of PsaC were determined by immunoblotting and laser densitometry of x-ray films exposed to different intensities and normalized from the levels of PsaB. Densitometric values of PsaC in the wild type membranes without any treatment (control) were considered as 100%. Results from three independent experiments are averaged. Bars indicate the standard deviation. Representative immunoblots are shown in the insets.
When wild type membranes were incubated
with 0.05% Triton X-100, PsaC was not significantly extracted, but when
the ADC4 membranes were incubated similarly for 10 min, about 50% of
the PsaC protein was removed (Fig. 2). EPR studies of a
similarly treated sample illuminated during freezing showed no removal
of F or F
from wild type, but the loss of more
than 50% of the F
and F
acceptors from the ADC4
membranes (data not shown). The concurrent loss of the F
and F
clusters is consistent with the
detergent-mediated removal of the PsaC protein rather than the in
situ destruction of the iron-sulfur clusters. When the ADC4
membranes were incubated with Nostoc sp. PCC 8009 PsaD and
subsequently treated with 0.05% Triton X-100, there was no loss of
F
and F
. There was no change in the appearance
of the EPR spectrum when the concentration of Triton X-100 was
increased to 1% (data not shown).
Figure 3:
EPR spectra of the PsaD-less thylakoids
isolated from the ADC4 strain of Synechocystis sp. PCC 6803
before and after the addition of the Nostoc sp. PCC 8009 PsaD
protein. The spectra in A and B were taken after
freezing in darkness and illumination at 15 K. The spectra in C and D were taken after illuminating the sample during
freezing to 15 K. The resonances were resolved by subtracting the
light-off (before light-on) from the light-on spectrum. Samples were
suspended in STN, containing 1 mM sodium ascorbate and 30
µM DCPIP at 600 µg ml Chl.
Spectrometer conditions: temperature, 15 K; microwave power, 20
milliwatts; microwave frequency, 9.456 GHz; receiver gain, 2.0
10
; modulation amplitude, 10 G at 100
kHz.
When recombinant Nostoc sp. PCC 8009 PsaD is added
to the ADC4 thylakoids, resonances characteristic of F at g = 2.045, 1.941, and 1.857 predominate when the sample
is frozen in darkness and illuminated at 15 K (Fig. 3B). There is also a small amount of F
photoreduced, as shown by the resonances at g =
2.066, 1.930, and 1.882. The line widths and g values of the
F
and F
resonances, and the pattern of
photoreduction at 15 K, are very similar to those observed in wild type Synechocystis sp. PCC 6803 membranes. When the reconstituted
sample is illuminated during freezing, the low-field and high-field
resonances of F
and F
merge at g = 2.047 and 1.886, and the mid-field resonances are nearly
identical to those in the wild type Synechocystis sp. PCC 6803
thylakoids and PS I complexes (Fig. 3D).
Figure 4:
EPR
spectra of the PS I complex reconstituted with Synechococcus sp. PCC 7002 PsaC, Nostoc sp. PCC 8009 PsaD, Synechococcus sp. PCC 7002 PsaE, and the Synechococcus sp. PCC 6301 P700-F core in the presence of
FeCl
, Na
S and
-mercaptoethanol. The
spectra in A and B were taken after freezing in
darkness and illumination at 15 K. The spectra in C and D were taken after illuminating the sample during freezing to 15 K.
The resonances were resolved by subtracting the light-off (before
light-on) from the light-on spectrum. Samples were suspended in 50
mM Tris-HCl buffer, pH 8.3, containing 1 mM ascorbate
and 30 µM DCPIP at a Chl concentration of 500 µg
ml
. Spectrometer conditions identical to Fig. 3.
When Nostoc sp. PCC 8009 PsaD is added to the PsaC and PsaE-reconstituted PS I
complex, resonances characteristic of F appear at g = 2.046, 1.943, and 1.857 after illumination at 15 K (Fig. 4B). A small amount of F
is reduced,
as shown by the resonances at g = 2.070, 1.931, and
1.884. In the presence of PsaD, the ratio of F
to F
reduced by illumination at 15 K is about 4:1. The line widths and g values of the F
and F
resonances, as
well as the pattern of photoreduction at 15 K, are very similar to
those observed in wild type Synechocystis sp. PCC 6803
complexes (Fig. 4B). When the reconstituted sample is
illuminated during freezing, the F
and F
clusters undergo magnetic interaction, resulting in a set of
resonances at g = 2.047, 1.939, 1.918, and 1.884 and
thus appear similar to wild type complexes isolated from Synechocystis sp. PCC 6803 thylakoids (Fig. 4D).
All psaD mutant strains could grow under photoautotrophic
conditions, with doubling times comparable with K106K strain. In
contrast, the ADC4 strain grew significantly slower than the wild type
and K106K strains. We examined the accumulation of mutant PsaD proteins
in the membranes using Western analysis (Fig. 5). On an equal
Chl basis, all strains contained similar levels of PsaD in the
membranes. Within the PS I complex, PsaD interacts with PsaA-PsaB,
PsaE, PsaC, and PsaL. When the accumulation of these PS I subunits was
examined by Western analysis, membranes of the mutant and K106K strains
contained similar levels of PsaB, PsaC, PsaE, and PsaL (Fig. 5).
Therefore Lys mutations in PsaD do not affect
accumulation of PsaD and PsaD-interacting subunits in the membrane.
Figure 5:
Accumulation of PS I subunits in the
membranes of Lys mutants cells. Photosynthetic membranes
were isolated from different cyanobacterial strains, proteins were
separated by Tricine-urea-SDS-PAGE, and transferred to Immobilon
membrane. Immunodetection was performed using horseradish
peroxidase-conjugated secondary antibody and enhanced chemiluminescence
reagents.
To
investigate the surface-exposed domains in PsaD, purified PS I
complexes from different strains were treated with thermolysin (Fig. 6). Previously, we have reported that incubation of wild
type PS I complexes with thermolysin resulted in accumulation of
distinct fragments from proteolysis at sites in the C-terminal 3
kDa of PsaD. Proteolytic patterns of Lys
mutant proteins
could be classified into three types. First, K106R and K106D proteins
showed fragments that were similar in size and abundance as the wild
type. Second, level of K106G protein declined more rapidly during
proteolysis than in the wild type PsaD. However, the number and sizes
of the fragments that were immunoreactive with anti-PsaD antibody were
remarkably similar to those in wild type, suggesting that these
mutations did not expose new proteolytic sites on the major fragments
of PsaD. Third, K106E, K106N, K106S, and K106G mutant proteins degraded
to one additional fragment that was 0.9 kDa smaller than the smallest
fragment generated from wild type PsaD. Therefore, these mutations at
best altered only slightly the conformation of the surface-exposed
domain of PsaD.
Figure 6: Thermolysin accessibility and nearest neighbors of PsaD in mutant and wild type PS I complexes. The PS I complexes were purified from different cyanobacterial strains and incubated with thermolysin at 50 µg of protease/mg of Chl for 30 min at 37 °C. For chemical cross-linking of PS I subunits, PS I complexes were incubated with 10 mM glutaraldehyde on ice. The reactions were terminated by addition of EDTA (20 mM) and glycine (100 mM). The control (lanes C), thermolysin-treated (lanes T), and glutaraldehyde-treated (lanes G) samples (5 µg of Chl/lane) were analyzed using Tricine-urea-SDS-PAGE and Western blotting. Immunodetection was performed using anti-PsaD antibody.
Modification with NHS-biotin has been used to
investigate exposure of protein surfaces to the aqueous phase.
NHS-biotin reacts with the N terminus and the -amino group of
lysyl residues(29) . We have shown previously that one or more
of four lysyl residues in the C-terminal 6-kDa region of PsaD are
present on the aqueous surface of PS I(34) . To examine if
lysyl residues other than Lys
are exposed on the PS I
surface, the PS I complexes from the K106K and K106G strains were
biotinylated and protein modification was detected using avidin
peroxidase and a chromogenic substrate for peroxidase. In both
complexes, PsaA-PsaB, PsaF, PsaL, and PsaE showed significant amounts
of biotin incorporation (Fig. 7). PsaD was biotinylated in PS I
complexes from K106K and to a lesser extent in K106G PS I complexes.
Therefore, besides Lys
, additional lysyl residues in PsaD
are exposed on the surface and may be involved in ionic interactions
observed between PS I and ferredoxin.
Figure 7: Mutant PsaD can be labeled with NHS-biotin. The biotinylated PS I from KD1 (K106K) and K106G equivalent to 10 µg of Chl were solubilized, and the proteins were separated by Tricine-urea-SDS-PAGE, electroblotted on Immobilon-P membranes, and stained with Coomassie Blue. A replica of the blot was probed with avidin-peroxidase conjugate, and the immunoreaction was visualized using 4-chloro-1-naphthol and hydrogen peroxide.
Figure 8:
EPR spectra of thylakoids from the
Lys mutant strains, K106D and K106E. The spectra in A and C were taken after freezing in darkness and
illumination at 15 K. The spectra in B and D were
taken after illuminating the sample during freezing to 15 K. The
resonances were resolved by subtracting the light-off (before light-on)
from the light-on spectrum. Samples were suspended in STN, containing 1
mM sodium ascorbate and 30 µM DCPIP at 1 mg
ml
Chl. Spectrometer conditions: temperature, 15 K;
microwave power, 20 milliwatts; microwave frequency, 9.647 GHz;
receiver gain, 2.0
10
; modulation amplitude, 10 G
at 100 kHz.
Thylakoids from the Lys mutant strains were used to
estimate the reductase activity of PS I in three different assays:
direct measurement of the rates of flavodoxin reduction,
NADP
reduction mediated by flavodoxin, and
NADP
reduction mediated by ferredoxin (Table 4).
The K106K membranes reduced flavodoxin or NADP
mediated by flavodoxin at rates of 940 and 525 µmol/mg of
Chl/h, respectively. PsaD-less membranes were able to reduce flavodoxin
and NADP
via flavodoxin at rates 40-50% that of
wild type membranes. All Lys
mutations showed normal
electron transfer rates to flavodoxin. This suggests that although PsaD
may enhance the interaction of flavodoxin with PS I, it is not an
essential component when flavodoxin functions as an electron acceptor
of PS I. These results also show that Lys
is not involved
in flavodoxin-PS I interactions. The K106K membranes reduced
NADP
at the rate of 490 µmol/mg/Chl/h when
ferredoxin was used as a mediator of electron transfer to
ferredoxin-NADP
reductase. The PsaD-less membranes
showed a drastic decrease in their ability to reduce NADP
via ferredoxin. The replacement of Lys
in K106R,
K106G, K106S, and K106N mutants reduced the rates of electron transfer
via ferredoxin by 10-34%. Disruptive replacement of Lys
by aspartate or glutamate led to 43 and 54% reduction in the
ferredoxin-mediated NADP
photoreduction rates,
respectively, compared with the K106K membranes. These results imply
that Lys
is important, but not essential, for the
interaction between PS I and ferredoxin.
The function of PsaD has been studied previously using
chemical cross-linking, biochemical resolution, and reconstitution and
targeted mutagenesis of the cyanobacterium Synechocystis sp.
PCC 6803. These studies have indicated that PsaD has multiple roles: it
can be cross-linked to ferredoxin, its absence in the ADC4
cyanobacterial mutant results in loss of NADP photoreduction, and its absence affects the EPR spectral
properties of the F
/F
clusters of PsaC. In the
present study, we investigated further the different roles of PsaD and
examined whether they were correlated using a mutant of Synechocystis sp. PCC 6803. The PsaD-less mutant organism can
synthesize and assemble a PS I reaction center containing PsaC and
PsaE(3) . This result agrees with in vitro reconstitution experiments, which showed that PsaC was able to
bind to a P700-F
core in the absence of added
PsaD(14) . We found that low concentrations of Triton X-100 led
to release of PsaC from membranes isolated from the PsaD-less strain of Synechocystis sp. PCC 6803. This result is also consistent
with those from in vitro reconstitution experiments, where it
was shown that Triton X-100 removed a large fraction of the added
recombinant PsaC from a P700-F
core(14) . Addition
of PsaD to the ADC4 thylakoids (this work) and to the
PsaC-reconstituted P700-F
core (14) led equally to
an enhanced resistance of PsaC to Triton X-100 extraction.
Low
temperature EPR studies of ADC4 membranes showed that F was
predominantly reduced at 15 K, and the line widths of the reduced
F
and F
clusters were broader than to the
control. The addition of PsaD to the ADC4 membranes resulted in the
photoreduction of F
when the sample was illuminated 15 K
and in a sharpening of the EPR line widths. This is also in close
agreement with the in vitro reconstitution experiments (14) and shows that Triton X-100 is not responsible for the
altered orientation of added PsaC. The presence of PsaE on the ADC4
membranes indicates that this protein does not require PsaD to assemble
on the PS I core. PsaE also does not influence the stability of PsaC or
the pattern of photoreduction of F
or F
, a
result which agrees with in vitro reconstitution experiments
performed on a P700-F
core in the presence of added
recombinant PsaE (this study).
The similarity of PsaC to P.
aerogenes ferredoxin (30) has led to the prediction that
the former contains a 2-fold symmetry axis related to the two
iron-sulfur binding sites(31, 32) . The altered
pattern of photoreduction of F and F
in the
ADC4 membranes could be explained if one function of PsaD were to
orient PsaC on the reaction center core. In the PsaD-less membranes,
PsaC would be misoriented; however, EPR studies on the membrane
fragments oriented on the thin mylar films have shown that the
orientation of principal g tensors of F
and
F
in ADC4 membranes are very similar with those of wild
type. This makes it unlikely that PsaC is oriented differently in ADC4;
the explanation of the altered pattern of photoreduction of F
and F
must hence be sought elsewhere.
The
interprotein electron transfer on the reducing side of PS I has been
studied using spectroscopy, chemical cross-linking, and
subunit-deficient cyanobacterial mutants. At least three proteins
(PsaC, PsaD, and PsaE) in PS I must interact with ferredoxin for
efficient electron transfer to occur(3, 4) . Recently,
kinetics of reduction of soluble cyanobacterial ferredoxin by
cyanobacterial PS I were investigated by flash absorption
spectroscopy(33) . This study revealed the existence of three
different first order components with t of
500 ns, 20 µs, and 100 µs. The 500-ns phase corresponds to
electron transfer from F
/F
to ferredoxin. These
analyses also pointed to the presence of at least two types of PS
I-ferredoxin complexes, all competent in electron transfer. Ferredoxin
accepts electrons from F
/F
of PsaC, implying
that these proteins should be in intimate contact with each other, yet
physical association between PsaC and ferredoxin has not been
demonstrated. The major obstacles in the association of PsaC and
ferredoxin are their unfavorable electrostatic interactions; both PsaC
and ferredoxin have strongly electronegative surfaces at the
physiological pH. Docking proteins may be required to facilitate the
interaction by providing clusters of amino acids with opposite charges.
PsaD and PsaE may fulfill this role, since they are required for the
interaction and reduction of ferredoxin(3, 4) .
Current evidence suggests that the interactions between ferredoxin
and PS I are electrostatic in nature. Spectroscopic study of ferredoxin
reduction suggests that complex formation precedes electron transfer
and the rate constants for complex formation depend on ion, especially
magnesium, concentrations(33, 36) . Lys of PsaD and Glu
of ferredoxin can be cross-linked
and thus provide one possible pair for ionic interaction. Changing
Lys
to uncharged amino acids, however, does not hinder
ferredoxin-mediated electron transfer. Clearly, an ionic interaction
between Lys
of PsaD and Glu
of ferredoxin is
not essential for docking of ferredoxin on the reaction center. Our
results show that one and more of the three lysyl residues (at
positions 117, 131, and/or 135) may also be exposed on the reducing
side of PS I. These residues along with three arginyl residues in the
C-terminal domain of PsaD could also provide ionic interactions during
docking of ferredoxin on PsaD. Alternatively, Lys
may
participate in ionic interactions between the wild type PS I and
ferredoxin, but its function can be replaced by other surface-exposed
lysines. Since Lys
of PsaD can be cross-linked to
Glu
of ferredoxin(18) , these residues come in
vicinity of each other. Close proximity between these residues may
facilitate docking, as the site-directed replacement of Lys
with negatively charged amino acids drastically reduces rates of
electron transfer to ferredoxin. Interestingly, a nonconservative
mutation in Anabaena ferredoxin at Glu
,
equivalent of Glu
in ferredoxin from Synechocystis sp. PCC 6803, does not affect its interaction with spinach PS
I(37) .
The absence of PsaD has a profound effect on PsaC
and its redox centers, which may be partly responsible for the
inability of PsaD-less membranes to carry out ferredoxin-mediated
NADP reduction. However, phenotypes of Lys
mutants show that PsaD has a distinct docking function that may
be its primary role in ferredoxin-mediated photoreduction. K106D and
K106E mutants contain normal amounts of PsaC and their
F
/F
clusters show EPR spectral characteristics
similar to similar to the wild type. These membranes also exhibit wild
type rates of flavodoxin reduction, again indicating normal electron
transfer within the PS I complex. Yet, membranes of these strains are
unable to support wild type rates of NADP
photoreduction using ferredoxin. Therefore, wild type PsaC and
its redox centers are not sufficient for obtaining wild type rates of
NADP
photoreduction. These results demonstrate the
requirement of PsaD in ferredoxin-mediated electron transfer and
provide a strong functional evidence for a ferredoxin-docking role of
PsaD.