From the
Department of Biochemistry, Biophysics,
and Molecular Biology, Iowa State University, Ames, Iowa 50011, the
Department of Chemistry, Brock University, St.
Catharines, Ontario L2S 3A1, Canada, the ¶Service
de Bioénergétique, CEA and URA 2096 CNRS, CEA/Saclay, 91191
Gif-sur-Yvette, Cedex, France, the ||Institut de
Biologie Physico-Chimique, Centre National de la Recherche Scientifique, UPR
1261, 13 Rue Pierre et Marie Curie, 75005 Paris, France, the
**Fachbereich Physik, Freie Universität, Berlin,
Arnimallee 14, D14195 Berlin, Germany, and the
Department of Biochemistry and
Molecular Biology, Penn State University, University Park, Pennsylvania
16802
Received for publication, March 24, 2003 , and in revised form, April 23, 2003.
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ABSTRACT |
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INTRODUCTION |
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Unlike in the bacterial RC, the two branches in Photosystem I (PS I) are difficult, if not impossible, to distinguish spectroscopically. As discussed in the previous paper (2), the use of point mutants and time-resolved EPR and optical spectroscopy allows this issue to be addressed in PS I. The initial electron transfer steps are difficult to observe because the trapping of the excitation from the antenna masks these early events. In contrast, the subsequent electron transfer from phylloquinone (A1) to the iron-sulfur center FX is easily observed and provides a convenient way to study the kinetics and pathway of electron transfer.
Early time-resolved optical studies of electron transfer from
A1 to FX appeared to be contradictory,
with kinetics from UV absorbance changes attributed to
A1 reoxidation reported to have a
t of 15 ns in PS I particles isolated from spinach
(3) and 200 ns in PS I
particles isolated from Synechococcus sp
(4), whereas transient EPR data
on PS I particles from Synechococcus sp. and spinach chloroplasts
both gave a value of
= 260 ns (t
= 180 ns)
(5,
6). Later studies using
so-called PS I-
particles from spinach showed a biphasic decay
attributed to A1 oxidation with
t
of 25 and 250 ns, and relative amplitudes of 65
and 35%, respectively (7). Both
kinetic phases were attributed to electron transfer from
A1 to FX. Studies at faster time
scales with PS I particles from Synechocystis sp. PCC 6803 also
showed an additional kinetic phase attributed to
A1 oxidation with a t
of 10 ns (8). Although the
faster of the two kinetic phases cannot be resolved directly by transient EPR,
changes in its relative amplitude are reflected in the observed spin
polarization (9,
10), which is sensitive to the
spin dynamics of short-lived precursor states with lifetimes as short as 500
ps.
Recently, this influence of the fast kinetic phase on the spin polarization
patterns was investigated (9),
and it was concluded that the amplitude of the fast phase in whole cells and
PS I particles of cyanobacteria could account for at most 20% of the
total amplitude, whereas the transient EPR spectra of PS I particles isolated
from spinach and Chlamydomonas rheinhardtii show a much larger
influence (9). Moreover, the
ratio of the fast to slow kinetic phases is not constant between different
preparations; rather, PS I particles isolated from spinach show a diminished
amplitude of the optically detected 25-ns kinetic phase when isolated using
less harsh conditions, down to 30% in a particle prepared without detergent
(7), whereas transient EPR
suggests at most a very small contribution from the fast kinetic phase in
spinach chloroplasts and cyanobacterial whole cells
(6,
9,
10).
In contrast, recent optical studies of whole cells of C.
reinhardtii (11) showed
biphasic kinetics attributed to the reoxidation of
A1 with t of 18 ns
and t
of 160 ns and of nearly equal amplitude.
Hence, it appears that the fast kinetic phase is a property inherent to PS I
but the ratio of the amplitudes of the fast and slow kinetic phases is
species-dependent and sensitive to environmental conditions such as the
presence of the detergent. Moreover, the sensitivity to detergent isolation is
much higher in eukaryotic PS I compared with cyanobacterial PS I. Whereas the
biphasic kinetics observed in the near UV are now generally thought to result
from electron transfer from A1 to FX,
the origin of the biphasic behavior remains controversial (see Ref.
12 for review). Sétif
and Brettel (7) suggested that
the redox potentials of A1 and FX are close and that the
fast kinetic phase reflects the establishment of a redox equilibrium between
A1 and FX. This proposal was made prior to detailed
knowledge about the pseudo C2 symmetry of PS I, and therefore
presupposes a unidirectional pathway of electron transfer. Joliot and Joliot
(11) suggested that the
biphasic kinetics could come about from either two conformational states that
differ by the reoxidation rate of A1 or two
phylloquinones that correspond to the two branches of the PS I heterodimer
involved in electron transfer. The former presupposes unidirectional electron
transfer and the latter presupposes bidirectional electron transfer.
The latter idea arose when the model of PS I based on the 6-Å crystal
structure (13) showed the
presence of a 2-fold axis of symmetry similar to the pseudo 2-fold axis of
symmetry in the purple bacterial RC. The atomic resolution structure based on
the 2.5-Å electron density map
(14,
15) shows that the position of
the electron transfer cofactors and the identity and positions of nearby amino
acids are highly similar on the PsaA side and the PsaB side polypeptides. In
particular, the phylloquinones on the PsaA side and the PsaB side are located
in similar environments that consist of: (i) a H-bond between a backbone Leu
and the stromal-facing carbonyl group of phylloquinone in the ortho position
relative to the phytyl tail, (ii) a
interaction with a Trp,
and (iii) an apparent lack of a H-bond to the other phylloquinone carbonyl
group in the meta position relative to the phytyl tail. One significant break
in symmetry is in the P700 chlorophyll a'/a
special pair; the chlorophyll a' has three H-bonds to PsaA side
amino acids, whereas there are no H-bonds to the chlorophyll a on the
PsaB side. Other significant symmetry breaks are also found further down the
electron transfer chain in the high resolution structure
(15).
The extent to which the two quinones are active in the electron transport chain, the origin of the biphasic kinetics of A1 reoxidation, and the role that the structural features of the binding site play in determining the electron transfer kinetics are all unresolved issues. In this paper, we report EPR and optical kinetic studies of mutants in and around the quinone binding sites of PS I. The premise of these experiments is that a change in the environment of the quinone should lead to a change in its redox potential, which, in turn, should translate to a change in the forward kinetics of electron transfer from the quinone to the iron-sulfur clusters. A network of H-bonded residues extends from the Met axial ligand of A0 through A1 to FX. The residues involved in this network are thus candidates for point mutations, which are expected to influence the rate of forward electron transfer.
In the preceding paper (2), we showed that the point mutations W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB cause only subtle structural and electronic changes so that they act as suitable markers for following the pathway of electron transfer. As discussed in the preceding paper (2), the W697FPsaA, W677FPsaB, S692CPsaA, and S672CPsaB, mutants affect the quinone binding sites and we expect only electron transfer in the branch containing the mutation to be affected. The R694APsaA and R674APsaB mutants on the other hand may affect both branches because each is involved in a salt bridge from the jk-surface loop to Gly572PsaB (Gly585PsaA) in the respective FX binding loop (16). In other words, this residue ties together the region of the quinone with that of the iron-sulfur cluster. Mutations of these residues should disrupt this arrangement and would be expected to cause a change in the properties of FX. The results presented in this paper address the issue of directionality and biphasic kinetics and can be interpreted to show that in Synechocystis sp. PCC 6803, the majority of electrons proceed along PsaA side cofactors in PS I. Whereas we cannot rule out the participation of PsaB side cofactors in PS I electron transfer, we place an upper limit on the fraction of electrons taking this pathway.
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MATERIALS AND METHODS |
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Flash-induced Transient Absorption Spectroscopy in the Near UV/Blue RegionFlash-induced absorbance changes of isolated PS I complexes were measured with a time resolution of about 2 ns with a set-up described previously (17) using 300-ps pulses of about 300 nJ/cm2 at 532 nm for excitation (repetition rate, 1 Hz) and the relatively flat top of a 50-µs Xe flash as measuring light. Stock solutions of PS I complexes were diluted in a buffer containing 50 mM Tris, pH 8.3, 10 mM sodium ascorbate, and 500 µM 2,6-dichloroindophenol, to a final Chl concentration of typically 60 and 150 µM for measurements at 380 and 480 nm, respectively. The optical path length for the measuring light was 2 mm. Between 1024 and 4096 transients were averaged for each sample and wavelength to improve the signal-to-noise ratio. A Marquardt least squares algorithm program was used for fitting of the absorbance change transients to a multiexponential decay. Time zero was defined as the midpoint of the rising edge of the transient, and fitting was started 2.5 ns after time zero.
Flash-induced absorbance changes in whole cells were measured with an optical setup described previously (18). Cells were centrifuged and resuspended in 20 mM Tris at pH 8.2 in the presence of 5% Ficoll. Dichlorodimethylurea (20 µM) and 2 mM hydroxylamine were added to inactivate Photosystem II and 20 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone was added to prevent accumulation of transmembrane potential.
X-band Transient EPR Spectroscopy at Room
TemperatureTransient EPR experiments at room temperature were
carried out using a modified Bruker ESP 200 spectrometer equipped with a
home-built, broadband amplifier (bandwidth >500 MHz) for direct detection
experiments. Light excitation was provided by a Continuum YAG/OPO laser system
operating at 680 or 532 nm and 10 Hz. The EPR signals were digitized using a
LeCroy LT322 500 MHz digital oscilloscope and transferred to a PC for storage
and analysis. The samples were measured using a flat cell and a Bruker
rectangular resonator and contained 1 mM sodium ascorbate and 50
µM phenazine methosulfate as external redox mediators. The
response time of the system is governed by the band-width of the resonator and
is estimated to be 50 ns; the decay of the spin polarization limits the
accessible time range to times shorter than a few microseconds. Complete
time/field data sets were collected and analyzed to determine the lifetimes of
the species and their decay-associated spectra. To ensure that the isolation
procedure does not influence the kinetics or polarization patterns, transient
EPR measurements of whole cells and isolated particles from wild-type
Synechocystis sp. PCC 6803 were compared. The observed spin
polarization patterns (not shown) and kinetic traces are identical
demonstrating that the isolation of the particles has no effect on the
kinetics.
X-band Transient EPR Spectroscopy at 260 KX-band transient
EPR experiments on frozen solutions at 260 K were carried out using a Bruker
ER046 XK-T microwave bridge equipped with a Flexline dielectric resonator
(6) and an Oxford liquid helium
gas-flow cryostat. The loaded Q-value for this dielectric ring
resonator was about Q = 3000, equivalent to a rise time of
r = Q/(2
x
mw)
50 ns. The samples were illuminated using a Spectra Physics Nd-YAG/MOPO laser
system operating at 10 Hz and contained 1 mM sodium ascorbate and
50 µM phenazine methosulfate.
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RESULTS |
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Time-resolved Optical Measurements in Whole Cells and Isolated PS I ComplexesThe advantage of studying kinetic processes in whole cells is that there is no potential damage to the acceptor cofactors as a result of detergent solubilization of the thylakoid membranes. The disadvantage is that the cells are actively growing and dividing, and PS I complexes may be present in a number of developmental states, including those undergoing assembly and those undergoing degradation. To minimize any potential for heterogeneity because of turnover and to reduce effects of background absorption and competing photoprocesses in larger antenna systems, studies were performed on whole cells and purified on PS I complexes. Ideally, the kinetic results should match in both studies.
The flash-induced difference spectrum of
A1/A1 shows a broad absorption
increase between 340 and 400 nm
(4,
19). Measurements on PS I
complexes were made at 380 and 480 nm, an absorption band in the visible
spectrum that reflects the reduction of A1. For technical reasons,
measurements were performed at 390 and 400 nm on whole cells.
Fig. 1 depicts the decay of the
relative flash-induced absorbance change at 400 nm on a logarithmic time scale
in whole cells of the wild-type in comparison with the W697FPsaA
(top) and W677FPsaB (bottom) mutants.
Table I summarizes the
lifetimes and relative amplitudes as obtained from a fit of the data to the
sum of a fast and slow kinetic phase. At detection wavelengths of 390 and 400
nm, two kinetic phases are found in the wild type with similar time constants
of = 10 and
= 300 ns in an amplitude ratio of about 2:3. In the
PsaA side mutant W697FPsaA the lifetime of the slow kinetic phase
is increased by a factor of about 4 but the lifetime of the fast kinetic phase
is relatively unchanged. In the corresponding (symmetric) PsaB side mutant
W677FPsaB the lifetime of the slow kinetic phase is not changed
significantly at either wavelength (see
Table I) but the lifetime of
the fast kinetic phase is increased by a factor of about 2.7 (see
Fig. 1, top).
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Examples of flash-induced absorption changes at 380 nm measured in isolated
PS I complexes with a time resolution of about 2 ns are shown in
Fig. 2. All samples studied
showed an instrument limited rise of absorption (including the formation of
A1
(4,
19)). As in whole cells, the
subsequent decay of the signal (attributed to electron transfer from
A1 to the ironsulfur clusters
(3,
4,
7,
8,
11)) was affected by the
mutations. The wild-type signal (Fig.
2) could be well fitted with two exponential decay phases of
= 10.6 and
= 240 ns at an amplitude ratio of about 1:3 (see
Table II), in line with a
previous report
(8).2
Compared with the measurements on whole cells from the wild type at 390 and
400 nm (see Table I), the
lifetimes of the two phases are rather similar, but the relative amplitude of
the faster phase appears to be smaller in isolated PS I (but only at 380 nm,
not at 480 nm). For the mutants W697FPsaA and W677FPsaB,
the kinetics in isolated PS I complexes followed the same trends as in whole
cells, i.e. the slower phase alone was slowed in the PsaA side
mutant, whereas the faster phase alone was slowed in the PsaB side mutant. The
relative amplitudes of the two phases were not significantly affected by these
mutations (data not shown; see Table
II for fit results). Similar results were observed in mutants of
C. reinhardtii (20),
in which the mutation of Trp to Phe on PsaA increased the lifetime of the slow
phase while the corresponding (symmetric) mutation on PsaB slowed down the
fast phase.
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The lifetimes and amplitudes as obtained from a fit of the data to a fast and slow kinetic phase in whole cells of the wild-type, S692CPsaA, and S672CPsaB mutants are summarized in Table I. In the PsaA side mutant S692CPsaA, the lifetime of the slow kinetic phase is increased by a factor of about 4, whereas within experimental error the lifetime of the fast kinetic phase does not appear to be lengthened. In the corresponding (symmetric) PsaB side mutant S672CPsaB, the life-time of the slow kinetic phase is not changed significantly at either wavelength (see Table I). Surprisingly, within experimental error the lifetime of the fast kinetic phase also does not appear to be lengthened at either detection wavelength. Note particularly that the large effect on the slow kinetic phase of the S692CPsaA mutant is not mirrored by that on the fast kinetic phase in the S672CPsaB mutant. However, because of the smaller number of data points that determine the fast kinetics phase and its relatively small amplitude the evaluated kinetic parameters are more reliable for the slow component than the fast component.
Consistent with the results on whole cells, isolated PS I from the mutant S672CPsaA showed a pronounced slowing of the slower phase and virtually no effect on the faster phase (Fig. 2). The fit results (Table II) appear to indicate some increase (compared with the wild type) of the relative amplitude of the fast phase. However, some evolution of the kinetics was observed upon prolonged experimentation with this sample (see Footnote a of Table II), so that the effect on the amplitude of the S672CPsaA mutation should be interpreted with caution. The corresponding PsaB side mutation S672CPsaB showed no significant effect on the slow phase, but there was an approximate 2-fold increase in the lifetime of the fast phase (Fig. 1). The latter observation deviates from the results on whole cells in which the same mutation in PsaB appeared to have less of an effect on the kinetics of A1 oxidation.
It has been shown previously (4, 19) that absorption changes associated with A1 can be observed around 480 nm, although the nature of this absorption band is not fully established (an electrochromic bandshift of a nearby carotenoid because of the negative charge on A1 has been suggested (4)). Fit results of 480 nm measurements on isolated PS I complexes from the wild type and the four mutants described above are also listed in Table II. In all cases, the mutation effects are similar to those observed at 380 nm, including some slowing of the faster phase in the S672CPsaB mutant. The changes in relative amplitudes differ at the two wavelengths and in all samples the relative amplitude of the faster phase is significantly higher at 480 nm compared with 380 nm. This is in line with previous reports comparing the absorbance difference spectra of the two phases: when normalizing the spectra in the 380-nm band, the 480-nm band was more pronounced for the faster phase than for the slower phase (7, 8, 11).
Together the data show that for all mutations expected to influence electron transfer in the PsaA branch, the slow kinetic phase is slowed significantly. It is less clear whether such a trend holds for the fast kinetic phase. The measured lifetimes for the Trp mutants suggest that the fast phase is only affected by mutations on the PsaB branch. However, for the Ser mutants, mutations in the PsaB branch showed a significant slowing of the fast phase only in isolated PS I and not in whole cells, although the corresponding mutations in the PsaA branch consistently affect the slow phase in both types of samples.
Room Temperature Electron Spin-polarized EPR Signals at
X-bandFig. 3 compares spin-polarized EPR transients of wild-type PS I and the
W697FPsaA (left) and W677FPsaB (right)
mutants, respectively. The transients are taken at selected field positions as
indicated in the spectra at the top of the figure. As the electron is
transferred from A1 to FX, the
emission/absorption/emission (E/A/E) polarization pattern of
P700+A1 changes to one with
net emission from P700+ in the
P700+(FeS) state where FeS is one of
the iron-sulfur centers. We refer to these two spectra as the early spectrum
(P700+A1) and the late
spectrum (P700+(FeS)). At field
position a, only the A1 contribution to
P700+A1 is observed, so
that the decay of the EPR signal at this field position reflects the forward
electron transfer, provided that it is significantly faster than the decay of
the spin polarization. At other field positions, contributions from both
P700+A1 and
P700+(FeS) occur. As is evident from
the kinetic traces, the transients from the W697FPsaA mutant
(Fig. 3, left) are
significantly different from the wild-type and forward electron transfer is
slower in this mutant. A fit of the data yields a lifetime for the electron
transfer of = 640 ± 50 ns in the W697FPsaA mutant
compared with
= 240 ± 50 ns in the wild-type. In contrast, the
transients and spectra from the W677FPsaB mutant are
indistinguishable from wild-type PS I. Fig.
4 shows corresponding transients for the S692CPsaA and
S672CPsaB mutants, which have similar kinetic behavior to the Trp
mutants (Fig. 3) except that
the effect of the PsaA mutation is even greater. A fit of the data set yields
a lifetime of
= 1290 ± 50 ns in the S692CPsaA mutant.
Again, the PsaB mutant is indistinguishable from the wild type. Thus,
consistent with the optical data, a slowing of the slow phase is observed as a
result of the mutations in the PsaA-branch quinone binding site. The
corresponding PsaB branch mutations do not cause any detectable change in the
spin polarization.
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Room temperature transients of the R694APsaA and
R674APsaB mutants are shown in
Fig. 5. Clearly the data from
the two mutants are very similar. An analysis of the data sets in the forward
electron transfer rate from A1 is slowed and
yields almost identical values of = 640 ± 50 ns and
= 680
± 50 ns for the respective mutants. The optical absorbance changes in
whole cells of the R674APsaB mutant also show an increase in the
lifetime of the slow phase (Table
I) but the lifetime obtained is roughly a factor of two longer.
The origin of this difference in the observed lifetimes is unclear but it may
be related to temperature differences between the two experiments (see below).
The fact that the slowing of the electron transfer rate obtained from the EPR
experiments is the same for both the PsaA- and PsaB-branch mutants can be
rationalized by making the reasonable assumption that the mutations primarily
affect the redox potential of FX (see "Discussion").
However, the optical data show no effect on the fast phase in the
R674APsaB mutant (see Table
I). In addition, the R694APsaA mutation leads to
changes in the spin density on A1 as observed in
low temperature electron nuclear double resonance and transient EPR
experiments (see previous paper, Ref.
2). Hence it is likely that
factors other than the redox potential of FX are also influenced by
mutation of these Arg residues.
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The values of the optically determined lifetimes of the slow phase for the mutants shown in Tables I and II are in reasonable qualitative agreement with the values determined from the transient EPR experiments summarized in Table III. However, the whole cell experiments in Table I give lifetimes that are consistently longer. This is likely a result of a difference in "room temperature" between the experiments. Spin relaxation can also influence the lifetimes obtained from EPR transients; however, relaxation effects are microwave power dependent (6) and no significant dependence on the microwave power was found for the electron transfer lifetimes. Hence different temperatures are the more likely cause of the difference in the lifetimes determined optically and from EPR.
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Both the optical and the transient EPR data show that the slow kinetic
phase is associated with electron transfer along the PsaA branch. However, no
clear influence from the fast kinetic phase observed optically is detectable
in the EPR data. As discussed in Ref.
9 if the two phases correspond
to two fractions in the sample with different rates of electron transfer from
A1 to FX, then both fractions
contribute to the spin polarization observed for
P700+(FeS) and they are sensitive to
both the relative magnitude of the two fractions and their lifetimes. In
spinach samples known to contain a large fraction of fast electron transfer
the influence of the fast phase is clearly evident
(10) in the transient EPR data
and it leads to a spectrum with net emission at early times. This raises the
question of how large an effect is expected in the transient EPR data of the
samples studied here based on the amplitudes and lifetimes observed optically.
Using the expressions discussed in Ref.
9 we estimate that the net
polarization observed in the
P700+(FeS) state at X-band is zero if
the lifetime of P700+A1 is
less than 1 ns, is maximum if the lifetime of
P700+A1 is greater than
100 ns, and most sensitive to changes in the rate of electron transfer
when the lifetime of
P700+A1 is roughly 50 ns.
Simulations of the spectra (not shown) indicate that for a lifetime of 10 ns
for the fast phase, its contribution to the transient EPR signals would be
within the signal to noise if it accounted for 20% or less of the reaction
centers. Thus, the lifetimes and amplitudes of the fast phase given in Tables
I and
II are expected to produce
signal contributions close to the detection limit of transient EPR and the
changes in the lifetime of the fast phase occur in a time region (10 to 20 ns)
in which the polarization is relatively insensitive to the lifetime.
EPR Studies of the Slowing of
A1 Oxidation at 260
KAt temperatures between 260 and 300 K, charge separation between
P700 and FA/FB remains reversible in
wild-type PS I. The slow phase of forward electron transfer as measured by
absorbance changes at 380 nm in PS I from Synechococcus elongatus
(21) follows Arrhenius
behavior with an activation energy of 220 ± 20 meV so that it is slowed
by a factor of about 3 at 260 K compared with room temperature. If the fast
and slow kinetic phases differ only in their activation energies then the fast
component would have a lower activation energy and therefore a weaker
temperature dependence. Transient EPR data taken below room temperature but
above 200 K allow the influence of the mutations on the activation energy
to be investigated and may show signal contributions to the spin polarization
from the fast phase, depending on how it changes with temperature. Spin
polarization patterns and transients taken at 260 K are shown in Figs.
6 and
7, respectively, and the
electron transfer lifetimes evaluated from the transient EPR data sets are
given in Table III. The
kinetics of A1 oxidation are slowed by roughly the same factor of
around 23 compared with room temperature for all of the samples. Thus,
none of the mutations appears to have a large impact on the activation energy
associated with the slow phase of electron transfer, and the kinetics of
A1 oxidation in the W677FPsaB and
S672CPsaB mutants remain identical to those of the wild-type at 260
K. The possible influence of the fast kinetic phase on the spin polarization
patterns is investigated directly in Fig.
6, which shows early spectra in four successive 16-ns time windows
for the wild-type and the S672CPsaB and S692CPsaA
mutants. In general, the polarization patterns of spin-correlated radical
pairs exhibit lifetime or Fourier broadening as well as coherence effects
because of transient mutations, quantum beats, and envelope modulation
(2225).
Coherence effects do not make significant contributions under our experimental
conditions and Fourier broadening dominates the short time behavior at X-band.
As is evident in Fig. 6, the
broadening causes an up-field shift of the up-field zero-crossing of the E/A/E
polarization pattern. This shift is very well reproduced in numerical
simulations (Fig. 6,
bottom) of the
P700+A1 spectrum based on
the spin-correlated radical pair model
(23,
26) in which the signal decay
is assumed to be negligibly slow. Thus, it is clear that
P700+A1 dominates the early
spectra. Moreover, within experimental error, the net polarization of the
experimental spectra is zero. Thus, no evidence is found for spin polarization
associated with P700+(FeS) from the
fast kinetic phase in the electron transfer between
A1 and the iron-sulfur clusters.
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This was also confirmed by the selected transients shown in Fig. 7 taken at 260 K for wild-type PS I and the S672CPsaB mutant. The transients at the maxima of the P700+A1 spectrum (field positions A, B, and C) are analogous to those measured at room temperature (see Fig. 3) and do not exhibit any significant difference between the mutant and the wild type. The transients taken up-field of the zero-crossing point of the P700+A1 spectrum (positions D, E, and F) demonstrate the influence of Fourier broadening by the short (<100 ns) initial, absorptive (positive) spike in transients D, E, and F generated by the shift of the zero-crossing point in the early spectra of Fig. 7. The spike at these field positions can only result from the P700+A1 state. Any early contribution from the P700+(FeS) state can only have an emissive (negative) amplitude. Because within experimental accuracy the spike does not change in amplitude between the wild-type and both mutants S672CPsaB and S692CPsaA (not shown), we conclude that the mutations do not cause a measurable change of the early signal for the time range >20 ns. If the fast kinetic phase were subject to the same activation process as the slow phase and if we extrapolate from the optically determined rates listed in Table II at room temperature, then the fast phase would slow to about 75 to 90 ns, which is within the time resolution of transient EPR. In addition, the spin polarization pattern of P700+ A1, as well as that of the subsequent P700+(FeS) state, would be characteristically different as predicted from the theoretical description of sequentially populated radical pairs (27, 28). However, if the fast phase was activationless and its relative amplitude remained the same or similar to that found at room temperature, it would remain difficult to detect at 260 K. Thus, the transient EPR data suggest that either the activation energies of the two phases are considerably different or the amplitude of the fast phase becomes smaller at low temperature.
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DISCUSSION |
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Evidence for Unidirectional Electron TransferEvidence favoring unidirectionality came first from continuous wave EPR experiments performed on deletion mutants of Synechocystis sp. PCC 6803. When wild-type PS I complexes are illuminated at 205 K in the presence of sodium hydrosulfite at pH 10.5, A1 is photoaccumulated within minutes. However, when PS I complexes from a PsaE/PsaF deletion mutant are illuminated under similar conditions, only A0 is photoaccumulated (29). The premise is that photoaccumulation of A0 can occur only when phylloquinone is doubly reduced; a necessary precondition for its appearance is that the semiquinone anion must be protonated prior to the second reduction step. The proton becomes available by the removal of PsaE/PsaF and the subsequent action of Triton X-100, which are proposed to open a water channel to the phylloquinone associated with the PsaA subunit. The logic behind the experiment is that if electron transfer were unidirectional, then there should have been either an all-or-none loss of the photoaccumulated A1 signal depending on which branch is active. However, if electron transfer were bidirectional, then there should have been a loss of that percentage of the photoaccumulated A1 signal associated with the slow phase. The total loss of photoaccumulated A1 and its replacement by A0 in the PsaE/PsaF mutant implies that electron transfer is unidirectional along PsaA subunit (29). This suggests that the mutation and the detergent alter the environment of the quinone on the PsaA side of PS I, perhaps by removing a chlorophyll molecule that would otherwise shield the quinone from the hydrophilic surroundings. A related issue is that it is very difficult to accumulate a second spin on the phylloquinone in PS I from Synechococcus sp. PCC 7002 (29) or Synechocystis sp. PCC 6803.3 Were electron transfer to be bidirectional, then it might be expected that once the PsaA side quinone is photoaccumulated as A1, then it should be relatively straightforward to photoaccumulate the PsaB side quinone. Yet, A1 becomes instead doubly reduced, and A0 is photoaccumulated (however, see Ref. 30 for experimental conditions under which a second quinone was reported to be photoaccumulated in PS I from C. reinhardtii).
The strength of the photoaccumulation protocol is that it subjects the sample to multiple turnovers, thereby providing a large number of opportunities for any given electron to be transferred through either potential pathway of cofactors. The weakness of this protocol is that the photoaccumulation of A1 is a largely kinetic trick that requires special conditions of prolonged illumination in the presence of a high mobility reductant at low temperature in a glassy solid. Its success rests largely on a favorable combination of kinetic rates, particularly on forward electron transfer between the sulfite ion and P700+ at that time when the reaction center is in the charge separated state P700+ and A1. Normally this is a low probability event, given the rapid charge recombination between P700+ and A1, but given enough time, it can succeed in trapping a large percentage of the reaction centers in the P700+A1 state. Given that these forward and backward rates are not well characterized as a function of temperature, it is not possible to predict with any confidence the effect on the ability to photoaccumulate A1 when the kinetic constants are altered. Nevertheless, the photoaccumulation experiment was the first to show that electron transfer occurred through the phylloquinone associated with the PsaA subunit (29). Also, this study, as well as a number of site-directed mutation experiments in a prokaryotic cyanobacterium and a eukaryotic alga (see next section) have confirmed that the optically detected slow A1 oxidation kinetics is associated with electron transfer along the PsaA side as is the EPR-detected transient P700+A1 state.
Evidence for Bidirectional Electron TransferEvidence
favoring bidirectionality is based largely on time-resolved optical studies of
site-directed mutants in C. reinhardtii
(20). These studies involve
changing the -stacked Trp on either the PsaA or PsaB subunits, and the
results are most easily explained if the source of the biphasic kinetics of
A1 oxidation is assumed to be the result of
electron transfer through the symmetrically located branches of cofactors in
PS I (11). The premise of this
experiment is that changing the environment of the quinone should alter the
redox potential, which because of the Frank-Condon term in the Marcus
equation, should result in a change in the rate of electron transfer. A Trp
Phe mutation on the PsaB side slowed the 18-ns fast kinetic phase to 97
ns, and a Trp
Phe mutation on the PsaA side slowed the 218-ns slow
kinetic phase to 609 ns (20).
Only the relative kinetics of electron transfer appeared to be changed, not
the amplitudes of the two kinetic phases. This is an important checkpoint
because, as a first approximation, the percentage of electrons that travel up
the PsaA and PsaB sides are already decided prior to the arrival of the
electron at the quinone, and only the lifetimes of electron transfer from
A1 to FX should be affected. This
experiment provided experimental support for the hypothesis that the two
kinetic phases represent electron transfer through the two branches of
cofactors in PS I. An EPR study of the PsaA side Trp
Leu and Trp
His mutations in C. reinhardtii showed a change of the relative
amplitudes of the biexponential (spin relaxation) decay of the out of phase
electron spin-echo signal arising from the
P700+A1 radical pair
(31). At this time, there are
no results reported on the PsaB side mutants. Thus the evidence is still
inconclusive and very indirect. The strength of the optical kinetic protocol
is that if one accepts the principles of Occam's razor, the biphasic kinetics
are most easily explained by electron transfer through both branches of
cofactors. The weakness of this protocol is that a number of factors are
capable of providing biphasic kinetics, including microheterogeneity along a
given branch of cofactors, and there is a reasonable probability that a point
mutation near a cofactor can have an effect on other cofactors. This can be
seen in this study in the counterintuitive effect on the fast and slow kinetic
phases in the Arg mutants.
Finally, a series of biochemical experiments have been reported in which one of the two phylloquinones could be extracted without influencing rates of steady-state forward electron transfer to NADP+ (32), or methyl viologen (33), the backreaction kinetics from [FA/FB] to P700+ (32) or the transfer of an electron to FA at either room or cryogenic temperatures (33). The removal of the second quinone eliminated electron transfer to NADP+ and resulted in a 20-ns backreaction from A0 to P700+ (32). The assumption is that the first extracted quinone was derived from one of the two chains of cofactors rather than representing the statistical removal of one-half of the quinones from each cofactor side. The implication is that either one side of electron transfer cofactors is inactive, or that when one side of cofactors is missing, electron transfer can occur entirely through the other side of cofactors at high efficiency.
Assignment of the Kinetic Phases of A1 ReoxidationWhile it is difficult to reconcile the results of all these experiments in a single, integrated model, there is full experimental agreement on one point: the slow kinetic phase of electron transfer occurs on the quinone associated with the PsaA subunit in PS I complexes from the eukaryotic organism C. reinhardtii (20) and from the prokaryotic organism Synechocystis sp. PCC 6803 (this work), and this is the same quinone that is observed by EPR in photoaccumulation experiments (29) and in low temperature spin-polarized EPR and electron nuclear double resonance experiments (2, 34).
The assignment of the fast phase is more problematic. The absorption
difference spectra associated with the two decay lifetimes (10 and 300 ns) in
whole cells of Synechocystis sp. PCC 6803 are nearly identical (data
not shown). In isolated PS I from the same organism, the spectra of the two
phases were found to be very similar between 330 and 440 nm, but deviated
significantly between 450 and 490 nm
(8). This would suggest that
the fast phase corresponds to an event similar to the one associated with the
slow phase: an electron transfer from a phylloquinone to an iron-sulfur
cluster. The optical kinetic data on the Trp Phe mutants in whole cells
and in PS I complexes show a trend in Synechocystis sp. PCC 6803
similar to that described in C. reinhardtii
(20). In both organisms, the
lifetime of the fast kinetic phase becomes longer when the mutation is made on
the PsaB side, and the lifetime of the slow kinetic phase becomes longer when
the mutation is made on the PsaA side. The rate constant and the relative
amplitude of the absorbance change are similar in whole cells and in PS I
complexes, implying that detergent isolation does not perturb the kinetics in
this set of mutants. The optical and EPR kinetic data on the Ser
Cys
mutants shows that the lifetime of the slow kinetic phase becomes longer in
the PsaA side mutant and the effect is larger than that observed for the Trp
Phe, again consistent with the assignment of the slow phase to electron
transfer in the PsaA branch. In contrast, it is not as evident that the
lifetime of the fast kinetic phase becomes longer in the Ser
Cys PsaB
side mutant. The discrepancy between the whole cells and the PS I particle
data (see Tables I and
II) may be a function of the
precision of the measurement, but the slowing is most likely not as strong as
that in the corresponding Trp
Phe mutant.
The optical kinetic data on the R674APsaB mutant appears to be hard to reconcile with the assignment of the fast phase as electron transfer in the PsaB branch. Despite the fact that the mutation is on the PsaB side of the reaction center between QK-B and FX, the lifetime of the fast kinetic phase remains unaltered, whereas the lifetime of the slow kinetic phase increases significantly. As outlined in Ref. 16, Arg694PsaA and Arg674PsaB have an important role in the structural environment of both FX and the respective phylloquinones QK-A and QK-B. The residue positions the flexible FX binding loop of PsaA as it crosses over the PsaB region by a pair of hydrogen bonds. Arg674PsaB may therefore interact with FX via a salt bridge from the B-jk surface helix to Gly585PsaA. Thus, it is possible that the redox potential of FX is decreased in this mutant and to the same extent in the C2-symmetric Arg694PsaA mutant. This hypothesis would explain the transient EPR data, which shows the same slowing of the (slow) kinetics in both the Arg694PsaA and Arg674PsaB mutants but is hard to reconcile with the fact that the fast phase is unaffected if the latter is associated with electron transfer along the PsaB branch. Alternately, if one assumes that the two kinetic phases are associated with different conformations of the protein rather than electron transfer through the two branches, then it is conceivable that the mutation has a more pronounced effect on one conformation than on the other. It is therefore possible that the changes in fast and slow kinetic phases are associated with electron transfer through only the PsaA side, and that the quinones associated with each kinetic phase are located in slightly different environments. However, the specific effect on the fast phase of mutations made in the close environment of the phylloquinone in the PsaB branch such as W677FPsaB is difficult to reconcile with this hypothesis. These data provide a good illustration of the difficulties associated with drawing conclusions from the changes induced by point mutations. Clearly, the effects of the mutations are likely to be more complex than just causing a small change in the local environment or the redox potential of a single cofactor. Thus, it is important to base conclusions on as much experimental data as possible.
We can summarize the above discussion as follows: all data are compatible with the assignment of the slow kinetic phase to electron transfer on the PsaA branch. In contrast, the assignment of the fast phase is less certain. The optical spectral characteristics reflect reoxidation of A1, and some correlation with mutations in the PsaB branch exists. None of the data rule out electron transfer in the PsaB branch. However, the lack of independent confirmation by a second spectroscopic technique and the fact that the R674APsaB mutation does not change the fast kinetic phase suggest that the assignment should be left as an open question at present.
Asymmetry of Electron TransferHaving discussed the
assignment of the kinetic phases, we now address the question of what fraction
of the electrons follows each kinetic phase. Whereas it is clear that the
optical data give a much better estimation of the fraction of fast kinetic
phase, there appears to be a problem of consistency with the EPR data.
Qualitatively, all of the data are consistent in showing that the slow kinetic
phase accounts for the major fraction of all of the electrons. The relative
amplitudes of the two phases obtained optically suggest that the slow phase
accounts for roughly 70% of the electrons promoted by P700. The EPR
data, on the other hand, do not show any clear indication of the fast phase at
either room temperature or at 260 K. As discussed above, the net polarization
of P700+ generated by the fast kinetic phase lasts for
many microseconds, so there is no issue of time resolution. However, if the
lifetime of P700+A1 is
sufficiently short (1020 ns) and the fraction of the fast component is
sufficiently small, <20%, this polarization is likely to be difficult to
detect underneath the much stronger polarization associated with the slow
kinetic phase. For the room temperature data on wild-type PS I, comparison
with simulated EPR spectra lead to the conclusion that at most 20% of the
reaction centers could have a 10-ns electron transfer time
(28). A qualitative
demonstration of the effect of the fast component suggested that for larger
fractions, the amplitude ratio of the late and the early spectrum would be
larger than observed. This ratio as extracted from the measured data depends
on the deconvolution of the data for the instrument response and on the choice
of the effective spin relaxation times of wA and
wB of the pairs
P700+A1 and
P700+(FeS), respectively (see Equation
1 in Ref. 28). Although care
has been taken to take these factors into account properly, some uncertainty
exists in the values used. Specifically, wA
cannot not be determined experimentally in intact PS I. The value
wA measured in a sample lacking all three
iron-sulfur centers is within error the same as
wB in intact samples measured under the same
conditions (6,
28). Thus, here we have used
the same relaxation rate for both radical pairs. However, the presence of the
iron-sulfur clusters in intact sample could conceivably increase the
relaxation rate and hence our value of wA would
be underestimated. Because the relative amplitude of the two signals depends
on the factor k + (wA
wB), an underestimation of
wA would lead to an underestimation of the
amplitude of the late spectrum and hence to an underestimation of a potential
fast phase of electron transfer. The values of wA
and wB are strongly microwave power dependent,
yet the decay of the
P700+A1 signal, which is
governed by k + wA, is independent of
the microwave power in wild-type PS I
(5) indicating that under the
conditions used here, k is much larger than
wA. Thus, although it is possible that the two
relaxation rates are not the same, it is unlikely that the uncertainty in
wA wB is
large enough to have any major influence on the relative amplitudes of the two
signals in wild-type PS I. In the PsaA mutants, however, the values of
k are considerably smaller and such effects could become important.
Thus, although the relative amplitudes of the early and late transient EPR
spectra do not give a clear indication of a fast component, they are not
inconsistent with the fraction estimated from the optical results.
Another quantifiable criterion is the amount of net polarization in the
early spectrum. For the pair
P700+A1, the net
polarization is virtually zero because of the extremely short lifetime
(30 ps) of the precursor state of
P700+A0
(27). By contrast,
P700+(FeS) shows pronounced emissive
polarization of P700+ (the contribution from
(FeS) is not visible because it relaxes very rapidly).
Hence, the putative formation of
P700+FX within about 10 ns
in a fraction of the reaction centers should be visible as a net emission of
the early EPR spectrum. In Fig. 4 of Ref.
27 a comparison of the
simulated P700+A1 spectrum
with the early spectrum extracted by fitting the experimental kinetic traces
of wild-type PS I is shown. From this comparison the early spectrum appears to
be slightly emissive, however, it is not clear whether this is because the
early and late spectra have not been separated completely by the fitting
procedure or whether it is because of an additional signal contribution at
early times. Therefore we have performed similar simulations (not shown) of
the total EPR spectrum (i.e. the sum of all expected contributions)
at several delay times following the laser flash and included various
fractions of the reaction centers with a 10-ns phase. According to these
simulations, 2030% of a 10-ns phase is compatible with the net
polarization of the measured room temperature transient EPR spectra in PS I
from wild-type Synechocystis sp. PCC 6803.
It is of note that in spinach samples known to contain a large fraction of
fast electron transfer the influence of the fast phase is clearly evident
(10) in the transient EPR data
resulting in a spectrum with net emission at early times. Thus, there is no
doubt that the presence of such a phase can be detected under the proper
conditions. Because the strength of the net polarization increases with the
lifetime of the precursor state, we had expected that the fast phase in our
cyanobacterial samples would become more easily observable perhaps even
directly by EPR by decreasing the temperature of the sample to 260 K. Yet, as
shown above no clear indication of the fast phase was found. However, this is
not surprising in light of a recent optical study on PS I from
Synechocystis sp. PCC 6803 that showed that the rate of the fast
phase was virtually independent of temperature and that its relative amplitude
decreased from 28% at room temperature to
20% at 260 K
(35). Three other observations
at 260 K are surprising, however. (i) The early time behavior of the spin
polarization could be simulated by a spin-correlated radical pair model with
the assumption that only
P700+A1 contributed to the
early spectra (see Fig. 6).
(ii) Within experimental error, the net polarization of the spectra at early
times was zero (Fig. 6). (iii)
EPR transients of wild-type and S692CPsaB mutant were virtually
identical (Fig. 7), although
the fast phase as detected optically at room temperature was found to be about
two times slower in isolated PS I from this mutant than in wild-type PS I.
All these observations can be most easily explained by the absence of a
signal contribution from a fast phase of A1
reoxidation. The more difficult question, however, is whether this is because
the phase is not present or whether the polarization associated with it is
simply too weak to observe clearly. Because it is difficult to predict
accurately whether 20% of a fast phase (with
10 ns in wild type and
20 ns in the S692CPsaB mutant) should produce effects
that exceed the experimental error of the EPR experiments, this must be left
as an open question at present.
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CONCLUSIONS |
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These findings raise a number of important and interesting issues. First, the origin and functional significance of the asymmetry in the electron transfer remains unknown. Clearly, the asymmetry is determined in the initial charge separation step, however, the nature of P700* is not known with certainty, and which of the chlorophylls acts as the initial acceptor is also unknown. It is generally assumed that this process is analogous to that in purple bacteria, for which asymmetric electron transfer is established. First, P* is localized on the special pair of chlorophylls, and the accessory chlorophyll acts as the acceptor. In PSI, the electronic structure and electrostatic environment of the P700 dimer are expected to be responsible for the asymmetry of electron transfer. Many of the properties of P700 display asymmetry; for example, the spin density distribution in P700+ and the triplet excitation at low temperature. The most striking asymmetry, however, is the fact that the two chlorophylls are two different structural isomers, chlorophyll a' and chlorophyll a. Furthermore, the chlorophyll a' has three H-bonds to the PsaA side polypeptides, whereas chlorophyll a has no H-bonds to the PsaB side polypeptides. None of these properties gives a direct measure of the properties of P700*, but they all suggest that the environment of the primary donor is conductive to electron transfer predominantly along one branch of acceptors.
Another issue is whether the asymmetry has any functional significance. Because both branches converge at FX there is no reason why one branch should be favored over the other. However, forward electron transfer is not the only process in which the cofactors are involved. Specifically, the reaction center must also accommodate energy transfer to P700 and secondary electron donation from plastocyanin or cytochrome to P700+. Given, the apparent redundancy in the electron transfer chain it is conceivable that the asymmetry in the electron transfer is a consequence of optimization of energy transfer or secondary electron transfer.
Because there is considerable species variability in these secondary processes this raises the question of whether the asymmetry in the electron transfer might also be species dependent. Indeed, it is well known that biphasic electron transfer from A1 to FX in spinach is very sensitive to the isolation procedure, whereas such effects are not observed in cyanobacteria. Preliminary investigations of several plants and green algae indicate that this sensitivity also shows significant species dependence among eukaryotes. We are currently investigating these and other related issues to come to a more complete understanding of what factors influence the appearance of the slow and fast kinetic phases.
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FOOTNOTES |
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To whom correspondence should be addressed. Fax: 814-863-7024; E-mail:
JHG5{at}psu.edu.
1 The abbreviations used are: RC, reaction center; PS, photosystem; EPR,
electron paramagnetic resonance; Chl, chlorophyll; phylloquinone,
2-methyl-3-phytyl-1,4-naphthoquinone or
2-methyl-3-(3,7,11,15-tetramethyl-2-hexadecenyl)-1,4-naphthalenedione.
2 A slight bleaching present at the end of the depicted time scale is
presumably from the state
P700+(FA/FB)
and/or a small number of antenna chlorophyll triplets.
3 B. Zybailov and J. H. Golbeck, unpublished results.
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REFERENCES |
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