Mutagenesis of Prochlorothrix Plastocyanin Reveals
Additional Features in Photosystem I Interactions*
Manuel
Hervás
,
Eugene
Myshkin§,
Nadejda
Vintonenko§,
Miguel A.
De la
Rosa
¶,
George S.
Bullerjahn§, and
José A.
Navarro
From the
Instituto de Bioquímica Vegetal y
Fotosíntesis, Centro de Investigaciones Científicas
"Isla de la Cartuja," Universidad de Sevilla y Consejo Superior de
Investigaciones Científicas, Américo Vespucio
s/n, 41092-Sevilla, Spain and the § Center for
Photochemical Sciences, Department of Biological Sciences, Bowling
Green State University, Bowling Green, Ohio 43403
Received for publication, November 22, 2002, and in revised form, December 19, 2002
 |
ABSTRACT |
Three surface residues of plastocyanin from
Prochlorothrix hollandica have been modified by
site-directed mutagenesis. Changes have been made in methionine
33, located in the hydrophobic patch of the copper protein, and in
arginine 86 and proline 53, both located in the eastern hydrophilic
area. The reactivity toward photosystem I of single mutants M33N, P53A,
P53E, R86Q, R86E, and the double mutant M33N/P14L has been studied by
laser flash absorption spectroscopy. All the mutations yield increased
reactivity of plastocyanin toward photosystem I as compared with wild
type plastocyanin, thus indicating that in
Prochlorothrix electron donation to photosystem I is not
optimized. The most drastic increases in the intracomplex electron
transfer rate are obtained with mutants in methionine 33, whereas
replacing arginine 86 only modestly affects the
plastocyanin-photosystem I equilibrium constant for complex formation.
Mutations at position 53 also promote major changes in the association
of plastocyanin with photosystem I, yielding a change from a mechanism
involving complex formation to a simpler collisional interaction.
Molecular dynamics calculations indicate that mutations at position 33 promote changes in the H-bond network around the copper center.
The comparative kinetic analysis of the reactivity of
Prochlorothrix plastocyanin mutants toward photosystem I
from other cyanobacteria reveals that mutations M33N, P53A, and P53E
result in enhanced general reactivity.
 |
INTRODUCTION |
Plastocyanin (Pc)1 is a
soluble type-I copper metalloprotein (molecular mass, ~10.5 kDa)
located inside the thylakoid lumen of photosynthetic organisms and
acting as a mobile electron carrier between the membrane-embedded
cytochrome b6f and photosystem I (PSI) complexes (1, 2). In eukaryotic Pc, two active sites have been
identified: site 1, an hydrophobic flat region around the copper
binding area, and site 2, a charged region referred to as the acidic
patch in plants and eukaryotic algae because it includes aspartate and
glutamate residues. Whereas site 1 is involved in vitro in
hydrophobic interactions of Pc with both redox partners and in the
electron transfer event itself, site 2 is responsible for the
electrostatic interactions and molecular recognition with complementary
positive areas both in PSI and cytochrome f (3). However,
the relevance of such electrostatic interactions in vivo can
be different, as is the case for the interaction between cytochrome
f and Pc (4). In cyanobacteria, site 2 can be either
negatively or positively charged (5). All these differences between
distinct organisms have led to different reaction mechanisms for PSI
reduction (5-7).
Prochlorophytes represent a diverse group of cyanobacteria containing
both chlorophyll a and b (8, 9). Recently, an analysis of the interaction of Pc with PSI from the prochlorophyte Prochlorothrix hollandica, both for WT and mutated Pc (10,
11), revealed that Prochlorothrix Pc reacts with PSI by
forming a transient complex with PSI that is stabilized by means of
hydrophobic interactions. The solution structure of
Prochlorothrix Pc has been solved by NMR spectroscopy (12),
showing that this Pc contains an altered hydrophobic patch due to the
presence of three unique residues, Tyr-12, Pro-14, and Met-33
(corresponding to the conserved Gly-10, Leu-12, and Asn-31 as numbered
in the spinach protein). Single (Y12G, Y12F, Y12W, and P14L) and double
(Y12G/P14L) mutations in the hydrophobic patch of
Prochlorothrix Pc do not alter the constant of complex
formation with PSI, but the electron transfer rate constant is
significantly affected (11). Most interesting changes are
obtained with mutants at Pro-14: reversion of the "unique" Pro-14
of Prochlorothrix Pc to the "standard" leucine of all
other Pcs enhances the reactivity of Pc toward PSI, thereby indicating
that Prochlorothrix Pc interaction with PSI is not optimized
(11). More recently, a computational simulation of Prochlorothrix Pc-PSI docking suggested that Tyr-12 in Pc
participates in hydrogen bonding with an asparagine residue in the PsaB
polypeptide of PSI. This model also shows a short
-helix in Pc
around position Pro-53 interacting with a small
-sheet extension of
the PsaA polypeptide in PSI (13).
In this study, we have extended our previous studies of
Prochlorothrix PSI reduction by Pc to analyze the reactivity
of Prochlorothrix Pc mutants at Met-33, in the hydrophobic
patch, as well as at Pro-53 and Arg-86, in the electrostatically
charged area. An arginine residue in an equivalent position to Arg-86
in Prochlorothrix Pc seems to be a specific feature of
prokaryotic Pc, and it has been shown that this residue plays a
fundamental role in the interaction with PSI (5, 14). The laser-flash
absorption spectroscopy analyses herein reported indicate that
replacing Met-33 by the standard asparagine (as is the case for
the replacement of Pro-14 by leucine) as well as Pro-53 substitution
make the copper protein react much more efficiently with PSI, whereas
Arg-86 replacement only promotes moderate effects on (Pc-PSI)
complex formation.
 |
EXPERIMENTAL PROCEDURES |
Expression and Characterization of Mutant Pc--
Mutant and WT
P. hollandica Pc were expressed as inclusion bodies in
Escherichia coli BL21(DE3) pLysS (Novagen, Madison, WI), as
previously described (15). Pc preparations were analyzed by absorption
and far-UV circular dichroism spectroscopy as described in Babu
et al. (15). Redox titration of Pc preparations was monitored by absorption spectroscopy at 602 nm in increasing ratios of
ferrocyanide/ferricyanide as previously described (16). Construction of
mutant Pc in our expression system employed the Stratagene QuikChange
kit, and custom mutagenic primers were obtained from Invitrogen.
The primer sequences directing the following mutations were obtained:
for M33N, 5'-CCGTTGAGTTCGTGAACAACAAGGTTGGTCCC-3'; R86Q,
5'-CGCCCCACCAGGGCGCTGGCATGGTCGGC-3'; R86E,
5'-CGCCCCACGAAGGCGCTGGCATGGTCGGC-3'; P53A,
5'-GCCGGTGAGAGCGCCGCCGCTCTGTCCAACACC-3'; P53E,
5'-GCCGGTGAGAGCGCCGAAGCTCTGTCCAACACC-3'. Double mutant M33N/P14L
employed the P14L mutant expression plasmid as a template for the M33N
primer (11).
PSI Purification and Kinetic Studies--
PSI particles from
Prochlorothrix were obtained by
-dodecyl maltoside
solubilization as described by Rögner et al. (17) and
modified in Navarro et al. (11). The P700 content in PSI samples was calculated from the photoinduced absorbance changes at 820 nm using the absorption coefficient of 6.5 mM
1 cm
1 determined by Mathis
and Sétif (18). Chlorophyll concentration was determined
according to Arnon (19). The chlorophyll/P700 ratio of the resulting
preparation was 150/1. Spinach, Synechocystis, and
Anabaena PSI were purified as previously described (6).
Kinetics of flash-induced absorbance changes in PSI were followed at
820 nm as described in Hervás et al. (6), except that
the setup was optimized by replacing the measuring light by an
attenuated laser diode (830 nm, 150 mW, model LD 1361 from Laser 2000 Ltd.). Unless otherwise stated, the standard reaction mixture
contained, in a final volume of 0.2 ml, 20 mM Tricine-KOH, pH 7.5, 0.03%
-dodecyl maltoside, 10 mM
MgCl2, an amount of PSI-enriched particles equivalent to
0.35 mg of chlorophyll ml
1, 0.1 mM methyl
viologen, 2 mM sodium ascorbate and Pc at the indicated
concentration. All the experiments were performed at 22 °C in a 1-mm
path-length cuvette. Each kinetic trace was the average of 5-10
independent measurements with 30 s spacing between flashes. For
most experiments, the estimated error in the observed rate constants
(kobs) was less than 10%, based on
reproducibility and signal-to-noise ratios. Data collection and
analysis were as previously described (6, 20).
Molecular Dynamics Simulations--
Mutations were introduced
into the NMR solution structure of the WT Prochlorothrix Pc
via SWISS-PDB Viewer (21) and saved as a (Protein Data Bank)
file. Only those rotamers lacking steric clashes with other amino acids
were selected. Charges of most atoms were taken from the AMBER 6.0 force field (22). Charges and other force field parameters for the
copper atom and its ligands were obtained from NMR structural data and
compared with similar Pc simulations in the literature (23, 24). To
preserve the geometry of the copper site, the bonds between the copper
atom and its ligands were treated as covalent (25). Topology and parameter files for both mutants were generated using the xLeap module
of the AMBER 6.0 package. Hydration was treated explicitly by including
the protein in a 10-Å water box according to the TIP3P model (26).
Proteins were subjected to 1000 steps of steepest descent energy
minimization to relax large steric overlaps or electrostatic
inconsistencies. Next, the system was equilibrated for 30 ps with 500 kcal/mol constraints applied on the protein structure. The temperature
was gradually increased from 100 K to 300 K. During the equilibration
phase the potential, kinetic, and total energies were monitored. The
water box reached density 1.00 after 4 ps. After equilibration, the
system was minimized during 1000 steps of the steepest descent
algorithm. Next, the molecular dynamics was run for 1000 ps at 300 K,
during which the data were collected every 1000 fs. No constraints were
applied on the Pc secondary structure to allow for the better
exploration of conformational space. Non-bonded van der Waals
interactions were cut off beyond 9 Å. The long-range electrostatics
were treated with the Particle Mesh Ewald method (27). The SHAKE option
(28) was used to constrain all bonds, which enables a 2-fs time step. The 950 ps of data were used for analysis by the CARNAL block of AMBER.
Hydrogen bonding was defined by geometric criteria: the cut-off
distance between hydrogen and acceptor was 3.5 Å (for sulfur, 4 Å)
and the deviation of donor-acceptor distance from linearity was less
than 60o (29). All H-bonds with occupancy greater than 25%
were considered as maintained during the simulation.
 |
RESULTS |
The variant residue Met-33 at the hydrophobic patch of
Prochlorothrix Pc (Fig. 1) was
chosen to be mutated to the standard asparagine in other Pcs in order
to study its role in the reactivity of Pc toward PSI, because it has
been previously reported that replacement in this area of the also
atypical Pro-14 by the standard leucine drastically enhances
Prochlorothrix Pc electron transfer to PSI (11). A double
mutant with reversion of both methionine and proline to the standard
residues asparagine and leucine, respectively, was also constructed
(Fig. 1). Proline at position 53, located in a loop proposed to
interact with PSI (13), was replaced by either alanine or glutamic
acid. Additionally, arginine at position 86 was replaced both by
glutamine and glutamate (Fig. 1), because an equivalent arginine has
been shown to be crucial in the donor-PSI interaction in other
cyanobacteria (5).

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Fig. 1.
Space-filling model of
Prochlorothrix plastocyanin (PDB code
1B3I) showing the residues modified by
mutagenesis. The molecule is oriented with the electrostatic area
around Tyr-81 (or site 2) to the right and the hydrophobic patch around
His-85 (or site 1) at the top (A) or by rotating
90o and showing the site 1 from a top view (B).
The mutated residues are depicted in black. His-85 and
Tyr-81 are in gray. The numbering corresponding to that of
spinach Pc is shown between parentheses.
|
|
Most of the mutations do not significantly alter the midpoint redox
potential of the copper protein (
Em
20 mV;
Table I), with the exception of the R86E
mutant, whose redox potential is about 30 mV lower than WT. In general,
changes in redox potential should slightly increase the driving force
for electron transfer to PSI, except in the case of the M33N mutant,
whose redox potential is about 20 mV higher than WT (Table I). All
mutant oxidized Pc yield an absorption peak at 602 nm, identical to
that of WT.
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Table I
Midpoint redox potentials (Em) of wild-type and mutant
plastocyanins and association rate constants (KA) and
electron transfer rate constants (ket) for
Prochlorothrix photosystem I reduction by the different mutants
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As previously described for the WT Pc (10, 11), the laser-flash-induced
kinetic traces of PSI reduction by all the mutated Pcs are
monoexponential (Fig. 2). However, the
efficiency of the electron transfer process changes from one protein to
another. Thus, as shown in Fig. 2 for some mutants, kinetic traces for PSI reduction by the P53A and M33N mutants are significantly faster as
compared with WT Pc, whereas the R83E mutant shows slightly slower
kinetics (Fig. 2). The dependence of the observed pseudo first-order
rate constant (kobs) upon donor protein
concentration shows a saturation profile for all mutants at positions
33 and 86 but not for those at position 53, which present linear
concentration dependences (Fig. 3). These
findings suggest the formation of a bimolecular (Pc-PSI) transient
complex prior to electron transfer for mutants at positions 33 and 86, as previously described for the WT system as well as for other mutants
of Prochlorothrix Pc (10, 11). However, the behavior of the
proteins mutated at position 53 can be better described by assuming
just a second-order collisional process, with no formation of any
detectable electron transfer complex (6).

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Fig. 2.
Kinetic traces showing
Prochlorothrix photosystem I reduction by wild type
and mutated plastocyanins. Absorbance changes were recorded at 820 nm and 50 µM plastocyanin concentration. Other conditions
were as described under "Materials and Methods."
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Fig. 3.
Dependence of the observed rate constant
(kobs) for Prochlorothrix
photosystem I reduction by wild type and mutated plastocyanins
upon donor protein concentration. Lines correspond to the
theoretical fits as described under "Results." Other
conditions were as described under "Materials and Methods."
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|
From the saturation profiles in Fig. 3, and applying the formalism
developed in Meyer et al. (20), it is possible to estimate both KA (the equilibrium constant for complex
formation) and ket (the intracomplex electron
transfer first-order rate constant). The bimolecular second-order rate
constant (k2) for the interaction between P53A
and P53E Pc and PSI can be directly calculated from linear dependences
shown in Fig. 3. Table I shows the values for all these constants, as
well as the previously reported values for the P14L mutant for
comparative purposes (11). There are no significant changes in
KA for the mutants M33N and M33N/P14L as
compared with WT Pc. Replacing Arg-86 by glutamine promotes a moderate
decrease (to 60%) in KA, whereas the most
drastic decrease is observed for the R86E mutant (to 15%), in which a
negative charge is introduced. Concerning the electron transfer step,
Table I shows that all the mutations significantly enhance the
efficiency of Pc in donating electrons to PSI, with the most relevant
increases being obtained for both mutants in Met-33, in which ~5 × increases in ket are observed (Table I).
Although it is not possible to estimate a ket
value for P53A and P53E mutants, the kobs values shown in Fig. 3 at high Pc concentration (up to 8,500 s
1)
are much higher than any ket value from Table
I.
To check whether the hydrophobic nature of the interaction between Pc
and PSI in Prochlorothrix is altered by mutations, the kinetics of PSI reduction were also followed at high ionic strength. As
can be seen in Table I, none of the mutants shows significant changes
in KA, ket, or
k2 upon increasing salt concentration, as
previously described for the WT and other mutated Pcs (10, 11).
It has been previously reported that Prochlorothrix WT Pc
exhibits a very low reactivity in cross reactions with PSI from either
spinach or the cyanobacteria Anabaena and
Synechocystis (10). However, the P14L mutant, designed to
revert the "exclusive" proline in the hydrophobic patch of
Prochlorothrix to the standard leucine, showed a general
enhanced reactivity toward PSI from different sources (11). Here we
have also checked the reactivity of mutants toward heterospecific PSI
(Table II). In all cases, linear
dependences were observed when plotting kobs
versus protein concentration (not shown), thus allowing the
estimation of k2 for the different Pc/PSI
systems (Table II). None of the mutants show reactivity with spinach
PSI, as deduced by the low k2 values shown in
Table II for this photosystem, and k2 values for
spinach Pc could not be calculated because this Pc follows a three-step reaction mechanism with its PSI (6). When studying cyanobacterial PSI,
replacement of Met-33 by the standard asparagine, either in the single
mutant or in the M33N/P14L, promotes significant increases in the
bimolecular rate constant of PSI reduction, both with
Synechocystis and Anabaena PSI. Whereas the R86Q
mutant does not significantly change its reactivity against
cyanobacterial PSI, the R86E mutant shows a lower efficiency with both
cyanobacterial photosystems (Table II). Regarding mutations in position
53, both mutants show a significantly higher efficiency with both
Synechocystis and Anabaena photosystems,
rendering similar values to those obtained with the M33N Pc (Table
II).
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Table II
Bimolecular rate constants (k2) for the reduction of
photosystem I from different sources by Prochlorothrix wild-type and
mutant plastocyanins
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Molecular Dynamics Studies--
To determine structural changes in
the protein introduced by these mutations and observe their dynamic
behavior, we have run 1 ns molecular dynamics (MD) simulations for WT
and the P14L, M33N, and P53E mutants. The stability of the simulation
was checked by monitoring temperature, density, and total energy
parameters. The calculated average root mean standard deviation
(r.m.s.d.) for all atoms was less than 2 Å (Table
III), suggesting that proteins are able
to maintain their global structure. The introduction into the protein
of non-native amino acids leads to the rearrangement of protein
structure, which can be monitored through the changes in the r.m.s.d.
In case of Pc, the copper center determines its function, thus the
alteration of the copper center geometry by mutations can be assessed
by r.m.s.d. of its ligands. The calculated data are presented in
Table III. It can be seen that in the
P14L mutant there is slight displacement of all copper ligands,
suggesting that this mutant has a slightly altered geometry, which in
its turn affects midpoint redox potential of the protein as reported previously (Table I and Ref. 11). On the contrary, in the M33N mutant
the three ligands have the same position except for His-39, which is
spatially adjacent to residue 33. The higher r.m.s.d. for His-39 in WT
is a result of fluctuations of the Met-33 side chain. Hydrogen bond
analysis suggests that there is a hydrogen bond from sulfur to the
backbone HN hydrogen of His-39 (Table IV and Fig.
4). In the case of asparagine, which has
a shorter side chain and smaller radius of oxygen compared with that of sulfur, there is a change in H-bonding pattern around the copper center. Lastly, MD analysis of the P53E mutation shows that Glu-53 yields alterations in the protein backbone of the short
-helix proposed to interact with the PsaA protein of the PSI core (Table III) (13). The WT and other mutants adopt similar conformations in this helical region (Table III).
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Table III
Summary of the structural statistics for selected Cu center ligands
and the eastern face residues 51-55 from WT and P14L, M33N and
P53E mutant Pc
Data were averaged from a molecular dynamics simulation of 950 ps
duration (see under "Experimental Procedures"). r.m.s.d. values are
provided in Å.
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Table IV
Percentage of the occupied hydrogen bonds for H-bond donors and
acceptors of WT and M33N mutants, calculated from MD simulations
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Fig. 4.
Hydrogen bond from the sulfur atom of the
methionine at position 33 to HN hydrogen of the backbone of
histidine at position 39. See the "Results" section for more
details.
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 |
DISCUSSION |
Mutations in Met-33--
Reverting the exclusive methionine at
position 33 in Prochlorothrix Pc to the standard asparagine
in other Pcs yields enhanced ket while
maintaining unaltered the association constant for (Pc-PSI) complex
formation. This effect is qualitatively similar to that previously
observed for mutations reverting the unique proline in
Prochlorothrix Pc to the standard leucine (11), although about two-times higher ket values are reported
here (Table I). Similarly, the increased efficiency of the P14L mutant
in electron transfer toward heterospecific PSI (11) is also seen in
Met-33 mutants toward cyanobacterial PSI (Table II). Taken together, these results indicate that reverting the peculiar hydrophobic patch of
Prochlorothrix Pc to the standard configuration promotes significantly increased electron transfer efficiencies, in agreement with the proposed role of this area as providing an adequate
hydrophobic surface for the electron transfer step (3).
We have carried out a MD study in order to better explain the greater
reactivity of the M33N mutant as compared with WT Pc (Table III). The
residue at position 33 is adjacent to the copper-ligand His-39. As a
result, there is a slight displacement of His-39 (r.m.s.d. 0.217) in WT
compared with M33N (0.147) because of the bigger side chain of
methionine. Also, there is a hydrogen bond from S.D. of Met-33 to the
backbone N of His-39, which is absent in Asn-33 because of its small
side chain and smaller oxygen radius (Fig. 4). Replacement of
methionine by asparagine rearranges the H-bonding network within the
protein by formation of novel bonds (Table IV). Thus, the MD studies
indicate that when Met-33 is changed to the canonical asparagine, the
internal organization (H-bond network) changes around the Cu site. Such
changes are not reflected in significantly altered redox potential, so
the increases in rates are probably because of decreased donor/acceptor distance, yielding subtle changes in conformation with a better fit to
PSI. The similar kinetic behavior of M33N and M33N/P14L mutants can be
explained by assuming that the optimization of the PSI-Pc interaction
promoted by the M33N mutation cannot be improved by additional mutation
of Pro-14.
Mutations in Pro-53--
A GRAMM (30) computational simulation
suggested an interaction between the Prochlorothrix Pc
eastern face and a short
-sheet in the PsaA subunit of PSI. Pro-53
is located in the middle of a short
-helix in Pc (13). In fact,
altering Pc at this site abolished complex formation with PSI in both
P53A and P53E mutants, which surprisingly also leads to an increased
electron transfer efficiency. Thus, it is clear that Pro-53 is
important in the Pc-PSI interaction, acting by fixing the
electron transfer complex in a non-optimized configuration. Replacement
of this proline group appears to promote the breakdown of specific
interactions with PSI, allowing enhanced electron transfer. This seems
to be a general feature of Prochlorothrix Pc, because
replacement of Pro-53 also promotes increased electron transfer
efficiencies toward other cyanobacterial PSIs, although not toward the
spinach photosystem. MD analyses reveal that the P53E mutation affects the geometry of the protein backbone within the interacting Pc
-helix (residues 51-55, Table IV), likely yielding a more flexible, less hydrophobic structure that may not interact with the PsaA
-sheet. Breaking this interaction site may result in collisional interactions that minimize donor-acceptor distance and hence improve reactivity.
Mutations in Arg-86--
Cyanobacterial Pcs contain just one
arginine residue (numbered Arg-88 in spinach) proposed to be crucial in
PSI interaction because its substitution makes Anabaena Pc
unable to reduce PSI (5, 14). Mutating the equivalent Arg-86 in
Prochlorothrix promotes a decrease in
KA but to a lesser extent increases
ket, the R86E mutant yielding a more pronounced
effect. Although ionic strength effects are not observed in both
mutants, overall these data suggest a role for electrostatics in
helping to establish a docked conformation. The effects are not as
pronounced as when such mutations are introduced into
Anabaena, showing that in the peculiar
Prochlorothrix Pc the role played by Arg-86 is not essential for the interaction with PSI. Furthermore, weakening specific interactions of this arginine with PSI groups by its substitution actually optimizes the electron transfer process. On the other hand,
introducing a negative charge at position 86 has a negative effect on
the bimolecular rate constants with heterospecific PSI, yielding the
lower k2 values of all the
Prochlorothrix Pc mutants tested until now (Table II) (11).
These data confirm a role for this arginine in helping to establish an
efficient Pc-PSI interaction in other cyanobacteria distinct from
Prochlorothrix.
Concluding Remarks--
The data presented here confirm previous
investigations indicating that Prochlorothrix Pc presents an
unique electron donating mechanism to PSI. This process is based in the
formation of an electron transfer complex mainly stabilized by
hydrophobic interactions of PSI with an altered site 1 in Pc. However,
the electron transfer complex is not optimized in a productive
configuration; mutations in the hydrophobic patch promote increased
electron transfer rates without altering Pc-PSI association constants,
and mutations in the east face decrease KA while
increasing ket. Thus, by reverting the Pc
hydrophobic patch to the standard configuration, and/or weakening
complex formation, the reactivity of Pc toward its PSI is enhanced.
 |
FOOTNOTES |
*
Research at Sevilla was supported by the Dirección
General de Investigación Científica y Técnica
(Grant BMC2000-0444), European Union (Networks ERB-FMRX-CT98-0218 and
HPRN-CT1999-00095), and Junta de Andalucía (CVI-198). Work at
Bowling Green was supported by National Science Foundation Grants
MCB-9634049 and BIR-0070334.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
34-954-489-506; Fax: 34-954-460-065; E-mail: marosa@us.es.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M211913200
 |
ABBREVIATIONS |
The abbreviations used are:
Pc, plastocyanin;
KA, equilibrium constant for complex formation;
ket, first-order rate constant for electron
transfer;
kobs, observed pseudo first-order rate
constant;
k2, bimolecular rate constant;
MD, molecular dynamics;
PSI, photosystem I;
WT, wild type;
r.m.s.d., root
mean standard deviation.
 |
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