Site-directed Mutagenesis of Cytochrome
c6 from Synechocystis sp. PCC
6803
THE HEME PROTEIN POSSESSES A NEGATIVELY CHARGED AREA THAT MAY BE
ISOFUNCTIONAL WITH THE ACIDIC PATCH OF PLASTOCYANIN*
Berta
De la Cerda,
Antonio
Díaz-Quintana,
José A.
Navarro,
Manuel
Hervás, and
Miguel A.
De la Rosa
From the Instituto de Bioquímica Vegetal y
Fotosíntesis, Universidad de Sevilla y CSIC, Centro Isla de la
Cartuja, Américo Vespucio s/n,
41092 Sevilla, Spain
 |
ABSTRACT |
This paper reports the first site-directed
mutagenesis analysis of any cytochrome c6, a
heme protein that performs the same function as the copper-protein
plastocyanin in the electron transport chain of photosynthetic
organisms. Photosystem I reduction by the mutants of cytochrome
c6 from the cyanobacterium
Synechocystis sp. PCC 6803 has been studied by laser flash
absorption spectroscopy. Their kinetic efficiency and thermodynamic
properties have been compared with those of plastocyanin mutants from
the same organism. Such a comparative study reveals that aspartates at
positions 70 and 72 in cytochrome c6 are
located in an acidic patch that may be isofunctional with the well
known "south-east" patch of plastocyanin. Calculations of surface
electrostatic potential distribution in the mutants of cytochrome
c6 and plastocyanin indicate that the changes
in protein reactivity depend on the surface electrostatic potential
pattern rather than on the net charge modification induced by
mutagenesis. Phe-64, which is close to the heme group and may be the
counterpart of Tyr-83 in plastocyanin, does not appear to be involved
in the electron transfer to photosystem I. In contrast, Arg-67, which
is at the edge of the cytochrome c6 acidic
area, seems to be crucial for the interaction with the reaction center.
 |
INTRODUCTION |
Cytochrome c6
(Cyt)1 and plastocyanin (Pc)
are soluble metalloproteins, located inside the thylakoid lumen of
photosynthetic organisms, that carry electrons from cytochrome
b6f to photosystem I (PSI), which are
both membrane-anchored complexes. Although cyanobacteria and eukaryotic
green alga can synthesize both Pc and Cyt, Pc seems to have been able
to replace the primitive Cyt along evolution of photosynthetic
organisms as the copper-protein is the only electron carrier in higher
plants. The two metalloproteins are now well characterized, both at the
structural and functional levels. Their three-dimensional structures
have been solved by x-ray crystallography and NMR spectroscopy in
several organisms, and their reaction mechanisms have been widely
investigated (see Ref. 1 for a recent review).
Alignment of eukaryotic Pc and Cyt molecules according to their dipole
moment and surface charge distribution has allowed us the observation
of areas in the heme protein similar to those previously reported in
Pc: i) a hydrophobic region around the surface-exposed heme edge that
could be equivalent to the north patch involving the copper ligand
His-87 in Pc, and ii) a negatively charged area in Cyt similar to the
east acidic patch around Tyr-83 in Pc (1, 2). It should be noted that
such an acidic patch is significantly smaller in the metalloproteins
isolated from prokaryotes, namely the cyanobacterium
Synechocystis sp. PCC 6803 in which the acidic patch is
rather south-east-facing, just below Tyr-87.
In eukaryotic systems, site-directed mutagenesis of Pc has supplied
relevant information on the role of specific residues in site 1 (or the
hydrophobic north pole) and site 2 (or the acidic east face), which
both have been proposed to be involved in the interaction with PSI
(3-6). Whereas negative residues in the east face seem to be
responsible for electrostatic interactions and complex formation with
the positively charged PsaF subunit of PSI, the transfer of electrons
from the Pc copper center to the chlorophyll dimer P700+ in
PSI could take place via the imidazole ring of His-87 (one of the four
ligands to the copper atom) in the north pole.
In Synechocystis, in which wild type (WT) Cyt and Pc
interact with PSI according to a simple oriented collisional reaction mechanism (7), we have recently carried out a functional site-directed mutagenesis analysis of Pc (8). This study showed that most of the
mutants react following the same model as the WT protein, with the only
exception of the double mutant D44R/D47R which is able to form a
kinetically detectable electrostatic complex with PSI at low ionic
strength, as do the eukaryotic copper proteins (9, 10). These findings
indicate that the reaction mechanism of PSI reduction can be
drastically modified by changing specific surface amino acids, mainly
the acidic residues in the east face of the protein (8).
This paper describes the kinetic and thermodynamic properties of a set
of mutants of Synechocystis Cyt, which has been modified in
two specific residues located in a region equivalent to the east face
of Pc, as well as in two others close to the heme environment. To the
best of our knowledge, this is the first mutagenesis study of any Cyt
reported up to now. The thermodynamic parameters of PSI reduction by
Cyt mutants are also analyzed in a comparative way with a number of
mutants of Pc modified in the east face.
 |
EXPERIMENTAL PROCEDURES |
DNA Techniques--
The petJ gene coding for Cyt was
cloned in pBluescript II (SK+) (Stratagene) as described
previously (11), thereafter being used as a template for the
site-directed mutagenesis protocol. The mutant genes were constructed
using the polymerase chain reaction in two steps (12). Cloning and
sequencing of the modified petJ genes were carried out as
reported previously (8) for Pc mutagenesis. Cloning and sequencing of
the modified petE genes encoding Pc have already been
published (8). The numbering of residues of Synechocystis
Cyt herein used is that corresponding to the heme protein from
Monoraphidium braunii (2).
Recombinant Proteins and PSI Particles--
The procedures for
production and purification of mutant metalloproteins (both Cyt and Pc)
were those previously described (8, 11) except that the recombinant Cyt
was produced in Escherichia coli MC1061 cells (rather than
in DH5
cells); the final yield of Cyt was 5-10-fold higher in the
MC1061 strain. PSI particles were purified by
-dodecyl maltoside
solubilization as described previously (7).
Redox Titrations--
Redox titrations of WT Cyt and its mutants
were performed in a dual wavelength spectrophotometer as described
previously (8) for Pc, except that differential absorbance changes were
monitored at 552
570 nm. Errors in experimental determinations were
less than 5 mV.
Laser Flash Absorption Spectroscopy--
Kinetics of
flash-induced absorbance changes in PSI were followed at 820 nm as
described previously (9). The standard reaction mixture and
experimental conditions were as described previously (8), the buffer
being 20 mM Tricine/KOH, pH 7.5; for experiments at pH 5.5, the buffer used was 20 mM MES/NaOH. In all cases, the standard reaction mixture contained 10 mM
MgCl2, which was omitted in experiments for the analysis of
the ionic strength effect. For the thermodynamic analyses, experiments
were run at varying temperatures (10). Data collection, as well as
kinetic and thermodynamic analyses were as described previously (9,
10). Apparent thermodynamic parameters were estimated as in
Díaz et al. (13). Electrostatic potential energies
were obtained by fitting the calculated apparent activation energies to
the Watkins equation (14). The errors in estimated values for the
observed pseudo first-order rate constant (kobs)
were less than 10%.
Computer Simulations--
The structures of WT Cyt and Pc, as
well as that of mutants obtained by specific changes of residues were
modeled using the SYBYL program (Tripos Inc.) in a SGI RC10000
workstation. The structure of WT Cyt from Synechocystis was
modeled using the three-dimensional crystal structure of Cyt from the
green alga M. braunii (2) as a template. The original PDB
file was modified to fit the Synechocystis Cyt sequence with
the BIOPOLYMER module of SYBYL. The resulting file was first submitted
to energy minimization in vacuo up to an RMS energy gradient
of 0.41 kJ mol
1 Å
1, using the SANDER
module of AMBER 4.1 (15), and then solvated with TIP3T water molecules
using the BLOB option of the EDIT module. Solvent was energy minimized
and submitted to a 9-ps molecular dynamics (MD) calculation. The whole
system was again energy minimized and submitted to a 750-ps MD run at
300 K, from which the last 200 ps of trajectory were extracted and
analyzed with CARNAL. The resulting structure (2.4 Å of RMSD from
original) was again submitted to energy minimization. The quality of
the structure was tested using the PROCHECK program (16). Force field
parameters for the heme moiety were those included in the AMBER package
(17). Models of the modified proteins were obtained by changing the residues with the BIOPOLYMER module of SYBYL and further energy minimization in vacuo. Surface electrostatic potentials were
estimated by using the algorithm of Nicholls and Honig (18), as
indicated in the MOLMOL program (19).
The structure of WT Pc from Synechocystis was modeled using
the three-dimensional crystal structure of the triple mutant
A42D/D47P/A63L (20). The structure was first submitted to restrained
energy minimization, for which the cut-off radius was 6 Å for the full simulation region around the modified residue and 12 Å for the buffer
region. Minimizations were performed first by the steepest descent
method and second by conjugated gradient minimization until an RMS
energy gradient of 0.12 kJ mol
1 Å
1 was
reached. TRIPOS force field parameters (21) were used. Bond lengths and
dihedral angles were restrained for the copper site. A
distance-dependent dielectric constant was used to simulate solvation. Surface electrostatic potentials were likewise calculated by
using the algorithm of Nicholls and Honig (18), as indicated in the
MOLMOL program (19).
 |
RESULTS AND DISCUSSION |
A site-directed mutagenesis study of Synechocystis Cyt
has been performed here to investigate its structural and functional analogies with Pc. First, the three-dimesional structure of Cyt was
modeled so that the mutants could properly be designed. Modeling makes
evident that Cyt does possess an acidic cluster at the south-east face
formed by Asp-70, Asp-72, Glu-74, Asp-75, and Asp-2. On the other hand,
Tyr-83 has widely been reported to be a highly conserved residue in the
east face of Pc (in the Introduction). Interestingly, Ullmann et
al. (22) have recently proposed not only that an aromatic residue
at position 64 in eukaryotic Cyt may be the counterpart of Pc Tyr-83,
but also that the cation-
system between the aromatic ring of Phe-64
and the guanidinium group of Arg-67 could play a special role in
electron transfer. On the basis of these data, we have mutated four
specific residues in Cyt, two of them in the acidic area and the two
others just outside or at the border: Asp-70 and Asp-72 were replaced
by arginines; Phe-64, which is close to the heme group, was substituted
by alanine; and Arg-67, which is at the edge of the east face, was
changed to aspartate (Fig. 1).

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Fig. 1.
Space-filling representation of the modeled
structures of cytochrome c6
(left) and plastocyanin (right)
showing the location of residues modified by site-directed
mutagenesis. The two molecules are depicted in a similar
orientation, with their respective "east" faces just in front. The
cytochrome molecule is oriented in such a way that the heme propionates
are at the top, with the heme plane laying almost parallel to the
paper. The copper protein is oriented with the so-called north
hydrophobic pole at the top and the Tyr-83-surrounding area in front.
The two structures were modeled as described under "Experimental
Procedures."
|
|
Table I shows that the redox potential
value of the heme group is not significantly affected by replacement of
any one of the two aspartates by arginines, but it decreases in ~30
mV when Phe-64 or Arg-67 are mutated. In all cases, however, the
comparative analysis of Cyt mutants by UV/visible spectroscopy reveals
no changes in the heme environment (data not shown).
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Table I
Midpoint redox potential of wild-type (WT) and mutant cytochrome
c6 at pH 7.0 (Em,7), and bimolecular rate constants
(kbim and k ) for the overall reaction of
photosystem I reduction
|
|
The capability of Cyt mutants to reduce the photooxidized chlorophyll
molecule P700 in PSI particles was determined by laser flash-induced
absorption spectroscopy. With the four mutants, the PSI reduction
kinetic trace is monoexponential, as is it with WT Cyt, but the rate
constant is drastically changed depending on the residue modified. As
can be inferred from the kinetic profiles in Fig.
2 (upper), R67D is impaired
for the reduction of PSI, but the D70R mutant is even more efficient
than WT Cyt. Fig. 2 (lower) shows that the observed rate
constant (kobs) for PSI reduction with all
mutants increases linearly with increasing donor protein concentration
up to 200 µM. As is the case not only with WT Cyt but
also with WT Pc (7), such a linear dependence is interpreted by
assuming that there is no formation of any kinetically detectable (or
long-lived) intermediate complex between the metalloprotein and PSI.
The reaction mechanism should thus involve a simple oriented collision
of Cyt molecules with the membrane-anchored PSI complex. The
experiments in Fig. 2 were run at pH 7.5, but similar data were
obtained at pH 5.5 (not shown).

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Fig. 2.
Kinetic traces showing laser flash-induced
photosystem I reduction by wild-type (WT) and mutant
species of cytochrome c6
(upper) and dependence of the observed rate
constant (kobs) upon heme protein
concentration (lower). The kinetic traces were
recorded at pH 7.5 with cytochrome c6 at 200 µM final concentration.
|
|
The bimolecular rate constant (kbim) for the
overall reaction can be obtained from the linear plots in Fig. 2. The
resulting values are presented in Table I, both at pH 5.5 and 7.5. As
can be seen, the mutant F64A behaves like the WT molecule, but the modification of charges in other mutants alters their rate constants: D70R and D72R yield values for kbim that are 2- or 3-fold higher than those with WT Cyt, whereas the
kbim values with R67D are 5-8-fold lower than
those with the WT protein. These findings indicate that electrostatic
repulsions between residues 70 and 72 and their counterparts in PSI are
hindering the interaction of the WT Cyt and that Arg-67 plays a
critical role in the interaction with PSI (see below).
Taking into account the electrostatic nature of the reaction between
Cyt and PSI, the effect of ionic strength on the second-order rate
constant was analyzed. As can be seen in Fig.
3, kbim with WT
Cyt increases with increasing NaCl concentration up to reach a maximum
value at high ionic strength, indicative of repulsive interactions
between the heme protein and PSI (9). The F64A mutant shows an ionic
strength dependence similar to WT Cyt, as would be expected for a
mutation that does not involve any charged residue. The mutant R67D, in
its turn, is much less efficient in transferring electrons to PSI.
Replacement of aspartate by arginine at positions 70 and 72 renders the
mutant proteins slightly ionic strength-dependent, but such
a substitution in position 70 makes the Cyt molecule exhibit an ionic
strength dependence that is opposite that of WT Cyt.

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Fig. 3.
Effect of ionic strength on the second-order
rate constant (kbim) for the overall
reaction of photosystem I reduction by wild-type (WT)
and mutant species of cytochrome
c6. The ionic strength was
adjusted at the desired values by adding small amounts of a
concentrated NaCl solution. Solid lines state for the
fitting of experimental data (except those with D70R) to the Watkins
equation (see "Results and Discussion" for further details).
|
|
By applying the formalism developed by Watkins (14), the bimolecular
rate constant extrapolated to infinite ionic strength, that is the
diffusion-limited rate constant (k
), can be calculated, a parameter that provides information on the intrinsic reactivity of redox partners in the absence of electrostatic
interactions. The k
values in Table I
indicate that F64A is the only mutant that approaches WT Cyt, thereby
suggesting that the other mutations involve changes that are not only
electrostatic in nature. The small salt dependence of
kbim for D70R impeded an accurate estimation of
k
, and the slow kinetics of the R67D mutant did not allow us to get a precise value for
k
.
A thermodynamic analysis was also performed to get further insights
into the reaction mechanism of PSI reduction by WT and mutant Cyt. In
all cases, the temperature dependence of the observed rate constant
yields linear Eyring plots with no breakpoints, from which the values
for the apparent activation enthalpy (
H
),
entropy (
S
), and free energy
(
G
) for the overall reaction can be
calculated. As can be seen in Table II,
the most drastic difference in the activation parameters is observed
with R67D: its free energy value at pH 7.5 is ~5 kJ mol
1 higher than that of WT Cyt, as expected from its
rather inefficient interaction with PSI. Such an increase in free
energy is mainly because of the change in enthalpy (but not in entropy)
of the system, thereby indicating that the mutation is altering just the electrostatic interactions between reaction partners. Noteworthy also is the increase in the entropic term (~14 J mol
1
K
1) with the D70R mutant.
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Table II
Apparent activation parameters and changes in electrostatic energy
( Vel) for photosystem I reduction by wild-type (WT) and
mutant cytochrome c6 and plastocyanin
In all cases, the Eyring plots yielded linear regression coefficients
higher than 0.99. G was calculated at 298 K. Errors in the determination of the G values
were in the order of 0.25 kJ mol 1. The values for
Vel were obtained from the experimental data in
Fig. 4.
|
|
As reported previously (23), the bulk electrostatic effect of
substitutions of charged residues can be roughly approximated by
assuming that the change in the activation free energy of the overall
reaction with mutants as compared with that with the WT protein
(
G
) is directly proportional to the net
charge difference between WT and mutant metalloproteins
(
q). Thus, the real effect of the mutations can be
estimated by using the Watkins equation (14),
|
(Eq. 1)
|
where Vii is the net electrostatic
potential for the interaction, which depends on the electrostatic
charges of the reaction partners; Xk is the
Debye-Hückel term that accounts for the effect of charge
screening as a function of ionic strength; and
k
is the diffusion-limited kinetic rate
constant. We can thus write that,
|
(Eq. 2)
|
where
Vel(
Vel =
Vii · RT) stands for the difference in
electrostatic energy between the WT and mutant proteins. From Equation 2, the difference between the apparent activation free energies for WT
and mutant proteins must be proportional to
Xk.
As expected, the experimental data in Fig.
4 (left) indicate that

G
changes with ionic strength according
to Equation 2, but such an energetic term does not approach 0 at high
ionic strength with some Cyt mutants. This finding suggests that
k
is being affected not just by purely
electrostatic changes, in good agreement with the
entropy-dependent change in
G
observed with D70R (see above). To account for these discrepancies, a
new term should be added to Equation 2,
|
(Eq. 3)
|
in which the relative value of k
with
mutants as compared with that with the WT protein is considered. The
experimental data in Fig. 4 were thus fitted to Equation 3, from which
the values for
Vel with the mutants D70R and
D72R were estimated (Table II). F64A exhibits a 0 value for
Vel, as expected from its kinetic behavior
close to the WT Cyt; with D67R, in turn, it was not possible to
determine a value for
Vel as

G
depends linearly on ionic strength
(see Fig. 4).

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Fig. 4.
Ionic strength dependence of the changes in
the apparent activation free energy
( G ) for
photosystem I reduction by mutants of cytochrome
c6 (left) and
plastocyanin (right). The values for
 G were obtained by subtracting those
for G with each mutant from
G with the WT metalloprotein. Solid
lines indicate fitting of experimental data to Equation 2 (see
"Results and Discussion" for further details).
|
|
A comparative thermodynamic study was carried out with a set of mutants
of Synechocystis Pc, which was modified by replacing specific residues in the east patch. We have previously reported that
the values of kobs for PSI reduction, at the
ionic strengths used throughout this work, depend linearly on Pc
concentration with all mutants of Synechocystis Pc
even
with the double mutant D44R/D47R, which reaches a saturation plateau at
lower ionic strength that suggests the formation of an electrostatic
transient complex with PSI (8).
The activation parameters of the electron transfer reaction between
modified Pc and PSI have thus been analyzed here in a similar way than
those with Cyt and its mutants. Fig. 4 (right) shows the
effect of ionic strength on 
G
for the
mutants of Synechocystis Pc. The experimental data indicates that 
G
changes with ionic strength
according to Equation 2, thereby indicating that the changes in this
area alter the rate constants as a consequence of the changes in the
bulk electrostatics of the interaction. In principle, the relative
effect of amino acid substitutions can directly be related to the net
change in charge
q. Actually, the values for

G
at low ionic strength with D44R/D47R
(
q = 4) are approximately 2-fold higher than those
with D44K and D44A/D47A (
q = 2).
Table II shows the apparent activation parameters, as calculated from
the Eyring plots under standard conditions, for the reduction of PSI by
WT and mutant Pc. It also shows the values for
Vel obtained by fitting the experimental data
in Fig. 4 to Equation 2. As expected, the differences in
G
between WT and any mutant Pc are
proportional to
Vel, the D44R/D47R mutant in
fact showing the largest value for
Vel as
well as the smallest one for
G
.
The
Vel values change from one to another Pc
mutant, even when there is no difference in
q. The
mutations are thus affecting in a different manner the electrostatic
interaction energy, with the relative effect of mutation on
Vel depending not only on
q but
also on the specific location of the modified residue in the protein
surface. Actually, when mutations involving the same net charge
difference are performed in residues Asp-44 and/or Asp-47, the
Vel value with mutants at position 44 is
always larger than that with mutants at position 47 (see Table II).
Such a finding indicates that the changes in reactivity mainly depend
on the net charge difference
q, but it is specifically
the local negative potential in the proximity of residue 44 that
determines the repulsion between reaction partners.
A computer simulation of the distribution of surface electrostatic
potential in the mutants of Cyt was compared with that in the mutants
of Pc (Fig. 5). Asp-70 in Cyt and Asp-44
in Pc are located near the interphase between the acidic patch and a positively charged region, whereas Asp-72 in Cyt and Asp-47 in Pc are
just located in the middle of the acidic patch (see also Fig. 1).
Hence, the positive charge introduced upon mutation of any one of the
latter residues to arginine is screened by the surrounding negative
ones, whereas mutations at the interphase between the two regions
result in a rather large modification of the electrostatic potential.
In Cyt, the replacement of Asp-70 by arginine induces a spread of the
positively charged region, whereas the mutation D72R has a reduced
effect on the electrostatic potential. In Pc, the single mutation in
D44K as well as the double replacement in D44A/D47A induce a
significant increase in the positively charged area closer to the
hydrophobic north pole. In addition, the double substitution in
D44R/D47R induces an inversion of the electrostatic potential at the
eastern patch of Pc, so enabling a large, long range electrostatic
attraction toward the reaction center.

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Fig. 5.
Electrostatic potential distribution on the
surface of wild-type (WT) and mutants of cytochrome
c6 (upper) and
plastocyanin (lower). Surface electrostatic
potential was calculated as indicated under "Experimental
Procedures." Simulations were performed assuming an ionic strength of
40 mM at pH 7. Negative and positive potential regions are
depicted in red and blue, respectively. The
relative orientation of the heme and copper proteins is as described in
the legend to Fig. 1.
|
|
In the MD model of Synechocystis Cyt, Phe-64 points toward
inside the protein, with the aromatic ring dwelling close to the heme
group in such a way that a small reorganization of the Arg-67 side
chain could bring the guanidinium group toward the aromatic ring of
Phe-64. However, the much lower reactivity of the Cyt R67D mutant
cannot be explained on Ullmann's hypothesis (see above). In fact, the
replacement of Phe-64 by alanine yields a mutant with the same kinetic
efficiency as the WT molecule, thus indicating not only that the
aromatic residue is not crucial for the interaction of Cyt with PSI but
also that the cation-
interaction is not necessary for electron
transfer to PSI. We should however bear in mind that Cyt has to
interact also with the cytochrome b6f complex, for which the aromatic residue would be required (22, 24). In
a similar way, Tyr-83 in Pc has been reported to be involved in the
interaction with cytochrome f but not with PSI (4, 25, 26).
In this context, Sebban-Kreuzer et al. (27) have shown that
a tyrosine residue at position 64 is essential for electron transfer
between Desulfovibrio vulgaris cytochrome c553 and its formate dehydrogenase. Whether or
not Phe-64 plays any role in the redox interaction with cytochrome
f, its vicinity both to the heme group and to Arg-67 would
explain why the mutation in Phe-64 leads to changes in the redox
potential value (see Table I).
We conclude that Cyt possesses a negatively charged cluster that is
isofunctional with the "south-east" patch of Pc. Despite the large
positively charged region on the north-east face of WT Cyt, such an
acidic patch should be responsible for the repulsive interactions with
PSI at low ionic strength.
 |
ACKNOWLEDGEMENT |
The authors thank Fernando P. Molina-Heredia
for help in expressing the recombinant heme proteins.
 |
FOOTNOTES |
*
This work was supported by the Dirección General de
Investigación Científica y Técnica (DGICYT, Grant
PB96-1381), European Union (EU, CHRX-CT94-0540 and ERB-FMRX-CT98-0218),
and Junta de Andalucía (PAI, CVI-0198).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 489506;
Fax: +34 954 460065; E-mail: marosa{at}cica.es.
 |
ABBREVIATIONS |
The abbreviations used are:
Cyt, cytochrome
c6;
kbim, bimolecular
rate constant for the overall reaction;
kobs, observed pseudo first-order rate constant;
k
, diffusion-limited rate constant;
Pc, plastocyanin;
PSI, photosystem I;
G
,
H
, and
S
, activation free energy, enthalpy, and
entropy of the overall reaction;

G
, change in the activation free energy of PSI reduction by mutant
proteins as compared with that with the wild-type molecule;
q, net charge difference between wild-type and mutant
metalloproteins;
Vel, difference in
electrostatic energy between the wild-type and mutant proteins;
Vii, net electrostatic potential for the redox
interaction;
WT, wild-type;
Xk, Debye-Hückel term accounting for charge screening;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
MES, 4-morpholineethanesulfonic acid;
MD, molecular dynamics;
r.m.s., root
mean square.
 |
REFERENCES |
-
Navarro, J. A.,
Hervás, M.,
and De la Rosa, M. A.
(1997)
J. Biol. Inorg. Chem.
2,
11-22[CrossRef]
-
Frazão, C.,
Soares, C. M.,
Carrondo, M. A.,
Pohl, E.,
Dauter, Z.,
Wilson, K. S.,
Hervás, M.,
Navarro, J. A.,
De la Rosa, M. A.,
and Sheldrick, G.
(1995)
Structure (Lond.)
3,
1159-1169[Medline]
[Order article via Infotrieve]
-
Nordling, M.,
Sigfridsson, K.,
Young, S.,
Lundberg, L. G.,
and Hansson, Ö.
(1991)
FEBS Lett.
291,
327-330[CrossRef][Medline]
[Order article via Infotrieve]
-
Haehnel, W.,
Jansen, T.,
Gause, K.,
Klösgen, R. B.,
Stahl, B.,
Michl, D.,
Huvermann, B.,
Karas, M.,
and Herrmann, R. G.
(1994)
EMBO J.
13,
1028-1038[Abstract]
-
Hippler, M.,
Reichert, J.,
Sutter, M.,
Zak, E.,
Altschmied, L.,
Schröer, U.,
Herrmann, R. G.,
and Haehnel, W.
(1996)
EMBO J.
15,
6374-6384[Abstract]
-
Sigfridsson, K.,
Young, S.,
and Hansson, Ö.
(1996)
Biochemistry
35,
1249-1257[CrossRef][Medline]
[Order article via Infotrieve]
-
Hervás, M.,
Ortega, J. M.,
Navarro, J. A.,
De la Rosa, M. A.,
and Bottin, H.
(1994)
Biochim. Biophys. Acta
1184,
235-241
-
De la Cerda, B.,
Navarro, J. A.,
Hervás, M.,
and De la Rosa, M. A.
(1997)
Biochemistry
36,
10125-10130[CrossRef][Medline]
[Order article via Infotrieve]
-
Hervás, M.,
Navarro, J. A.,
Díaz, A.,
Bottin, H.,
and De la Rosa, M. A.
(1995)
Biochemistry
34,
11321-11326[Medline]
[Order article via Infotrieve]
-
Hervás, M.,
Navarro, J. A.,
Díaz, A.,
and De la Rosa, M. A.
(1996)
Biochemistry
35,
2693-2698[CrossRef][Medline]
[Order article via Infotrieve]
-
Hervás, M.,
Navarro, F.,
Navarro, J. A.,
Chávez, S.,
Díaz, A.,
Florencio, F. J.,
and De la Rosa, M. A.
(1993)
FEBS Lett.
319,
257-260[CrossRef][Medline]
[Order article via Infotrieve]
-
Giebel, L. B.,
and Spritz, R. A.
(1990)
Nucleic Acids Res.
18,
4947[CrossRef][Medline]
[Order article via Infotrieve]
-
Díaz, A.,
Hervás, M.,
Navarro, J. A.,
De la Rosa, M. A.,
and Tollin, G.
(1994)
Eur. J. Biochem.
222,
1001-1007[Abstract]
-
Watkins, J. A.,
Cusanovich, M. A.,
Meyer, T. E.,
and Tollin, G.
(1994)
Protein Sci.
3,
2104-2114[Abstract/Free Full Text]
-
Pearlman, D. A.,
Case, D. A.,
Cadwell, G. C.,
Siebel, G. L.,
Singh, U. C.,
Weiner, P.,
and Kollman, P. A.
(1995)
AMBER 4.1., University of California, San Francisco, CA
-
Laskowski, R. A.,
MacArthur, M. W.,
Moss, D. S.,
and Thornton, J. M.
(1993)
J. Appl. Crystallogr.
26,
283-291[CrossRef]
-
Giammona, D. A.
(1984)
Ph. D. Thesis., University of California, Davis, CA
-
Nicholls, A.,
and Honig, B.
(1991)
J. Comput. Chem.
12,
435-445
-
Koradi, R.,
Billeter, M.,
and Wuthrich, K.
(1996)
J. Mol. Graph.
14,
51-55[CrossRef][Medline]
[Order article via Infotrieve]
-
Romero, A.,
De la Cerda, B.,
Varela, P. F.,
Navarro, J. A.,
Hervás, M.,
and De la Rosa, M. A.
(1998)
J. Mol. Biol.
275,
327-336[CrossRef][Medline]
[Order article via Infotrieve]
-
Clark, M.,
Cramer, R. D.,
and Van Opdenbosh, N.
(1989)
J. Comput. Chem.
10,
982-1012
-
Ullmann, G. M.,
Hauswald, M.,
Jensen, A.,
Kostic, N. M.,
and Knapp, E.-W.
(1997)
Biochemistry
36,
16187-16196[CrossRef][Medline]
[Order article via Infotrieve]
-
Kannt, A.,
Young, S.,
and Bendall, D. S.
(1996)
Biochim. Biophys. Acta
1277,
115-126
-
Ullmann, G. M.,
Knapp, E.-W.,
and Kostic, N. M.
(1997)
J. Am. Chem. Soc.
119,
42-52[CrossRef]
-
Modi, S.,
Nordling, M.,
Lundberg, L. G.,
Hannson, Ö.,
and Bendall, D. S.
(1992)
Biochim. Biophys. Acta
1102,
85-90[Medline]
[Order article via Infotrieve]
-
Modi, S.,
He, S.,
Gray, J. C.,
and Bendall, D. S.
(1992)
Biochim. Biophys. Acta
1101,
64-68
-
Sebban-Kreuzer, C.,
Blackledge, M.,
Dolla, A.,
Marion, D.,
and Guerlesquin, F.
(1998)
Biochemistry
37,
8331-8340[CrossRef][Medline]
[Order article via Infotrieve]
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