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
Received for publication, August 4, 2000, and in revised form, September 29, 2000
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ABSTRACT |
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Positively charged plastocyanin from
Anabaena sp. PCC 7119 was investigated by
site-directed mutagenesis. The reactivity of its mutants toward
photosystem I was analyzed by laser flash spectroscopy. Replacement of
arginine at position 88, which is adjacent to the copper ligand
His-87, by glutamine and, in particular, by glutamate makes
plastocyanin reduce its availability for transferring electrons to
photosystem I. Such a residue in the copper protein thus appears to be
isofunctional with Arg-64 (which is close to the heme group) in
cytochrome c6 from Anabaena
(Molina-Heredia, F. P., Díaz-Quintana, A., Hervás,
M., Navarro, J. A., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 33565-33570) and
Synechocystis (De la Cerda, B., Díaz-Quintana, A.,
Navarro, J. A., Hervás, M., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 13292-13297). Other
mutations concern specific residues of plastocyanin either at its
positively charged east face (D49K, H57A, H57E, K58A, K58E, Y83A, and
Y83F) or at its north hydrophobic pole (L12A, K33A, and K33E).
Mutations altering the surface electrostatic potential distribution
allow the copper protein to modulate its kinetic efficiency: the more positively charged the interaction site, the higher the rate constant. Whereas replacement of Tyr-83 by either alanine or phenylalanine has no
effect on the kinetics of photosystem I reduction, Leu-12 and
Lys-33 are essential for the reactivity of plastocyanin.
Plastocyanin (Pc),1 a
small single copper protein, and cytochrome c6
(Cyt), a monoheme protein, function as alternative mobile electron
carriers between the two membrane complexes
b6f and photosystem I (PSI)
(cf. Refs. 1-3 for reviews). The isoelectric point of Pc
and Cyt varies widely depending on the organism; the two molecules exhibit the same value of ~9 in the cyanobacterium
Anabaena sp. PCC 7119 (4).
Their respective three-dimensional structures have repeatedly been
solved using proteins from different organisms, and their structure-function relationships have been comparatively
analyzed. In Pc, which was investigated first, two active sites were
identified: site 1 (or the so-called north hydrophobic pole), located
in a flat region around the copper ligand His-87, and site 2 (or the so-called east face), which is referred to as the acidic patch in
eukaryotic organisms because it includes aspartic and glutamic residues
at positions 42-45 and 59-61 surrounding the solvent-exposed Tyr-83
(1, 2). Recent data indicate that site 2 is responsible for the
electrostatic interactions with cytochrome f and PSI, whereas site 1, in particular His-87, is involved in electron transfer
itself (3, 5).
We have recently reported that Cyt from Synechocystis (6)
and Anabaena (7) possesses two areas equivalent to those of Pc: site 1, which is a hydrophobic region at the edge of the heme pocket providing the contact surface for electron transfer, and site 2, which is a charged patch driving the electrostatic movement toward its
two membrane-anchored partners. Our mutagenesis studies of these two
cyanobacterial Cyts revealed the existence of a highly conserved
arginine residue at position 64, which is located on the protein
surface at the frontier between sites 1 and 2, very close to the heme
group, that is essential for electron transfer to PSI (6, 7). Arg-64 is
the only arginine residue in Anabaena Cyt, as is Arg-88 in
Anabaena Pc. Arg-64 in Cyt could thus be the counterpart of
Arg-88 in Pc.
Another peculiarity of Pc and Cyt from Anabaena, as compared
with the proteins from other sources, is their high isoelectric point
(see above) because of their high lysine content. In fact, site 2 is
positively charged in both Pc and Cyt, whereas it is typically negative
in other cyanobacteria and, in particular, in higher plants (2).
Considering that it is site 2 that mainly drives the attractive
movement of these two mobile metalloproteins toward the membrane
complexes, it was of interest to investigate in Anabaena Pc
(as previously done in Cyt) how the surface electrostatic potential
distribution and redox kinetics are altered by mutations.
This work was thus aimed at comparing the role of specific amino acids
in Anabaena Pc with those of its counterpart Cyt from the
same organism. Pc was modified by mutagenesis of specific residues
either at site 1 or site 2, with special attention being paid to
Arg-88. The kinetic mechanism of PSI reduction by mutant Pcs was
analyzed by laser flash absorption spectroscopy.
Purification of Native Plastocyanin--
Pc from
Anabaena sp. PCC 7119 was purified as described previously
(8), with the following two exceptions. i) Pc samples were
applied onto the CM-cellulose column after oxidation with potassium
ferricyanide, and ii) elution of the adsorbed proteins was performed
with a linear gradient of 2-30 mM potassium phosphate buffer, pH 7.0, containing 50 µM potassium ferricyanide.
Pc concentration was determined spectrophotometrically using an
absorption coefficient of 4.5 mM Construction of Mutants--
The mutant petE genes
were constructed by the polymerase chain reaction with the QuickChange
kit (Stratagene) using oligonucleotides of 26-38 bases, 15 ng of DNA
templates, and 16 cycles of 12 min in extension time. The construction
for the Anabaena petE gene previously described was used as
a template (4). The DNA fragments were sequenced to check the
mutations. Other molecular biology protocols were standard (10).
Production of Recombinant Proteins and Purification
Procedures--
Transformed cells of Escherichia coli
MC1061 were grown in a 40-liter fermentor (Biostat C, B. Braun Biotech)
at 37 °C for 18 h under aerobic conditions. The fermentor was
filled with 20 liters of standard Luria-Bertani medium (10)
supplemented with 100 µg/ml ampicillin and 200 µM
CuSO4. The air flow was adjusted automatically to keep the
concentration of dissolved oxygen at ~95% of its saturation value.
The culture was stirred at 250 rpm. The pH value was kept constant at
~6.0 by addition of small amounts of 1 N HCl. To prevent
loss of the expression vector, E. coli cells were
transformed and immediately used to inoculate the reactor.
Cells were collected by tangential filtration in a Sartocon cross-flow
filtration system (Sartorius), and the periplasmic fraction was
extracted by freezing the cell paste at Redox Titrations--
The redox potential value for each Pc
mutant was determined as reported previously (4, 11) by following the
differential absorbance changes at 597 minus 500 nm. Errors in the
experimental determinations were less than 10 mV.
Preparation of PSI Particles--
PSI particles were isolated
from Anabaena cells by Laser-flash Absorption Spectroscopy--
Kinetics of
flash-induced absorbance changes in PSI were followed at 820 nm as
described previously (16). The standard reaction mixture and other
experimental conditions have been reported (17). The buffer used
throughout this work was 20 mM Tricine/KOH, pH 7.5. Data
collection, as well as kinetic and thermodynamic analyses, were carried
out as reported previously (16, 18). Apparent thermodynamic parameters
were estimated as in Díaz et al. (19) by fitting the
experimental data to the equation of Watkins et al.
(20). Experimental errors were less than 10% for both the kinetic
constants and thermodynamic parameters.
Structure Simulations--
Structure and surface electrostatic
potential distribution of Pc mutants were modeled using the
Swiss-Pdb Viewer program (21). The solution structure of reduced
Pc from Anabaena variabilis (22), whose amino acid sequence
exhibits 100% identity with that of Pc from Anabaena sp.
PCC 7119, was used as a template. The quality of the modeled structures
for each Pc mutant was tested using the PROCHECK program (23).
Seven residues of Anabaena Pc were chosen to be
mutated, two at the north hydrophobic patch and five at the east
charged area (Fig. 1). The steric
hindrance and nature of interactions at site 1 were investigated by
replacing Leu-12 by Ala, and Lys-33 by Ala or Glu. The
functional equivalence of site 2 in Anabaena Pc and in the
negatively charged copper proteins was analyzed by substituting
residues at positions 49, 57, and 58; Asp-49 was replaced by Lys, and
both His-57 and Lys-58 were replaced by either Ala or Glu. Tyr-83 was
changed to Ala and Phe to check whether Tyr-83 is involved in
redox reactions, a role that was first proposed by He et al.
(24) but later discarded by Bendall et al. (25) in other
Pcs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1
cm
1 at 597 nm for the oxidized protein
(9).
20 °C. The frozen cell
paste was resuspended in 100 ml of deionized water and centrifuged. The
resulting suspension was extensively dialyzed against 2 mM
potassium phosphate, pH 7.0, with the exception of mutants K33E, H57E,
K58E, and R88E, which were dialyzed against 2 mM Tris-HCl,
pH 8.0. From this point on, the purification procedure for most of the
mutants was identical to that for native Pc, with minor changes
in elution gradients. The mutants K33E, H57E, and K58E were applied
onto a DEAE-cellulose column equilibrated with 2 mM
Tris-HCl, pH 8.0. Elution of the adsorbed proteins was performed with a
linear gradient of 0-100 mM NaCl in the same buffer. R88E was purified by gel filtration in a Sephadex G-50 column. In all cases,
50 µM potassium ferricyanide was added to the gradient buffers to keep Pc oxidized. Protein concentration was determined as
described above.
-dodecyl maltoside solubilization
(12, 13). The chlorophyll:P700 ratio of the resulting PSI preparations
was ~140:1. 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
(14). Chlorophyll concentration was determined according to the method
of Arnon (15).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (85K):
[in a new window]
Fig. 1.
Space-filling model of Anabaena
plastocyanin showing the residues modified by mutagenesis.
The molecule is oriented with its typical east face (electrostatic area
around Tyr-83; site 2) in front, whereas the north
hydrophobic patch (or site 1) is at the top. The mutant
residues are depicted in gray, and His-87 is depicted in
black. Each residue is identified with two numbers, the
first corresponding to its position in the copper protein from higher
plants and the second (between parentheses) corresponding to its place
in the amino acid sequence of Anabaena plastocyanin.
Our analysis of all primary sequences of Pcs deposited at the Protein Data Bank with the BLAST search program (26) revealed that Arg-88 is conserved in cyanobacteria but not in higher plants or in green algae. The reason for such conservation of Arg-88 in cyanobacterial but not in eukaryotic Pcs was thus investigated by replacing Arg-88 with either glutamine, as it is in spinach, or with glutamate.
Neither the electronic absorption spectrum nor the midpoint redox potential of Anabaena Pc was altered by the mutations mentioned above, thereby revealing that the copper center was not distorted. As shown in Table I, the only exceptions were the R88E and K33E mutants, whose midpoint redox potential values are 25-30 mV lower than that of WT Pc.
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The kinetic profile of PSI reduction under standard conditions was
monoexponential with all mutants, as it is with WT Pc (16), but the
rate constants varied greatly. The observed pseudo first-order rate
constant (kobs) of PSI reduction by any mutant is linearly dependent on Pc concentration, as is the case with the WT
molecule (Fig. 2). This finding can be
interpreted by assuming that there is no formation of a detectable
transient complex between PSI and Pc, in agreement with a collisional
kinetic model (3, 16).
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The bimolecular rate constant for the overall reaction (kbim) can be calculated under standard conditions from the linear plots of kobs versus Pc concentration. Table I shows that most mutants yield kbim values smaller than WT Pc, with the exception of D49K, which exhibits a higher reactivity, and the two mutants at Tyr-83, which are slightly affected. As expected, all mutants of the hydrophobic patch suffer a significant decrease in their kbim value. Electrostatic mutations at positions 33, 57, and 58 cause Anabaena Pc to decrease its availability to reduce PSI as much as its global charge is made more negative. Major changes are obtained with the Arg-88 mutants, because replacement of Arg-88 with glutamine or glutamate induces a decrease in kbim of ~8 and 24 times, respectively (Table I). Fig. 2 shows how inefficient the R88Q and R88E mutants are, even at high concentrations, compared with the WT molecule.
Taking into account the electrostatic nature of the interactions between Pc and PSI, a detailed analysis of the effect of ionic strength on kbim was performed. The kbim values of WT Pc and all its mutants decrease steadily with increasing NaCl concentration, indicating the existence of attractive electrostatic interactions between the reaction partners that are overwhelmed at high ionic strength. The only exception is the R88E mutant, for which the kbim values are independent of ionic strength (see Fig. 4).
The formalism developed by Watkins et al. (20) facilitates
the analysis of the intrinsic reactivity of redox partners in the
absence of electrostatic interactions. By applying the Watkins equation
to our data on the ionic strength dependence of
kbim, values for the bimolecular rate constant
extrapolated to infinite ionic strength (kinf)
can be calculated. As shown in Table I, all mutants, except L12A and
R88E, exhibit kinf values similar to that of WT
Pc, indicating that the minor reactivity of Pc mutants under standard
conditions is mainly due to electrostatic rather than hydrophobic or
structural changes. In the case of L12A, however, the
kinf value is 7-fold lower than that for WT Pc.
This suggests that such an isoelectric mutation may indeed induce
structural changes hindering the interaction between Pc and PSI, as
previously observed in Synechocystis (17) and spinach (27).
The Watkins equation, however, cannot be applied as such to R88E
because its interactions with PSI are independent of ionic strength.
The kbim value for R88E at any NaCl
concentration (1.46 × 106
M1 s
1)
is 4-fold lower than the kinf value for WT Pc
(see Table I). This suggests that the electrostatic change induced by
mutation is not the only factor affecting the reactivity of R88E toward PSI. Worth mentioning is the finding that the R64E mutant of
Anabaena Cyt exhibits a kinf value
that is also 4- or 5-fold lower than that for WT Cyt (7).
The nature of interactions between PSI and Pc was further investigated
by performing a thermodynamic analysis of PSI reduction by the copper
protein mutants. In all cases, the temperature dependence of the
observed rate constant yielded linear Eyring plots with no breakpoints,
from which the values for the apparent activation enthalpy
(H
), entropy
(
S
), and free energy
(
G
) of the overall reaction can be
calculated. As shown in Table I, the most significant difference is
observed with the R88E mutant, whose free energy change is 8.0 kJ
mol
1 higher than that of WT Pc, as expected
from its impaired reactivity toward PSI. Such a difference in
G
is mainly due to a decrease of 25.6 J
mol
1 K
1 in the
entropic term. Also interesting is the L12A mutant, which exhibits a
free energy change of 4.3 kJ mol
1 higher than
that of WT Pc that is mainly due to the decrease in the entropic term.
The observed changes in
G
with the other
mutants can be attributed to shifts in the enthalpic and/or entropic terms.
To elucidate whether the lower reactivity of R88E can be ascribed to
reasons other than the electrostatic change itself, its availability to
transfer electrons to PSI from spinach and Synechocystis was
analyzed in comparison with Anabaena WT Pc. The
kbim value for spinach PSI reduction was
3.5 × 105 M1
s
1 with R88E, or 4.3-fold lower than that
with Anabaena WT Pc (1.5 × 106
M
1 s
1).
The kbim value for Synechocystis PSI
reduction was 1.9 × 106
M
1 s
1
with R88E, or 15-fold lower than that with Anabaena WT Pc
(2.8 × 107 M
1
s
1). These findings allow us to conclude that
Arg-88 is a crucial residue not only for long range electrostatic
attractions between Pc and PSI but also for electron transfer. It
should be noted that glutamine at position 88 of spinach Pc has been
replaced with asparagine, glutamate, lysine, and tyrosine with no
significant changes in the kinetics of PSI reduction (27, 28).
Divalent cations like Mg2+ have previously been reported to be specifically involved in Pc/PSI interactions in other organisms, but not Anabaena (8). As such, an analysis of the effect of Mg2+ cations on the bimolecular rate constant of PSI reduction by WT and mutant Pcs was performed. The experimental results showed that the three mutants with one positive charge replaced by a negative residue (K33E, K58E, and R88E) are the only ones altered by magnesium cations. With these mutants the bimolecular rate constant exhibits higher values with MgCl2 than with NaCl at the same ionic strength (data not shown). All these findings can be interpreted by assuming not only that the added negative charges in Pc mutants facilitate the formation of new salt bridges with positive counterparts in PSI (16), but also that the original positive residues in WT Pc interact with an acidic area in PSI.
The positively charged site 2 in Anabaena Pc does appear to
play the same role as the negatively charged site in other Pcs. The
surface electrostatic potential distribution of Anabaena Pc was then calculated for its WT species as well as for the D49K and R88E
molecules. As can be seen in Fig. 3,
their respective electrostatic charges at site 2 correlate well with
their relative efficiency as electron donors to PSI; the more positive
the local surface charge, the higher the kinetic rate constant of
photosystem reduction. A similar correlation had previously been
observed with a number of mutants at site 2 of Cyt from
Anabaena (7) and Synechocystis (6).
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The close similarity between Pc and Cyt from Anabaena was
investigated by comparing the structures of the two molecules and the
relative position of critical surface residues. The three-dimensional structures of the metalloproteins were thus modelled (Fig.
4, upper panel). Assuming that
His-87 in Pc plays the same redox role as the heme group in Cyt (29),
the comparison of the two protein structures makes Arg-88 and Asp-49 of
Pc occupy positions equivalent to Arg-64 and Asp-72 in Cyt. In
addition, the high number of lysine residues in both molecules could
contribute to site 2 being so positively charged.
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Such a structural coincidence between Pc and Cyt from Anabaena is indeed supported by the experimental findings concerning the kinetic behavior of mutants affected at the arginine and aspartate residues. Fig. 4 (lower panels) shows how the kbim values at low ionic strength for the D49K mutant of Pc are significantly higher than those for WT Pc, as are the values for the D72K mutant of Cyt compared with WT Cyt. The kbim values for R88E of Pc and R64E of Cyt are, however, lower than those for their respective WT species. Also worth noting is the parallel ionic strength dependence of kbim with the WT and mutant proteins, i.e. the decrease in kbim with increasing NaCl concentration.
Several possibilities concerning the role of the single arginyl residue in both Cyt and Pc can be devised. The interaction of these two metalloproteins with PSI should involve the formation of a transient electrostatic complex, which has in fact kinetically been detected with Cyt but not with Pc, probably due to its low association constant. The arginyl residue could thus be required for appropriate orientation of the redox centers within the transient complex. Specific electrostatic interactions of Arg-88 in Pc and Arg-64 in Cyt with negatively charged groups in PSI could be made evident as soon as a high resolution structure for PSI is made available. In addition, the proximity of the arginyl residue to the prosthetic group in each protein can explain why the arginine mutants of both Pc and Cyt exhibit values for the rate constant extrapolated to infinite ionic strength lower than those of the respective WT species. In this context, conformational changes of Arg-88 related to changes in the redox state of Pc have been described recently (30).
To conclude, we can say that site 1 of Anabaena Pc is
similar to site 1 of other Pcs, but site 2 is positively charged in Anabaena, whereas it is negatively charged in other
organisms. In addition, not only Pc but also Cyt from
Anabaena contains a single arginine residue between sites 1 and 2 that appears to play the same function in the two molecules.
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ACKNOWLEDGEMENTS |
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We thank S. Alagaratnam, A. Balme, A. Díaz, and S. Murdoch for critically reading the manuscript.
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FOOTNOTES |
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* This research was supported by the Dirección General de Investigación Científica y Técnica (Grant PB96-1381), European Union (Networks ERB-FMRX-CT98-0218 and HPRN-CT1999-00095), 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.: 34 954 489 506;
Fax: 34 954 460 065; E-mail: marosa@cica.es.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M007081200
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ABBREVIATIONS |
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The abbreviations used are: Pc, plastocyanin; Cyt, cytochrome c6; PSI, photosystem I; WT, wild-type.
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