Tài liệu Báo cáo Y học: Role of electrostatics in the interaction between plastocyanin and photosystem I of the cyanobacterium Phormidium laminosum ppt

Role of electrostatics in the interaction between plastocyanin
and photosystem I of the cyanobacterium
Phormidium laminosum
Beatrix G. Schlarb-Ridley
1
, Jose
´
A. Navarro
2
, Matthew Spencer
1
, Derek S. Bendall
1
, Manuel Herva
´
s
2
,
Christopher J. Howe
1
and Miguel A. De la Rosa
2
1
Department of Biochemistry and Cambridge Centre for Molecular Recognition, University of Cambridge, UK;
2
Instituto de
Bioquı
´
mica Vegetal y Fotosı
´
ntesis, Centro de Investigaciones Cientı

´
ficas Isla de la Cartuja, Universidad de Sevilla y CSIC, Spain
The interactions between photosystem I and five charge
mutants of plastocyanin from the cyanobacterium Phormi-
dium laminosum were investigated in vitro. The dependence
of the overall rate constant of reaction, k
2
, on ionic strength
was investigated using laser flash photolysis. The rate con-
stant of the wild-type reaction increased with ionic strength,
indicating repulsion between the reaction partners. Remov-
ing a negative charge on plastocyanin (D44A) accelerated the
reaction and made it independent of ionic strength; removing
a positive charge adjacent to D44 (K53A) had little effect.
Neutralizing and inverting the charge on R93 slowed the
reaction down and increased the repulsion. Specific effects of
MgCl
2
were observed for mutants K53A, R93Q and R93E.
Thermodynamic analysis of the transition state revealed
positive activation entropies, suggesting partial desolvation
of the interface in the transition state. In comparison with
plants, plastocyanin and photosystem I of Phormidium
laminosum react slowly at low ionic strength, whereas the two
systems have similar rates in the range of physiological salt
concentrations. We conclude that in P. laminosum, in con-
trast with plants in vitro, hydrophobic interactions are more
important than electrostatics for the reactions of plastocya-
nin, both with photosystem I (this paper) and with cyto-
chrome f [Schlarb-Ridley, B.G., Bendall, D.S. & Howe, C.J.

(2002) Biochemistry 41, 3279–3285]. We discuss the impli-
cations of this conclusion for the divergent evolution of
cyanobacterial and plant plastocyanins.
Keywords: cyanobacteria; electron transfer; photosystem I;
plastocyanin; weak interaction.
Electron-transfer chains like that of oxygenic photosyn-
thesis impose special restraints on the proteins involved.
Reactions must be fast to allow rapid turnover of the
chain. Binding between the reaction partners must be
transient, while at the same time sufficient specificity needs
to be retained. Surface properties of proteinaceous reac-
tion partners play a crucial role in meeting these criteria.
The aim of our research was to increase our understand-
ing of how one property of the protein surface, the charge
pattern, influences the rate constant of the overall reaction
and how it may have evolved. Our model protein is
plastocyanin, a soluble photosynthetic redox protein
which accepts an electron from cytochrome f in the
cytochrome bf complex and passes it on to P
700
+
in
photosystem I. In a previous study [1], we mutated
negatively and positively charged residues on the proposed
interaction site of plastocyanin with cytochrome f and
analysed the reaction of these mutants with the soluble
redox-active domain of cytochrome f (Cyt f) in vitro.This
paper presents results on the interaction in vitro between a
representative subset of these charge mutants with the
physiological electron acceptor of plastocyanin, photosys-

tem I. Hence, we can compare two sets of protein–protein
interactive surfaces operating in the same compartment
with similar functional selection pressures, with the aim of
identifying common features.
The organism from which plastocyanin and both its
reaction partners, Cyt f [1] and photosystem I (this paper),
were taken is a moderately thermophilic cyanobacterium,
Phormidium laminosum. Studying these photosynthetic
electron-transfer reactions of cyanobacteria is of evolu-
tionary interest: whereas the overall three-dimensional
structure of plastocyanin is highly conserved among plants
and cyanobacteria, the surface charge pattern varies
greatly [1]. Comparing cyanobacterial data with the wealth
of information available for the higher plant reaction [2–5]
reveals which functional aspects are variable. Further-
more, the type I copper protein plastocyanin can be
replaced by cytochrome c
6
, a redox protein of similar size
but entirely different folding, in a number of eukaryotic
algae and cyanobacteria including P. laminosum [6,7].
Hence two more sets of protein–protein interactive
surfaces with the same function as Cyt f – plastocyanin
and plastocyanin–photosystem I – are available for identi-
fication of features common to interprotein electron-
transfer reactions [4,7]. To our knowledge, this is the first
Correspondence to B. G. Schlarb-Ridley, Department of Biochemistry,
University of Cambridge, Building O, The Downing Site,
Cambridge CB2 1QW, UK.
Fax: + 44 1223 333345, Tel.: + 44 1223 333684,

E-mail:
Abbreviations: Cyt f, soluble redox-active domain of cytochrome f;
k
obs
, observed first-order rate constant; k
on
, rate constant of protein
association; k
off
, rate constant of complex dissociation before electron
transfer has taken place; k
et
, rate constant of intracomplex electron
transfer; k
2
, bimolecular rate constant of the overall reaction; k
¥
, k
2
at
infinite ionic strength.
(Received 10 June 2002, revised 5 September 2002,
accepted 15 October 2002)
Eur. J. Biochem. 269, 5893–5902 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03314.x
case in which kinetic data of the interaction of plastocya-
nin with both Cyt f and photosystem I have been
collected in a homologous cyanobacterial system. This is
essential for informed discussion of evolutionary relation-
ships.
The structure and charge properties of plastocyanin have

been described previously in detail (Introduction in [1]). Its
primary electron acceptor is P
+
700
of photosystem I, a
photo-oxidized chlorophyll a-dimer. The crystal structure
of a cyanobacterial photosystem I has been solved at a
resolution of 2.5 A
˚
[8]. In higher plants, the positively
charged N-terminal lumenal helix of PsaF has been shown
to be involved in binding of plastocyanin [9,10]. In
cyanobacteria, deletion of PsaF did not change the kinetics
of photosystem I reduction by either plastocyanin or
cytochrome c
6
[10,11]. Schubert et al. [12] suggest that, in
cyanobacteria, subunits PsaA and PsaB are largely respon-
sible for binding plastocyanin or cytochrome c
6
in a shallow
pocket.
In the reaction between photosystem I and plastocya-
nin from different organisms, three different types of
kinetics have been observed, which may represent vari-
ations on a single reaction scheme [13,14]. Type I kinetics
are characterized by monophasic decay of the absorbance
of photo-oxidized P
+
700

at 820 nm on reduction by
plastocyanin, and linear dependence of the observed
pseudo-first-order rate constant k
obs
on the plastocyanin
concentration. This type is observed for weak interac-
tions: in a range of experimentally reasonable plastocy-
anin concentrations, no sign of saturation is apparent.
Type II also exhibits monophasic kinetics; however, k
obs
approaches a saturating value at high plastocyanin
concentrations, which provides explicit evidence for
complex formation followed by intracomplex electron
transfer. Type III shows biphasic kinetics, which provides
evidence for the formation of an additional reaction
complex (compared to Type II) so that rearrangement
must occur before intracomplex electron transfer. The
reaction between plastocyanin and photosystem I of
P. laminosum is of Type I [7].
Determination of the ionic strength dependence of rates is
an important method of studying electrostatic interactions
[1]. The salt commonly added to increase ionic strength is
NaCl. However, it has been reported that bivalent cations
can play a specific role in the reaction in vitro between
photosystem I and both plastocyanin [15–17] and cyto-
chrome c
6
[13,18–21] by forming electrostatic bridges
between negative charges on the interacting surfaces. In
this study, we investigated the dependence of the second-

order rate constant of the overall reaction, k
2
, on both NaCl
and MgCl
2
concentration.
Information about the thermodynamic parameters of
the transition state can be obtained by measuring the
temperature dependence of k
2
. This analysis has been
performed for the interactions of plastocyanin and/or
cytochrome c
6
with their respective homologous photo-
system I from various plants, green algae and cyanobac-
teria [14,15,19,22] (including P. laminosum wild-type [7]).
We determined the activation parameters and their
dependence on NaCl and MgCl
2
concentration for the
reaction of P. laminosum photosystem I with P. lamino-
sum plastocyanin wild-type as well as five charge
mutants.
MATERIALS AND METHODS
Molecular biology and mutagenesis
Molecular biological methods were essentially as described
by Schlarb-Ridley et al.[1].
Protein methods
Expression, purification and characterization of wild-type

and mutant plastocyanins were carried out essentially as in
Schlarb et al. [23].
Photosystem I preparations
P. laminosum photosystem I particles were obtained by
solubilization with b-dodecyl maltoside as described by
Ro
¨
gner et al. [24] and Herva
´
s et al. [21]. The chlorophyll/
P
700
ratio of the resulting photosystem I preparation was
150 : 1. The P
700
content in photosystem I samples was
calculated from the photoinduced absorbance increase at
820 nm using an absorption coefficient of 6.5 m
M
)1
Æcm
)1
[25]. Chlorophyll concentration was determined by the
method of Arnon [26].
Kinetic analysis
The second-order rate constant, k
2
, and its ionic strength
dependence were measured using laser-flash-induced
absorbance changes of photosystem I at 820 nm. Unless

stated otherwise, the experimental setup and programmes
used in the analysis were as in Herva
´
s et al.[13].The
standard experimental conditions were as described by
De la Cerda et al. [27]. Measurements of the dependence of
k
obs
on the concentration of plastocyanin were carried out in
the following buffer: 20 m
M
tricine/KOH (pH 7.5), 10 m
M
MgCl
2
, 100 l
M
methyl viologen and 0.03% (w/v) b-dodecyl
maltoside to which photosystem I-enriched particles
(0.39 mg chlorophyll per ml) were added. The same reaction
mixture but without the 10 m
M
MgCl
2
was used for
measuring the dependence of k
2
on ionic strength. The ionic
strength was adjusted with small aliquots of concentrated
solutions of NaCl or MgCl

2
, and correction was made for
the resulting dilution of the reaction mixture. All experi-
ments were carried out at 278, 283, 288, 293 and 298 K.
Thermodynamic activation parameters DH
à
, DS
à
and DG
à
were obtained according to the transition state theory by
fitting plots of k
2
/T vs. T to the Eyring equation:
k
2
T
¼
k
B
h
expðÀDG
z
=RTÞ
¼
k
B
h
expðÀDH
z

=RTÞexpðÀDS
z
=RÞð1Þ
where k
B
is the Boltzmann constant, h is the Planck constant,
and R is the gas constant. Nonlinear regression by the least-
squares method gave the standard error of DG
à
. To obtain an
independent error estimate for each of the correlated
parameters DH
à
and DS
à
, the Exhaustive Search Method
[28,29] was applied. Plots of rate constants, k
2
, against ionic
strength were fitted to the monopole–monopole version of
the Watkins equation (Eqn 2) by a nonlinear least-squares
method (
KALEIDAGRAPH
TM
version 3.51; Synergy Software):
5894 B. G. Schlarb-Ridley et al.(Eur. J. Biochem. 269) Ó FEBS 2002
k
2
¼ k
1

exp½ÀV
ii
expðÀ0:3295q
ffiffi
I
p
Þ=ð1 þ0:3295q
ffiffi
I
p
Þ
ð2Þ
where q is the radius of the interactive site (in A
˚
), and the
factor 0.3295
ffiffi
I
p
is the Debye-Hu
¨
ckel parameter j at 298 K
[30]. The allowable error was set to 10
)4
%. For the criteria
used to determine the data range, see the Discussion.
Overall errors in the experimental determination of kinetic
constants were estimated to be 10%.
Electrostatic potentials
Electrostatic potentials of wild-type and mutant plastocy-

anins in the reduced form were calculated by a finite
difference solution of the linear Poisson–Boltzmann equa-
tion with
DELPHI
II [31]. The
SWISS
-
PDBVIEWER
was used to
add polar and aromatic ring hydrogens to chain A of pdb
file 1baw, and was also used to introduce mutations. Atomic
radii and partial charges were assigned from the PARSE list
of Sitkoff et al. [32].
RESULTS
Concentration dependence of
k
obs
and standard thermodynamic analysis
Five charge mutants of plastocyanin from P. laminosum
were chosen for analysis with wild-type photosystem I
isolated from the same organism (Fig. 1). All of them were
in a surface patch shown to interact with photosystem I in
the plant case [33]. One mutant neutralized a negative
charge (D44A), one neutralized an adjacent positive charge
(K53A), and three neutralized or inverted the charge on R93
(R93A, R93Q, R93E), a residue situated close to the charge
cluster that includes D44 and K53 and at the edge of the
hydrophobic flat end of the protein surrounding the copper
ligand H92. R93 has been shown to be essential for the
interaction of plastocyanin with photosystem I in Anabaena

[15], and is highly conserved in cyanobacterial plastocya-
nins. Mutagenesis, expression, purification and character-
ization of the plastocyanins has been described [1].
Representations of the electrostatic surfaces showing the
changes introduced by the mutations are displayed in Fig. 1.
The decay of the flash-induced absorbance of P
700
+
at
820 nm was monoexponential for all proteins at each of the
five temperatures (278, 283, 288, 293 and 298 K). In the
range of concentrations and temperatures used in this study,
k
obs
showed no sign of rate saturation. The best interpret-
ation of the results as a whole was a linear response to
plastocyanin concentration through the origin. Examples at
293 K and 298 K are shown in Fig. 2. Thus wild-type and
Fig. 1. Representations of the electrostatic surface potentials of wild-
type and mutant P. laminosum plastocyanin drawn with GRASP [50].
The molecular surface (probe radius 1.4 A
˚
) is coloured according to
electrostatic potential on a scale of red (acidic) to blue (basic). The
orientation is similar to that of Fig. 2 of [1].
Fig. 2. Dependence of k
obs
on plastocyanin concentration: wild-type and
mutant P. laminosum plastocyanin reacting with wild-type P. laminosum
photosystem I at (A) 293 K and (B) 298 K. The data were fitted to the

equation k
obs
¼ k
2
[plastocyanin].
Ó FEBS 2002 Electrostatics in electron transfer: Pc–PSI (Eur. J. Biochem. 269) 5895
all mutants were treated as following kinetic Type I. Balme
et al. [7] have already reported Type I behaviour for the
wild-type protein. From the slopes of the linear regressions
in Fig. 2A the bimolecular rate constants for the overall
reaction, k
2
, were determined (Table 1). The rate constant
increased when a negatively charged residue was neutralized
(D44A), hardly changed when an adjacent positively
charged residue was neutralized (K53A), but decreased
markedly when the charge of R93 was neutralized (R93A,
R93Q), and even more so when it was inverted (R93E). The
results are summarized in Table 1 and are qualitatively
similar to those obtained in the reaction with Cyt f [1].
Balme et al. [7] have previously reported a slightly higher
value for k
2
of the wild-type reaction, and we attribute this
to the use of different photosystem I preparations.
The thermodynamic parameters obtained from tempera-
ture-dependence measurements of k
obs
at 10 m
M

MgCl
2
show that DG
à
decreases slightly for D44A compared with
wild-type, remains essentially unchanged for K53A, and
increases for all three R93 mutants, most markedly for
R93E (Table 1). Owing to the correlation between DH
à
and
DS
à
, their independent errors, determined by the Exhaustive
Search Method, are large. Hence in all but one case (DH
à
of
R93E), DS
à
and DH
à
lie within the 67% confidence interval
of the wild-type values. However, the trends parallel those
seen for DG
à
: a decrease relative to wild-type for D44A, no
change for K53A, and an increase for all three R93 mutants,
again most pronounced in R93E. It is noteworthy that, with
67% confidence, all DS
à
values are positive under these

conditions. Implications for the structure of the transition
state are described in the Discussion.
Ionic strength dependence
Response to NaCl. The dependence of the second-order
rate constant, k
2
, on the concentration of NaCl was
investigated at five different temperatures (278, 283, 288,
293 and 298 K). Figure 3 shows the result for all proteins at
298 K; the other temperatures gave analogous results. For
wild-type plastocyanin, the rate increased with increasing
salt concentration, as observed by Balme et al.[7].Thisisin
clear contrast with the reaction of wild-type plastocyanin
with Cyt f, where the rate decreases with increasing ionic
strength [1]. The mutant D44A showed no dependence on
ionic strength, but K53A reacted slightly more slowly than
wild-type and exhibited a shallower dependence on NaCl
concentration. R93A and R93Q were slower still with a
similar steepness, and again R93E showed the most
pronounced effect. Experimental results were fitted to the
Watkins equation (see Materials and methods), as shown in
Fig. 3, to obtain estimates of k
2
at infinite ionic strength (k
¥
)
(Table 1). Modification of charge at positions 44 and 53 had
no significant effect on k
¥
, but values were significantly

lower for mutants of R93.
Response to MgCl
2
. In some systems, enhancement effects
have been reported when bivalent rather than univalent
cations were used in measurements of ionic strength
dependence (see the Introduction). Hence, the dependence
of k
2
of wild-type and all mutants on the concentration of
MgCl
2
was investigated at 278, 283, 288, 293 and 298 K.
Table 1. Kinetic and thermodynamic parameters of the reaction between wild-type and mutant P. laminosum plastocyanin with wild-type P. laminosum
photosystem I. Errors given are either standard errors obtained from curve fitting by least squares (k
2
,k
¥
, DG
à
) or 67% confidence limits derived by
the Exhaustive Search Method (DH
à
, DS
à
).
Plastocyanin
k
2
at 298 K

a
(l
M
)1
Æs
)1
)
k
¥
at 298 K
b
(l
M
)1
Æs
)1
)
k
¥
at 298K
c
(l
M
)1
Æs
)1
)
DG
àa
(kJÆmol

)1
)
DH
àa
(kJÆmol
)1
)
DS
àa
(JÆmol
)1
ÆK
)1
)
Wild-type 7.1 ± 0.5 10.6 ± 0.5 10.0 ± 0.7 34.06 ± 0.08 40.2 (34.2–46.5) 20.8 (0.3–42.5)
D44A 12.1 ± 0.5 10.9 ± 2.1 11.7 ± 0.2 32.66 ± 0.06 37.9 (34.8–41.1) 17.9 (7.3–28.8)
K53A 7.8 ± 0.1 12.4 ± 0.7 12.3 ± 1.0 33.74 ± 0.08 39.8 (34.5–45.4) 20.7 (2.5–39.8)
R93A 3.3 ± 0.2 5.9 ± 0.5 7.6 ± 1.3 36.00 ± 0.12 47.5 (42.7–52.5) 39.2 (22.9–56.4)
R93Q 4.1 ± 0.1 7.0 ± 0.6 6.7 ± 0.5 35.45 ± 0.11 46.2 (44.4–48.0) 36.6 (30.5–42.9)
R93E 1.3 ± 0.1 8.5 ± 3.4 3.4 ± 0.6 38.37 ± 0.12 50.4 (47.9–52.9) 40.9 (32.4–49.6)
a
Buffer used contained 10 m
M
MgCl
2
.
b
Buffer contained no MgCl
2
; ionic strength was adjusted with NaCl. The first datapoint was not

included in the fit (see Discussion).
c
Buffer contained no NaCl; ionic strength was adjusted with MgCl
2
. The first datapoint was not included
in the fit (see Discussion).
Fig. 3. Ionic strength dependence (NaCl) of k
2
: wild-type and mutant
P. laminosum plastocyanin reacting with wild-type P. laminosum photo-
system I at 298 K. All measured data points are shown; for the fits to
the Watkins equation the first data point was excluded (see Discus-
sion). Values for k
¥
obtained from the fit are given in Table 1.
5896 B. G. Schlarb-Ridley et al.(Eur. J. Biochem. 269) Ó FEBS 2002
Wild-type plastocyanin showed little or no significant
difference in its ionic strength dependence whether NaCl
or MgCl
2
was used (Fig. 4A; this has also been reported in
[7]). The same was the case for the mutants D44A and
R93A. For mutants K53A, R93Q and R93E, however, the
rate constant increased faster with ionic strength when
MgCl
2
rather than NaCl was added (Fig. 4B,C). Figure 4
shows the results obtained at 298 K, and analogous effects
were observed at the other temperatures.
Activation parameters

Nonlinear Eyring plots of the effect of temperature on k
2
at
each salt concentration were used to determine the effect of
ionic strength on the activation enthalpy, entropy and free
energy. No significant difference was observed between the
thermodynamics of the NaCl and MgCl
2
dependencies.
Figure 5A–C shows DH
à
and –TDS
à
at 298 K plotted
against the square root of ionic strength (using MgCl
2
)for
wild-type, K53A and R93E. The noise in the wild-type data
buries any trend, if there is one. Although there is still
considerable noise in the K53A data, a trend in both DH
à
(increasing with ionic strength) and –TDS
à
(decreasing with
increasing ionic strength) is emerging. For R93E, this trend
is clear and considerably larger than any noise. These trends
have also been observed for DH
à
and –TDS
à

of plastocyanin
and cytochrome c
6
from Synechocystis sp. PCC 6803, and a
trend of opposite sign has been reported for plastocyanin
from Anabaena (each reacting with their respective homo-
logous photosystem I), whereas Anabaena sp. PCC 7119
cytochrome c
6
showed an increase for both DH
à
and –TDS
à
[14].
Comparison between
P. laminosum
and spinach
The response to ionic strength of the reaction between
P. laminosum plastocyanin and photosystem I was in
marked contrast with the behaviour of the homologous
system in spinach. A direct comparison of the two systems
at 298 K is shown in Fig. 6 (spinach data taken from [14]).
Below 100 m
M
NaCl, the plant system reacted at least one
order of magnitude faster than that of the cyanobacterium,
but with increasing NaCl concentration the difference
diminished; the point of intersection of the two curves can
be extrapolated to %270 m
M

NaCl. Eyring plots can be
used to extrapolate k
2
to 318 K [7], the temperature at
which P. laminosum is cultured. When the resulting data
were plotted together with the spinach data at 298 K (an
acceptable growth temperature for spinach), the point of
intersection moved to %150 m
M
NaCl. To our knowledge,
the ionic strength of the thylakoid lumen has not been
determined. Published values of the ionic strength in the
stroma of chloroplasts vary from 130 m
M
to 200 m
M
[34,35], and it seems reasonable to assume that the lumenal
ionic strength lies within a similar range. Hence, at
physiological ion concentrations and temperatures, the
plant and cyanobacterial systems show similar rates.
DISCUSSION
To our knowledge, the work described here and in the
related publications [1,7] is the first kinetic analysis of the
in vitro interactions Cyt f–plastocyanin and plastocyanin–
Fig. 4. Comparison of ionic strength curves obtained by using NaCl or
MgCl
2
: wild-type and mutant P. laminosum plastocyanin reacting with
wild-type P. laminosum photosystem I at 298 K. (A) wild-type; (B)
K53A; (C) R93Q and R93E.

Ó FEBS 2002 Electrostatics in electron transfer: Pc–PSI (Eur. J. Biochem. 269) 5897
photosystem I from the same cyanobacterium. Here, we
will discuss the new results presented in the context of these
two directly related publications [1,7].
Concentration and ionic strength dependence
Comparison with the reaction Cyt f–plastocyanin [1]. In
all cases but one, the concentration dependence of k
obs
for
the mutant plastocyanins reacting with photosystem I
showed qualitatively the same effect as that observed in
the reaction with Cyt f, i.e. neutralizing acidic residues
speeded the reaction up, and neutralizing or inverting the
charge on basic residues slowed it down (Fig. 3 in [1], Fig. 2
in this paper). This indicates that the interacting sites on
plastocyanin used for the two reactions were similar. The
exception is the mutant K53A, which was slower than wild-
type plastocyanin in reaction with Cyt f, but appeared to be
virtually identical with wild-type plastocyanin in reaction
with photosystem I. This difference may be due to specific
effects of Mg
2+
(see below).
Response to NaCl. In the NaCl-based ionic strength
dependence of k
2
, the effects of the mutations relative to
wild-type plastocyanin resembled those observed in reaction
with Cyt f, confirming that similar interactive sites were
used ([1] and Fig. 3). However, the wild-type curve differed

dramatically between the reaction with photosystem I and
with Cyt f. Whereas the reaction with Cyt f showed an
overall attraction between the reaction partners, the reaction
with photosystem I exhibited a repulsion. The attraction
between wild-type plastocyanin and Cyt f could be virtually
abolished by neutralizing a single positive charge (K53A),
whereas the repulsion between wild-type plastocyanin and
Fig. 5. Ionic strength dependence (MgCl
2
)ofDH
à
and –TDS
à
at 298 K.
(A) wild-type; (B) K53A: (C) R93E. Error bars indicate 67% confid-
ence limits obtained by the Exhaustive Search Method [28,29].
Fig. 6. Comparison between P. laminosum and a plant: ionic strength
dependence (NaCl) of k
2
for wild-type P. laminosum plastocyanin
reacting with wild-type P. laminosum photosystem I and wild-type
spinach plastocyanin reacting with wild-type spinach photosystem I.
Experimental data were collected at 298 K; for P. laminosum the rate
constantswerealsoextrapolatedto318K,thetemperatureatwhich
P. laminosum is cultured. The lines represent an interpolation between
the data points.
5898 B. G. Schlarb-Ridley et al.(Eur. J. Biochem. 269) Ó FEBS 2002
photosystem I was abolished by neutralizing a single
negative charge (D44A). The plastocyanin-interaction sites
of Cyt f and photosystem I therefore appear to have a

difference in charge of 2.
These conclusions can be drawn from the shape of the
data curve without using curve fitting. To extrapolate to
infinite ionic strength and so obtain k
¥
, the monopole–
monopole version of the Watkins equation was applied [30].
The Watkins equation is based on a model that assumes that
onlychargesoftheproteinattheactivesitearerelevant,and
these can be represented by a disc of fixed radius and
uniform charge. The same analysis has been used for the
interaction between Cyt f and plastocyanin [1], and for an
in-depth discussion of the advantages and limitations of the
Watkins model the reader is referred to [1]. As described in
[1], deviations from the overall curvature are observed at low
ionic strength, probably due to changes in Debye length (the
distance over which the electrostatic field around a charge is
reduced to a value of 1/e of what it would have been in the
absence of electrostatic screening, and thus a measure of the
radius of effective electrostatic influence of a charge), which
the Watkins model does not accommodate. Hence in both
analyses (Fig. 4 in [1], Fig. 3 in this paper), omission of
datapoints at low ionic strength led to better fits and more
reliable k
¥
values (Table 1 in [1] and in this paper). Although
the curves are shallow, making extrapolation more difficult,
and the number of datapoints is smaller than in the case of
the interaction between Cyt f and plastocyanin [1], the
values shown in Table 1 confirm the qualitative conclusion

that changes in position 93 have a more pronounced and
specific effect. For all three R93 mutants, k
¥
is significantly
slower than that of wild-type or the other mutants,
indicating that in addition to the electrostatic effect, which
leads to low rates at low ionic strength, another nonelectro-
static factor, e.g. altered structure of the complex, reduces
the rate at infinite ionic strength.
Response to MgCl
2
. For three mutants, the ionic strength
dependence using MgCl
2
was markedly different from that
using NaCl (Fig. 4B,C). For macromolecular systems,
electrostatic theory, such as the Gouy–Chapman theory
applied to a model membrane [36], can predict stronger
effects for bivalent ions compared with univalent ions at
equivalent ionic strength. However, the fact that not all
plastocyanins in this study show an enhancement effect
suggests a different cause, e.g. binding. A bivalent cation
such as Mg
2+
can function as a bridge between two
negative charges on two interaction sites more effectively
than a univalent cation, and may thus speed up a reaction
by neutralizing repelling acidic groups. Figure 1 shows that,
in comparison with wild-type plastocyanin, where little or
no enhancement effect occurs (Fig. 4A), K53A has gained a

strongly acidic region, as one of the two basic residues
counteracting D44 and D45 has been lost. Binding of Mg
2+
to this region would counteract its repulsive effect, leading
to the enhancement observed. The reaction buffer for the
concentration dependence with photosystem I contained
10 m
M
MgCl
2
, whereas the buffer for the analogous
reaction with Cyt f contained only NaCl (90 m
M
[1]). This
may be the explanation for the fact that K53A reacted as
fast as wild-type with photosystem I, but slower than wild-
type with Cyt f in the concentration dependence of k
obs
.The
enhancement seen for R93E (Fig. 4C) can be explained in
an analogous way. It is less obvious why R93Q exhibits an
effect (Fig. 4C) whereas R93A does not, especially as the
representations of the electrostatic surface of both mutants
(Fig. 1) show very little difference. However, Gln is polar
and also protrudes further into the solvent than the
hydrophobic Ala. Mg
2+
may bind to the partially negat-
ively charged oxygen of Gln, leading to the observed effect.
Analysis of activation parameters

This analysis was based on the transition state theory of
Eyring [37]. The interpretation of the activation parameters
in Table 1 depends on whether the reaction is diffusion-
limited or activation-limited. In the former case, the
transition state would be that of association (k
on
). One
would then expect to see a fast phase with a rate constant
independent of plastocyanin concentration in the experi-
mental traces, which was not observed in this study. Hence
the reaction is likely to be activation controlled, and DG
à
,
DH
à
and DS
à
listed in Table 1 represent more than one
transition state, i.e. that of binding (k
on
and k
off
) and that of
electron transfer (k
et
). With the information to hand, the
magnitude or even sign of each contribution to the measured
parameters cannot be precisely determined. It has to be
remembered that transition state theory was developed for
elementary chemical reaction steps, not for the interaction of

macromolecules in solution. Furthermore, the magnitude
(but not the sign) of the thermodynamic parameters of
activation measured with the same experimental setup can
vary with different photosystem I preparations (compare
Table 2 in [7] and Table 1 in this paper). In what follows we
summarize the expected contributions of binding and
electron transfer to DH
à
and DS
à
and estimate their relative
importance in the light of the data in Table 1.
The contributions to DH
à
from binding are expected to
be positive as repulsion between the reaction partners has to
be overcome. The contribution of solvent effects on DH
à
is
determined by the molecular structure of the protein–
protein interface and the degree of desolvation in the
transition state. If this contribution were negative, it would
be expected to be small. The nuclear factor of k
et
also
contributes positively to DH
à
(equation 35 in [38,39]). Hence
there is no definite source of negative DH
à

, and it is not
surprising that the measured values for DH
à
were positive
for wild-type and all mutants (Table 1). However, the
situation is different for DS
à
. The loss of translational and
rotational degrees of freedom on complex formation and
the electronic factor make a negative contribution to DS
à
[38]. The only source of positive DS
à
is solvent exclusion
from the complex interface, as water molecules gain degrees
of freedom when leaving the ordered protein solvation shell
and joining the bulk solvent. The fact that DS
à
was positive
for wild-type and all mutants (within at least 67% confid-
ence; Table 1) indicates that solvent effects play an import-
ant role in the interaction between plastocyanin and
photosystem I. When copper proteins react with small
inorganic reagents where little solvent exclusion occurs, DS
à
is negative [40], but when plastocyanin reacts with Cyt f,
involving a relatively large interface [3], it is positive [41]. We
can conclude that the transition state for binding in the
reaction between plastocyanin and photosystem I is parti-
ally desolvated. The importance of desolvation of the

encounter complex in protein–protein association has been
Ó FEBS 2002 Electrostatics in electron transfer: Pc–PSI (Eur. J. Biochem. 269) 5899
stressed by others [42,43]. The increase in both DH
à
and DS
à
with increasing ionic strength, which was most clearly seen
for R93E (Fig. 5C), has also been observed for the
cyanobacterium Synechocystis sp. PCC 6803 [14] and the
green alga Monoraphidium braunii. These three systems are
characterized by repulsion between plastocyanin and phot-
osystem I.
In contrast, both DH
à
and DS
à
decreased for Anabaena
sp. PCC 7119 plastocyanin [14], where plastocyanin and
photosystem I attract each other. All reactions mentioned
show no fast phase in their laser flash kinetics; hence they
are expected to be activation controlled and should have
activation parameters that represent transition states of
binding (k
on
and k
off
) and electron transfer (k
et
) as discussed
above. The qualitative analysis applied above to the values

in Table 1 is, by itself, inadequate to explain the direction of
the changes in DH
à
and DS
à
. Most notably, for repelling
reaction partners, an increase in ionic strength would be
expected to decrease the positive DH
à
of binding; any
solvent effect would go in the same direction. The inverse is
thecasefortheattractioninthecaseofAnabaena. Also, if
the structure of the final complex in which electron transfer
takes place remained unaltered with increasing ionic
strength, no significant changes would be expected for the
activation parameters of electron transfer for both P. lami-
nosum and Anabaena. Hence, in both cases, the measured
trend is the opposite of what one would expect.
The observed effect could be explained, however, if one
assumed that increasing ionic strength modifies the structure
of the final complex [17,19]. In the case of P. laminosum,the
final complex may be tighter at high ionic strength, as
repelling electrostatic forces between the reaction partners
have been screened out. In this case, the activation
parameters associated with k
off
and k
et
would experience
additional changes. For k

off
, a tighter final binding complex
would mean that both DH
à
and DS
à
become more negative,
as a greater degree of re-solvation implies liberation of extra
solvation energy and loss in degrees of freedom for the
additional molecules of bulk solvent re-solvating the
protein. The overall effect on the measured parameters
would be an increase of both DH
à
and DS
à
. This would have
to overcompensate the decrease in DH
à
and DS
à
with
increasing ionic strength predicted for k
on
.ForAnabaena
plastocyanin, the converse of the above would lead to the
measured decrease for DH
à
and DS
à
.

Modifications in the structure of the final complex may
also influence the transition state of electron transfer. The
most important effects are likely to be on the electronic
factor. If the complex became tighter with increasing ionic
strength, this contribution to DS
à
would become less
negative. The inverse would apply for Anabaena. Hence
for both organisms, the changes in DS
à
of the electronic
factor would have the same sign as the measured trend.
Further discussion of the effects of ionic strength on
thermodynamic parameters can be found in Dı
´
az et al.[19]
and Herva
´
s et al. [14].
Evolutionary implications
Comparison of Cyt f–photosystem I. In the case of the
reaction of plastocyanin with Cyt f, it is clear that the acidic
residues D44 and D45 slow the reaction down and that R93
has a more specific role than K46 or K53. The experiments
reported here were intended to clarify if R93 has the same
specific role in reaction with photosystem I (as is the case for
the analogous residue in Anabaena sp. PCC 7119, R88 [15])
and if the interaction with photosystem I is the reason for
having acidic residues in the interface (especially the better
conserved D44; see alignment in [44]). The results of this

study indicate that similar interaction sites are being used
for both the reactions with Cyt f and photosystem I. R93
has a specific role in both interactions, and D44 slows the
reaction down in both cases. Electrostatics seem to play a
minor role in both reactions.
Whereas it is not surprising that R93 is being used in both
interactions, one may ask why the acidic residues (especially
D44, see above) have been conserved. Reasons for conser-
ving surface residues fall into two broad categories. The first
category comprises Ôexternal reasonsÕ such as modification
of interactions with other proteins (enhancing the interac-
tion with reaction partners, discouraging unfavourable
contacts) or with the membrane (avoiding sticking to it
while enabling two-dimensional diffusion). D44 clearly does
not enhance the reaction with either Cyt f or photosystem I,
but may do so with cytochrome oxidase, another potential
reaction partner of plastocyanin in cyanobacteria. It
remains to be clarified if the acidic residues serve to
discourage futile reactions (e.g. with photosystem II) and/or
modify interactions with the membrane. The second
category of causes for conserving surface residues comprises
Ôinternal reasonsÕ, for example to enhance stability of the
protein. Networks of surface salt bridges have been reported
to enhance protein stability [45]; this may be one role of the
negative charges on D44 and D45. It is interesting to note
that the D72K mutant of cytochrome c
6
from Anabaena sp.
PCC 7119 shows increased activity (faster rates with its
endogenous reaction partners), but decreased stability

(C. Lange, personal communication).
Comparison of cyanobacterial and plant systems. Figure 6
reveals that, although the reaction between plant plastocy-
anin and photosystem I is faster than that of P. laminosum
at low ionic strength values, the difference disappears in the
region of physiological salt concentrations. This is in
accordance with the results obtained for the reaction
Cyt f–plastocyanin. We have argued previously [1] that
in vitro the plant and cyanobacterial systems reach similar
rates in different ways: the plant system uses mainly
electrostatic interactions, as indicated by the steep decrease
in the rate constant with increasing ionic strength and its
low k
8
. P. laminosum relies on hydrophobic interactions, as
indicated by its shallow ionic strength dependence and the
high k
8
. The importance of hydrophobic interactions has
also been shown in the plastocyanin–photosystem I system
of Synechocystis [14] and Prochlorothrix [46]. It seems
reasonable to conclude from Fig. 6 by extrapolation that k
8
of the plant plastocyanin–photosystem I reaction is lower
than that of P. laminosum. Qualitatively this would lead to
the same conclusion as that drawn for the Cyt f–plastocy-
anin interaction in vitro. The behaviour in vivo, however,
which is of key evolutionary relevance, remains to be
investigated. Studies of both reactions in the green alga
Chlamydomonas reinhardtii have led to the conclusion that,

under favourable growth conditions the charge properties of
plastocyanin seem to be without kinetic consequence, but
may become so under some conditions of stress, thus
5900 B. G. Schlarb-Ridley et al.(Eur. J. Biochem. 269) Ó FEBS 2002
accounting for their conservation [10,47–49]. The question
remains why and when such a shift between hydrophobic
and electrostatic enhancement of the rate has happened
during evolution. One possibility is that primitive cyano-
bacteria at the time of chloroplast origin had poorly defined
interactions between Cyt f and plastocyanin and/or plasto-
cyanin and photosystem I, and subsequently the nature of
the interactions in the two lineages (hydrophobic in
cyanobacteria, electrostatic in chloroplasts) diverged as a
result of different environmental conditions. In cyanobac-
teria there may have been a greater degree of exposure to
environmental fluctuations in ionic strength compared with
chloroplasts. This is highlighted in the extant cyanobacte-
rium Gleobacter, where the photosynthetic apparatus is
located in the cytoplasmic membrane. However, selective
pressures for formation of acidic patches on plastocyanin
have still to be identified.
ACKNOWLEDGEMENTS
We are extremely grateful to Alexis Balme for his expert help and
advice, to O
¨
rjan Hansson, Wolfgang Haehnel, Hualing Mi, William
Teale and Ju
¨
rgen Wastl for fruitful discussions, and to Barry Honig for
making available the programs DelPhi and Grasp. This work was

supported by the Biotechnology and Biological Sciences Research
Council, UK, the Oppenheimer Fund, University of Cambridge, UK,
Corpus Christi College Cambridge, UK, Ministerio de Ciencia y
Tecnologı
´
a, Junta de Andalucı
´
a, Spain and the Research Training
Network ÔTRANSIENTÕ in the Programme Human Potential and
Mobility of Researchers of the European Commission (HPRN-CT-
1999-00095).
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