De la Rosa2 1 Department of Biochemistry and Cambridge Centre for Molecular Recognition, University of Cambridge, UK;2Instituto de Bioquı´mica Vegetal y Fotosı´ntesis, Centro de Investig
Trang 1Role of electrostatics in the interaction between plastocyanin
Beatrix G Schlarb-Ridley1, Jose´ A Navarro2, Matthew Spencer1, Derek S Bendall1, Manuel Herva´s2, Christopher J Howe1and Miguel A De la Rosa2
1
Department of Biochemistry and Cambridge Centre for Molecular Recognition, University of Cambridge, UK;2Instituto 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 laminosumwere investigated in vitro The dependence
of the overall rate constant of reaction, k2, 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
MgCl2were 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 laminosumreact 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 c6, 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 toB 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: bgs9@mole.bio.cam.ac.uk
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)
Trang 2case 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 c6[10,11] Schubert et al [12] suggest that, in
cyanobacteria, subunits PsaA and PsaB are largely
respon-sible for binding plastocyanin or cytochrome c6in a shallow
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 kobs 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, kobs
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 c6 [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, k2, on both NaCl
and MgCl2concentration
Information about the thermodynamic parameters of
the transition state can be obtained by measuring the
temperature dependence of k2 This analysis has been
performed for the interactions of plastocyanin and/or
cytochrome c6 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 MgCl2 concentration for the
reaction of P laminosum photosystem I with P
lamino-sum plastocyanin wild-type as well as five charge
mutants
M A T E R I A L S A N D M E T H O D S
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/
P700ratio of the resulting photosystem I preparation was
150 : 1 The P700 content in photosystem I samples was calculated from the photoinduced absorbance increase at
820 nm using an absorption coefficient of 6.5 mM )1Æcm)1 [25] Chlorophyll concentration was determined by the method of Arnon [26]
Kinetic analysis The second-order rate constant, k2, 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
kobson the concentration of plastocyanin were carried out in the following buffer: 20 mMtricine/KOH (pH 7.5), 10 mM
MgCl2, 100 lMmethyl 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 mM MgCl2 was used for measuring the dependence of k2on ionic strength The ionic strength was adjusted with small aliquots of concentrated solutions of NaCl or MgCl2, 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, DSand DG were obtained according to the transition state theory by fitting plots of k2/T vs T to the Eyring equation:
k2
T ¼kB
h expðDGz=RTÞ
¼kB
h expðDHz=RTÞ expðDSz=RÞ ð1Þ where kBis 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 To obtain an independent error estimate for each of the correlated parameters DH and DS, the Exhaustive Search Method [28,29] was applied Plots of rate constants, k2, against ionic strength were fitted to the monopole–monopole version of the Watkins equation (Eqn 2) by a nonlinear least-squares method ( TMversion 3.51; Synergy Software):
Trang 3k2¼ k1exp½Viiexpð0:3295q ffiffi
I
p Þ=ð1 þ 0:3295q ffiffi
I p 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 withDELPHIII [31] TheSWISS-PDBVIEWERwas 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]
R E S U L T S
Concentration dependence ofkobs
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,
kobsshowed 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 ¼ k [plastocyanin].
Trang 4all 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, k2, 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 k2of 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 kobs at 10 mM MgCl2
show that DGdecreases 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 DHand
DS, their independent errors, determined by the Exhaustive
Search Method, are large Hence in all but one case (DHof
R93E), DSand DHlie within the 67% confidence interval
of the wild-type values However, the trends parallel those
seen for DG: 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 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, k2, 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] This is in
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 k2at 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 MgCl2 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 k2of wild-type and all mutants on the concentration of MgCl2 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) or 67% confidence limits derived by the Exhaustive Search Method (DH , DS ).
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 a
(kJÆmol)1)
DH a
(kJÆmol)1)
DS a
(JÆmol)1ÆK)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.
Trang 5Wild-type plastocyanin showed little or no significant
difference in its ionic strength dependence whether NaCl
or MgCl2was 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
MgCl2rather 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 k2at
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 MgCl2 dependencies
Figure 5A–C shows DH and –TDS at 298 K plotted
against the square root of ionic strength (using MgCl2) 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
(increasing with ionic strength) and –TDS(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 DHand –TDSof plastocyanin
and cytochrome c6from 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 c6showed an increase for both DHand –TDS
[14]
Comparison betweenP 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 mMNaCl, 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 mM NaCl Eyring plots can be
used to extrapolate k2 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 mMNaCl 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 mM to 200 mM
[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
D I S C U S S I O N
To our knowledge, the work described here and in the
related publications [1,7] is the first kinetic analysis of the
in vitrointeractions 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.
Trang 6photosystem 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 kobsfor 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 Mg2+(see below)
Response to NaCl In the NaCl-based ionic strength dependence of k2, 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 ) of DH and –TDS 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 constants were also extrapolated to 318 K, the temperature at which
P laminosum is cultured The lines represent an interpolation between the data points.
Trang 7photosystem 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
only charges of the protein at the active site are relevant, 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 MgCl2 For three mutants, the ionic strength
dependence using MgCl2was 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 Mg2+ 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 Mg2+
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 mM MgCl2, whereas the buffer for the analogous
reaction with Cyt f contained only NaCl (90 mM[1]) This
may be the explanation for the fact that K53A reacted as
fast as type with photosystem I, but slower than
wild-type with Cyt f in the concentration dependence of kobs 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 Mg2+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-diffusion-limited In the former case, the transition state would be that of association (kon) 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,
DH and DS listed in Table 1 represent more than one transition state, i.e that of binding (konand koff) and that of electron transfer (ket) 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 DHand DSand estimate their relative importance in the light of the data in Table 1
The contributions to DHfrom binding are expected to
be positive as repulsion between the reaction partners has to
be overcome The contribution of solvent effects on DHis 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 ket also contributes positively to DH(equation 35 in [38,39]) Hence there is no definite source of negative DH, and it is not surprising that the measured values for DHwere positive for wild-type and all mutants (Table 1) However, the situation is different for DS The loss of translational and rotational degrees of freedom on complex formation and the electronic factor make a negative contribution to DS [38] The only source of positive DS 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 DSwas 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
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
Trang 8stressed by others [42,43] The increase in both DHand DS
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 DHand DSdecreased 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 (konand koff) and electron transfer (ket) 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 DHand DS Most notably, for repelling
reaction partners, an increase in ionic strength would be
expected to decrease the positive DH of binding; any
solvent effect would go in the same direction The inverse is
the case for the attraction in the case of Anabaena 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-nosumand 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 koffand ket would experience
additional changes For koff, a tighter final binding complex
would mean that both DHand DSbecome 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 DHand DS This would have
to overcompensate the decrease in DH and DS with
increasing ionic strength predicted for kon For Anabaena
plastocyanin, the converse of the above would lead to the
measured decrease for DHand DS
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 would become less
negative The inverse would apply for Anabaena Hence
for both organisms, the changes in DS 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 external reasons such as modification
of interactions with other proteins (enhancing the interac-tion with reacinterac-tion 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
internal reasons, 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 c6from 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 vitrothe 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 k8 P laminosum relies on hydrophobic interactions, as indicated by its shallow ionic strength dependence and the high k8 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 k8
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 reinhardtiihave 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
Trang 9accounting 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
A C K N O W L E D G E M E N T S
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 TRANSIENT in the Programme Human Potential and
Mobility of Researchers of the European Commission
(HPRN-CT-1999-00095).
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