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: 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) 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. 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Biochem. 269) Ó FEBS 2002 . 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. the first kinetic analysis of the in vitro interactions Cyt f plastocyanin and plastocyanin Fig. 4. Comparison of ionic strength curves obtained by using