The Unique Proline of the Prochlorothrix hollandica Plastocyanin Hydrophobic Patch Impairs Electron Transfer to Photosystem I*

A number of surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in amino acids located in the amino-terminal hydrophobic patch of the copper protein, which presents a variant structure as compared with other plastocyanins. The single mutants Y12G, Y12F, Y12W, P14L, and double mutant Y12G/P14L have been produced. Their reactivity toward photosystem I has been analyzed by laser flash absorption spectroscopy. Plots of the observed rate constant with all mutants versus plastocyanin concentration show a saturation profile similar to that with wild-type plastocyanin, thus suggesting the formation of a plastocyanin-photosystem I transient complex. The mutations do not induce relevant changes in the equilibrium constant for complex formation but induce significant variations in the electron transfer rate constant, mainly with the two mutants at proline 14. Additionally, molecular dynamics calculations indicate that mutations at position 14 yield small changes in the geometry of the copper center. The comparative kinetic analysis of the reactivity

Plastocyanin (Pc) 1 is a small redox protein (molecular mass, ϳ10.5 kDa) that functions in photosynthesis as a mobile electron carrier between the two membrane-embedded complexes cytochrome b 6 f and photosystem I (PSI) (1)(2)(3). Whereas higher plants produce just Pc, there is a number of intermediate species, both cyanobacteria and eukaryotic algae, that are able to synthesize cytochrome c 6 as an alternative redox carrier under copper deficiency (4). The interaction between these two metalloproteins and PSI has been studied by laser-flash absorption spectroscopy in a wide variety of evolutionarily differentiated organisms, including prokaryotic and eukaryotic systems (5,6). All these comparative kinetic analyses have allowed us to propose different reaction mechanisms for PSI reduction (7,8).
Recently, a comparative analysis of the interaction of Pc and cytochrome c 6 with PSI from the prochlorophyte Prochlorothrix hollandica has been carried out (9). Prochlorophytes represent a deeply branched group of cyanobacteria containing both chlorophyll a and b (10,11). These studies have shown that Prochlorothrix Pc reacts with PSI according to a two-step reaction mechanism involving complex formation and electron transfer, the complex being mainly hydrophobic in nature. Cytochrome c 6 , in its turn, follows a three-step reaction mechanism with rearrangement of redox partners within an intermediate electrostatic complex before electron transfer (9). Such a difference in the kinetic mechanisms reflects interesting differences not only in electrostatic charge surface distribution but also in dynamic properties.
The solution structure of Prochlorothrix Pc has been recently solved by NMR spectroscopy (12). Despite the relatively low number of conserved residues shared with other Pcs, the Prochlorothrix molecule has a similar overall folding pattern, including the classical two-sheet ␤-barrel tertiary structure. Interestingly, Prochlorothrix Pc has an altered hydrophobic patch, a region that is thought to be crucial in Pc interaction with its redox counterparts. Whereas the backbone and loops at the hydrophobic area of Prochlorothrix Pc are as those of other Pc molecules, the presence of two unique residues (Tyr-12 and Pro-14 in Prochlorothrix Pc versus Gly-10 and Leu-12 in all other Pcs) yields a structurally different hydrophobic surface, with Tyr-12 protruding outwards from this patch (12).
In this paper, we are extending our previous studies of Prochlorothrix PSI reduction by wild-type (WT) Pc to analyze the reactivity of Pc mutants at Tyr-12 and Pro-14. The laser-flash absorption spectroscopy analyses herein reported indicate that the replacement of Pro-14 by leucine, which is the "standard" residue in all other Pcs, makes the copper protein react much more efficiently with PSI.

EXPERIMENTAL PROCEDURES
Expression and Reconstitution of Wild-type and Mutant Plastocyanins-Mutant and WT P. hollandica Pc was expressed as inclusion bodies in Escherichia coli BL21(DE3) pLysS (Novagen, Madison, WI) as previously described (13). Specifically, the Prochlorothrix Pc expression plasmid, pVAPC10, was used as a template for polymerase chain reaction-mediated mutagenesis (Stratagene QuikChange kit, La Jolla, CA). Custom mutagenic PCR primers were obtained from Life Technologies, * Research at Universidad de Sevilla was supported by the Dirección General de Investigación Científica y Técnica (MCYT Grant BMC2000-0444), European Union (Networks ERB-FMRX-CT98-0218 and HPRN-CT1999-00095), and Junta de Andalucía (PAI, . Work at Bowling Green was supported by National Science Foundation Grants MCB-9634049 and BIR-0070334. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Characterization of Reconstituted Plastocyanin-Refolded Pc preparations were analyzed by absorption and far-UV circular dichroism spectroscopy as described previously (13). Redox titration of Pc preparations was monitored by absorption spectroscopy at 602 nm in increasing ratios of ferrocyanide/ferricyanide as previously described (14). Alternatively, redox potentials were determined by cyclic voltammetry (15).
Preparation of Photosystem I Particles-PSI particles from Prochlorothrix were obtained by ␤-dodecyl maltoside solubilization as described by Rögner et al. (16) and modified by Hervá s et al. (17). The following modification was indeed made. The preparation of PSI-enriched particles obtained after the discontinuous sucrose gradient was washed with buffer D (20 mM MES, pH 6.5, 10 mM CaCl 2 , 10 mM MgCl 2 , 0.5 M D-mannitol, 20% glycerol) to remove sucrose and then diluted 50% with buffer A (20 mM MES, pH 6.5, 10 mM CaCl 2 , 10 mM MgCl 2 ) plus 0.25 M D-mannitol before being applied to a second continuous sucrose gradient (10 -25%). The lower, darker, green band containing PSI was washed and concentrated as previously described (17). The P700 content in PSI samples was calculated from the photoinduced absorbance changes at 820 nm using the absorption coefficient of 6.5 mM Ϫ1 cm Ϫ1 determined by Mathis and Sétif (18). Chlorophyll concentration was determined according to Arnon (19). The chlorophyll/P700 ratio of the resulting PSI preparations was 150/1. Spinach, Synechocystis and Anabaena PSI were purified as previously described (7).
Laser-flash Absorption Spectroscopy-Kinetics of flash-induced absorbance changes in PSI were followed at 820 nm as described in Hervá s et al. (7). Unless otherwise stated, the standard reaction mixture contained, in a final volume of 0.2 ml, 20 mM Tricine-KOH, pH 7.5, 0.03% ␤-dodecyl maltoside, 10 mM MgCl 2 , an amount of PSI-enriched particles equivalent to 0.35 mg of chlorophyll ml Ϫ1 , 0.1 mM methyl viologen, 2 mM sodium ascorbate, and Pc at the indicated concentration. All the experiments were performed at 22°C in a 1-mm path-length cuvette. Each kinetic trace was the average of 5-10 independent measurements with 30-s spacing between flashes. For most experiments, the estimated error in the observed rate constants (k obs ) was less than 10%, based on reproducibility and signal-to-noise ratios.
Kinetic Analyses-Data collection was as previously described (7). Oscilloscope traces were treated as sums of several exponential components; exponential analyses were performed using the Marquardt method with the software devised by P. Sétif (Saclay, France). Kinetic analyses were carried out according to the reaction mechanisms previously proposed (7).
Molecular Dynamics Studies-The structures of WT and P14L mutant Prochlorothrix Pcs were analyzed using the SWISS-PDB Viewer. Charges of most atoms were taken from the AMBER 6.0 force field. Charges and other force field parameters for the copper atom and its ligands were obtained from NMR structural data and compared with similar Pc simulations in the literature (20,21). Topology and parameter files for both proteins were generated using the xLeap module of the AMBER 6.0 package. Hydration was treated explicitly by including the proteins in a 10-Å water box according to the TIP3P model. Proteins were subjected to 500 steps of steepest descent energy minimization to relax large steric overlaps or electrostatic inconsistencies. Molecular dynamics (MD) was employed to study conformational changes (21). The starting configurations were subjected to MD runs of 10 5 steps (1-fs increments) at 300 K. The SHAKE option was used to constrain all hydrogen bonds. The data were analyzed using the CARNAL block of AMBER. MD data were obtained by sampling every 100 fs, yielding 1000 conformations.

RESULTS AND DISCUSSION
The two variant residues at the hydrophobic patch of Prochlorothrix Pc, namely Tyr-12 and Pro-14 ( Fig. 1), were chosen to be mutated to investigate their role in the reactivity of the copper protein toward PSI. By studying the properties of this natural variant of Pc, the minimum structural requirements for complex formation and efficient electron transfer to PSI could be investigated. Tyrosine at position 12 was thus replaced with two different aromatic residues, phenylalanine and tryptophan, as well as with glycine, the latter being a con-served residue at this position in virtually all known sequences of Pc. Additionally, proline at position 14 was replaced with leucine, which is also a conserved residue in all other Pcs. A double mutant with reversion of both tyrosine and proline to the standard residues glycine and leucine, respectively, was likewise constructed.
Description of the Mutant Plastocyanins-Most of the mutations do not significantly alter the midpoint redox potential value of the copper protein, with the exception of the P14L mutant and the Y12G/P14L double mutant, whose redox potentials are ϳ15 mV lower (Table I). Thus, only those mutations affecting position 14 yield measurable changes in the environment of the copper center, and such changes in redox potential slightly increase the driving force for electron transfer to PSI. Nonetheless, such modifications do not significantly alter the electronic absorption spectra, as all mutant Pcs yield an absorption peak at 602 nm identical to the WT (13).
Electron Transfer Kinetics to Prochlorothrix Photosystem I-The laser-flash-induced kinetic traces of PSI reduction by WT and mutated Pcs are monoexponential, even at high donor protein concentration (not shown). The dependence of the observed pseudo first-order rate constant (k obs ) upon donor protein concentration shows a saturation profile (Fig. 2). This finding suggests the formation of a bimolecular transient Pc⅐PSI complex before electron transfer, as previously described for the WT system (9) according to the following minimal two-step reaction mechanism.
Pc red ϩ PSI ox -| 0 in which K A stands for the equilibrium constant of complex formation, and k et , which can be experimentally inferred from the limiting k obs at infinite Pc concentration, denotes the subsequent intracomplex electron transfer first-order rate constant. Table I (22). As can be seen in Table I, there are no significant changes in K A with all mutants. This is in contrast to the results obtained from mutations at the amino-terminal patch of other Pcs, from which it has been inferred that both the hydrophobic and charged patches seem to be involved in the interaction of Pc with PSI (3, 6). Thus, the protein-protein interactions mediating complex formation in Prochlorothrix are likely located elsewhere on the Pc surface. To check if the hydrophobic nature of the interaction between Pc and PSI in Prochlorothrix (9) is altered by mutations, the kinetics of PSI reduction were also followed at varying ionic strength. As can be seen in Table I, none of the mutants shows significant changes in K A or k et upon increasing salt concentration, as previously described for the WT Pc (9).
Concerning the electron transfer step, Table I shows that none of the mutations at position 12 significantly alters the efficiency of Pc in donating electrons to PSI, with the exception of the replacement of tyrosine by glycine, which induces a decrease of about 30% in k et . These results suggest that in Prochlorothrix Pc the surface conformation of the hydrophobic patch at position 12 is not essential for productive electron transfer. Much more drastic is the effect obtained by replacing proline at position 14 by leucine, as the k et value increases up to three times with respect to the WT species (Table I). This can be explained in part by assuming that Pro-14 in some way distorts the interaction site, its replacement thus making the copper site be ϳ1-Å closer to the acceptor (23).
Indeed, modeling the hydrophobic patch of the P14L mutant protein suggests subtle alterations in the position of His-85 relative to the WT (Fig. 3). MD calculations also reveal alter-ations in the copper site, yielding a 0.25-Å displacement of the axial copper ligand Cys-82 in the P14L mutant compared with the WT (Fig. 4). This conformational change is confirmed by the complementary 0.2-Å displacement of the axial ligand, Met-90 (Fig. 4, bottom). By contrast, MD calculations do not reveal such pronounced alterations in the His ligands of the copper center (Table II). Overall, such structural changes may be the basis for the slightly lower redox potential of the leucine mutant. This small increase in driving force may also contribute to the increased k et . Additionally, residues Tyr-12, Met-33, and Ala-88 in or near the hydrophobic patch also exhibit structural alterations that may affect donor-acceptor distance (summarized in Table II). Last, the double mutant Y12G/P14L behaves as expected from the compensating effect of individual mutations. In fact, this mutant yields a k et value that doubles that with WT and is 67% that with the single mutant P14L, a decrease that compares fairly well with that observed with the single mutant Y12G with respect to WT (see above).
Electron Transfer Kinetics to Heterospecific Photosystem I-It has been previously reported that Prochlorothrix WT Pc does not form any electron transfer complex with PSI from either spinach or the cyanobacteria Anabaena and Synechocys-  2. Dependence of the observed rate constant (k obs ) for Prochlorothrix photosystem I reduction by wild-type and mutated plastocyanins upon donor protein concentration. The continuous line corresponds to the theoretical fitting to the two-step formalism described in Meyer et al. (22). Other conditions were as described under "Experimental Procedures." tis. The copper protein does exhibit a very low reactivity in all cross-reactions (9). Because the mutants constructed in this study are aimed to revert the "exclusive" hydrophobic patch of Prochlorothrix to the standard configuration, we have also checked the reactivity of mutants toward heterospecific PSI. In all cases, linear dependences were observed when plotting the observed rate constants versus protein concentration, as shown in Fig. 5 for Synechocystis PSI. Because no complex formation was observed, the bimolecular rate constants (k 2 ) for PSI reduction were calculated (Table III). Replacement of Pro-14 with leucine makes the bimolecular rate constant of PSI reduction both with eukaryotic and prokaryotic photosystems increase by 1 order of magnitude. Whereas the mutants at Tyr-12, including that with the reversion to the standard glycine, do not significantly change their reactivity, the double mutant Y12G/ P14L shows a significant increased efficiency with spinach PSI and minor changes with cyanobacterial photosystems (Table  III). Relatively small increases in reactivity are also observed with the mutant Y12W. These results with mutants at position 12 are in clear contrast with the previously proposed requirement for a flat surface in the area of Gly-12 at the hydrophobic patch of Pc to ensure efficient electron transfer to PSI (24). In case of cross-reactions with Synechocystis PSI, the mutant P14L was shown to be even more reactive than the Synechocystis WT Pc (Fig. 5 and Table III). Overall, these observations provide some agreement with the suggestion made by Sigfridsson and co-workers (25), who demonstrated that replacement of leucine in higher plant Pc with the less bulky alanine decreased k et despite a modest increase in the driving force (0.024 eV) for electron transfer to P700. These authors suggested that a bulky hydrophobic residue might yield a better fit to PSI (25).
Concluding Remarks-The fact that WT Pc from Prochlorothrix possesses a residue that is impairing its redox interaction with its physiological electron acceptor may indicate that this organism is using a divergent protein that appeared before evolution selected for leucine at position 14. We can thus say that reversion of Pro-14 to the standard leucine makes Prochlorothrix Pc much more reactive toward PSI from any organism, including Prochlorothrix.
Last, the influence of distance versus driving force for the  3.6 ϫ 10 6 Anabaena Pc 7.0 ϫ 10 7 a k 2 could not be calculated because spinach plastocyanin follows a three-step reaction mechanism (7). enhanced reactivity observed with the P14L mutant should be addressed. Given the fact that the driving force in the mutant increased by 0.015 eV, k et would be expected to increase slightly (ϳ30%) (26) via this parameter alone. However, the large (10-fold) increases in k 2 seen in reactions with spinach and Synechocystis PSI suggest that a decrease in distance to the acceptor in the mutant plays an important role in enhancing electron transport.