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J. Biol. Chem., Vol. 275, Issue 23, 17590-17595, June 9, 2000

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Dynamic Interaction of Plastocyanin with the Cytochrome bf Complex^*

Jürgen Illerhaus, Lothar Altschmied^§¶, Jan Reichert, Elena Zak^§, Reinhold G. Herrmann^§, and Wolfgang Haehnel

From the  Institut für Biologie II/Biochemie, Albert-Ludwigs-Universität Freiburg, Schänzlestrasse 1, D-79104 Freiburg and ^§ Botanisches Institut, Ludwig-Maximilians-Universität, Menzinger Strasse 67, D-80638 Munich, Germany

Received for publication, July 8, 1999, and in revised form, February 14, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The interaction between plastocyanin and the intact cytochrome bf complex, both from spinach, has been studied by stopped-flow kinetics with mutant plastocyanin to elucidate the site of electron transfer and the docking regions of the molecule. Mutation of Tyr-83 to Arg or Leu provides no evidence for a second electron transfer path via Tyr-83 of plastocyanin, which has been proposed to be the site of electron transfer from cytochrome f. The data found with mutations of acidic residues indicate that both conserved negative patches are essential for the binding of plastocyanin to the intact cytochrome bf complex. Replacing Ala-90 and Gly-10 at the flat hydrophobic surface of plastocyanin by larger residues slowed down and accelerated, respectively, the rate of electron transfer as compared with wild-type plastocyanin. These opposing effects reveal that the hydrophobic region around the electron transfer site at His-87 is divided up into two regions, of which only that with Ala-90 contributes to the attachment to the cytochrome bf complex. These binding sites of plastocyanin are substantially different from those interacting with photosystem I. It appears that each of the two binding regions of plastocyanin is split into halves, which are used in different combinations in the molecular recognition at the two membrane complexes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The function of the cytochrome (cyt)¹ bf and cyt bc₁ complexes is essential for energy conversion in photosynthesis and respiration, respectively. Although the general features have been known for nearly two decades, a detailed understanding of the processes in these complexes has been gained only recently with the application of molecular biology and the resolution of the atomic structure of the cyt bc₁ complexes from bovine and chicken heart mitochondria (1, 2). The intracomplex routes of electron transfer have found a structural basis and an intriguing mechanism involving the switching of the position of the Rieske iron sulfur protein between cyt b and cyt c₁ in the electron transfer cycle (1, 3, 4). The structure of the cyt bf complex has not been solved at atomic resolution, except for fragments of cyt f from turnip (5) and of the Rieske iron-sulfur protein (6) truncated at the C and N terminus, respectively. The structural and phylogenetic relationship between the cyt bc₁ and the cyt bf complex suggests that a similar mechanism operates in both membrane assemblies. In plants, most algae and some cyanobacteria it is plastocyanin which accepts electrons from cyt f and transfers them to P700, the reaction center chlorophyll of photosystem I (PSI) (7-9). While the interaction of plastocyanin with PSI has been studied extensively by different groups with consistent results (8, 10-14), that with the cyt bf complex is considerably less understood and controversial.

Two regions of plastocyanin have been proposed to represent potential recognition and docking sites for its reaction partners (15). One is the flat hydrophobic area around the solvent-exposed copper ligand His-87, and the other is composed of two conserved acidic patches, Asp-42/Glu-43/Asp-44/Glu-45 and Glu-59/Glu-60/Asp-61, both adjacent to Tyr-83. To investigate the role of these elements in the interaction with PSI, cross-linking between plastocyanin and PSI (8, 9, 16), as well as measurements of electron transfer kinetics with site-directed plastocyanin mutants (9, 10, 12, 14), have been conducted. The results suggest that in the complex the electron transfer occurs via the copper ligand His-87 (10, 12) and that, apart from the interaction of the hydrophobic area, only the negative patch of residues 42-45 contributes substantially an electrostatic attraction to a positive domain of the PsaF subunit of PSI in plants (9, 13, 14). Subsequent mutations within the PsaF domain in Chlamydomonas reinhardtii uncovered the importance of a single Lys residue (17). The electron transfer from plastocyanin to PSI involves a fast and tight binding of reduced plastocyanin and a considerably weaker binding of oxidized plastocyanin, which is released from PSI after the electron transfer to P700 in a step that is rate-limiting for the oxidation of cyt f (18).

In contrast to these details, much less is known about the interaction of plastocyanin with the cyt bf complex. The role of surface regions of plastocyanin in the interaction with cyt f has been studied by chemical modification of either cyt f (19) or plastocyanin (20-23), and by site-directed mutagenesis of plastocyanin (13, 24-27) and cyt f (28-30). In the majority of these studies, the C-terminal trans-membrane helix of cyt f was removed to convert this cytochrome to a soluble protein. Furthermore, cyt f and plastocyanin were generally from heterologous sources, except for a recent study with the proteins from C. reinhardtii (29). Lee et al. (13) have reported that exclusively mutations of the negative patch of residues 42-45 exerted an effect on the interaction with the solubilized cyt f. On the other hand, Kannt et al. (26) found that both acidic patches are involved in this interaction and concluded that binding and not the rate of the intracomplex electron transfer is affected. A substitution of Leu-12 in the hydrophobic patch (25) by Asn and Glu was found to accelerate and slow down the second-order rate constant, respectively, a finding that is not fully understood but may suggest a role of this site. For the interaction with truncated, soluble cyt f, it is widely accepted that the electron is transferred to the copper center of plastocyanin via Tyr-83 (20, 24, 25, 27), which seems to be favored by the high electron density at the thiolate of the copper ligand Cys-84 (31).

Apart from the fact that molecular interactions may not be precisely tuned with heterologous components, reactions with a truncated solubilized cyt f may differ from those with cyt f embedded in the complex, in which an interaction of cyt f with the Rieske iron-sulfur protein may also depend on the redox state. To examine these objections and to contribute to the understanding of molecular recognition and the specific interactions of plastocyanin with PSI and the cyt bf complex, we have studied the interaction of a set of mutant plastocyanins with the cyt bf complex from a homologous source (spinach).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Proteins-- The cyt bf complex was isolated from spinach as described (32) using 30 mM octylglucoside and 0.5 g of cholate in 100 ml. The complex harvested from the sucrose gradient after ultracentrifugation was concentrated by ultrafiltration with a 100-kDa cutoff membrane (YM100, Amicon). This sample was diluted by a factor of 20 with buffer and the complex concentrated again by ultrafiltration. The molecular mass of the complex was determined by size exclusion chromatography on Sepharose Cl-6B (Amersham Pharmacia Biotech) after calibration with protein standards MW GF-1000 (Sigma). Wild-type plastocyanin was purified from spinach leaves (33). Recombinant plastocyanin was obtained by expression of a truncated precursor gene in Escherichia coli as described (12) and purified from the medium by ion exchange chromatography. The mutations were verified by electrospray mass spectrometry of the proteins (9). The mutants G10L, A90L, and Y83L have been previously described (12). Fig. 1 shows the position of the mutated residues at the surface of plastocyanin.

View larger version (94K): [in this window] [in a new window]   Fig. 1.   Van der Waals surface of poplar plastocyanin (15) with amino acids mutated in the spinach protein. Red, acidic residues; cyan, His-87; yellow, Gly-10 and Ala-90; green, Tyr-83 and Leu-12. The presentation was generated with the program GRAMS. Single-letter codes are used on figure.

Spectroscopic Measurements-- UV/Vis spectra were recorded with a Shimadzu (UV 2101-PC) spectrophotometer at 1-cm optical path length. The concentration of plastocyanin was determined from the absorbance of the oxidized protein at 597 nm and the extinction coefficient of 4.9 mM¹ cm¹ (34). The concentrations of cyt f and cyt b₆ were determined from the absorbance difference between the reduced and oxidized proteins at 554 minus 543.3 and 563.3 minus 577 nm using the difference extinction coefficient of 25 (35) and 21.5 mM¹ cm¹ (36), respectively. Chlorophyll was determined after acetone extraction (37).

Redox Titration-- The redox titration of plastocyanin at a concentration of approximately 0.5 mM was performed at 298 K as described (38, 39) with the following modifications. The electrochemical cell with an optical path length of 40 µm was made of Lucite. The ambient potential was adjusted with a home-built potentiostat. Potassium ferricyanide, N,N-dimethyl-p-phenylenediamin, benzoquinone, and diaminodurene, each at a concentration of 100 µM, were used as redox mediators (40). In addition, the solution contained 10 mM MOPS buffer, pH 7.0, and 100 mM KCl.

Kinetic Measurements-- Kinetic measurements were performed at 278 K (5 °C) with a stopped-flow apparatus (SFM-3, Bio-Logic, France) equipped with a diode array photometer (J&M, Aalen, Germany), which monitors spectra between 200 and 620 nm with 512 diodes and a resolution of 2 nm (full width at half-height) at time intervals of 1.3 ms. The absorbance difference of 554.3 minus 543.7 nm at the -band, and of 421.7 minus 410.2 nm at the Soret band, were extracted from the spectra as a function of time. The traces of four individual experiments were averaged for each data analysis. Immediately before the measurements the cyt bf complex was reduced with sodium ascorbate, plastocyanin oxidized with ferricyanide, and both samples passed over short desalting columns (PD10, Amersham Pharmacia Biotech). The concentration of the cyt bf complex and plastocyanin (Pc) was adjusted to about 0.15 and 1 µM, respectively, to ensure a pseudo first-order reaction with a rate constant k' equal to the product k₂[Pc] of the second-order rate constant k₂ and the plastocyanin concentration [Pc]. In addition the reaction mixture contained 20 mM octylglucoside and 10 mM sodium phosphate buffer, pH 7.0. Further additions are given in the figure legends. The kinetics were fitted to a single-exponential and, if necessary, to a two-exponential time course.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fig. 2 shows the absorbance spectrum of the isolated cyt bf complex in the oxidized state with prominent peaks at 417 nm resulting from the superimposed Soret bands of the cytochromes and chlorophyll associated with the complex and at 669 nm from chlorophyll alone. The difference of the spectra in the reduced minus oxidized state of cyt f and cyt b₆ are shown in the inset as traces B and C, respectively. From these spectra, we estimate a molar ratio of cyt b₆ to cyt f of 2 ± 0.1, and of chlorophyll to cyt f of 1.3 ± 0.4. Table I summarizes the characteristics of four samples a, b, c, and d, which were isolated from different batches of spinach. For sample d, the step of the isolation procedure including dilution and ultrafiltration was repeated three times in an attempt to remove the chlorophyll bound to the complex. This procedure reduced the molar ratio to 0.5 chlorophyll per cyt f but decreased also the rate of electron transfer from decyl-plastoquinol to cyt c. The decreased activity may be due to the extended isolation time and the formation of monomeric complex (41). Therefore, only samples a, b, and c were chosen for measurements of stopped-flow kinetics. Size exclusion chromatography of the complex yielded a molecular mass of approximately 230 kDa consistent with a dimeric cyt bf complex (data not shown). The electron transfer activity from the donor decyl-plastoquinol was also measured with oxidized plastocyanin in the place of cyt c as in Table I and yielded values of 50 s¹.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Representative spectra of the cyt bf complex after sucrose gradient centrifugation. A, untreated sample; B, ascorbate reduced minus ferricyanide oxidized; C, dithionite reduced minus ascorbate reduced sample.

The redox titration of wild-type and the mutant plastocyanins D42N/E43Q/D44N/E45Q, E59Q/E60Q/D61N, and A90T showed midpoint potentials E_m of 376, 372, 396, and 377 mV, respectively. The value of 376 mV for wild-type plastocyanin is in line with published data ranging between 370 and 390 mV (13, 34, 42). It is noteworthy that the midpoint potential of the mutant E59Q/E60Q/D61N is significantly higher than that of wild-type plastocyanin or of the mutant of the other acidic patch. This is attributed to the effect of the negative charges of residues 59-61 being closer to the copper center than the other negative patch (9). All other mutants exhibited midpoint potentials that resemble that of wild-type plastocyanin (data not shown).

Fig. 3 presents the oxidation kinetics of cyt f after stopped-flow mixing at the final concentrations of 0.15 µM reduced cyt bf complex and 1 µM oxidized wild-type plastocyanin at two different ionic strengths at 5 °C. The measurements at the Soret band and at the -band (inset) illustrate an excellent agreement and similar signal to noise ratios. The time course has been fitted to a two-exponential decay. The fast kinetic component had a half-life, which decreased at increasing concentrations of plastocyanin as expected for a second-order reaction (data not shown). The values of the second-order rate constant k₂ were 40 × 10⁶ and 130 × 10⁶ M¹ s¹ at 96 and 32 mM NaCl (i.e. an ionic strength of 0.12 and 0.05 M), respectively. The slow component showed no dependence on the concentration of plastocyanin and followed first-order kinetics with rate constants of 9 and 16 s¹ and a relative amplitude of 25 and 9%, respectively. It was not observed in all samples and hence originates probably from a small fraction of inactive complex. Therefore, only the fast kinetic component was used to compare the mutant plastocyanins.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Time course of the absorbance change at 421.7 minus 410.2 nm and in the inset at 554.3 minus 543.7 nm after mixing 0.15 µM reduced cyt bf complex with 1 µM oxidized plastocyanin (wild-type). The traces were averaged from four stopped-flow experiments. Traces A, 30 mM; traces B, 100 mM NaCl.

The second-order rate constant as a function of the temperature is shown by an Arrhenius plot in Fig. 4. An activation energy of 31 kJ × mol¹ is estimated from the slope of the curve. In most of our experiments, the temperature was set to 5 °C to slow down the kinetics relative to that at room temperature and to enable the resolution of the time course at low ionic strength. At 25 °C the value of the rate constant is 62 × 10⁶ M¹ s¹ in the presence of 150 mM NaCl. Assuming the same ionic strength dependence of the reaction rate at 5 (see below) and 25 °C, this value extrapolates to a rate constant of 185 × 10⁶ M¹ s¹ at 90 mM NaCl, which is by a factor of 4 faster than that reported for truncated cyt f under these conditions (24, 25, 27) but comparable to the value of 100 × 10⁶ M¹ s¹ found recently (29).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Second-order rate constant of cyt f oxidation by plastocyanin as a function of the temperature. The reaction mixture contained 0.15 µM cyt bf complex, 1 µM plastocyanin, and 150 mM NaCl.

To analyze the surface regions of plastocyanin involved in the rate-limiting step of the reaction with the cyt bf complex, the role of residues in the hydrophobic and acidic patches of spinach plastocyanin was probed using site-directed mutagenesis. One set includes mutations of Tyr-83 and the neighboring acidic regions with the amino acids Asp-42/Glu-43/Asp-44/Glu-45 and Glu-59/Glu-60/Asp-61. Site-directed mutations were also made at residues Gly-10, Leu-12, and Ala-90 that cover a large fraction of the hydrophobic patch around the copper ligand His-87 (cf. Fig. 1).

The Acidic Patches of Plastocyanin-- Fig. 5 shows the second-order rate constants of cyt f oxidation by wild-type and mutant plastocyanin with a decreased number of negative charges as a function of the NaCl concentration. The strong decrease of the rate constant at increasing ionic strength is consistent with an electrostatic attraction between the negative and positive charges of plastocyanin and cyt f, respectively. In the mutants D42N/E43Q/D44N/E45Q and E59Q/E60Q/D61N, all acidic residues of a patch have been replaced by their neutral amides; in the mutant Y83R, a positive charge has been inserted between these two patches. The removal of the four and three negative charges or even introducing only one positive charge in this region leads to a remarkable decrease of the electron transfer rate. The decreased number of negative charges results also in a diminished dependence of the rate constant on the ionic strength. Therefore, at ionic strengths below 50 mM, the rate constant of plastocyanin with a neutral patch of residues 42-45 is more than an order of magnitude smaller than that of wild-type plastocyanin. At ionic strengths higher than 150 mM, the relative effect of these mutations is masked by screening of the charged residues consistent with previous results found with mutant cyt f (28).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Second-order rate constant of the reaction of 0.15 µM reduced cyt bf complex with 1 µM oxidized plastocyanin as a function of the sodium chloride concentration. Figures shows wild-type () and mutants D42N/E43Q/D44N/E45Q (), E59Q/E60Q/D61N (), Y83L (), and Y83R ().

Does Tyr-83 Play a Significant Role in the Electron Transfer to Cytochrome f?-- To reexamine the deduced function of the aromatic amino acid Tyr-83 of plastocyanin as the electron transfer site to cyt f (24), this residue was also mutated to Leu. For wild-type, Y83L-, and Y83R-plastocyanin, the second-order rate constants k₂ were 47 × 10⁶, 27 × 10⁶, and 15 × 10⁶ M¹ s¹, respectively, at 90 mM NaCl. These values and the results shown in Fig. 5 indicate a decrease of the rate constant with the Y83L-plastocyanin by a factor of 1.7 relative to wild-type plastocyanin. This is significantly different from published data (24, 27), which indicated a decrease of the second-order rate constant by the factor of 40 after this mutation. Thus, in our experiments with an intact homologous cyt bf complex, the effect of the Leu residue is rather small and that of Arg is in line with the changed charges. This does not support an essential role of Tyr-83 in the electron transfer.

The Flat Hydrophobic Surface of Plastocyanin-- A set of mutant plastocyanins was designed where the small side chains of Gly-10 and Ala-90 in the flat hydrophobic surface region were changed to larger hydrophobic residues. As with PSI (12), this could impair the docking of plastocyanin and cyt f in a transient complex and decrease the electron transfer rate by an increased distance (11). The second-order rate constants are shown in Fig. 6 at different concentrations of NaCl. Remarkably, the mutants G10V and G10L and, to a smaller extent, G10M showed an increased second-order rate constant as compared with wild-type plastocyanin, in contrast to the mutants A90L and A90T, which show a diminished rate-constant. In mutant L12A, the electron transfer rate was by a factor of 1.9 smaller than that of the wild type at 90 mM NaCl in agreement with the data of Modi et al. (25).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Second-order rate constant of the reaction of 0.15 µM reduced cyt bf complex with 1 µM oxidized plastocyanin as a function of the sodium chloride concentration. Figure shows wildtype () and mutants A90L (), A90T (), G10L (), G10M (), G10V (), and L12A ().

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The recognition process of proteins involves the interaction of specific surface regions. A pre-orientation by long range electrostatic effects and the dipole momentum of the molecules is followed by the complex formation. The selective binding of plastocyanin to two different complexes represents a special case of a general mechanism involving the docking of a protein to different reaction partners. An obvious problem is how the two potential recognition and binding areas of the rather small plastocyanin molecule are used to ensure stable complex formation and a preferential binding at different redox states for efficient electron transfer at the two complexes.

An electrostatic interaction between the negatively charged patches of plastocyanin and positive residues of cyt f has been suggested for the reaction between plastocyanin and the cyt bf complex (5, 43). During the binding process, a properly oriented pair of the redox partners must be formed to optimize the electron transfer between the active sites (44). Assuming that the actual electron transfer is fast (45), the rate constant reflects that of the rate-limiting step during this docking process, k_on. In this study the interaction between cyt f and plastocyanin has been investigated for the first time with site-directed plastocyanin mutants and intact cyt bf complex from the same organism.

Electrostatic Interaction-- The effect of the ionic strength on the electron transfer from the cyt bf complex to plastocyanin indicates the attraction of two oppositely charged regions in the rate-determining step in good agreement with most studies using soluble cyt f and plastocyanin from heterologous (19, 26) or homologous sources (29). It is not consistent with results showing no effect of mutated charges of residues 59-61 (13, 14) or an increase of the rate constant at increasing ionic strength below 40 mM (45) (cf. Fig. 4). Our data demonstrate unambiguously that both acidic patches of plastocyanin are involved in the interaction with the cyt bf complex. Even a positive charge at Tyr-83, which bridges the surface between the two patches (Fig. 1), has an effect similar to that of the neutralized acidic residues. The decreased number of negative charges in these three mutants results in a corresponding decrease of the rate constant in agreement with other mutants of plastocyanin (26). The electrostatic attraction is emphasized by the analogous results found with mutants of cyt f with a diminished number of positive charges (28-30).

Site of Electron Transfer-- The electron transfer path between cyt f and plastocyanin has been proposed to include Tyr-83 of plastocyanin (20, 21, 24, 25, 27). However, in contrast to previous results with a Y83L mutant, we did not find a decrease of the second-order rate constant relative to wild-type plastocyanin by a factor of 40 but only by a factor not higher than 1.7 at 90 mM NaCl. This finding (46) has recently been confirmed even with the system used previously (47). The difference is not understood but may originate in the heterologous source and the solubility of the truncated cyt f used in the previous studies. Since even the mutation of Tyr-83 to Arg leads to a decrease in the rate by a factor not higher than 3, we conclude that Tyr-83 is not involved in the electron transfer path. The direct contact of this residue to both acidic patches (see Fig. 1) suggests that the mutation may cause deviations from the proper orientation by small structural changes or the removal of a H-bond in the complex. Thus, the second electron transfer route different from that via His-87 to and from the copper center in the plastocyanin molecule is an unlikely one. This solves also a previously not understood difference between the structure of plastocyanin and cyt c₆, which does not exhibit the possibility of a second electron transfer route but can replace plastocyanin in many algae and cyanobacteria. The electron transfer from the iron center of cyt c₆ via the surface exposed edge of the heme is analogous to that from the copper center via the surface exposed His-87 (48, 49).

The Hydrophobic Surface-- The electron transfer via the copper ligand His-87 in the center of the hydrophobic surface of plastocyanin has been favored for PSI (12). A tight fit of the hydrophobic surfaces of plastocyanin and cyt f could contribute to the formation of the electron transfer complex as well. In analogy, replacing amino acid residues with small side chains in the hydrophobic surface (Fig. 1) by larger ones should inhibit the binding and the electron transfer as found for the interaction of plastocyanin with PSI (12). The six mutations of plastocyanin (A90L, A90T, G10L, G10M, G10V, and L12A) exert a small but noticeable effect on the second-order rate constant of the electron transfer from the cyt bf complex. Remarkably, mutations of Gly-10 lead to an increase that of Ala-90 to a decrease of the rate constant. These effects indicate that this region, as the acidic patch, contributes to the interaction with the cyt bf complex. Bulky residues at the position of Ala-90 impair the docking in this region as observed with PSI. In the mutant L12A the flat hydrophobic surface should form a trough, which diminishes the contribution of the hydrophobic surface to the binding. The increase of the rate by hydrophobic residues of intermediate size at the position of Gly-10 was surprising since it indicates an improved contact with the cyt bf complex opposite to that at PSI. This can be understood if a small gap exists in the complex at this position. It is relevant to note that the observed effects are consistent in all details with the orientation of plastocyanin in the bound state found recently by NMR measurements with truncated cyt f (50). The coincidence with our kinetic data suggests that this bound state is very similar to the complex formed in the rate-determining step of the second-order process with the intact cyt bf complex. The bound state (50) involves only part of the hydrophobic surface around Ala-90 and electrostatic interactions of both negative patches with positive residues very likely including Arg-209 and Lys-187 of cyt f. Fig. 7A shows a schematic cross-section of this complex. This places bound plastocyanin close to but not in contact with the positive residues Lys-58, Lys-65, and Lys-66 at the large domain of cyt f (numbering refers to amino acid residues of turnip cyt f (Ref. 5)). A decrease of the rate constant determined by stopped-flow experiments after mutation of these residues to neutral or negative ones indicate their effect on the complex formation. However, in intact C. reinhardtii, these mutations showed only minor effects on the oxidation kinetics of cyt f after laser-induced oxidation of P700 (29). This difference is not fully understood but may partially be explained by the release of oxidized plastocyanin from PSI being rate-limiting for the oxidation of cyt f (18).

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Schematic view outlining the orientation of plastocyanin in the electron transfer complex with the cyt bf complex (A) and PSI (B). The view of PSI and its subunit PsaF is derived from our previous work (9, 12, 17), the surface of cyt f traced by a red line from Ref. 5. The red and green squares indicate the approximate position of the heme and P700, respectively, the blue circle the copper center of Pc, and yellow lines hydrophobic contacts. C gives a schematic view of the two characteristic surface areas of plastocyanin, the hydrophobic surface with the site of electron transfer at His-87 and the negative patch with Tyr-83, and their use in the interaction. Light brown symbolizes docking areas used by both complexes, red and green the areas used exclusively by cyt f and PSI, respectively. Single-letter codes are used on figure. For further details, see "Discussion."

Comparison of the Docking Regions of Plastocyanin at the Cytochrome bf Complex and Photosystem I-- The outlined findings suggest the following interactions in the step being rate-determining for the electron transfer. (i) Both groups of negative residues of the negative patch including Tyr-83 exert electrostatic interactions with a positive region of cyt f. (ii) The effect of the hydrophobic residues support strongly that Ala-90 and Leu-12, but not Gly-10 are in close contact with cyt f. The resulting orientation for the electron transfer from the heme to plastocyanin places the copper ligand His-87 close to Tyr-1 of cyt f and the propionate side chains of the heme both providing short pathways for electron transfer. The electron transfer is controlled not only by the distance but also by the redox potential difference (i.e. the driving force) and the reorganization energy (44). The hydrophobic contact in the electron transfer region is in favor of a low reorganization energy. Since the value of the driving force is only about 100 and 10 mV for the reaction with P700 and cyt f, respectively, a low reorganization energy is an advantage for a fast electron transfer rate being maximal at equal values of the driving force and the reorganization energy (44).

An extension of the hydrophobic surface at Gly-10 by larger residues improves the electron transfer from cyt f. This raises the question why this mutation has not been realized by evolution. However, such a change inhibits efficiently the docking at PSI (12). Fig. 7B shows an orientation proposed for the complex between plastocyanin and PSI in our previous work. A comparison with Fig. 7A highlights that the docking is optimized for two different configurations. The complex at PSI involves the whole hydrophobic surface of plastocyanin while that at cyt f only the section around Ala-90.

Split Usage of Characteristic Surface Areas of Plastocyanin-- The use of either the full hydrophobic patch or a section of it is similar to that of the negatively charged area of plastocyanin with its two patches. In contrast to cyt f with PSI, only residues 42-45 are involved in the complex formation. This is sketched in a schematic of plastocyanin in Fig. 7C. Each area contributes a section which is used with both complexes (colored light brown). The sections exclusively used with PSI and the cyt bf complex are colored green and red, respectively. There may be several reasons for such a selection. (i) A complex with a tight fit of both areas may be too stable for efficient turnover in electron transport. (ii) A rapid and precise positioning and orientation may need the electrostatic in addition to the hydrophobic interaction. The electron transfer changes the electrostatic interaction in different ways in the two complexes. Whether the alternatives reflect a balance of binding and release for optimized turn-over in electron transport will require further study.

	ACKNOWLEDGEMENTS

We gratefully acknowledge the expert technical assistance of Carolin Schraff and Gabriele Burkhard. We thank Friedel Drepper for help with the graphics program.

	FOOTNOTES

^* This work was supported by German Research Foundation Grants SFB 184 (to R. G. H.) and SFB 388/A1 (to W. H.), by the Fonds der Chemischen Industrie, and by European Community Contract BIO2CT-930077 (to W. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Current address: Inst. of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany.

To whom correspondence should be addressed: Inst. für Biologie II/Biochemie, Albert-Ludwigs-Universität Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany. Tel.: 49-761-2032690; Fax: 49-761-2032601; E-mail: haehnel@uni-freiburg.de.

	ABBREVIATIONS

The abbreviations used are: cyt, cytochrome; Pc, plastocyanin; PSI, photosystem I; MOPS, 3-(N-morpholino)propanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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