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J Biol Chem, Vol. 274, Issue 47, 33565-33570, November 19, 1999

Site-directed Mutagenesis of Cytochrome c₆ from Anabaena Species PCC 7119
IDENTIFICATION OF SURFACE RESIDUES OF THE HEMEPROTEIN INVOLVED IN PHOTOSYSTEM I REDUCTION^*

Fernando P. Molina-Heredia, Antonio Díaz-Quintana, Manuel Hervás, José A. Navarro, and Miguel A. De la Rosa

From the Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla y Consejo Superior de Investigaciones Científicas, Centro Isla de la Cartuja, Américo Vespucio s/n, 41092 Sevilla, Spain

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A number of surface residues of cytochrome c₆ from the cyanobacterium Anabaena sp. PCC 7119 have been modified by site-directed mutagenesis. Changes were made in six amino acids, two near the heme group (Val-25 and Lys-29) and four in the positively charged patch (Lys-62, Arg-64, Lys-66, and Asp-72). The reactivity of mutants toward the membrane-anchored complex photosystem I was analyzed by laser flash absorption spectroscopy. The experimental results indicate that cytochrome c₆ possesses two areas involved in the redox interaction with photosystem I: 1) a positively charged patch that may drive its electrostatic attractive movement toward photosystem I to form a transient complex and 2) a hydrophobic region at the edge of the heme pocket that may provide the contact surface for the transfer of electrons to P₇₀₀. The isofunctionality of these two areas with those found in plastocyanin (which acts as an alternative electron carrier playing the same role as cytochrome c₆) are evident.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In cyanobacteria and green algae, cytochrome (Cyt)¹ c₆ acts as a soluble redox carrier that can replace plastocyanin in the transport of electrons between the two membrane-embedded complexes Cyt b₆f and photosystem I (PSI) (see Refs. 1 and 2 for reviews). In higher plants, however, the copper protein is the only electron carrier. The cyanobacterium Anabaena, like some other organisms, is able to synthesize either Cyt c₆ or plastocyanin (which both exhibit a basic isoelectric point of ~9) as a function of copper concentration in the growing medium (3).

The structures and functions of these two metalloproteins have been extensively studied in a wide range of organisms (4-11). According to our laser flash-induced kinetic studies, the reaction mechanism of PSI reduction follows three different models: type I, which involves an oriented collision between the two redox partners; type II, which proceeds through the formation of a transient complex prior to electron transfer; and type III, which requires an additional rearrangement step so as to make the redox centers orientate properly within the complex (9). During the evolution of photosynthetic organisms, interaction between a positively charged Cyt c₆ and PSI was first optimized (as is the case with Anabaena Cyt c₆, which follows the type III mechanism) and only later in evolution was a more complex kinetic mechanism developed with plastocyanin (2).

The three-dimensional structures of Cyt c₆ from the two eukaryotic green algae Monoraphidium (12) and Chlamydomonas (13) and from the cyanobacterium Synechococcus (14) have been solved. The analysis of the Cyt c₆ molecule compared with the plastocyanin structure allowed us to identify a hydrophobic region around the solvent-exposed heme propionates that resembles the north pole of plastocyanin as well as a negatively charged patch in eukaryotic Cyt c₆ similar to the east face of eukaryotic plastocyanin (12). In Anabaena, neither Cyt c₆ nor plastocyanin exhibits the acidic patches at their east face, which is rather positively charged (2).

Extensive mutagenesis studies of plastocyanin have supplied relevant information on the role of specific residues located both in its north and east faces (5, 8, 15-17). Recently, we performed a site-directed mutagenesis analysis of Synechocystis Cyt c₆ (18). Aspartates 70 and 72 appear to be located in a negatively charged region of Cyt c₆ that may be isofunctional with the well known "south-east" acidic patch of plastocyanin. In addition, Phe-64 (which is close to the heme group and could be the counterpart of Tyr-83 in plastocyanin (19)) does not appear to be involved in the electron transfer to PSI. In contrast, Arg-67, which is located at the edge of the Cyt c₆ acidic area, seems to be crucial.

This paper reports the kinetic and thermodynamic characterization of PSI reduction by a set of site-directed mutants of Anabaena Cyt c₆. The results demonstrate that a single mutation of specific residues in the hydrophobic or positively charged area of Cyt c₆ can promote drastic changes in the reaction mechanism. The two functional areas of Cyt c₆ involved in PSI photoreduction have been identified.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of Native Cytochrome c₆-- The hemeprotein from Anabaena sp. PCC 7119 was purified as described previously (11), but with slight modifications. Cyt c₆ samples were applied to a CM-cellulose column after oxidation with potassium ferricyanide. Elution of the adsorbed proteins was performed with a 50 µM potassium ferricyanide solution in a linear gradient of 2-30 mM potassium phosphate buffer, pH 7.0. The fractions containing pure Cyt c₆ were pooled, concentrated on an Amicon pressure-dialysis cell fitted with a YM-10 membrane, and stored at 80 °C. Cyt c₆ concentration was determined spectrophotometrically using an absorption coefficient of 26.2 mM¹ cm¹ at 553 nm for the reduced protein (20).

Construction of Mutants-- The mutant petJ genes were constructed by polymerase chain reaction with the QuickChange kit (Stratagene) using oligonucleotides of 32 base pairs, 15 ng of DNA templates, and 12 min of extension time. The previously described expression construction for the petJ gene from Anabaena (21) was used as a template. The DNA sequencing service MediGenomix carried out the nucleotide sequence analysis. Other molecular biology protocols were standard (22).

Production of Recombinant Proteins and Purification Procedures-- Escherichia coli MC1061-transformed cells were grown in M9 medium (22) supplemented with 1 g/liter Tryptone, 6 mg/liter Fe(III) ammonium citrate, and 100 µg/ml ampicillin. Cells from 10-liter microaerobic cultures were collected, and the periplasmic fraction was extracted according to the method of Hoshino and Kageyama (23) as modified by Eftekhar and Schiller (24). The resulting suspension was extensively dialyzed against 2 mM potassium phosphate, pH 7.0. From this point, the purification procedure was that for native Cyt c₆ (see above), with the exception of mutants K66E and D72K, which were eluted with buffer gradients ranging from 1 to 10 mM and from 2 to 120 mM, respectively. In all cases, 50 µM potassium ferricyanide was added to the gradient solutions to keep Cyt c₆ oxidized. Protein concentration was determined as described previously (21).

Redox Titrations-- The redox potential value for the heme group in each Cyt c₆ mutant was determined as reported previously (21, 25), for which the differential absorbance changes at 553 minus 570 nm were followed. Errors in the experimental determinations were less than ±5 mV.

Preparation of PSI Particles-- PSI particles were isolated from Anabaena cells by -dodecyl maltoside solubilization (26, 27). The chlorophyll/P₇₀₀ ratio of the resulting PSI preparations was 140:1. The P₇₀₀ content in PSI samples was calculated from the photoinduced absorbance changes at 820 nm using the absorption coefficient of 6.5 mM¹ cm¹ determined by Mathis and Sétif (28). Chlorophyll concentration was determined according to Arnon (29).

Laser Flash Absorption Spectroscopy-- The kinetics of flash-induced absorbance changes in PSI were followed at 820 nm as described previously (9, 18). Experimental conditions and the standard reaction mixture were also as reported previously (17); the buffer used throughout this work was 20 mM Tricine/KOH, pH 7.5. Unless otherwise indicated, low and high ionic strengths refer to the absence and addition of 10 mM MgCl₂, respectively. Data collection and kinetic and thermodynamic analyses were carried out as reported by Hervás et al. (9, 10). Apparent thermodynamic parameters were estimated as described Díaz et al. (6) by fitting the experimental data to the Watkins equation (30). The values for the rate constant for electron transfer and K_A (see below) were determined according to the formalism by Meyer et al. (31).

Structure Simulation-- The structures of WT and mutant Cyt c₆ were modeled using the SYBYL program (Tripos Associates) in an SGI RC10000 workstation. The three-dimensional crystal structure of Cyt c₆ from the green alga Monoraphidium braunii (12) was used as a template. Sequence alignment and subsequent amino acid substitution were performed with the BIOPOLYMER module of SYBYL Version 6.4. Force field parameters for the heme moiety were those in the AMBER package (32). The resulting file was first submitted to energy minimization in vacuo up to a root mean square energy gradient of 0.41 kJ mol¹ Ź using the SANDER module of AMBER Version 4.1 (33). During these calculations, the backbone heavy atoms of -helix regions were restrained at their position by a harmonic force of 62.7 kJ mol¹ Ź. Then, the whole system was solvated with three-point water molecules using the BLOB option of the EDIT module. Solvent was energy-minimized and submitted to a 9-ps molecular dynamics calculation. The whole system was again energy-minimized and submitted to a 1250-ps molecular dynamics run at 300 K. A total of 10 samples from the last 400 ps of trajectory were quenched by freezing the system in six steps of 1.5 ps. The qualities of the resulting structures were tested using the PROCHECK program (34). Surface electrostatic potentials were estimated using the algorithm of Nicholls and Honig (35), as indicated in the MOLMOL program (36).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To analyze the role of specific residues of Anabaena Cyt c₆ in the reaction mechanism of PSI reduction, six amino acids (two near the heme group and four in the positively charged east face) were chosen for mutations (Fig. 1). At the edge of the heme crevice, modifications were made at residues 25 and 29: Val-25, which is located near the heme -meso-position, was substituted by alanine and glutamate; and Lys-29, which exhibits its side chain lying close to propionate 7, was replaced with histidine. Val-25 is located in the middle of the hydrophobic patch, but Lys-29 is not. However, mutation of the latter to histidine was considered to be interesting because the residue at position 29 is lysine in all Cyts c₆ with the exception of that from Monoraphidium, in which it is histidine (actually, the EPR spectra of Monoraphidium Cyt c₆ suggested an unusual histidine-histidine axial coordination for the heme iron, a ligand system that is not possible in the rest of Cyts c₆ with just one histidine residue (37)). In addition, basic residues at positions 28-30 in type I cytochromes have been proposed to control the redox potential of the heme group through stabilization of propionate 7 (38). On the east face, modifications included the replacement of Lys-62 and Lys-66 by glutamates; Asp-72, which is also located in the middle of the east patch, was changed to lysine. Finally, Arg-64, which is at the edge of the east patch and has been shown to be crucial in Synechocystis Cyt c₆ (18), was replaced with glutamate.

View larger version (71K): [in this window] [in a new window]   Fig. 1.   Space-filling model of cytochrome c6 showing the residues modified by mutagenesis. The molecule is oriented to show the six residues mutated; the "east" positively charged patch (equivalent to the acid face of eukaryotic cytochrome c6 and plastocyanin) is in front, whereas the "north" hydrophobic patch is on the left. The heme group is depicted in green.

The EPR and electronic absorption spectra of Anabaena Cyt c₆ were not changed by the mutations (data not shown), but its midpoint redox potential (E_m) could be significantly affected (Table I). The E_m value was unchanged when the residue mutated was at the east face, including Arg-64, but it was ~50 mV lower when the mutations were located near the heme group. Only minor differences were observed in the ¹H NMR spectra and nuclear Overhauser effect intensities of heme resonances with protons from other residues.²

The kinetics of PSI reduction by WT Cyt c₆ are biphasic, but those with the mutants (with the exception of D72K) are monoexponential, lacking the fast phase typically observed with the WT species. As shown in Fig. 2, the kinetics corresponding to D72K and WT Cyt c₆ exhibited a sharp initial fast phase (with a rate constant that was independent of donor protein concentration) followed by a slower decay. In contrast, the oscilloscope traces with mutants V25A and V25E fit to single exponential curves.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Kinetic traces showing PSI reduction by WT and mutant cytochrome c6. The hemeprotein concentration was 150 µM, and the pH value was 7.5. The oscilloscope traces obtained with the WT molecule and D72K fit to biphasic kinetics, whereas those corresponding to mutants V25A and V25E fit to single exponential curves.

Fig. 3 shows that the observed pseudo first-order rate constant (k_obs) of PSI reduction by any mutant (with the exception of D72K) varied linearly with Cyt c₆ concentration. This can be interpreted by assuming that there is no formation of any stable complex between PSI and Cyt c₆, and the reaction thus follows a collisional kinetic mechanism (type I). With mutant D72K, however, the protein concentration dependence of k_obs exhibited a saturation profile, which indicates the formation of a bimolecular Cyt c₆·PSI complex prior to electron transfer. The extrapolated rate constant at infinite D72K concentration is similar to its electron transfer rate constant, which is obtained directly from the fast kinetic phase, thus suggesting a type II mechanism.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Dependence upon cytochrome c6 concentration of kobs for PSI reduction by the WT species and a set of mutants of the hemeprotein. The solid line for mutant D72K corresponds to the theoretical fitting described by Meyer et al. (31). Experimental conditions were as described in the legend to Fig. 2.

Such a saturation profile with D72K was even more evident at lower ionic strengths, which increase attractive electrostatic interactions. As shown in Fig. 4, the D72K mutant showed efficient complex formation, with an equilibrium constant (K_A) of 1.06 × 10⁵ M¹ at low ionic strength. Extrapolation of the observed rate constant to infinite D72K concentration yielded a value that approached one-half the experimental electron transfer rate constant, which indicates that this mutant may follow a type III mechanism at low ionic strength. This finding also suggests that the rearrangement step (see above) is not limiting at high ionic strength.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Dependence upon hemeprotein concentration of kobs for PSI reduction by WT cytochrome c6 and its mutants V25A and D72K at low ionic strength. The solid lines for the mutants correspond to theoretical fittings according to Meyer et al. (31). The experimental conditions were as described in the legend to Fig. 2, except that magnesium chloride was omitted from the reaction mixture.

Fig. 4 also shows that the V25A mutant was likewise able to form a transient complex with PSI at low ionic strength, even though its kinetic profiles of PSI reduction were monophasic at any ionic strength. The K_A value for complex formation between V25A and PSI is 1.07 × 10⁴ M¹, which is 10 times lower than that with D72K. These data indicate that V25A follows a type II mechanism in the absence of MgCl₂ and a type I mechanism when 10 mM MgCl₂ is added.

The bimolecular rate constant for the overall reaction (k_bim) of PSI reduction, which can be calculated from the linear plots in Fig. 3, is smaller with any mutant than with WT Cyt c₆ (Table I). In the hydrophobic patch, replacement of Val-25 with alanine or glutamate promoted a decrease in k_bim of ~2 and 60 times, respectively, whereas mutation of Lys-29 to histidine involved a parallel decrease of ~50% as compared with WT Cyt c₆. Substitution of Arg-64, in its turn, by glutamate induced the rate constant to decrease by a factor of 5. In the east face, replacement of Lys-62 and Lys-66 with glutamate made the k_bim value decrease to half that of WT Cyt c₆. In the case of D72K, it should be noted that the bimolecular rate constant in Table I refers to its association constant, which is kinetically different from the other k_bim values in the table. Its electron transfer rate constant, calculated directly from the fast phase, compares well with that of WT Cyt c₆, as does the maximum percentage of fast phase (Table I).

Taking into account the electrostatic nature of the interaction of Cyt c₆ with PSI, a detailed analysis of the effect of ionic strength on k_bim was performed. Fig. 5 shows that the k_bim values with WT Cyt c₆ monotonically diminished with increasing NaCl concentration, thereby indicating the existence of attractive electrostatic interactions between the reaction partners as described previously (10, 11). A similar effect of ionic strength on k_bim was observed with all mutants, but some differences could be found among them. Actually, the k_bim values with mutants V25A and D72K are significantly higher than that with WT Cyt c₆ at low ionic strength, but they decreased drastically upon small additions of NaCl. Mutant K29H showed an ionic strength dependence similar to that of WT Cyt c₆, although its electron transfer efficiency was lower in the whole range analyzed. The k_bim values with all the other mutants are lower than that with WT Cyt c₆, indicating that the changes in net electrostatic charge alter the attractive interactions between PSI and mutant proteins.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effect of ionic strength on kbim for PSI reduction by WT cytochrome c6 and its mutants. Experimental conditions were as described in the legend to Fig. 4, but the ionic strength was adjusted to the desired value by adding small amounts of a concentrated NaCl solution.

Using the Watkins equation (30), the bimolecular rate constant extrapolated to infinite ionic strength (k_inf) (which facilitates the analysis of the intrinsic reactivity of redox partners in the absence of electrostatic interactions) can be calculated from the experimental data. As shown in Table I, the k_inf values with Cyt c₆ mutated at the positively charged patch are very similar to that with WT Cyt c₆, a fact that can be explained by assuming that the changes in reactivity induced by these mutations are mainly due to electrostatic, and not structural, effects. The only exception is R64E, for which the k_inf value is 4-5 times lower than that with WT Cyt c₆, a finding suggesting that the change in electrostatic charge is not the only factor affecting its reactivity toward PSI, as reported previously for Synechocystis Cyt c₆ (18). The effect of the K29H mutation seems to be merely electrostatic in nature, as k_inf is similar to that with WT Cyt c₆. The two mutations at position 25 involve modifications that make k_inf lower (the V25E mutant, in particular, exhibits a k_inf value that is 70 times lower than that with WT Cyt c₆).

To gain further insights into the nature of the interactions between PSI and Cyt c₆, a thermodynamic analysis of PSI reduction by the Cyt c₆ mutants was performed. In all cases, the temperature dependence of the observed rate constant (k_obs) yielded linear Eyring plots with no breakpoints, from which the values for the apparent activation enthalpy (H), entropy (S) and free energy (G) of the overall reaction could be calculated. For comparative purposes, Table II shows the differences in such activation parameters between WT Cyt c₆ and every mutant. The greatest difference was observed with V25E, whose free energy change is 9.41 kJ mol¹ higher than that of WT Cyt c₆, as expected from its inefficient interaction with PSI. This difference is due mainly to a decrease in the entropic term by ~28 J mol¹ K¹. Also interesting is mutant R64E, whose free energy change is 4.15 kJ mol¹ higher than that of WT Cyt c₆, a fact that is due to changes in both the enthalpic and entropic terms. Mutant V25A behaved differently than any other mutant; in fact, the free energy term of its reaction is similar to that of WT Cyt c₆ despite the fact that both entropy and enthalpy show dramatic changes as compared with the thermodynamic parameters of WT Cyt c₆.

To check whether the differences in G between WT and mutant Cyt c₆ (G) were due to electrostatic interactions, the experimental data were fitted to the Watkins equation (30). As shown in Fig. 6, the G values with D72K and V25A perfectly fit the Watkins equation, and those of K29H, K62E, and K66E roughly fit it. Mutants V25E and R64E showed a linear NaCl dependence of G at high ionic strength that caused the data to deviate from the Watkins equation.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Effect of ionic strength on G for PSI reduction by mutants of cytochrome c6 as compared with the WT molecule. Experimental conditions were as described in the legend to Fig. 5.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Up to now, the only site-directed mutational analysis of any Cyt c₆ was recently reported by De la Cerda et al. (18) in the cyanobacterium Synechocystis. This hemeprotein, which is almost neutral, reacts with PSI according to a simple collisional model (type I). On the contrary, Anabaena Cyt c₆ is a positively charged protein that exhibits strong electrostatic attractions toward PSI (9, 11) and that reacts with the photosystem following a more complex three-step mechanism (type III). The goal of this study was to elucidate the role played by some specific amino acids of Anabaena Cyt c₆ in such an attractive interaction with PSI and to investigate the possible involvement of its hydrophobic north area in the type III reaction mechanism.

Mutations of Val-25 indicate that this residue may contribute to the specific topology of the north hydrophobic area of Cyt c₆ in its interaction with PSI. Similar conclusions were inferred from mutants at the north pole of eukaryotic plastocyanin (5, 8), in which the fast phase of electron transfer to PSI cannot be detected. This suggests that the mutant proteins (both Cyt c₆ and plastocyanin) are unable to reach the optimal orientation required for their redox centers to transfer electrons to PSI. It is interesting to compare the drastic effect induced by mutation of Val-25 to glutamate with mutation to alanine; the severe kinetic phenotype of V25E can be attributed to the presence of a negative charge in the middle of a normally hydrophobic region.

The K29H mutant possesses a redox potential value that is 65 mV more negative than that of WT Cyt c₆. This is probably due to the proximity of Lys-29 to heme propionate 7, in agreement with previous reports (38). Even though the driving force of electron transfer from K29H to PSI is higher, the kinetic profile of PSI reduction by the mutant loses the first fast phase, and its k_bim value is about half that of WT Cyt c₆. This can be ascribed in part to electrostatic effects, as the dependence of G on ionic strength fits the Watkins equation (30), and the value for k_inf compares well with that of WT Cyt c₆. The change in redox potential induced by the mutation clearly does not affect reactivity with PSI, suggesting that complex formation is the rate-limiting step of the overall reaction.

Mutations of Lys-62 and Lys-66 at the positively charged patch of Cyt c₆ demonstrate that this region is responsible for the attractive electrostatic interactions with PSI. In Anabaena, the PsaF subunit does not seem to be directly involved in the interaction of PSI with its donor proteins, as is the case in Synechocystis (16, 39). Hence, the positive charges of Anabaena Cyt c₆ may interact with certain negatively charged areas in the PsaA/PsaB heterodimer of PSI to form a transient electrostatic complex. The surface electrostatic potential of WT and mutant Cyt c₆ was then calculated. Fig. 7 shows that the WT molecule has an extensive area of positive potential at its west face, i.e. close to the hydrophobic patch at the edge of the heme cleft. Mutants K66E, K62E, and R64E present a reduction in charge of the positive patch, although the change in the orientation of the dipole moment is <10°. This is consistent with the kinetic behavior exhibited by these three mutants, in which the ionic strength dependence of the interaction with PSI is not so much evident.

View larger version (85K): [in this window] [in a new window]   Fig. 7.   Distribution of the surface electrostatic potential of WT cytochrome c6 and its mutants. Electrostatic potential was calculated as indicated under "Experimental Procedures." Simulations were performed assuming an ionic strength of 40 mM at pH 7.0. Negative and positive potential regions are depicted in red and blue, respectively.

It is noteworthy that the effect of such mutations on the reaction rates can be closely correlated to changes in the positive electrostatic potential at the edge of the hydrophobic area. In fact, replacement of Arg-64 (which is located in the basic patch, adjacent to the hydrophobic region) by an acidic residue promotes a drastic reduction in size of the positive patch. Mutation of Lys-66 to glutamate likewise induces a diminution of the basic patch, but it does not alter the electrostatic surface potential at the edge of the hydrophobic area.

Mutant D72K is of special relevance as it clearly supports our proposal that the positively charged patch of Cyt c₆ is responsible for the interaction with PSI. Replacement of Asp-72 by a positive residue like lysine makes the positive potential region spread out (Fig. 7), thus favoring the attractive interactions with any negatively charged area of PSI. Like WT Cyt c₆, D72K is the only mutant that exhibits the fast kinetic phase of PSI reduction.

The thermodynamic analysis of PSI reduction by the mutants of Cyt c₆ revealed that all mutations induce changes in H and S, but only some of them (V25A, V25E, and R64E) are significant. The large entropic changes of such mutants suggest that the solvent molecules are tuning the reactivity of the donor proteins toward PSI. The kinetic behavior of V25A indicates that the electrostatic interactions are predominant over hydrophobic forces.

To conclude, the experimental data reported here reveal that in Anabaena Cyt c₆, there are at least two interaction sites (which would be isofunctional with two similar areas in plastocyanin) for electron transfer to PSI: (i) a positively charged area, responsible for the electrostatic interactions forming the transient complex with the photosystem; and (ii) a hydrophobic region surrounding the heme pocket, responsible for the formation of the contact interphase with PSI allowing electrons to go from the heme iron to P₇₀₀⁺. The main difference with respect to Synechocystis Cyt c₆ (18) is found at the electrostatically charged patch, which explains the differences in their respective reaction mechanisms of PSI photoreduction (10).

	FOOTNOTES

^* This work was supported by Dirección General de Investigación Científica y Técnica Grant PB96-1381, European Union Grant ERB-FMRX-CT98-0218, and Junta de Andalucía Grant CVI-0198.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.

To whom correspondence should be addressed. Tel.: 34-954-489506; Fax: 34-954-460065; E-mail: marosa@cica.es.

² A. Díaz-Quintana, M. Hervás, J. A. Navarro, and M. A. De la Rosa, unpublished data.

	ABBREVIATIONS

The abbreviations used are: Cyt, cytochrome; PSI, photosystem I; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild-type; k_bim, bimolecular rate constant for the overall reaction; k_inf, diffusion-limited rate constant.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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