The Unique Proline of the Prochlorothrix hollandica Plastocyanin Hydrophobic Patch Impairs Electron Transfer to Photosystem I
2001; Elsevier BV; Volume: 276; Issue: 40 Linguagem: Inglês
10.1074/jbc.m105367200
ISSN1083-351X
AutoresJosé A. Navarro, Eugene Myshkin, Miguel Á. De la Rosa, George S. Bullerjahn, Manuel Hervás,
Tópico(s)Plant and animal studies
ResumoA number of surface residues of plastocyanin fromProchlorothrix 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 versusplastocyanin 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 of plastocyanin mutants toward photosystem I from different organisms (plants and cyanobacteria) reveals that reversion of the unique proline ofProchlorothrix plastocyanin to the conserved leucine of all other plastocyanins at this position enhances the reactivity of theProchlorothrix protein. A number of surface residues of plastocyanin fromProchlorothrix 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 versusplastocyanin 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 of plastocyanin mutants toward photosystem I from different organisms (plants and cyanobacteria) reveals that reversion of the unique proline ofProchlorothrix plastocyanin to the conserved leucine of all other plastocyanins at this position enhances the reactivity of theProchlorothrix protein. plastocyanin equilibrium constant for complex formation first-order rate constant for electron transfer observed pseudo first-order rate constant molecular dynamics photosystem I wild-type 4-morpholineethanesulfonic acid N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Plastocyanin (Pc)1 is a small redox protein (molecular mass, ∼10.5 kDa) that functions in photosynthesis as a mobile electron carrier between the two membrane-embedded complexes cytochromeb6f and photosystem I (PSI) (1Chitnis P.R. Xu Q. Chitnis V.P. Nechushtai R. Photosynth. Res. 1995; 44: 23-40Crossref PubMed Scopus (101) Google Scholar, 2Navarro J.A. Hervás M. De la Rosa M.A. J. Biol. Inorg. Chem. 1997; 2: 11-22Crossref Scopus (61) Google Scholar, 3Sigfridsson K. Photosynth. Res. 1998; 57: 1-28Crossref Scopus (76) Google Scholar). Whereas higher plants produce just Pc, there is a number of intermediate species, both cyanobacteria and eukaryotic algae, that are able to synthesize cytochrome c6 as an alternative redox carrier under copper deficiency (4Ho K.K. Krogmann D.W. Biochim. Biophys. Acta. 1984; 766: 310-316Crossref Scopus (112) Google Scholar). 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 (5Hope A.B. Biochim. Biophys. Acta. 2000; 1456: 5-26Crossref PubMed Scopus (193) Google Scholar, 6Molina-Heredia F.P. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 2001; 276: 601-605Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). All these comparative kinetic analyses have allowed us to propose different reaction mechanisms for PSI reduction (7Hervás M. Navarro J.A. Dı́az A. Bottin H. De la Rosa M.A. Biochemistry. 1995; 34: 11321-11326Crossref PubMed Scopus (128) Google Scholar, 8Hervás M. Navarro J.A. Dı́az A. De la Rosa M.A. Biochemistry. 1996; 35: 2693-2698Crossref PubMed Scopus (46) Google Scholar).Recently, a comparative analysis of the interaction of Pc and cytochrome c6 with PSI from the prochlorophyteProchlorothrix hollandica has been carried out (9Navarro J.A. Hervás M. Babu C.R. Molina-Heredia F.P. Bullerjahn G.S. De la Rosa M.A. Garab G. Photosynthesis: Mechanisms and Effects. III. Kluwer Academic Publishers Group, Dordrecht, The Netherlands1998: 1605-1608Google Scholar). Prochlorophytes represent a deeply branched group of cyanobacteria containing both chlorophyll a and b (10Matthijs H.C.P. van der Staay G.W.M. Mur L.R. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer Academic Publishers Group, Dordrecht, The Netherlands1994: 49-64Google Scholar, 11Nelissen B. Van der Peer Y. Wilmotte A. De Wachter R. Mol. Biol. Evol. 1995; 12: 1166-1173PubMed Google Scholar). 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 c6, in its turn, follows a three-step reaction mechanism with rearrangement of redox partners within an intermediate electrostatic complex before electron transfer (9Navarro J.A. Hervás M. Babu C.R. Molina-Heredia F.P. Bullerjahn G.S. De la Rosa M.A. Garab G. Photosynthesis: Mechanisms and Effects. III. Kluwer Academic Publishers Group, Dordrecht, The Netherlands1998: 1605-1608Google Scholar). 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 (12Babu C.R. Volkman B.F. Bullerjahn G.S. Biochemistry. 1999; 38: 4988-4995Crossref PubMed Scopus (33) Google Scholar). Despite the relatively low number of conserved residues shared with other Pcs, theProchlorothrix molecule has a similar overall folding pattern, including the classical two-sheet β-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 (12Babu C.R. Volkman B.F. Bullerjahn G.S. Biochemistry. 1999; 38: 4988-4995Crossref PubMed Scopus (33) Google Scholar).In this paper, we are extending our previous studies ofProchlorothrix 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.RESULTS AND DISCUSSIONThe two variant residues at the hydrophobic patch ofProchlorothrix 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 conserved 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 PlastocyaninsMost 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 ∼15 mV lower (TableI). 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 (13Babu C.R. Arudchandran A. Hille R. Gross E.L. Bullerjahn G.S. Biochem. Biophys. Res. Commun. 1997; 235: 631-635Crossref PubMed Scopus (5) Google Scholar).Table IMidpoint redox potentials (Em) of wild-type and mutant plastocyanins as well as association rate constants (KA) and electron transfer rate constants (ket) for photosystem I reduction by the different copper proteinsProteinEmKAKA (+0.1 mNaCl)ketket (+ 0.1m NaCl)mVm−1m−1s−1s−1WT3701.4 × 1041.6 × 10413901450Y12G3672.7 × 1042.5 × 10410001150Y12F3681.5 × 1042.0 × 10415001200Y12WND1-aND, not determined.2.8 × 1042.0 × 10411601300P14L3550.9 × 1040.6 × 10439004050Y12G/P14L3560.6 × 1040.8 × 104260024001-a ND, not determined. Open table in a new tab Electron Transfer Kinetics to Prochlorothrix Photosystem IThe 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 (kobs) upon donor protein concentration shows a saturation profile (Fig.2). This finding suggests the formation of a bimolecular transient Pc·PSI complex before electron transfer, as previously described for the WT system (9Navarro J.A. Hervás M. Babu C.R. Molina-Heredia F.P. Bullerjahn G.S. De la Rosa M.A. Garab G. Photosynthesis: Mechanisms and Effects. III. Kluwer Academic Publishers Group, Dordrecht, The Netherlands1998: 1605-1608Google Scholar) according to the following minimal two-step reaction mechanism.Figure 2Dependence of the observed rate constant (kobs) for Prochlorothrixphotosystem I reduction by wild-type and mutated plastocyanins upon donor protein concentration. The continuous linecorresponds to the theoretical fitting to the two-step formalism described in Meyer et al. (22Meyer T.E. Zhao Z.G. Cusanovich M.A. Tollin G. Biochemistry. 1993; 32: 4552-4559Crossref PubMed Scopus (115) Google Scholar). Other conditions were as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)Pcred+PSIox⇄KA[Pcred…PSIox]→ketPcox+PSIredEquation 1 in which KA stands for the equilibrium constant of complex formation, and ket, which can be experimentally inferred from the limiting kobsat infinite Pc concentration, denotes the subsequent intracomplex electron transfer first-order rate constant. Table I shows the values for KA and ket with WT and mutant Pcs at pH 7.5 as calculated from experimental data in Fig. 2by using the formalism described by Meyer et al. (22Meyer T.E. Zhao Z.G. Cusanovich M.A. Tollin G. Biochemistry. 1993; 32: 4552-4559Crossref PubMed Scopus (115) Google Scholar). As can be seen in Table I, there are no significant changes inKA 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 (3Sigfridsson K. Photosynth. Res. 1998; 57: 1-28Crossref Scopus (76) Google Scholar, 6Molina-Heredia F.P. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 2001; 276: 601-605Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). 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 (9Navarro J.A. Hervás M. Babu C.R. Molina-Heredia F.P. Bullerjahn G.S. De la Rosa M.A. Garab G. Photosynthesis: Mechanisms and Effects. III. Kluwer Academic Publishers Group, Dordrecht, The Netherlands1998: 1605-1608Google Scholar) 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 KA orket upon increasing salt concentration, as previously described for the WT Pc (9Navarro J.A. Hervás M. Babu C.R. Molina-Heredia F.P. Bullerjahn G.S. De la Rosa M.A. Garab G. Photosynthesis: Mechanisms and Effects. III. Kluwer Academic Publishers Group, Dordrecht, The Netherlands1998: 1605-1608Google Scholar).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% inket. These results suggest that inProchlorothrix 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 theket 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 ∼1-Å closer to the acceptor (23Moser C.C. Keske J.M. Warncke K. Farid R.S. Dutton P.L. Nature. 1992; 355: 796-802Crossref PubMed Scopus (1613) Google Scholar).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 alterations 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 increasedket. 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 ket 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).Figure 3Comparison of the structures of the wild-type and P14L mutant hydrophobic patches. Structures were modeled from averaging the MD simulation described under “Experimental Procedures.” The positions of Pro-14, Leu-14, and His-85 are indicated by arrows.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4MD analysis of the conserved Cys-82 (top) and Met-90 (bottom) copper ligand. Distances shown are the root mean square deviation over a 105-fs simulation covering 100 fs per frame.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IISummary of the structural statistics for selected copper center and hydrophobic patch residues of the wild type and P14L mutant plastocyaninsWTP14LAverage r.m.s.d.DeviationAverage r.m.s.d.Deviation(Å)(Å)Tyr-120.4770.1770.6900.140Met-330.8270.0600.4790.083Val-360.2470.0810.3620.082Pro-380.2010.1160.150.058His-390.2950.0560.2610.045Cys-820.2730.0710.4850.071His-850.2060.0500.2110.044Ala-880.2930.1320.4190.036Met-900.3470.0240.1950.039Val-36 and Met-33 are unique residues in Prochlorothrix Pc that border the hydrophobic patch. r.m.s.d., root mean square deviation. Open table in a new tab Electron Transfer Kinetics to Heterospecific Photosystem IIt 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 Synechocystis. The copper protein does exhibit a very low reactivity in all cross-reactions (9Navarro J.A. Hervás M. Babu C.R. Molina-Heredia F.P. Bullerjahn G.S. De la Rosa M.A. Garab G. Photosynthesis: Mechanisms and Effects. III. Kluwer Academic Publishers Group, Dordrecht, The Netherlands1998: 1605-1608Google Scholar). Because the mutants constructed in this study are aimed to revert the “exclusive” hydrophobic patch ofProchlorothrix 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 (k2) 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 (24Hippler M. Reichert J. Sutter M. Zak E. Altschmied L. Schröer U. Herrmann R.G. Haehnel W. EMBO J. 1996; 15: 6374-6384Crossref PubMed Scopus (126) Google Scholar). 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 (25Sigfridsson K. Young S. Hansson Ö. Biochemistry. 1996; 35: 1249-1257Crossref PubMed Scopus (76) Google Scholar), who demonstrated that replacement of leucine in higher plant Pc with the less bulky alanine decreased ket 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 (25Sigfridsson K. Young S. Hansson Ö. Biochemistry. 1996; 35: 1249-1257Crossref PubMed Scopus (76) Google Scholar).Figure 5Dependence of the observed rate constant (kobs) for Synechocystisphotosystem I reduction by Prochlorothrixwild-type and mutated plastocyanins as well as bySynechocystis plastocyanin upon donor protein concentration. Other conditions were as in Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIIBimolecular rate constants (k2) for the reduction of photosystem I from different organisms by Prochlorothrix wild-type and mutant plastocyaninsPSI/proteink2m−1 s−1Spinach PSIWT2.0 × 105P14L1.8 × 106Y12G/P14L6.5 × 105Y12W3.6 × 105Y12G2.7 × 105Y12F2.2 × 105Spinach Pc3-ak2 could not be calculated because spinach plastocyanin follows a three-step reaction mechanism (7).—Synechocystis 6803 PSIWT1.2 × 106P14L1.3 × 107Y12G/P14L3.0 × 106Y12W2.8 × 106Y12G2.2 × 106Y12F1.7 × 106Synechocystis Pc9.0 × 106Anabaena 7119 PSIWT2.8 × 106P14L1.5 × 107Y12G/P14L3.3 × 106Y12W5.7 × 106Y12G1.8 × 106Y12F3.6 × 106Anabaena Pc7.0 × 1073-a k2 could not be calculated because spinach plastocyanin follows a three-step reaction mechanism (7Hervás M. Navarro J.A. Dı́az A. Bottin H. De la Rosa M.A. Biochemistry. 1995; 34: 11321-11326Crossref PubMed Scopus (128) Google Scholar). Open table in a new tab Concluding RemarksThe fact that WT Pc fromProchlorothrix 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 makesProchlorothrix Pc much more reactive toward PSI from any organism, including Prochlorothrix.Last, the influence of distance versus driving force for the 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, ket would be expected to increase slightly (∼30%) (26Moser C.C. Dutton P.L. Biochim. Biophys. Acta. 1992; 1101: 171-176Crossref PubMed Scopus (9) Google Scholar) via this parameter alone. However, the large (10-fold) increases in k2 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. Plastocyanin (Pc)1 is a small redox protein (molecular mass, ∼10.5 kDa) that functions in photosynthesis as a mobile electron carrier between the two membrane-embedded complexes cytochromeb6f and photosystem I (PSI) (1Chitnis P.R. Xu Q. Chitnis V.P. Nechushtai R. Photosynth. Res. 1995; 44: 23-40Crossref PubMed Scopus (101) Google Scholar, 2Navarro J.A. Hervás M. De la Rosa M.A. J. Biol. Inorg. Chem. 1997; 2: 11-22Crossref Scopus (61) Google Scholar, 3Sigfridsson K. Photosynth. Res. 1998; 57: 1-28Crossref Scopus (76) Google Scholar). Whereas higher plants produce just Pc, there is a number of intermediate species, both cyanobacteria and eukaryotic algae, that are able to synthesize cytochrome c6 as an alternative redox carrier under copper deficiency (4Ho K.K. Krogmann D.W. Biochim. Biophys. Acta. 1984; 766: 310-316Crossref Scopus (112) Google Scholar). 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 (5Hope A.B. Biochim. Biophys. Acta. 2000; 1456: 5-26Crossref PubMed Scopus (193) Google Scholar, 6Molina-Heredia F.P. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 2001; 276: 601-605Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). All these comparative kinetic analyses have allowed us to propose different reaction mechanisms for PSI reduction (7Hervás M. Navarro J.A. Dı́az A. Bottin H. De la Rosa M.A. Biochemistry. 1995; 34: 11321-11326Crossref PubMed Scopus (128) Google Scholar, 8Hervás M. Navarro J.A. Dı́az A. De la Rosa M.A. Biochemistry. 1996; 35: 2693-2698Crossref PubMed Scopus (46) Google Scholar). Recently, a comparative analysis of the interaction of Pc and cytochrome c6 with PSI from the prochlorophyteProchlorothrix hollandica has been carried out (9Navarro J.A. Hervás M. Babu C.R. Molina-Heredia F.P. Bullerjahn G.S. De la Rosa M.A. Garab G. Photosynthesis: Mechanisms and Effects. III. Kluwer Academic Publishers Group, Dordrecht, The Netherlands1998: 1605-1608Google Scholar). Prochlorophytes represent a deeply branched group of cyanobacteria containing both chlorophyll a and b (10Matthijs H.C.P. van der Staay G.W.M. Mur L.R. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer Academic Publishers Group, Dordrecht, The Netherlands1994: 49-64Google Scholar, 11Nelissen B. Van der Peer Y. Wilmotte A. De Wachter R. Mol. Biol. Evol. 1995; 12: 1166-1173PubMed Google Scholar). 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 c6, in its turn, follows a three-step reaction mechanism with rearrangement of redox partners within an intermediate electrostatic complex before electron transfer (9Navarro J.A. Hervás M. Babu C.R. Molina-Heredia F.P. Bullerjahn G.S. De la Rosa M.A. Garab G. Photosynthesis: Mechanisms and Effects. III. Kluwer Academic Publishers Group, Dordrecht, The Netherlands1998: 1605-1608Google Scholar). 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 (12Babu C.R. Volkman B.F. Bullerjahn G.S. Biochemistry. 1999; 38: 4988-4995Crossref PubMed Scopus (33) Google Scholar). Despite the relatively low number of conserved residues shared with other Pcs, theProchlorothrix molecule has a similar overall folding pattern, including the classical two-sheet β-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 (12Babu C.R. Volkman B.F. Bullerjahn G.S. Biochemistry. 1999; 38: 4988-4995Crossref PubMed Scopus (33) Google Scholar). In this paper, we are extending our previous studies ofProchlorothrix 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. RESULTS AND DISCUSSIONThe two variant residues at the hydrophobic patch ofProchlorothrix 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 conserved 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 PlastocyaninsMost 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 ∼15 mV lower (TableI). 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 (13Babu C.R. Arudchandran A. Hille R. Gross E.L. Bullerjahn G.S. Biochem. Biophys. Res. Commun. 1997; 235: 631-635Crossref PubMed Scopus (5) Google Scholar).Table IMidpoint redox potentials (Em) of wild-type and mutant plastocyanins as well as association rate constants (KA) and electron transfer rate constants (ket) for photosystem I reduction by the different copper proteinsProteinEmKAKA (+0.1 mNaCl)ketket (+ 0.1m NaCl)mVm−1m−1s−1s−1WT3701.4 × 1041.6 × 10413901450Y12G3672.7 × 1042.5 × 10410001150Y12F3681.5 × 1042.0 × 10415001200Y12WND1-aND, not determined.2.8 × 1042.0 × 10411601300P14L3550.9 × 1040.6 × 10439004050Y12G/P14L3560.6 × 1040.8 × 104260024001-a ND, not determined. Open table in a new tab Electron Transfer Kinetics to Prochlorothrix Photosystem IThe laser
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