SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2040316218> ?p ?o ?g. }

Showing items 1 to 98 of 98 with 100 items per page.

W2040316218 endingPage "605" @default.
W2040316218 startingPage "601" @default.
W2040316218 abstract "Positively charged plastocyanin from Anabaena sp. PCC 7119 was investigated by site-directed mutagenesis. The reactivity of its mutants toward photosystem I was analyzed by laser flash spectroscopy. Replacement of arginine at position 88, which is adjacent to the copper ligand His-87, by glutamine and, in particular, by glutamate makes plastocyanin reduce its availability for transferring electrons to photosystem I. Such a residue in the copper protein thus appears to be isofunctional with Arg-64 (which is close to the heme group) in cytochrome c6 from Anabaena(Molina-Heredia, F. P., Dı́az-Quintana, A., Hervás, M., Navarro, J. A., and De la Rosa, M. A. (1999)J. Biol. Chem. 274, 33565–33570) and Synechocystis (De la Cerda, B., Dı́az-Quintana, A., Navarro, J. A., Hervás, M., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 13292–13297). Other mutations concern specific residues of plastocyanin either at its positively charged east face (D49K, H57A, H57E, K58A, K58E, Y83A, and Y83F) or at its north hydrophobic pole (L12A, K33A, and K33E). Mutations altering the surface electrostatic potential distribution allow the copper protein to modulate its kinetic efficiency: the more positively charged the interaction site, the higher the rate constant. Whereas replacement of Tyr-83 by either alanine or phenylalanine has no effect on the kinetics of photosystem I reduction, Leu-12 and Lys-33 are essential for the reactivity of plastocyanin. Positively charged plastocyanin from Anabaena sp. PCC 7119 was investigated by site-directed mutagenesis. The reactivity of its mutants toward photosystem I was analyzed by laser flash spectroscopy. Replacement of arginine at position 88, which is adjacent to the copper ligand His-87, by glutamine and, in particular, by glutamate makes plastocyanin reduce its availability for transferring electrons to photosystem I. Such a residue in the copper protein thus appears to be isofunctional with Arg-64 (which is close to the heme group) in cytochrome c6 from Anabaena(Molina-Heredia, F. P., Dı́az-Quintana, A., Hervás, M., Navarro, J. A., and De la Rosa, M. A. (1999)J. Biol. Chem. 274, 33565–33570) and Synechocystis (De la Cerda, B., Dı́az-Quintana, A., Navarro, J. A., Hervás, M., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 13292–13297). Other mutations concern specific residues of plastocyanin either at its positively charged east face (D49K, H57A, H57E, K58A, K58E, Y83A, and Y83F) or at its north hydrophobic pole (L12A, K33A, and K33E). Mutations altering the surface electrostatic potential distribution allow the copper protein to modulate its kinetic efficiency: the more positively charged the interaction site, the higher the rate constant. Whereas replacement of Tyr-83 by either alanine or phenylalanine has no effect on the kinetics of photosystem I reduction, Leu-12 and Lys-33 are essential for the reactivity of plastocyanin. plastocyanin cytochrome c6 photosystem I wild-type Plastocyanin (Pc),1 a small single copper protein, and cytochrome c6(Cyt), a monoheme protein, function as alternative mobile electron carriers between the two membrane complexes b6 f and photosystem I (PSI) (cf. Refs. 1Gross E.L. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 413-429Google Scholar, 2Navarro J.A. Hervás M. De la Rosa M.A. J. Biol. Inorg. Chem. 1997; 2: 11-22Crossref Scopus (61) Google Scholar, 3Hope A.B. Biochim. Biophys. Acta. 2000; 1456: 5-26Crossref PubMed Scopus (193) Google Scholar for reviews). The isoelectric point of Pc and Cyt varies widely depending on the organism; the two molecules exhibit the same value of ∼9 in the cyanobacterium Anabaena sp. PCC 7119 (4Molina-Heredia F.P. Hervás M. Navarro J.A. De la Rosa M.A. Biochem. Biophys. Res. Commun. 1998; 243: 302-306Crossref PubMed Scopus (40) Google Scholar). Their respective three-dimensional structures have repeatedly been solved using proteins from different organisms, and their structure-function relationships have been comparatively analyzed. In Pc, which was investigated first, two active sites were identified: site 1 (or the so-called north hydrophobic pole), located in a flat region around the copper ligand His-87, and site 2 (or the so-called east face), which is referred to as the acidic patch in eukaryotic organisms because it includes aspartic and glutamic residues at positions 42–45 and 59–61 surrounding the solvent-exposed Tyr-83 (1Gross E.L. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 413-429Google Scholar, 2Navarro J.A. Hervás M. De la Rosa M.A. J. Biol. Inorg. Chem. 1997; 2: 11-22Crossref Scopus (61) Google Scholar). Recent data indicate that site 2 is responsible for the electrostatic interactions with cytochrome f and PSI, whereas site 1, in particular His-87, is involved in electron transfer itself (3Hope A.B. Biochim. Biophys. Acta. 2000; 1456: 5-26Crossref PubMed Scopus (193) Google Scholar, 5Sigfridsson K. Photosynth. Res. 1998; 57: 1-28Crossref Scopus (76) Google Scholar). We have recently reported that Cyt from Synechocystis (6De la Cerda B. Dı́az-Quintana A. Navarro J.A. Hervás M. De la Rosa M.A. J. Biol. Chem. 1999; 274: 13292-13297Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and Anabaena (7Molina-Heredia F.P. Dı́az-Quintana A. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 1999; 274: 33565-33570Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) possesses two areas equivalent to those of Pc: site 1, which is a hydrophobic region at the edge of the heme pocket providing the contact surface for electron transfer, and site 2, which is a charged patch driving the electrostatic movement toward its two membrane-anchored partners. Our mutagenesis studies of these two cyanobacterial Cyts revealed the existence of a highly conserved arginine residue at position 64, which is located on the protein surface at the frontier between sites 1 and 2, very close to the heme group, that is essential for electron transfer to PSI (6De la Cerda B. Dı́az-Quintana A. Navarro J.A. Hervás M. De la Rosa M.A. J. Biol. Chem. 1999; 274: 13292-13297Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 7Molina-Heredia F.P. Dı́az-Quintana A. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 1999; 274: 33565-33570Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Arg-64 is the only arginine residue in Anabaena Cyt, as is Arg-88 in Anabaena Pc. Arg-64 in Cyt could thus be the counterpart of Arg-88 in Pc. Another peculiarity of Pc and Cyt from Anabaena, as compared with the proteins from other sources, is their high isoelectric point (see above) because of their high lysine content. In fact, site 2 is positively charged in both Pc and Cyt, whereas it is typically negative in other cyanobacteria and, in particular, in higher plants (2Navarro J.A. Hervás M. De la Rosa M.A. J. Biol. Inorg. Chem. 1997; 2: 11-22Crossref Scopus (61) Google Scholar). Considering that it is site 2 that mainly drives the attractive movement of these two mobile metalloproteins toward the membrane complexes, it was of interest to investigate in Anabaena Pc (as previously done in Cyt) how the surface electrostatic potential distribution and redox kinetics are altered by mutations. This work was thus aimed at comparing the role of specific amino acids in Anabaena Pc with those of its counterpart Cyt from the same organism. Pc was modified by mutagenesis of specific residues either at site 1 or site 2, with special attention being paid to Arg-88. The kinetic mechanism of PSI reduction by mutant Pcs was analyzed by laser flash absorption spectroscopy. Pc from Anabaena sp. PCC 7119 was purified as described previously (8Medina M. Dı́az A. Hervás M. Navarro J.A. Gómez-Moreno C. De la Rosa M.A. Tollin G. Eur. J. Biochem. 1993; 213: 1133-1138Crossref PubMed Scopus (37) Google Scholar), with the following two exceptions. i) Pc samples were applied onto the CM-cellulose column after oxidation with potassium ferricyanide, and ii) elution of the adsorbed proteins was performed with a linear gradient of 2–30 mm potassium phosphate buffer, pH 7.0, containing 50 μm potassium ferricyanide. Pc concentration was determined spectrophotometrically using an absorption coefficient of 4.5 mm−1cm−1 at 597 nm for the oxidized protein (9Hervás M. Navarro F. Navarro J.A. Chávez S. Dı́az A. Florencio F.J. De la Rosa M.A. FEBS Lett. 1993; 319: 257-260Crossref PubMed Scopus (36) Google Scholar). The mutant petE genes were constructed by the polymerase chain reaction with the QuickChange kit (Stratagene) using oligonucleotides of 26–38 bases, 15 ng of DNA templates, and 16 cycles of 12 min in extension time. The construction for the Anabaena petE gene previously described was used as a template (4Molina-Heredia F.P. Hervás M. Navarro J.A. De la Rosa M.A. Biochem. Biophys. Res. Commun. 1998; 243: 302-306Crossref PubMed Scopus (40) Google Scholar). The DNA fragments were sequenced to check the mutations. Other molecular biology protocols were standard (10Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Transformed cells of Escherichia coliMC1061 were grown in a 40-liter fermentor (Biostat C, B. Braun Biotech) at 37 °C for 18 h under aerobic conditions. The fermentor was filled with 20 liters of standard Luria-Bertani medium (10Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) supplemented with 100 μg/ml ampicillin and 200 μmCuSO4. The air flow was adjusted automatically to keep the concentration of dissolved oxygen at ∼95% of its saturation value. The culture was stirred at 250 rpm. The pH value was kept constant at ∼6.0 by addition of small amounts of 1 n HCl. To prevent loss of the expression vector, E. coli cells were transformed and immediately used to inoculate the reactor. Cells were collected by tangential filtration in a Sartocon cross-flow filtration system (Sartorius), and the periplasmic fraction was extracted by freezing the cell paste at −20 °C. The frozen cell paste was resuspended in 100 ml of deionized water and centrifuged. The resulting suspension was extensively dialyzed against 2 mmpotassium phosphate, pH 7.0, with the exception of mutants K33E, H57E, K58E, and R88E, which were dialyzed against 2 mm Tris-HCl, pH 8.0. From this point on, the purification procedure for most of the mutants was identical to that for native Pc, with minor changes in elution gradients. The mutants K33E, H57E, and K58E were applied onto a DEAE-cellulose column equilibrated with 2 mmTris-HCl, pH 8.0. Elution of the adsorbed proteins was performed with a linear gradient of 0–100 mm NaCl in the same buffer. R88E was purified by gel filtration in a Sephadex G-50 column. In all cases, 50 μm potassium ferricyanide was added to the gradient buffers to keep Pc oxidized. Protein concentration was determined as described above. The redox potential value for each Pc mutant was determined as reported previously (4Molina-Heredia F.P. Hervás M. Navarro J.A. De la Rosa M.A. Biochem. Biophys. Res. Commun. 1998; 243: 302-306Crossref PubMed Scopus (40) Google Scholar, 11Ortega J.M. Hervás M. Losada M. Eur. J. Biochem. 1988; 199: 239-243Google Scholar) by following the differential absorbance changes at 597 minus 500 nm. Errors in the experimental determinations were less than 10 mV. PSI particles were isolated from Anabaena cells by β-dodecyl maltoside solubilization (12Hervás M. Ortega J.M. Navarro J.A. De la Rosa M.A. Bottin H. Biochim. Biophys. Acta. 1994; 1184: 235-241Crossref Scopus (64) Google Scholar, 13Rögner M. Nixon P.J. Dinner B.A. J. Biol. Chem. 1990; 265: 6189-6196Abstract Full Text PDF PubMed Google Scholar). 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 mm−1cm−1 determined by Mathis and Sétif (14Mathis P. Sétif P. Isr. J. Chem. 1981; 21: 316-320Crossref Scopus (117) Google Scholar). Chlorophyll concentration was determined according to the method of Arnon (15Arnon D.I. Plant Physiol. 1949; 24: 1-15Crossref PubMed Google Scholar). Kinetics of flash-induced absorbance changes in PSI were followed at 820 nm as described previously (16Hervás M. Navarro J.A. Dı́az A. Bottin H. De la Rosa M.A. Biochemistry. 1995; 34: 11321-11326Crossref PubMed Scopus (130) Google Scholar). The standard reaction mixture and other experimental conditions have been reported (17De la Cerda B. Navarro J.A. Hervás M. De la Rosa M.A. Biochemistry. 1997; 36: 10125-10130Crossref PubMed Scopus (34) Google Scholar). The buffer used throughout this work was 20 mm Tricine/KOH, pH 7.5. Data collection, as well as kinetic and thermodynamic analyses, were carried out as reported previously (16Hervás M. Navarro J.A. Dı́az A. Bottin H. De la Rosa M.A. Biochemistry. 1995; 34: 11321-11326Crossref PubMed Scopus (130) Google Scholar, 18Hervás M. Navarro J.A. Dı́az A. De la Rosa M.A. Biochemistry. 1996; 35: 2693-2698Crossref PubMed Scopus (48) Google Scholar). Apparent thermodynamic parameters were estimated as in Dı́az et al. (19Dı́az A. Hervás M. Navarro J.A. De la Rosa M.A. Tollin G. Eur. J. Biochem. 1994; 222: 1001-1007Crossref PubMed Scopus (28) Google Scholar) by fitting the experimental data to the equation of Watkins et al.(20Watkins J.A. Cusanovich M.A. Meyer T.E. Tollin G. Protein Sci. 1994; 3: 2104-2114Crossref PubMed Scopus (70) Google Scholar). Experimental errors were less than 10% for both the kinetic constants and thermodynamic parameters. Structure and surface electrostatic potential distribution of Pc mutants were modeled using the Swiss-Pdb Viewer program (21Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9493) Google Scholar). The solution structure of reduced Pc from Anabaena variabilis (22Badsberg U. Jørgensen A.M.M. Gesmar H. Led J.J. Hammerstad J.M. Jespersen L.-L. Ulstrup J. Biochemistry. 1996; 35: 7021-7031Crossref PubMed Scopus (73) Google Scholar), whose amino acid sequence exhibits 100% identity with that of Pc from Anabaena sp. PCC 7119, was used as a template. The quality of the modeled structures for each Pc mutant was tested using the PROCHECK program (23Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Seven residues of Anabaena Pc were chosen to be mutated, two at the north hydrophobic patch and five at the east charged area (Fig. 1). The steric hindrance and nature of interactions at site 1 were investigated by replacing Leu-12 by Ala, and Lys-33 by Ala or Glu. The functional equivalence of site 2 in Anabaena Pc and in the negatively charged copper proteins was analyzed by substituting residues at positions 49, 57, and 58; Asp-49 was replaced by Lys, and both His-57 and Lys-58 were replaced by either Ala or Glu. Tyr-83 was changed to Ala and Phe to check whether Tyr-83 is involved in redox reactions, a role that was first proposed by He et al.(24He S. Modi S. Bendall D.S. Gray J.C. EMBO J. 1991; 10: 4011-4016Crossref PubMed Scopus (115) Google Scholar) but later discarded by Bendall et al. (25Bendall D.S. Wagner M.J. Scharlab B. Söllick T.-R. Ubbink M. Howe C.J. Peschek G.A. Löffelhardt W. Schmetterer G. The Phototrophic Prokaryotes. Kluwer Academic/Plenum Publishers, New York1999: 315-328Crossref Google Scholar) in other Pcs. Our analysis of all primary sequences of Pcs deposited at the Protein Data Bank with the BLAST search program (26Altschul S.F. Madden T.L. Scgäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59329) Google Scholar) revealed that Arg-88 is conserved in cyanobacteria but not in higher plants or in green algae. The reason for such conservation of Arg-88 in cyanobacterial but not in eukaryotic Pcs was thus investigated by replacing Arg-88 with either glutamine, as it is in spinach, or with glutamate. Neither the electronic absorption spectrum nor the midpoint redox potential of Anabaena Pc was altered by the mutations mentioned above, thereby revealing that the copper center was not distorted. As shown in Table I, the only exceptions were the R88E and K33E mutants, whose midpoint redox potential values are 25–30 mV lower than that of WT Pc.Table IMidpoint redox potential (E m) of wild-type and mutant plastocyanins, as well as bimolecular rate constants and activation parameters for the overall reaction of PSI reduction by the different copper proteinsPlastocyaninEm , pH 7.0kbim × 10−7kinf × 10−6ΔG ‡ΔH ‡ΔS ‡mVm −1 s −1m −1 s −1kJ mol −1kJ mol −1J mol −1 K −1Wild-type3557.685.9627.936.227.7Mutants at the east face R88E3250.32ND1-aND, not determined, because this mutant shows a kbim independent of ionic strength.35.936.52.1 R88Q3360.995.7833.041.433.0 D49K3539.736.2927.535.326.2 H57A3451.706.0931.739.124.7 H57E3413.765.9429.639.332.5 K58A3463.033.4830.341.136.3 K58E3391.594.1931.742.937.4 Y83A3404.446.1829.344.049.2 Y83F3434.735.2229.134.016.5Mutants at the north pole L12A3711.370.8432.231.0−4.2 K33A3552.553.7530.740.231.9 K33E3301.353.1032.241.932.31-a ND, not determined, because this mutant shows a kbim independent of ionic strength. Open table in a new tab The kinetic profile of PSI reduction under standard conditions was monoexponential with all mutants, as it is with WT Pc (16Hervás M. Navarro J.A. Dı́az A. Bottin H. De la Rosa M.A. Biochemistry. 1995; 34: 11321-11326Crossref PubMed Scopus (130) Google Scholar), but the rate constants varied greatly. The observed pseudo first-order rate constant (kobs) of PSI reduction by any mutant is linearly dependent on Pc concentration, as is the case with the WT molecule (Fig. 2). This finding can be interpreted by assuming that there is no formation of a detectable transient complex between PSI and Pc, in agreement with a collisional kinetic model (3Hope A.B. Biochim. Biophys. Acta. 2000; 1456: 5-26Crossref PubMed Scopus (193) Google Scholar, 16Hervás M. Navarro J.A. Dı́az A. Bottin H. De la Rosa M.A. Biochemistry. 1995; 34: 11321-11326Crossref PubMed Scopus (130) Google Scholar). The bimolecular rate constant for the overall reaction (kbim) can be calculated under standard conditions from the linear plots of kobs versus Pc concentration. Table I shows that most mutants yield kbim values smaller than WT Pc, with the exception of D49K, which exhibits a higher reactivity, and the two mutants at Tyr-83, which are slightly affected. As expected, all mutants of the hydrophobic patch suffer a significant decrease in their kbim value. Electrostatic mutations at positions 33, 57, and 58 cause Anabaena Pc to decrease its availability to reduce PSI as much as its global charge is made more negative. Major changes are obtained with the Arg-88 mutants, because replacement of Arg-88 with glutamine or glutamate induces a decrease in kbim of ∼8 and 24 times, respectively (TableI). Fig. 2 shows how inefficient the R88Q and R88E mutants are, even at high concentrations, compared with the WT molecule. Taking into account the electrostatic nature of the interactions between Pc and PSI, a detailed analysis of the effect of ionic strength on kbim was performed. The kbim values of WT Pc and all its mutants decrease steadily with increasing NaCl concentration, indicating the existence of attractive electrostatic interactions between the reaction partners that are overwhelmed at high ionic strength. The only exception is the R88E mutant, for which the kbimvalues are independent of ionic strength (see Fig. 4). The formalism developed by Watkins et al. (20Watkins J.A. Cusanovich M.A. Meyer T.E. Tollin G. Protein Sci. 1994; 3: 2104-2114Crossref PubMed Scopus (70) Google Scholar) facilitates the analysis of the intrinsic reactivity of redox partners in the absence of electrostatic interactions. By applying the Watkins equation to our data on the ionic strength dependence of kbim, values for the bimolecular rate constant extrapolated to infinite ionic strength (kinf) can be calculated. As shown in Table I, all mutants, except L12A and R88E, exhibit kinf values similar to that of WT Pc, indicating that the minor reactivity of Pc mutants under standard conditions is mainly due to electrostatic rather than hydrophobic or structural changes. In the case of L12A, however, the kinf value is 7-fold lower than that for WT Pc. This suggests that such an isoelectric mutation may indeed induce structural changes hindering the interaction between Pc and PSI, as previously observed in Synechocystis (17De la Cerda B. Navarro J.A. Hervás M. De la Rosa M.A. Biochemistry. 1997; 36: 10125-10130Crossref PubMed Scopus (34) Google Scholar) and spinach (27Sigfridsson K. Young S. Hansson Ö Biochemistry. 1996; 35: 1249-1257Crossref PubMed Scopus (76) Google Scholar). The Watkins equation, however, cannot be applied as such to R88E because its interactions with PSI are independent of ionic strength. The kbim value for R88E at any NaCl concentration (1.46 × 106m−1 s−1) is 4-fold lower than the kinf value for WT Pc (see Table I). This suggests that the electrostatic change induced by mutation is not the only factor affecting the reactivity of R88E toward PSI. Worth mentioning is the finding that the R64E mutant of Anabaena Cyt exhibits a kinf value that is also 4- or 5-fold lower than that for WT Cyt (7Molina-Heredia F.P. Dı́az-Quintana A. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 1999; 274: 33565-33570Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The nature of interactions between PSI and Pc was further investigated by performing a thermodynamic analysis of PSI reduction by the copper protein mutants. In all cases, the temperature dependence of the observed rate constant yielded linear Eyring plots with no breakpoints, from which the values for the apparent activation enthalpy (ΔH ‡), entropy (ΔS ‡), and free energy (ΔG ‡) of the overall reaction can be calculated. As shown in Table I, the most significant difference is observed with the R88E mutant, whose free energy change is 8.0 kJ mol−1 higher than that of WT Pc, as expected from its impaired reactivity toward PSI. Such a difference in ΔG ‡ is mainly due to a decrease of 25.6 J mol−1 K−1 in the entropic term. Also interesting is the L12A mutant, which exhibits a free energy change of 4.3 kJ mol−1 higher than that of WT Pc that is mainly due to the decrease in the entropic term. The observed changes in ΔG ‡ with the other mutants can be attributed to shifts in the enthalpic and/or entropic terms. To elucidate whether the lower reactivity of R88E can be ascribed to reasons other than the electrostatic change itself, its availability to transfer electrons to PSI from spinach and Synechocystis was analyzed in comparison with Anabaena WT Pc. The kbim value for spinach PSI reduction was 3.5 × 105m−1s−1 with R88E, or 4.3-fold lower than that with Anabaena WT Pc (1.5 × 106m−1 s−1). The kbim value for Synechocystis PSI reduction was 1.9 × 106m−1 s−1with R88E, or 15-fold lower than that with Anabaena WT Pc (2.8 × 107m−1s−1). These findings allow us to conclude that Arg-88 is a crucial residue not only for long range electrostatic attractions between Pc and PSI but also for electron transfer. It should be noted that glutamine at position 88 of spinach Pc has been replaced with asparagine, glutamate, lysine, and tyrosine with no significant changes in the kinetics of PSI reduction (27Sigfridsson K. Young S. Hansson Ö Biochemistry. 1996; 35: 1249-1257Crossref PubMed Scopus (76) Google Scholar, 28Ivkovic-Jensen M.M. Ullmann G.M. Young S. Hansson Ö. Crnogorac M.M. Ejdebäck M. Kostic N.M. Biochemistry. 1998; 37: 9557-9569Crossref PubMed Scopus (33) Google Scholar). Divalent cations like Mg2+ have previously been reported to be specifically involved in Pc/PSI interactions in other organisms, but not Anabaena (8Medina M. Dı́az A. Hervás M. Navarro J.A. Gómez-Moreno C. De la Rosa M.A. Tollin G. Eur. J. Biochem. 1993; 213: 1133-1138Crossref PubMed Scopus (37) Google Scholar). As such, an analysis of the effect of Mg2+ cations on the bimolecular rate constant of PSI reduction by WT and mutant Pcs was performed. The experimental results showed that the three mutants with one positive charge replaced by a negative residue (K33E, K58E, and R88E) are the only ones altered by magnesium cations. With these mutants the bimolecular rate constant exhibits higher values with MgCl2 than with NaCl at the same ionic strength (data not shown). All these findings can be interpreted by assuming not only that the added negative charges in Pc mutants facilitate the formation of new salt bridges with positive counterparts in PSI (16Hervás M. Navarro J.A. Dı́az A. Bottin H. De la Rosa M.A. Biochemistry. 1995; 34: 11321-11326Crossref PubMed Scopus (130) Google Scholar), but also that the original positive residues in WT Pc interact with an acidic area in PSI. The positively charged site 2 in Anabaena Pc does appear to play the same role as the negatively charged site in other Pcs. The surface electrostatic potential distribution of Anabaena Pc was then calculated for its WT species as well as for the D49K and R88E molecules. As can be seen in Fig. 3, their respective electrostatic charges at site 2 correlate well with their relative efficiency as electron donors to PSI; the more positive the local surface charge, the higher the kinetic rate constant of photosystem reduction. A similar correlation had previously been observed with a number of mutants at site 2 of Cyt from Anabaena (7Molina-Heredia F.P. Dı́az-Quintana A. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 1999; 274: 33565-33570Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and Synechocystis (6De la Cerda B. Dı́az-Quintana A. Navarro J.A. Hervás M. De la Rosa M.A. J. Biol. Chem. 1999; 274: 13292-13297Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The close similarity between Pc and Cyt from Anabaena was investigated by comparing the structures of the two molecules and the relative position of critical surface residues. The three-dimensional structures of the metalloproteins were thus modelled (Fig.4, upper panel). Assuming that His-87 in Pc plays the same redox role as the heme group in Cyt (29Frazão C. Soares C.M. Carrondo M.A. Pohl E. Dauter Z. Wilson K.S. Hervás M. Navarro J.A. De la Rosa M.A. Sheldrick G. Structure. 1995; 3: 1159-1169Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), the comparison of the two protein structures makes Arg-88 and Asp-49 of Pc occupy positions equivalent to Arg-64 and Asp-72 in Cyt. In addition, the high number of lysine residues in both molecules could contribute to site 2 being so positively charged. Such a structural coincidence between Pc and Cyt from Anabaena is indeed supported by the experimental findings concerning the kinetic behavior of mutants affected at the arginine and aspartate residues. Fig. 4 (lower panels) shows how the kbim values at low ionic strength for the D49K mutant of Pc are significantly higher than those for WT Pc, as are the values for the D72K mutant of Cyt compared with WT Cyt. The kbim values for R88E of Pc and R64E of Cyt are, however, lower than those for their respective WT species. Also worth noting is the parallel ionic strength dependence of kbim with the WT and mutant proteins,i.e. the decrease in kbim with increasing NaCl concentration. Several possibilities concerning the role of the single arginyl residue in both Cyt and Pc can be devised. The interaction of these two metalloproteins with PSI should involve the formation of a transient electrostatic complex, which has in fact kinetically been detected with Cyt but not with Pc, probably due to its low association constant. The arginyl residue could thus be required for appropriate orientation of the redox centers within the transient complex. Specific electrostatic interactions of Arg-88 in Pc and Arg-64 in Cyt with negatively charged groups in PSI could be made evident as soon as a high resolution structure for PSI is made available. In addition, the proximity of the arginyl residue to the prosthetic group in each protein can explain why the arginine mutants of both Pc and Cyt exhibit values for the rate constant extrapolated to infinite ionic strength lower than those of the respective WT species. In this context, conformational changes of Arg-88 related to changes in the redox state of Pc have been described recently (30Inoue T. Sugawara H. Hamanaka S. Tsukui H. Suzuki E. Kohzuma T. Kai Y. Biochemistry. 1999; 38: 6063-6069Crossref PubMed Scopus (59) Google Scholar). To conclude, we can say that site 1 of Anabaena Pc is similar to site 1 of other Pcs, but site 2 is positively charged in Anabaena, whereas it is negatively charged in other organisms. In addition, not only Pc but also Cyt from Anabaena contains a single arginine residue between sites 1 and 2 that appears to play the same function in the two molecules. We thank S. Alagaratnam, A. Balme, A. Dı́az, and S. Murdoch for critically reading the manuscript." @default.
W2040316218 created "2016-06-24" @default.
W2040316218 creator A5049125752 @default.
W2040316218 creator A5058167768 @default.
W2040316218 creator A5062143235 @default.
W2040316218 creator A5069186338 @default.
W2040316218 date "2001-01-01" @default.
W2040316218 modified "2023-10-16" @default.
W2040316218 title "A Single Arginyl Residue in Plastocyanin and in Cytochrome c6 from the Cyanobacterium Anabaenasp. PCC 7119 Is Required for Efficient Reduction of Photosystem I" @default.
W2040316218 cites W1974907033 @default.
W2040316218 cites W1979449963 @default.
W2040316218 cites W1986191025 @default.
W2040316218 cites W1986384608 @default.
W2040316218 cites W1990197565 @default.
W2040316218 cites W1996062078 @default.
W2040316218 cites W2001988030 @default.
W2040316218 cites W2003411022 @default.
W2040316218 cites W2003750440 @default.
W2040316218 cites W2015642465 @default.
W2040316218 cites W2030055944 @default.
W2040316218 cites W2034288826 @default.
W2040316218 cites W2038093030 @default.
W2040316218 cites W2044017071 @default.
W2040316218 cites W2052668496 @default.
W2040316218 cites W2057636971 @default.
W2040316218 cites W2062712118 @default.
W2040316218 cites W2069425547 @default.
W2040316218 cites W2074779648 @default.
W2040316218 cites W2082345507 @default.
W2040316218 cites W2100655798 @default.
W2040316218 cites W2145064110 @default.
W2040316218 cites W2152639174 @default.
W2040316218 cites W2158714788 @default.
W2040316218 cites W2346509692 @default.
W2040316218 cites W9623643 @default.
W2040316218 doi "https://doi.org/10.1074/jbc.m007081200" @default.
W2040316218 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11013249" @default.
W2040316218 hasPublicationYear "2001" @default.
W2040316218 type Work @default.
W2040316218 sameAs 2040316218 @default.
W2040316218 citedByCount "43" @default.
W2040316218 countsByYear W20403162182013 @default.
W2040316218 countsByYear W20403162182014 @default.
W2040316218 countsByYear W20403162182015 @default.
W2040316218 countsByYear W20403162182016 @default.
W2040316218 countsByYear W20403162182018 @default.
W2040316218 countsByYear W20403162182020 @default.
W2040316218 countsByYear W20403162182021 @default.
W2040316218 countsByYear W20403162182022 @default.
W2040316218 countsByYear W20403162182023 @default.
W2040316218 crossrefType "journal-article" @default.
W2040316218 hasAuthorship W2040316218A5049125752 @default.
W2040316218 hasAuthorship W2040316218A5058167768 @default.
W2040316218 hasAuthorship W2040316218A5062143235 @default.
W2040316218 hasAuthorship W2040316218A5069186338 @default.
W2040316218 hasBestOaLocation W20403162181 @default.
W2040316218 hasConcept C154828652 @default.
W2040316218 hasConcept C181199279 @default.
W2040316218 hasConcept C183688256 @default.
W2040316218 hasConcept C185592680 @default.
W2040316218 hasConcept C2780768313 @default.
W2040316218 hasConcept C2781338088 @default.
W2040316218 hasConcept C55493867 @default.
W2040316218 hasConcept C75297921 @default.
W2040316218 hasConcept C80298142 @default.
W2040316218 hasConcept C96806796 @default.
W2040316218 hasConceptScore W2040316218C154828652 @default.
W2040316218 hasConceptScore W2040316218C181199279 @default.
W2040316218 hasConceptScore W2040316218C183688256 @default.
W2040316218 hasConceptScore W2040316218C185592680 @default.
W2040316218 hasConceptScore W2040316218C2780768313 @default.
W2040316218 hasConceptScore W2040316218C2781338088 @default.
W2040316218 hasConceptScore W2040316218C55493867 @default.
W2040316218 hasConceptScore W2040316218C75297921 @default.
W2040316218 hasConceptScore W2040316218C80298142 @default.
W2040316218 hasConceptScore W2040316218C96806796 @default.
W2040316218 hasIssue "1" @default.
W2040316218 hasLocation W20403162181 @default.
W2040316218 hasLocation W20403162182 @default.
W2040316218 hasOpenAccess W2040316218 @default.
W2040316218 hasPrimaryLocation W20403162181 @default.
W2040316218 hasRelatedWork W1973428813 @default.
W2040316218 hasRelatedWork W1979468667 @default.
W2040316218 hasRelatedWork W1980344142 @default.
W2040316218 hasRelatedWork W1991203969 @default.
W2040316218 hasRelatedWork W2027794765 @default.
W2040316218 hasRelatedWork W2051340966 @default.
W2040316218 hasRelatedWork W2061877635 @default.
W2040316218 hasRelatedWork W2084093130 @default.
W2040316218 hasRelatedWork W2098444755 @default.
W2040316218 hasRelatedWork W647591513 @default.
W2040316218 hasVolume "276" @default.
W2040316218 isParatext "false" @default.
W2040316218 isRetracted "false" @default.
W2040316218 magId "2040316218" @default.
W2040316218 workType "article" @default.