75 research outputs found
Influence of the axial ligands on the spectral properties of P700 of photosystem I: A study of site-directed mutants
No full textTwo histidines provide the axial ligands of the two chlorophyll a (Chl a) molecules which form the primary electron donor (P700) of photosystem I (PSI). Histidine 676 in the protein subunit PsaA, His(A676), and histidine 656 in subunit PsaB, His(B656), were replaced in the green algae Chlamydomnas reinhardtii by site-directed mutagenesis with nonpolar, uncharged polar, acidic, and basic amino acid residues. Only the substitutions with uncharged polar residues led to a significant accumulation of PSI in the thylakoid membranes. These PSI complexes were isolated and the physical properties of the primary donor characterized. The midpoint potential of P700(+.)/P700 was increased in all mutants (up to 140 mV) and showed a dependence on size and polarizability of the residues when His(B656) was substituted. In the light-minus-dark absorbance spectra, all mutations in PsaB exhibited an additional bleaching band at 665 nm at room temperature comparable with the published spectrum for the replacement of His(B656) with asparagine [Webber, A. N., Su Hui, Bingham, S. E., Kass, H., Krabben, L., Kuhn, M., Jordan, R., Schlodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857-12863]. Substitutions of His(A676) showed an additional shoulder around 680 nm. In the low-temperature absorbance difference spectra of P700(+.)/P700, a blue shift of the main bleaching band by 2 nm and some changes in the spectral features around 660 nm were observed for mutations of His(B656) in PsaB. The analogous substitution in PsaA showed only a shift of the main bleaching band. Similar effects of the mutations were found in the (3)P700/P700 absorbance difference spectra at low temperatures (T = 2 K). The zero-field splitting parameters of (3)P700 were not significantly changed in the mutated PSI complexes. The electron spin density distribution of P700(+.), determined by ENDOR spectroscopy, was only changed when His(B656) was replaced. In all measurements, two general observations were made. (i) The replacement of His(B656) had a much stronger impact on the physical properties of P700 than the mutation of His(A676). (ii) The exchange of His(B656) with glutamine induces the smallest changes in the spectra or the midpoint potential, whereas the other replacements exhibited a stronger but very similar influence on the spectroscopic features of P700. The data provide convincing evidence that the unpaired electron in the cation radical and the triplet state of P700 are mainly localized on the Chl a of the dimer which is axially coordinated by His(B656)
Site-directed mutagenesis of Thr A739 of photosystem I in Chlamydomonas reinhardtii alters significantly the excitonic and electronic coupling of the primary electron donor P700
No full textThe primary electron donor P700 of photosystem I is a dimer comprised of one chlorophyll (Chl) a (PB) and one Chl a ́ (PA). To investigate the influence of protein-cofactor interactions on the properties of P700, we constructed a series of site-directed mutants in the surrounding of P700. The most interesting effects were obtained for the replacement of Thr A739, a possible hydrogen bond donor to the 9-keto group of PA, against Val. The low-energy exciton absorption band of P700 observed as a shoulder at about 700 nm in the absorption spectrum of PS I complexes from wild type is not visible in the mutant TV A739. The main bleaching band in the (P700+-P700) and (3P700-P700) absorption difference spectra is blue shifted by 9 nm. Both results imply that the excitonic coupling of P700 is severely disturbed. A similar blue shift is observed for the main bleaching in the Soret region. Redox titrations yielded a decrease of the midpoint potential for the oxidation of P700 by 32 mV. ENDOR spectroscopy revealed a change of the electron spin densitiy distribution of P700+. The data provide evidence that P700 is a Chl-dimer with an asymmetric spin/charge density distributio
Hydrogen bonding to P700: Site-directed mutagenesis of threonine A739 of photosystem I in Chlamydomonas reinhardtii
No full textThe primary electron donor P700 of photosystem I is a dimer comprised of chlorophyll a (P-B) and chlorophyll a' (P-A). P-A is involved in a hydrogen bond network with several surrounding amino acid residues and a nearby water molecule. To investigate the influence of hydrogen bond interactions on the properties of P700, the threonine at position A739, which donates a putative hydrogen bond to the 13(1)-keto group Of P-A, was replaced with valine, histidine, and tyrosine in Chlamydomonas reinhardtii using site-directed mutagenesis. Growth of the mutants was not impaired. (i) The (P700+. - P700) FTIR difference spectra of the mutants lack a negative band at 1634 cm(-1) observed in the wild-type spectrum and instead exhibit a new negative band between 1658 and 1672 cm(-1) depending on the mutation. This band can therefore be assigned to the 13(1)-keto group of P-A which is upshifted to higher frequencies upon removal of the hydrogen bond. (ii) The main bleaching band in the Q(y) region of the (P700(+.) - P700) and ((3)P700 - P700) absorption difference spectra is blue shifted for the mutants by similar to6 nm compared to that of the wild type. A blue shift is also observed for the main bleaching in the Soret region. (iii) The (P700(+.) - P700) CD difference spectrum of the wild type reveals two bands at 694 nm (positive CD) and 680 nm (negative CD) of approximately equal area. For each mutant, these two components are blue-shifted to the same extent. The results strongly suggest that a blue shift of the Qy absorption band Of P-A is responsible for a blue shift of the exciton bands. (iv) Redox titrations yielded a decrease in the midpoint potential for the oxidation of P700 by 32 mV for the exchange of Thr against Val. (v) ENDOR spectroscopy shows that the hfc of the methyl protons at position 12 of the spin-carrying Chl P-B is decreased due to the removal of the hydrogen bond to P- A. This indicates a redistribution of spin density in P700(+.) compared to that in the wild type. This gives evidence for an electronic coupling between the two halves of the dimer in the wild type and mutants
Net charge oscillation and proton release during water oxidation in photosynthesis. An electrochromic band shift study at pH 5.5–7.0
No full textAbstractIn the S-state cycle of water oxidation, a local electric field was measured in states S2 and S3. This was indicated by the strongly retarded reduction kinetics of the oxidized primary electron donor of PS II in these states (Brettel, K., Schlodder, E. and Witt, H.T. (1984) Biochim. Biophys. Acta 766, 403–415) as well as by electrochromic band shifts in state S2 and S3 (Saygin, Ö. and Witt, H.T., FEBS Lett. 176 (1984) 83–87; 189 (1985) 224–226). The electric field oscillation of 0:0:1:1 in S0:S1:S2:S3 is strictly coupled with the pattern of manganese redox changes measured at 365 nm and of O2 evolution under very different conditions (Kretschmann, H. and Witt, H.T. (1993) Biochim. Biophys. Acta 1144, 331–345). In this work with PS II complexes from the cyanobacterium Synechococcus elongatus it is shown that the electric field oscillation as well as the pattern of redox changes of manganese are practically pH-independent between pH 5.5 and pH 7.0; i.e., in the range in which the pattern of O2 evolution and water oxidation, respectively, is pH-independent. It was suggested that a net charge created as charge difference between electron extraction and proton release from the catalytic center may be the origin of the electric field. With this explanation it follows that, with the S0→S1→S2→S3S0 transitions, a independent proton release of 1:0:1:2 from the catalytic center takes place. The proton release into the medium is, however, generally pH-dependent. For PS II complexes from cyanobacteria a mechanism is proposed which may be responsible for the modification of the supposed pH-independent proton release from the catalytic center into the pH-dependent proton release into the medium. It is proposed that in the pH 5–7 range an amino acid residue with a pK value of approx. 6 releases a proton induced by a pK shift through electrostatic interaction with the local electric field set up in state S2. When, subsequently, the created base traps a proton released from the catalytic center in the S3→S0 transition, this results in a pH-dependent non-integer oscillation of the proton release into the medium. The predicted values have been compared with the directly measured ones
Species-specific differences of the spectroscopic properties of P700 - Analysis of the influence of non-conserved amino acid residues by site-directed mutagenesis of photosystem I from Chlamydomonas reinhardtii
No full textWe applied optical spectroscopy, magnetic resonance techniques, and redox titrations to investigate the properties of the primary electron donor P700 in photosystem I (PS I) core complexes from cyanobacteria (Thermosynechococcus elongatus, Spirulina platensis, and Synechocystis sp. PCC 6803), algae (Chlamydomonas reinhardtii CC2696), and higher plants (Spinacia oleracea). Remarkable species-specific differences of the optical properties of P700 were revealed monitoring the ((3)P700-P700) and (P700(+.)-P700) absorbance and CD difference spectra. The main bleaching band in the Q(y) region differs in peak position and line width for the various species. In cyanobacteria the absorbance of P700 extends more to the red compared with algae and higher plants which is favorable for energy transfer from red core antenna chlorophylls to P700 in cyanobacteria. The amino acids in the environment of P700 are highly conserved with two distinct deviations. In C. reinhardtii a Tyr is found at position PsaB659 instead of a Trp present in all other organisms, whereas in Synechocystis a Phe is found instead of a Trp at the homologous position PsaA679. We constructed several mutants in C. reinhardtii CC2696. Strikingly, no PS I could be detected in the mutant YW B659 indicating steric constraints unique to this organism. In the mutants WA A679 and YA B659 significant changes of the spectral features in the ((3)P700 - P700), the (P700(+.)-P700) absorbance difference and in the (P700(+.)-P700) CD difference spectra are induced. The results indicate structural differences among PS I from higher plants, algae, and cyanobacteria and give further insight into specific protein-cofactor interactions contributing to the optical spectra
Recombination Kinetics of the Radical Pair, P680+I‒, in Closed Reaction centers of PS II as Function of Temperature
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Initial Kinetics of ATP-Synthesis and of Conformational Changes in the Chloroplast ATPase Studied by External Electric Field Pulses
No full textOptical characterization of the immediate electron donor to chlorophyll a+II in O2-evolving photosystem II complexes Tyrosine as possible electron carrier between chlorophyll aII and the water-oxidizing manganese complex
No full textAbstractThe number and chemical nature of the electron carrier(s) between Chl aII and the water-oxidizing enzyme, S, were analyzed through flash-induced absorption changes in the UV with nanosecond time resolution. (i) At all wavelengths where the reaction of the donor with Chl a+II has been characterized, this donor is oxidized in the nanosecond time range in exact accordance with the reduction kinetics of Chl a+II. The donor is in turn re-reduced with t12 > 10,μs, i.e. in the range where S is oxidized. From this time course it is concluded that there exists only one electron carrier between Chl a+II and S. (ii) The UV-diference spectrum due to the electron transfer from the immediate donor to Chl a+II in the nanosecond time range in O2-evolving PS II complexes is characterized by a maximum around 260 nm and smaller minimum around 310 nm. This spectrum is identical with that observed for the reaction of the donor with Chl a+II in the microsecond time range in Tris-treated PS II. Therefore, the donors in both reactions must be of the same chemical nature. (iii) This result, together with the well-established similarity of EPR signal IIf of the oxidized donor in Tris-treated PS II to the EPR signal IIIs, recently assigned to Tyr-160 of the D2 protein of PS II [(1988) Proc. Natl. Acad. Sci. USA 85, 427–430], provides strong evidence that the immediate donor to Chl a+II in water-oxidizing PS II is also a tyrosine. (iv) It is shown that the UV-difference spectra of the oxidation of the immediate donor in O2-evolving as well as that of Tris-treated PS II complexes are similar to the in vitro difference spectrum of the oxidation of tyrosine in water. This independent result supports the conclusion that the donor is a tyrosine
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