Plastoquinol diffusion in linear photosynthetic electron transport

The diffusion of plastoquinol and its binding to the cytochrome bf complex, which occurs during linear photosynthetic electron transport and is analogous to reaction sequences found in most energy-converting membranes, has been studied in intact thylakoid membranes. The flash-induced electron transfer between the laterally separated photosystems II and photosystems I was measured by following the sigmoidal reduction kinetics of P-700⁺ after previous oxidation of the intersystem electron carriers. The amount of flash-induced plastoquinol produced at photosystem II was (a) reduced by inhibition with dichlorophenyl-dimethylurea and (b) increased by giving a second saturating flash. These signals were simulated by a new model which combines a deterministic simulation of reaction kinetics with a Monte Carlo approach to the diffusion of plastoquinol, taking into account the known structural features of the thylakoid membrane. The plastoquinol molecules were assumed to be oxidized by either a diffusion-limited or a nondiffusion-limited step in a collisional mechanism or after binding to the cytochrome bf complex. The model was able to account for the experimental observations with a nondiffusion-limited collisional mechanism or with a binding mechanism, giving minimum values for the diffusion coefficient of plastoquinol of 2 × 10^-8 cm²s^-1 and 3 × 10^-7 cm²s^-1, respectively.     

The ratio of the two light reactions and their coupling in chloroplasts.

The kinetics of the absorbance changes of chlorophyll alphaI (P-700) and plastoquinone induced by xenon flashes of saturating intensity were studied in spinach chloroplasts. 1. The total amount of chlorophyll alphaI is compared with that amount being reduced via the rate-limiting step between the light reactions. This is based on the amplitudes of the absorbance changes of chlorophyll alphaI after chemical reduction and after a group of flashes following far-red preillumination. It is concluded that only 75% of chlorophyll alphaI is coupled to chlorophyll alphaII via linear electron transport and that the remaining 25% is functionally isolated. 2. A ratio of 0.85 for coupled chlorophyll alphaI to chlorophyll alphaII is estimated from the time course of the absorbance changes of plastoquinone and chlorophyll alphaI in two independent ways. 3. The oxygen yield per flash is used to calculate the difference extinction coefficient of chlorophyll alphaI at the maximum of the red absorbance band in spinach chloroplasts: delta xi703 = (6.7 +/- 0.7)-10(4) M-1-cm-1. The assumption of a quantitative electron transfer from water via plastoquinone to coupled chlorophyll alphaI is supported by the same extinction coefficient reported by Hiyama and Ke for Photosystem I particles. The location and function of the different chlorophylls alphaI is discussed in detail.     

A large fraction of PsaF is nonfunctional in photosystem I complexes lacking the PsaJ subunit

PsaJ is a small hydrophobic subunit of the photosystem I complex (PSI) whose function is not yet fully understood. Here we describe mutants of the green alga Chlamydomonas reinhardtii, in which the psaJ chloroplast gene has been inactivated either in a wild-type or in a PsaF-deficient nuclear background. Cells lacking one or both subunits grow photoautotrophically and contain normal levels of PSI. Flash-absorption spectroscopy performed with isolated PSI particles isolated from the PsaJ-deficient strain indicates that only 30% of the PSI complexes oxidize plastocyanin (Pc) or cytochrome c6 (Cyt c6) with kinetics identical to wild type, whereas the remaining 70% follow slow kinetics similar to those observed with PsaF-deficient PSI complexes. This feature is not due to partial loss of PsaF, as the PsaJ-less PSI complex contains normal levels of the PsaF subunit. The N-terminal domain of PsaF can be cross-linked to Pc and Cyt c6 indicating that in the absence of PsaJ, this domain is exposed in the lumenal space. Therefore, the decreased amount of functional PsaF revealed by the electron-transfer measurements is best explained by a displacement of the N-terminal domain of PsaF which is known to provide the docking site for Pc and Cyt c6. We propose that one function of PsaJ is to maintain PsaF in a proper orientation which allows fast electron transfer from soluble donor proteins to P700(+).     

The hydrophobic recognition site formed by residues PsaA-Trp651 and PsaB-Trp627 of photosystem I in Chlamydomonas reinhardtii confers distinct selectivity for binding of plastocyanin and cytochrome c6

On the lumenal side of photosystem I (PSI), each of the two large core subunits, PsaA and PsaB, expose a conserved tryptophan residue to the surface. PsaB-Trp(627) is part of the hydrophobic recognition site that is essential for tight binding of the two electron donors plastocyanin and cytochrome c(6) to the donor side of PSI (Sommer, F., Drepper, F., and Hippler, M. (2002) J. Biol. Chem. 277, 6573-6581). To examine the function of PsaA-Trp(651) in binding and electron transfer of both donors to PSI, we generated the mutants PsaA-W651F and PsaA-W651S by site-directed mutagenesis and biolistic transformation of Chlamydomonas reinhardtii. The protein-protein interaction and the electron transfer between the donors and PSI isolated from the mutants were analyzed by flash absorption spectroscopy. The mutation PsaA-W651F completely abolished the formation of a first order electron transfer complex between plastocyanin (pc) and the altered PSI and increased the dissociation constant for binding of cytochrome (cyt) c(6) by more than a factor of 10 as compared with wild type. Mutation of PsaA-Trp(651) to Ser had an even larger impact on the dissociation constant. The K(D) value increased another 2-fold when the values obtained for the interaction and electron transfer between cyt c(6) and PSI from PsaA-W651S and PsaA-W651F are compared. In contrast, binding and electron transfer of pc to PSI from PsaA-W651S improved as compared with PSI from PsaA-W651F and admitted the formation of an inter-molecular electron transfer complex, resulting in a K(D) value of about 554 microm that is still five times higher than observed for wild type. These results demonstrate that PsaA-Trp(651) is, such as PsaB-Trp(627), crucial for high affinity binding of pc and cyt c(6) to PSI. Our results also indicate that the highly conserved structural recognition motif that is formed by PsaA-Trp(651) and PsaB-Trp(627) confers a differential selectivity in binding of both donors to PSI.     

Design, synthesis and properties of synthetic chlorophyll proteins.

A chemoselective method is described for coupling chlorophyll derivatives with an aldehyde group to synthetic peptides or proteins modified with an aminoxyacetyl group at the epsilon-amino group of a lysine residue. Three template-assembled antiparallel four-helix bundles were synthesized for the ligation of one or two chlorophylls. This was achieved by coupling unprotected peptides to cysteine residues of a cyclic decapeptide by thioether formation. The amphiphilic helices were designed to form a hydrophobic pocket for the chlorophyll derivatives. Chlorophyll derivatives Zn-methyl-pheophorbide b and Zn-methyl-pyropheophorbide d were used. The aldehyde group of these chlorophyll derivatives was ligated to the modified lysine group to form an oxime bond. The peptide-chlorophyll conjugates were characterized by electrospray mass spectrometry, analytical HPLC, and UV/visible spectroscopy. Two four-helix bundle chlorophyll conjugates were further characterized by size-exclusion chromatography, circular dichroism, and resonance Raman spectroscopy.     

Regulation of the Minichromosome Maintenance Protein 3 (MCM3) Chromatin Binding by the Prolyl Isomerase Pin1

Human Pin1 is a peptidyl prolyl cis/trans isomerase with a unique preference for phosphorylated Ser/Thr-Pro substrate motifs.Here we report that MCM3 (minichromosome maintenance complex component 3) is a novel target of Pin1. MCM3 interacts directly with the WW domain of Pin1. Proline-directed phosphorylation of MCM3 at S112 and T722 are crucial for the interaction with Pin1. MCM3 as a subunit of the minichromosome maintenance heterocomplex MCM2–7 is part of the pre-replication complex responsible for replication licensing and is implicated in the formation of the replicative helicase during progression of replication. Our data suggest that Pin1 coordinates phosphorylation-dependently MCM3 loading onto chromatin and its unloading from chromatin, thereby mediating S phase control.     

Oxidative inactivation of glutamine synthetase from the cyanobacterium Anabaena variabilis.

In crude extracts of the cyanobacterium Anabaena variabilis, glutamine synthetase (GS) could be effectively inactivated by the addition of NADH. GS inactivation was completed within 30 min. Both the inactivated GS and the active enzyme were isolated. No difference between the two enzyme forms was seen in sodium dodecyl sulfate-gels, and only minor differences were detectable by UV spectra, which excludes modification by a nucleotide. Mass spectrometry revealed that the molecular masses of active and inactive GS are equal. While the Km values of the substrates were unchanged, the Vmax values of the inactive GS were lower, reflecting the inactivation factor in the crude extract. This result indicates that the active site was affected. From the crude extract, a fraction mediating GS inactivation could be enriched by ammonium sulfate precipitation and gel filtration. GS inactivation by this fraction required the presence of NAD(P)H, Fe3+, and oxygen. In the absence of the GS-inactivating fraction, GS could be inactivated by Fe2+ and H2O2. The GS-inactivating fraction produced Fe2+ and H2O2, using NADPH, Fe3+, and oxygen. Accordingly, the inactivating fraction was inhibited by catalase and EDTA. This GS-inactivating system of Anabaena is similar to that described for oxidative GS inactivation in Escherichia coli. We conclude that GS inactivation by NAD(P)H is caused by irreversible oxidative damage and is not due to a regulatory mechanism of nitrogen assimilation.     

Evidence for complexed plastocyanin as the immediate electron donor of P-700

The reduction of P-700 by its electron donors shows two fast phases with half-times of 20 and 200 μs in isolated spinach chloroplasts. We have studied this electron transfer and the oxidation kinetics of cytochrome f.

Incubation of chloroplasts with KCN or HgCl₂ decreased the amplitude of the 20 μs phase. This provides evidence for a function of plastocyanin as the immediate electron donor of P-700.

At low concentrations of salt and sugar the fast phases of P-700⁺ reduction were largely inhibited. Increasing concentrations of MgCl₂, KCl and sorbitol (up to 5, 150 and 200 mM, respectively) were found to increase the relative amplitudes of the fast phases to about one-third of the total P-700 signal. Addition of both 3 mM MgCl₂ and 200 mM sorbitol increased the relative amplitude of the 20 μs phase to 70%. The interaction between P-700 and plastocyanin is concluded to be favoured by a low internal volume of the thylakoids and compensation of surface charges of the membrane.

The half-time of 20 μs was not changed when the amplitude of this phase was altered either by salt and sorbitol, or by inhibition of plastocyanin. This is evidence for the existence of a complex between plastocyanin and P-700 with a lifetime long compared to the measuring time. The 200 μs phase exhibited changes in its half-time that indicated the participation of a more mobile pool of plastocyanin.

Cytochrome f was oxidized with a biphasic time course with half-times of 70–130 μs and 440–860 μs at different salt and sorbitol concentrations. The half-time of the faster phase and a short lag of 30–50 μs in the beginning of the kinetics indicate an oxidation of cytochrome f via the 20 μs electron transfer to P-700. An inhibition of this oxidation by MgCl₂ suggests that the electron transfer from cytochrome f to complexed plastocyanin is not controlled by negative charges in contrast to that from plastocyanin to P-700.     

Sodium ditiocarb as adjuvant immunotherapy for high risk breast cancer: A randomized study

Sixty-four patients with non metastatic high risk breast cancer were randomized in a double blind trial of adjuvant immunotherapy with sodium ditiocarb (DDC) versus placebo. All patients underwent prior surgery (mammectomy according to Patey) then adjuvant FAC chemotherapy +/- DDC. With a median follow-up of 5 years we observed 6 relapses and 5 deaths in DDC group; 13 relapses and 12 deaths in control group. At 6 years, overall survival is 81% in DDC group versus 55%. Disease free survival (DFS) is 76% in DDC group versus 55%. DDC associated to chemotherapy and locoregional treatment can improve survival and probably DFS in this high risk breast cancer subgroup.Abbreviations DDC sodium ditiocarb - DFS disease free survival - OS overall survival - N node - FAC 5 Fluorouracil, Adrianycin, Cyclopho-pharmide     

Electron transfer from plastocyanin to photosystem I.

Mutant plastocyanins with Leu at position 10, 90 or 83 (Gly, Ala and Tyr respectively in wildtype) were constructed by site-specific mutagenesis of the spinach gene, and expressed in transgenic potato plants under the control of the authentic plastocyanin promoter, as well as in Escherichia coli as truncated precursor intermediates carrying the C-terminal 22 amino acid residues of the transit peptide, i.e. the thylakoid-targeting domain that acts as a bacterial export signal. The identity of the purified plastocyanins was verified by matrix-assisted laser desorption/ionization mass spectrometry. The formation of a complex between authentic or mutant spinach plastocyanin and isolated photosystem I and the electron transfer has been studied from the biphasic reduction kinetics of P700+ after excitation with laser flashes. The formation of the complex was abolished by the bulky hydrophobic group of Leu at the respective position of G10 or A90 which are part of the conserved flat hydrophobic surface around the copper ligand H87. The rate of electron transfer decreased by both mutations to < 20% of that found with wildtype plastocyanin. We conclude that the conserved flat surface of plastocyanin represents one of two crucial structural elements for both the docking at photosystem I and the efficient electron transfer via H87 to P700+. The Y83L mutant exhibited faster electron transfer to P700+ than did authentic plastocyanin. This proves that Y83 is not involved in electron transfer to P700 and suggests that electron transfer from cytochrome f and to P700 follows different routes in the plastocyanin molecule. Plastocyanin (Y83L) expressed in either E. coli or potato exhibited different isoelectric points and binding constants to photosystem I indicative of differences in the folding of the protein. The structure of the binding site at photosystem I and the mechanism of electron transfer are discussed.