Stromal Side Research Articles

EPR studies of the oxygen-evolving complex reveal a light-adaption process in Photosystem II

Photosystem II utilizes solar energy to drive electrons from the Mn cluster at the lumenal side to the quinone at the stromal side of the thylakoid membrane. The source of electrons is H2O, which is split to oxygen by the OEC. The water-splitting process involves cycling of the Mn-cluster through four semi-stable oxidation states, termed the S0, S1, S2, and S3 states. The OEC can be trapped in the different S-states, and studied by EPR techniques. The S2 state is often obtained by exposing a dark-adapted (S1 state) sample to continuous illumination at low temperature, or to a single flash of light at room temperature followed by freezing. Both procedures capture the S2 state, as reached by a single oxidation of the OEC following a period of dark-adaptation. With a laser flash procedure, the S2 state can be obtained a second time after 5 flashes, when the S-cycle has been completed once. We have discovered differences between the S2 state formed after one flash and the S2 state formed after five flashes, in terms of the relaxation behavior of the S2 multiline EPR signal. These data indicate a change within the Mn cluster that builds up during the first turnovers after dark-adaptation. Pulsed field-swept spectra of samples given 0-5 flashes of light show a similar trend: the S1 state obtained by zero and four flashes differ significantly from one another. Based on the data presented and similar reports in the literature, we propose that the OEC undergoes a light-adaptation process during the first two turnovers after dark-adaptation, adjusting the system for efficient continuous water-splitting. We tentatively suggest that light-adaptation involves rearrangements of the proton network in the OEC, possibly as a means of setting up proton channels. (Less)

The position of cytochrome b559 relative to Q A in Photosystem II studied by electron–electron double resonance (ELDOR)

The electron–electron double resonance (ELDOR) method was applied to measure the dipole interaction between cytochrome (Cyt) b + 559 and the primary acceptor quinone (Q − A), observed at g=2.0045 with the peak to peak width of about 9 G, in Photosystem II (PS II) in which the non-heme Fe 2+ was substituted by Zn 2+. The paramagnetic centers of Cyt b + 559Y ⋅ DQ − A were trapped by illumination at 273 K for 8 min, followed by dark adaptation for 3 min and freezing into 77 K. The distance between the pair Cyt b + 559-Q − A was estimated from the dipole interaction constant fitted to the observed ELDOR time profile to be 40±1 Å. In the membrane oriented PS II particles the angle between the vector from Q A to Cyt b 559 and the membrane normal was determined to be 80±5°. The position of Cyt b 559 relative to Q A suggests that the heme plane is located on the stromal side of the thylakoid membrane. ELDOR was not observed for Cyt b + 559 Y ⋅ D spin pair, suggesting the distance between them is more than 50 Å.

Molecular characterization and subcellular localization of protoporphyrinogen oxidase in spinach chloroplasts.

Protoporphyrinogen oxidase (Protox) is the last common enzyme in the biosynthesis of chlorophylls and heme. In plants, there are two isoenzymes of Protox, one located in plastids and other in the mitochondria. We cloned the cDNA of spinach (Spinacia oleracea) plastidal Protox and purified plastidal Protox protein from spinach chloroplasts. Sequence analysis of the cDNA indicated that the plastid Protox of spinach is composed of 562 amino acids containing the glycine-rich motif GxGxxG previously proposed to be a dinucleotide binding site of many flavin-containing proteins. The cDNA of plastidal Protox complemented a Protox mutation in Escherichia coli. N-terminal sequence analysis of the purified enzyme revealed that the plastidal Protox precursor is processed at the N-terminal site of serine-49. The predicted transit peptide (methionine-1 to cysteine-48) was sufficient for the transport of precursors into the plastid because green fluorescent protein fused with the predicted transit peptide was transported to the chloroplast. Immunocytochemical analysis using electron microscopy showed that plastidal Protox is preferentially associated with the stromal side of the thylakoid membrane, and a small portion of the enzyme is located on the stromal side of the chloroplast inner envelope membrane.

A New Subunit of Cytochromeb6f Complex Undergoes Reversible Phosphorylation upon State Transition

A 15.2-kDa polypeptide, encoded by the nuclear gene PETO, was identified as a novel cytochrome b(6)f subunit in Chlamydomonas reinhardtii. The PETO gene product is a bona fide subunit, subunit V, of the cytochrome b(6)f complex, because (i) it copurifies with the other cytochrome b(6)f subunits in the early stages of the purification procedure, (ii) it is deficient in cytochrome b(6)f mutants accumulating little of the complex, and (iii) it colocalizes with cytochrome f, which migrates between stacked and unstacked membrane regions upon state transition. Sequence analysis and biochemical characterization of subunit V shows that it has a one transmembrane alpha-helix topology with two large hydrophilic domains extending on the stromal and lumenal side of the thylakoid membranes, with a lumenal location of the N terminus. Subunit V is reversibly phosphorylated upon state transition, a unique feature that, together with its topological organization, points to the possible role of subunit V in signal transduction during redox-controlled short term and long term adaptation of the photosynthetic apparatus in eukaryotes.

Carnitine palmitoyltransferases in pea leaf chloroplasts: partial purification, location, and properties

Carnitine palmitoyltransferase (EC 2.3.1.21), an enzyme that catalyses the reversible transfer of activated long-chain acyl groups between CoASH and L-carnitine, has been confirmed in pea leaf chloroplasts. This enzyme is bound to the chloroplast inner envelope membrane and has two isoforms, one bound to the outside (cytosol side) of the inner envelope and one bound to the inside (stromal side) of the inner envelope. Malonyl CoA inhibited the activity of the outer carnitine palmitoyltransferase, while stimulating the activity of the inner isoform and may be a regulator of these enzymes in vivo. Carnitine palmitoyltransferase was solubilized from the chloroplast envelope by detergent treatment and the two isoforms separated by Q-Sepharose anion exchange chromatography. Both proteins were immunochemically observed by probing Western blots of sodium dodecyl sulfate - polyacrylamide gel electrophoresis gels using an anti-beef heart mitochondrial carnitine palmitoyltransferase polyclonal antibody. The monomeric molecular mass of the protein recognized by this antibody was approximately 20 kDa. This 20-kDa protein also bound3H-carnitine. Both isoforms had broad acyl CoA substrate specificities, but showed increased activity with desaturated long-chain acyl CoAs, exhibiting a preference for linolenoyl CoA. A role for carnitine palmitoyltransferase in the shuttling of fatty acids across the chloroplast envelope is suggested.Key words: Pisum sativum, chloroplasts, carnitine palmitoyltransferase, fatty acid metabolism, eukaryotic pathway, membrane transport.

Light-induced stimulation of carbonic anhydrase activity in pea thylakoids

Stimulation of the bicarbonate dehydration reaction in thylakoid suspension under conditions of saturating light at pH 7.6–8.0 was discovered. This effect was inhibited by nigericin or the lipophilic carbonic anhydrase (CA) inhibitor ethoxyzolamide (EZ), but not by the hydrophilic CA inhibitor, acetazolamide. It was shown that the action of EZ is not caused by an uncoupling effect. It was concluded that thylakoid CA is the enzyme utilizing the light-generated proton gradient across the thylakoid membrane thus facilitating the production of CO 2 from HCO 3 − and that this enzyme is covered from the stroma side of thylakoids by a lipid barrier.

Amniotic Membrane Patching Promotes Healing and Inhibits Proteinase Activity on Wound Healing Following Acute Corneal Alkali Burn

Amniotic membrane (AM) contains basement membrane components and various proteinase inhibitors. Furthermore, when used as a graft, the basement membrane of AM could block inflammatory insults to a damaged corneal surface. Thus, we evaluated whether amniotic membrane patching could promote the healing process by inhibiting proteolytic damage. Alkali wounds were inflicted on the central corneas of rabbits by applying a round filter paper, 6.0 mm in diameter, soaked in 1 N NaOH for 30 sec. Amniotic membrane patching was performed over the perilimbal sclera immediately after wounding. A total of 115 rabbits were divided into four groups: (1) immediately covered by AM with the amnion cell side down up to the perilimbal sclera (n =26); (2) covered by AM with the stromal side down up to the perilimbal sclera (n =19); (3) anchored to the fornix (n =29); and (4) uncovered as a control (n =41). AM was removed 3 days postoperatively. During follow-ups, epithelial defects, corneal thickness and its opacity of each eye were measured. Some corneas were removed for histopathologic studies and for proteinase activity assay and zymography. The epithelial healing was faster and the corneal thickness was thicker in all three AM-covered groups than in the control (P<0.05). No significant difference was found between covered and anchored groups (P>0.05). Corneal opacity was least in the amnion cell side down group. Infiltration of polymorphonuclear leukocytes (PMNs) was much less in AM-covered groups than in the control. Pathological results were associated with zymographic findings, which revealed much higher proteinase activity in uncovered group than AM-covered groups. Immediate intervention for acute alkali burns with AM as a temporary patch promotes wound healing by inhibiting proteinase activity and PMNs infiltration.

Insertion of light-harvesting chlorophyll a/b protein into the thylakoid topographical studies.

The major light-harvesting chlorophyll a/b-binding protein (Lhcb1,2) of photosystem II is inserted into the thylakoid via the signal recognition particle dependent pathway. However, the mechanism by which the protein enters the membrane is at this time unknown. In order to define some topographical restrictions for this process, we constructed several recombinant derivatives of Lhcb1 carrying hexahistidine tags at either protein terminus or in the stromal loop domain. Additionally, green fluorescent protein (GFP) was fused to either terminus. None of the modifications significantly impair the pigment-binding properties of the protein in the in vitro reconstitution of LHCII. With the exception of the C-terminal GFP fusion, all mutants stably insert into isolated thylakoids in the absence of Ni2+ ions. The addition of low concentrations of Ni2+ ions abolishes the thylakoid insertion of C-terminally His-tagged mutants whereas the other His-tagged proteins fail to insert only at higher Ni2+ concentrations. The C-terminus of Lhcb1 must cross the membrane during protein insertion whereas the other sites of Lhcb1 modification are positioned on the stromal side of LHCII. We conclude that a Ni2+-complexed His tag and fusion to GFP inhibit translocation of the protein C-terminus across the thylakoid. Our observations indicate that the N-terminal and stromal domain of Lhcb1 need not traverse the thylakoid during protein insertion and are consistent with a loop mechanism in which only the C-terminus and the lumenal loop of Lhcb1 are translocated across the thylakoid.

Pea chloroplast carnitine acetyltransferase.

The purpose of this study was to resolve the controversy as to whether or not chloroplasts possess the enzyme carnitine acetyltransferase (CAT) and whether the activity of this enzyme is sufficient to support previously reported rates of fatty acid synthesis from acetylcarnitine. CAT catalyses the freely reversible reaction: carnitine + short-chain acylCoA <--> short-chain acylcarnitine + CoASH. CAT activity was detected in thc chloroplasts of Pisum sativum L. With membrane-impermeable acetyl CoA as a substrate. activity was only detected in ruptured chloroplasts and not with intact chloroplasts, indicating that the enzyme was located on the stromal side of the envelope. In crude preparations, CAT could only be detected using a sensitive radioenzymatic assay due to competing reactions from other enzymes using acetyl CoA and large amounts of ultraviolet-absorbing materials. After partial purification of the enzyme, CAT was detected in both the forward and reverse directions using spectrophotometric assays. Rates of 100 nmol of product formed per minute per milligram of protein were obtained, which is sufficient to support reported fatty acid synthesis rates from acetylcarnitine. Chloroplastic CAT showed optimal activity at pH 8.5 and had a high substrate specificity, handling C2-C4 acyl CoAs only. We believe that CAT has been satisfactorily demonstrated in pea chloroplasts.

An Arabidopsis thaliana protein homologous to cyanobacterial high-light-inducible proteins.

An Arabidopsis thaliana cDNA clone encoding a novel 110 amino acid thylakoid protein has been sequenced. The in vitro synthesized protein is taken up by intact chloroplasts, inserted into the thylakoid membrane and the transit peptide is cleaved off during this process. The mature protein is predicted to contain 69 amino acids, to form one membrane-spanning alpha-helix and to have its N-terminus at the stromal side of the thylakoid membrane. The protein showed similarity to the LHC, ELIP and PsbS proteins of higher plants, but more pronounced to the high-light-inducible proteins (HLIPs) of cyanobacteria and red algae, to which no homologue previously has been detected in higher plants. As for HLIP and ELIP, high light increases the mRNA levels of the corresponding gene. Sequence comparisons indicate that the protein may bind chlorophyll and form dimers in the thylakoid membrane. The level of expression of the protein seems to be far lower than that of normal PSI and PSII subunits.

Interaction of a plant 14-3-3 protein with the signal peptide of a thylakoid-targeted chloroplast precursor protein and the presence of 14-3-3 isoforms in the chloroplast stroma.

The 14-3-3 proteins are acidic, dimeric proteins that have been implicated in many eukaryotic cellular processes because of direct protein association with enzymes and other metabolic and regulatory proteins. 14-3-3 proteins are largely considered to be cytoplasmic, but a search for proteins that specifically interact with a plant 14-3-3 resulted in the isolation of a nuclear-encoded, thylakoid-targeted chloroplast precursor, the full-length Arabidopsis photosystem I N-subunit At pPSI-N (P.C. Sehnke, R.J. Ferl ¿1995 Plant Physiol 109: 1126). Using precursor truncations in the two-hybrid system, it was determined that the leader sequence is the site of PSI-N that associates with 14-3-3. This suggested the novel possibility that 14-3-3 would be found within chloroplasts. Immuno-electron microscopy of leaf tissue and western analysis of chloroplast fractions with monoclonal anti-14-3-3 antibodies localized 14-3-3 proteins to the chloroplast stroma and the stromal side of thylakoid membranes. Using peptide-generated, isoform-specific antibodies, GF14nu, GF14epsilon, GF14mu, and GF14upsilon were shown to be present in the chloroplast stromal extract. These isoforms represent two distinct phylogenetic 14-3-3 groupings. These data suggest a novel interorganellar role for these phylogenetically distinct 14-3-3 proteins.

Evidence that PSI-D, a chloroplast photosystem I protein, is in haustoria of the powdery mildew fungus Erysiphe pisi

Monoclonal antibody UB43 raised against isolated haustorial complexes (HC) of the pea powdery mildew fungus Erysiphe pisi, recognized an 18 kDa protein on Western blots of HC lysates but also of thylakoids isolated from pea chloroplasts. Microsequencing of the N-terminus of the antigen purified from chloroplasts, yielded a sequence of 13 amino acids with high homology to that of a photosystem I subunit (PSI-D). Co-identity of the UB43 antigen in chloroplasts and PSI-D, was confirmed by the use of authentic anti-PSI-D antibodies and immunoprecipitation procedures. These anti-PSI-D antibodies also bound to HCs and recognized an 18 kDa antigen. Immunofluorescence and EM-immunogold labelling showed that UB43 and the anti-PSI-D antibodies bound to the cytoplasm of haustorial lobes. Radiolabelled spinach PSI-D precursor produced by an in vitro transcription/translation system of PSI-D cDNA, was taken up by chloroplast-free HCs into a thermolysin-inaccessible compartment. PSI-D is located on the stromal side of the thylakoid membrane, and is thought to act as a docking site for the soluble ferredoxin. The nature and origin of the PSI-D related protein in HCs is discussed.

The solution structure of photosystem I accessory protein E from the cyanobacterium Nostoc sp. strain PCC 8009.

PsaE is a small basic subunit located on the stromal (cytoplasmic) side of photosystem I. In cyanobacteria, this subunit participates in cyclic electron transport and modulates the interactions of the complex with soluble ferredoxin. The PsaE protein isolated from the cyanobacterium Synechococcus sp. strain PCC 7002 adopts the beta topology of an SH3 domain, with five beta strands (betaA through betaE) and a turn of 3(10) helix between strands betaD and betaE [Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062]. The primary structure of the PsaE protein is strongly conserved across all oxygen-evolving photosynthetic organisms. However, variability in loop lengths, as well as N- or C-terminal extensions, suggests that the structure of a second representative PsaE subunit would be useful to characterize the interactions among photosystem I polypeptides. In this work, the solution structure of PsaE from the cyanobacterium Nostoc sp. strain PCC 8009 was determined by NMR methods. Compared to PsaE from Synechococcus sp. strain PCC 7002, this PsaE has a seven-residue deletion in the loop connecting strands betaC and betaD, and an eight-residue C-terminal extension. Angular and distance restraints derived from homonuclear and heteronuclear NMR experiments were used to calculate structures by a distance-geometry/simulated-annealing protocol. A family of 20 structures (rmsd of 0.24 A in the regular secondary structure) is presented. Differences between the two cyanobacterial proteins are mostly confined to the CD loop region; the C-terminal extension is disordered. The thermodynamic stability of Nostoc sp. strain PCC 8009 PsaE toward urea denaturation was measured by circular dichroism and fluorescence spectroscopy, and thermal denaturation was monitored by UV absorption spectroscopy. Chemical and thermal denaturation curves are modeled satisfactorily with two-state processes. The DeltaG degrees of unfolding at room temperature is 12.4 +/- 0.3 kJ mol(-1) (pH 5), and the thermal transition midpoint is 59 +/- 1 degrees C (pH 7). Interactions with other proteins in the photosystem I complex may aid in maintaining PsaE in its native state under physiological conditions.

Scanning Electron Microscopic Studies of the Basal Surface of the Corneal Endothelium and the Stromal and Endothelial Surfaces of Descemet's Membrane in Rats

Purpose: The basal surface of the corneal endothelium and the stromal and endothelial surfaces of Descemet's membrane in rats were studied by scanning electron microscopy. Methods: We compared the fine structures of the two surfaces of Descemet's membrane both after sputter-coating with platinum and without sputter-coating. Results: Fine structures were made clearly visible without metal coating by heating specimens to 300°C during observation. On the basal surface of the endothelium, pinocytotic vesicles and invagination of the basal cell membrane were visible. After sputter-coating, Descemet's membrane appeared to be composed of granular substances. The size of the granules was larger on the stromal side than on the endothelial side. Descemet's membrane observed without sputter-coating was composed of fine fibrous structures, showing a felt-like appearance on both stromal and endothelial sides. The diameter of the fibers on the stromal surface was thicker than on the endothelial side. Conclusion: These results may indicate a characteristic difference between the collagen types existing on the two sides of Descemet's membrane.

Comparison of wound healing after photorefractive keratectomy and laser in situ keratomileusis in rabbits

Purpose To evaluate and compare the corneal wound-healing process after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). Setting Kangnam St. Mary’s Hospital, Seoul, Korea. Methods Two surgical procedures, PRK with the VISX Star excimer laser and LASIK with a MicroTech microkeratome, were performed in 24 rabbit eyes. In the PRK group (n=12 eyes), the rabbit cornea was treated with a 20 μm ablation. In the LASIK group (n=12 eyes), a 20 μm laser ablation was performed after a 150 μm thick hinged corneal flap had been made. During both procedures, dichlorotriazinyl aminofluorescien (DTAF) dye was applied to the ablated stromal bed; in the LASIK group, the stromal side of the corneal flap was also stained with DTAF to differentiate regenerated collagen from normal stromal tissue. Corneal wound healing was evaluated postoperatively at 1, 4, 8, and 12 weeks using light, electron, and fluorescence microscopy. The amount of regenerated stromal tissue and the number of keratocytes were analyzed by an image-analysis system. Results In the PRK group, epithelial migration and regeneration were observed in the ablated area without any stromal regeneration 1 week postoperatively. However, newly regenerated, irregularly arranged stromal collagen, with epithelial hyperplasia in the ablated area, was observed 4 to 12 weeks postoperatively by light and fluorescence microscopy. The number of keratocytes in the surgical area was also increased. In ultrastructural observation using an electron microscope, the shape of keratocytes in the ablated area was changed, and the number of rough and smooth endoplasmic reticuli, ribosomes, mitochondria, and electron-dense vesicles in the cytoplasm were increased, suggesting that the cells were activated. In the LASIK group, there was no observed regenerated collagen between the corneal flap and the ablated stromal bed except in the wound margin. Lamellated, parallel collagen fibers in the cornealstroma were not disturbed. However, in the wound margin, corneal epithelial ingrowth between the flap and the stromal bed was observed, as was some regenerated stromal tissue. The amount of regenerated stromal tissue and the number of keratocytes in the wound area were statistically smaller than those in the PRK group ( P < .05). Observation by electron microscopy showed no activated keratocytes, unlike in the PRK group. The collagen fibers in the wound area were parallel. Conclusion Stromal wound healing in the LASIK group was minimal compared with that in the PRK group, except in the wound margin. These results may support the clinical findings of less corneal haze in the human cornea after LASIK.

Suppression of transforming growth factor-beta isoforms, TGF-beta receptor type II, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix.

Down-regulation of the transforming growth factor-beta (TGF-beta) signaling system is a strategy for preventing scarring during wound healing. Human corneal and limbal fibroblasts were cultured on the stromal matrix side of preserved human amniotic membrane. The levels of TGF-beta1, beta2, and beta3 and TGF-beta type II receptor transcripts and TGF-beta1 and beta2 proteins were suppressed as early as 8 hr and more dramatically at 24 hr after contact with an amniotic membrane. This suppressive effect was accompanied by down-regulation of alpha-smooth muscle actin, EDA spliced form of fibronectin, and integrin alpha5. It persisted even when challenged by 10 ng/ml TGF-beta1. In contrast with their counterparts grown on plastic or in collagen gel, such suppression in amniotic membrane cultures remained complete after 1 week of culturing. Cells cultured on amniotic membrane showed significantly reduced [3H]-thymidine incorporation compared to cells cultured on plastic and displayed no DNA fragmentation. These results reveal a novel mechanism by which the TGF-beta signaling system, DNA synthesis, and subsequent myofibroblast differentiation can be suppressed by an amnionic membrane matrix. This action explains in part the antiscarring results of amniotic membrane transplantation used for ocular surface reconstruction, a surgical technique applicable to other subspecialties. It may also explain in part why fetal wound healing is scarless.

Photosystem I, an Improved Model of the Stromal Subunits PsaC, PsaD, and PsaE

An improved electron density map of photosystem I (PSI) calculated at 4-A resolution yields a more detailed structural model of the stromal subunits PsaC, PsaD, and PsaE than previously reported. The NMR structure of the subunit PsaE of PSI from Synechococcus sp. PCC7002 (Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062) has been used as a model to interpret the region of the electron density map corresponding to this subunit. The spatial orientation with respect to other subunits is described as well as the possible interactions between the stromal subunits. A first model of PsaD consisting of a four-stranded beta-sheet and an alpha-helix is suggested, indicating that this subunit partly shields PsaC from the stromal side. In addition to the improvements on the stromal subunits, the structural model of the membrane-integral region of PSI is also extended. The current electron density map allows the identification of the N and C termini of the subunits PsaA and PsaB. The 11-transmembrane alpha-helices of these subunits can now be assigned uniquely to the hydrophobic segments identified by hydrophobicity analyses.

Proton to electron stoichiometry in electron transport of spinach thylakoids

According to the concept of the Q-cycle, the H +/e − ratio of the electron transport chain of thylakoids can be raised from 2 to 3 by means of the rereduction of plastoquinone across the cytochrome b 6 f complex. In order to investigate the H +/e − ratio we compared stationary rates of electron transport and proton translocation in spinach thylakoids both in the presence of the artificial electron acceptor ferricyanide and in the presence of the natural acceptor system ferredoxin+NADP. The results may be summarised as follows: (1) a variability of the H +/e − ratio occurs with either acceptor. H +/e − ratios of 3 (or even higher in the case of the natural acceptor system, see below) are decreased towards 2 if strong light intensity and low membrane permeability are employed. Mechanistically this could be explained by proton channels connecting the plastoquinol binding site alternatively to the lumenal or stromal side of the cytochrome b 6 f complex, giving rise to a proton slip reaction at high transmembrane ΔpH. In this slip reaction protons are deposited on the stromal instead of the lumenal side. In addition to the pH effect there seems to be a contribution of the redox state of the plastoquinone pool to the control of proton translocation; switching over to stromal proton deposition is favoured when the reduced state of plastoquinone becomes dominant. (2) In the presence of NADP a competition of both NADP and oxygen for the electrons supplied by photosystem I takes place, inducing a general increase of the H +/e − ratios above the values obtained with ferricyanide. The implications with respect to the adjustment of a proper ATP/NADPH ratio for CO 2 reduction are discussed.

Electron Transfer between QA and QB in Photosystem II Is Thermodynamically Perturbed in Phototolerant Mutants of Synechocystis sp. PCC 6803

Several random mutations have been generated in the psbA2 gene of Synechocystis sp. PCC 6803 [Narusaka, Y., Murakami, A., Saeki, J., Kobayashi, H., and Satoh, K. (1996) Plant Sci. 115, 261-266]. The phototolerant mutant (I6) carrying all the amino acid substitutions in the lumenal side of D1 protein (S322I, I326F, and F328S) and a site-directed mutant of the same phenotype (NDFS) substituted in the stromal side of the protein (N234D and F260S) were characterized by thermoluminescence measurements. We observed (1) no significant differences in their growth rates at either low or high light irradiance, (2) a downshifted B-band in the NDFS mutant, (3) an upshifted Q-band in the I6 mutant, and (4) a damped period four oscillation of thermoluminescence in the B-band of both mutants. By examining the possible implications of these results on the redox properties of the PS II components in the mutants, we concluded that equilibrium constants for sharing an electron between the primary (QA) and secondary acceptor plastoquinones (QB) are decreased in both mutants.

Three-dimensional Structure of Higher Plant Photosystem I Determined by Electron Crystallography

We describe the three-dimensional structure of higher plant photosystem I (PSI) as obtained by electron microscopy of two-dimensional crystals formed at the grana margins of thylakoid membranes. The negatively stained crystalline areas displayed unit cell dimensions a = 26.6 nm, b = 27.7 nm, and gamma = 90(o), and p22121 plane group symmetry consisting of two monomers facing upward and two monomers facing downward with respect to the membrane plane. Higher plant PSI shows several structural similarities to the cyanobacterial PSI complex, with a prominent ridge on the stromal side of the complex. The stromal ridge is resolved into at least three separate domains that are interpreted as representing the three well characterized stromal subunits, the psa C, D, and E gene products. The lumenal surface is relatively flat but exhibits a distinct central depression that may be the binding site for plastocyanin. Higher plant PSI is of dimensions 15-16 x 11-12.5 nm, and thus leaves a larger footprint in the membrane than its cyanobacterial equivalent (13 x 10.5 nm). It is expected that additional membrane-bound polypeptides will be present in the higher plant PSI. Both higher plant and cyanobacterial complexes span about 8-9 nm in the direction orthogonal to the membrane. This report represents the first three-dimensional structure for the higher plant PSI complex.