Complex Of Spinach Research Articles

Ultrafast excited-state dynamics in land plants Photosystem I core and whole supercomplex under oxidised electron donor conditions.

The kinetics of excited-state energy migration were investigated by femtosecond transient absorption in the isolated Photosystem I-Light-Harvesting Complex I (PSI-LHCI) supercomplex and in the isolated PSI core complex of spinach under conditions in which the terminal electron donor P700 is chemically pre-oxidised. It is shown that, under these conditions, the relaxation of the excited state is characterised by lifetimes of about 0.4ps, 4.5ps, 15ps, 35ps and 65ps in PSI-LHCI and 0.15ps, 0.3ps, 6ps and 16ps in the PSI core complex. Compartmental spectral-kinetic modelling indicates that the most likely mechanism to explain the absence of long-lived (ns) excited states is the photochemical population of a radical pair state, which cannot be further stabilised and decays non-radiatively to the ground state with time constants in the order of 6-8ps.

Biochemical and Spectroscopic Characterization of Highly Stable Photosystem II Supercomplexes from Arabidopsis

Photosystem II (PSII) is a large membrane supercomplex involved in the first step of oxygenic photosynthesis. It is organized as a dimer, with each monomer consisting of more than 20 subunits as well as several cofactors, including chlorophyll and carotenoid pigments, lipids, and ions. The isolation of stable and homogeneous PSII supercomplexes from plants has been a hindrance for their deep structural and functional characterization. In recent years, purification of complexes with different antenna sizes was achieved with mild detergent solubilization of photosynthetic membranes and fractionation on a sucrose gradient, but these preparations were only stable in the cold for a few hours. In this work, we present an improved protocol to obtain plant PSII supercomplexes that are stable for several hours/days at a wide range of temperatures and can be concentrated without degradation. Biochemical and spectroscopic properties of the purified PSII are presented, as well as a study of the complex solubility in the presence of salts. We also tested the impact of a large panel of detergents on PSII stability and found that very few are able to maintain the integrity of PSII. Such new PSII preparation opens the possibility of performing experiments that require room temperature conditions and/or high protein concentrations, and thus it will allow more detailed investigations into the structure and molecular mechanisms that underlie plant PSII function.

Promotion of nano-anatase TiO 2 on the spectral responses and photochemical activities of D1/D2/Cyt b559 complex of spinach

Previous researches approved that photocatalysis activity of nano-TiO 2 could obviously increase photosynthetic effects of spinach, but the mechanism of improving light energy transfer and conversion is still unclear. In the present we investigated effects of nano-anatase TiO 2 on the spectral responses and photochemical activities of D1/D2/Cyt b559 complex of spinach. Several effects of nano-anatase TiO 2 were observed: (1) UV–vis spectrum was blue shifted in both Soret and Q bands, and the absorption intensity was obviously increased; (2) resonance Raman spectrum showed four main peaks, which are ascribed to carotene, and the Raman peak intensity was as 6.98 times as that of the control; (3) the fluorescence emission peak was blue shifted and the intensity was decreased by 23.59%; (4) the DCPIP photoreduction activity showed 129.24% enhancement; (5) the oxygen-evolving rate of PS II was elevated by 51.89%. Taken together, the studies of the experiments showed that nano-anatase TiO 2 had bound to D1/D2/Cyt b559 complex, promoted the spectral responses, leading to the improvement of primary electron separation, electron transfer and light energy conversion of D1/D2/Cyt b559 complex.

Purification and crystallization of the cytochrome b6f complex in oxygenic photosynthesis.

The cytochrome b6f complex from the thermophilic cyanobacterium Mastigocladus laminosus and spinach chloroplasts has been purified as a dimeric species. It was found by electrospray ionization mass spectroscopy to contain eight and nine subunits, respectively, and dimeric masses of 217,070 and 286,454 Da. The subunits common to the complex from both sources are petA (cytochrome f), B (cytochrome b6), C (Rieske iron-sulfur protein), D (subunit IV), and small 3.2-4.2 kDa polypeptides petG,L,M, and N. The ninth polypeptide, the 35 kDa petH poly-peptide in the spinach complex, was identified as ferredoxin NADP reductase (FNR), which binds to the complex tightly at a stoichiometry of approx 0.9 (cyt f)-1. The spinach complex contains diaphorase activity diagnostic of FNR, and is active in facilitating ferredoxin-dependent electron transfer from NADPH to the cytochrome b6f complex. The purified cytochrome b6f complex contains stoichiometrically bound chlorophyll a and beta-carotene at a ratio of one per cytochrome f, and bound lipid, in which MGDG and PG are the most abundant species. The delipidated highly purified complexes are active immediately after preparation and for approx 1 wk if left on ice, transferring 300-350 electrons/cyt f/s. Both complexes are subject to proteolysis and associated loss of activity if left for extended periods (>1 wk) at room temperature. Addition of pure synthetic lipid to the delipidated M. laminosus complex (the "lipid augmentation" technique) allows rapid and ready formation of large (>0.2 mm) crystals suitable for x-ray diffraction analysis and structure determination, which diffract with good statistics to 3.0 A.

Full Subunit Coverage Liquid Chromatography Electrospray Ionization Mass Spectrometry (LCMS+) of an Oligomeric Membrane Protein: Cytochrome b6f Complex From Spinach and the Cyanobacterium Mastigocladus Laminosus

Highly active cytochrome b(6)f complexes from spinach and the cyanobacterium Mastigocladus laminosus have been analyzed by liquid chromatography with electrospray ionization mass spectrometry (LCMS+). Both size-exclusion and reverse-phase separations were used to separate protein subunits allowing measurement of their molecular masses to an accuracy exceeding 0.01% (+/-3 Da at 30,000 Da). The products of petA, petB, petC, petD, petG, petL, petM, and petN were detected in complexes from both spinach and M. laminosus, while the spinach complex also contained ferredoxin-NADP(+) oxidoreductase (Zhang, H., Whitelegge, J. P., and Cramer, W. A. (2001) Flavonucleotide:ferredoxin reductase is a subunit of the plant cytochrome b(6)f complex. J. Biol. Chem. 276, 38159-38165). While the measured masses of PetC and PetD (18935.8 and 17311.8 Da, respectively) from spinach are consistent with the published primary structure, the measured masses of cytochrome f (31934.7 Da, PetA) and cytochrome b (24886.9 Da, PetB) modestly deviate from values calculated based upon genomic sequence and known post-translational modifications. The low molecular weight protein subunits have been sequenced using tandem mass spectrometry (MSMS) without prior cleavage. Sequences derived from the MSMS spectra of these intact membrane proteins in the range of 3.2-4.2 kDa were compared with translations of genomic DNA sequence where available. Products of the spinach chloroplast genome, PetG, PetL, and PetN, all retained their initiating formylmethionine, while the nuclear encoded PetM was cleaved after import from the cytoplasm. While the sequences of PetG and PetN revealed no discrepancy with translations of the spinach chloroplast genome, Phe was detected at position 2 of PetL. The spinach chloroplast genome reports a codon for Ser at position 2 implying the presence of a DNA sequencing error or a previously undiscovered RNA editing event. Clearly, complete annotation of genomic data requires detailed expression measurements of primary structure by mass spectrometry. Full subunit coverage of an oligomeric intrinsic membrane protein complex by LCMS+ presents a new facet to intact mass proteomics.

Three-dimensional Electron Cryo-microscopy Study of the Extrinsic Domains of the Oxygen-evolving Complex of Spinach: ASSIGNMENT OF THE PsbO PROTEIN

Three independent three-dimensional reconstructions of the spinach photosystem II-light-harvesting complex supercomplex were derived from single particle analyses of non-stained, vitrified samples imaged by electron microscopy. Each reconstruction was found to differ significantly in the composition of the lumenal oxygen-evolving complex extrinsic proteins. From difference mapping, aided by electron microscopy of negatively stained selectively washed samples, regions of density were assigned to the PsbO and PsbP/PsbQ proteins. Interpretation of the density assigned to the PsbO protein was explored using computer-aided structural predictions. PsbO is calculated to be mainly a beta-protein (38% beta) composed of two domains within an overall elongated shape (Pazos, F., Heredia, P., Valencia, A., and De Las Rivas, J. (2001) Proteins Struct. Funct. Genet. 45, 372-381). The positioning and fitting of the proposed structural model for the PsbO protein within the three-dimensional map indicated that there is a single copy per reaction center. Moreover, the structural model derived for PsbO, together with difference mapping, indicates that this protein stretches across the surface of the reaction center with its N- and C-terminal domains located toward the CP47 and CP43 side, respectively. This structural assignment is discussed in terms of the recent x-ray-derived cyanobacterial model of PSII (Zouni, A., Witt, H.-T., Kern, J., Fromme, P., Krauss, N., Saenger, W., and Orth, P. (2001) Nature 409, 739-743).

Mass Spectrometry of the Subunits of Highly Active Cytochrome b6f Complex from Spinach and the Cyanobacterium, M. laminosus: Identification of PetA,B,C,D,G,L,M,N Subunits in both Complexes and FNR in the Spinach b6f Complex

Highly active cytochrome b6f complexes from spinach and the cyanobacterium M. laminosus have been analyzed by liquid-chromatography with electrospray-ionization mass spectrometry (LC-MS). Both size-exclusion and reverse-phase separations were used to separate proteins from other contaminants allowing measurement of protein molecular weights to an accuracy exceeding 0.01 % (+/- 3Da at 30000 Da). Four small subunits corresponding to the products of petG, petL, petM and petN were detected in both complexes. The M. laminosus complex had four larger subunits corresponding to the products of petA, petB, petC and petD, a total of eight subunits. The spinach complex had the same four large subunits and a fifth polypeptide of molecular weight 35314.0 Da, a total of nine subunits. Based upon the mass of CNBr fragments found in a fraction collected simultaneously to the elution of this component we identified the fifth polypeptide as ferredoxin-NADP oxidoreductase (FNR). This is the largest number and range of integral membrane subunits determined by MS techniques in an integral membrane protein. The excellent resolution afforded by ESI-MS revealed profiles that define the native covalent state of the components of the complex as well as their molecular heterogeneity. Supported by NIH AI-12601-24 (JW), NIH GM-18457 (WAC), and the WM Keck Foundation.

The Deletion of petG in Chlamydomonas reinhardtii Disrupts the Cytochrome bf Complex

The 4-kDa protein encoded by chloroplast petG copurifies with the cytochrome bf complex of spinach and is found in a number of other photosynthetic organisms, including the eukaryotic alga Chlamydomonas reinhardtii. To determine whether petG is involved in the function or assembly of the cytochrome bf complex, the gene was cloned from C. reinhardtii, excised from the DNA fragment, and replaced with a spectinomycin resistance cassette. A petG deletion strain of C. reinhardtii was then obtained by biolistic transformation. The resulting homoplasmic petG deletion strains are unable to grow photosynthetically, and immunoblot analysis shows markedly decreased levels of cytochrome b6, cytochrome f, the Rieske iron-sulfur protein, and subunit IV. To verify that this phenotype was due to the removal of petG, we also constructed a strain with a deletion in the open reading frame (ORF56), which is found 25 base pairs downstream of petG. The ORF56 deletion strain grew photosynthetically and had wild-type levels of the four major cytochrome bf subunits. We conclude that the absence of the PetG protein affects either the assembly or stability of the cytochrome bf complex in C. reinhardtii.

Comparison of structure and function of the photosynthetic oxygen-evolving complex of spinach and the green alga Scenedesmus obliquus

Comparison of structure and function of the photosynthetic oxygen-evolving complex of spinach and the green alga Scenedesmus obliquus

Spectral and thermodynamic properties of the two hemes of the D1D2cytochrome b-559 complex of spinach

In agreement with previous work [Shuvalov, Heber and Schreiber (1988) FEBS Lett. 258, 27-31] two hemes (low potential (LP) and extra low potential (XLP )) per two pheophytins were found in isolated D1D2Cyt b-559 complexes. Reductive and oxidative redox titrations demonstrate that the E m of the LP form is at about +150 mV. It is independent of pH between pH 7.2 and 9.4. The XLP heme is autoxidizable at pH 7.2 and displays, at this pH, an E m of − 45 mV. Both the LP and XLP hemes show absorption peaks at 559 nm. They are proposed to have bis-histidine ligation of the heme iron. At pH 9.4, the XLP heme splits into two forms. One of them has an E m of +40 mV, and absorption peaks at 559 nm showing the bis-histidine ligation. The other displays an E m of −220 mV and the peak is shifted to 562 nm. This last form is proposed to be due to the incorporation of OH − which occupies the 6th coordination position of the heme Fe(III) at high pH. The p K value for the conversion of the XLP heme is close to 7.7. In a structure simulation of the α-helices of α- and β-polypeptides, the β-polypeptide, but not the α-polypeptide, reveals a distance between the histidine N and the heme Fe which permits stable N-Fe coordination. In the α-polypeptide, OH − can be incorporated between N and Fe. The functional role of the two hemes of cyt b-559 is briefly discussed with respect to water oxidation and cyclic electron transfer.

Low molecular weight subunits associated with the cytochrome b 6 f complexes from spinach and Chlamydomonas reinhardtii

Cytochrome b 6 f complexes, prepared from spinach and Chlamydomonas thylakoids, have been examined for their content of low molecular weight subunits. The spinach complex contains two prominent low molecular weight subunits of 3.7 and 4.1 kD while a single prominent component of 4.5 kD was present in the Chlamydomonas complex. An estimation of the relative stoichiometry of these subunits suggests several are present at levels approximating one copy per cytochrome complex. The low molecular weight subunits were purified by reversed phase HPLC and N-terminal sequences obtained. Both the spinach and Chlamydomonas cytochrome complexes contain a subunit that is identified as the previously characterized petG gene product (4.8 kD in spinach and 4.1 kD in Chlamydomonas). A second subunit (3.8 kD in spinach and 3.7 kD in Chlamydomonas) appears to be homologous in the two complexes and is likely to be a nuclear gene product. The possible presence of other low molecular weight subunits in these complexes is also considered.

Polypeptide composition of higher plant photosystem I complex: Identification of psaI, psaJ and psaK gene products

High resolution gel electrophoresis of the native photosystem I complex retaining light-harvesting chlorophyll complex revealed the presence of three low-molecular-mass proteins of 7, 4.1 and 3.9 kDa in spinach, and 6.8, 4.4 and 4.1 kDa in pea, in addition to the other well-characterized higher-molecular-mass components. Upon further detergent treatment to deplete light-harvesting chlorophyll complex, the 7 kDa and 4.1 kDa proteins were removed from the photosystem I core complex of spinach, while the 3.9 kDa protein was retained. N-terminal sequencing demonstrated that the 4.1 kDa proteins from both spinach and pea correspond to the gene product of ORF42/44 in chloroplast genome of liverwort and higher plants, which was previously hypothesized as a photosystem I gene ( psaJ) based on sequence homology with the cyanobacterial photosystem I component of 4.1 kDa [(1989) FEBS Lett. 253, 257-263]. N-terminal sequence of the spinach 3.9 kDa and pea 4.4 kDa proteins fitted with chloroplast ORF36/40 ( psaI) although no homologue has been found in cyanobacteria. The spinach 7 kDa and pea 6.8 kDa proteins correspond to the nuclear-encoded psaK product and significantly matched with the N-terminal sequence of the cyanobacterial 6.5 kDa subunit. The evolutional conservation of the psaJ and psaK seems to suggest their intrinsic role(s) in photosystem I.

Chromatographic Purification and Determination of the Carboxy-Terminal Sequences of Photosystem II Reaction Center Proteins, Dl and D2

The D1 and D2 subunits of the reaction center of photosystem II are intrinsic proteins. A procedure was developed for the separation and purification of these two proteins on a large scale from the photosystem II reaction center complex of spinach by high-performance liquid chromatography on a gel-permeation column. The purified D1 and D2 proteins were subjected to determination of their carboxy-terminal sequences by digestion of the proteins with carboxypeptidase Y

The mode of binding of three extrinsic proteins of 33 kDa, 23 kDa and 18 kDa in the photosystem II complex of spinach

Binding of three extrinsic proteins of 33 kDa, 23 kDa and 18 kDa to the oxygen-evolving Photosystem II (PS II) complex was investigated under equilibrium conditions using spinach PS II membranes. Dissociation constants for the functional and stoichiometric binding of the extrinsic proteins were determined to be 12, 2.7 and 3.8 nM for the 33 kDa, 23 kDa and 18 kDa proteins, respectively. These values indicate the binding energy between the proteins and the PS II complex on the order of 10–11 kcal·mol −1 . Effect of removal of the extrinsic proteins and Mn atoms from the PS II complex on the dissociation constant and the number of binding sites for the proteins was also examined. The results indicate that the binding site for the 33 kDa protein is present in the PS II complex devoid of the functional Mn and that the Mn stabilizes the binding of the 33 kDa protein. The change in the binding energy of the 33 kDa protein afforded by the Mn was estimated to be about 1 kcal·mol −1 , indicating that the interaction between the 33 kDa protein and the functional Mn is, if any, very weak. It is also indicated that the 33 kDa protein bound to the PS II complex provides the high-affinity binding site for the 23 kDa protein in the complex and that the 23 kDa protein together with the 33 kDa protein does so for the 18 kDa protein. The measurement of the sedimentation coefficients of the extrinsic proteins and protein mixtures in an aqueous solution showed that the extrinsic proteins did not interact with each other when they were apart from the PS II complex. These observations suggest that the binding of the extrinsic proteins to the PS II complex alters the conformation of the extrinsic proteins themselves and/or that of the intrinsic part of the complex so as to create the binding sites for the other extrinsic proteins.

Evidence that the amino-terminus of the 33 kDa extrinsic protein is required for binding to the Photosystem II complex

Chymotrypsin and Staphylococcus aureus (strain V8) proteinase eliminated sixteen and eighteen amino acid residues, respectively, from the amino-terminal side of the extrinsic 33 kDa protein of the oxygen-evolving Photosystem II (PS II) complex of spinach. The carboxy-terminus of the resultant large fragments was found to be identical with that of the intact protein. Neither fragment could rebind to PS II membranes depleted of all the extrinsic proteins. Circular dichroism spectroscopy did not reveal any major conformational change within the two fragments. These results suggest that the amino-terminal region of the 33 kDa protein contains a domain essential for binding to the PS II complex.

Topological Considerations of the 9-kDa Polypeptide which Contains Centers A and B, Associated with the 14- and 19-kDa Polypeptides in the Photosystem I Complex of Spinach

Etude de la topographie du polypeptide de 9 KDa du PSI de l'epinard par des traitements avec des ions alcalins et chaotropiques, par digestions tryptiques et liaisons croisees d'anticorps diriges contre les polypeptides de 9, 14 et 19 KDa de la membrane des thylakoides. Le modele topologique presente ici est en desaccord avec celui presente par Munch et al. en 1988

Partial degradation of the extrinsic 23-kDa protein of the Photosystem II complex of spinach

Chymotrypsin eliminated nine amino acid residues at the amino-terminal side of the extrinsic 23-kDa protein of the oxygen-evolving Photosystem II complex of spinach. The resultant 22-kDa fragment was able to bind to the Photosystem II complex but with lowered binding affinity. However, once the 22-kDa fragment bound to the complex, it retained most functions of the 23-kDa protein; the fragment provided a binding site for the extrinsic 18-kDa protein, preserved a tight trap for Ca 2+ in the complex, and shifted the optimum Cl − concentration for oxygen evolution from 30 to 10 mM, although it was less effective in sustaining oxygen evolution at Cl − concentrations below 10 mM. These observations suggest that the elimination of nine amino acid residues at the amino-terminal region of the 23-kDa protein does not significantly alter the conformation of the protein, except for partial modification of its binding site and its interaction with Cl −.

Purification of the Chloroplast Pyruvate Dehydrogenase Complex from Spinach and Maize Mesophyll

Abstract This paper deals with the partial purification of the pyruvate dehydrogenase complex (PDC) from chloroplasts of spinach and maize mesophyll which hitherto has been isolated only from pea chloroplasts. Starting with membrane free suspensions of lyophilized chloroplasts, and following a high-speed (140000 Xg) centrifugation of this “stromal extract”, the initial specific PDC-activities were concentrated by a factor 10 in the sediment. While most of the purification procedures described earlier resulted in almost complete loss of enzyme activities, a rate zonal sedimentation on linear glycerol gradients allowed for an additional up to 100-fold enrichment of the labile multienzyme complex, albeit with low yields. In contrast to chloroplast PDC from maize mesophyll, inactivation of the spinach complex during glycerol fractionation was due to the dissociation of its loosely bound dihydrolipoyl dehydrogenase component which collected in a lower density fraction of the gradient. Its recombination with PDC constituents of the bottom layer nearly restored initial activities. The chloroplast complex has been identified as true PDC by its substrate specificity for pyru­vate, NAD+, and coenzyme A and the 1:1:1 stoichiometry of its reaction products NADH, CO2, and acetyl-CoA. The chloroplast PDC of both plant species showed the well known higher pH-and Mg-requirements than the mitochondrial complex. The observed species-specific differences in the stability of this multienzyme system suggest a connection with the aggregation state of its components. Apparently, the individual subcomplexes are able to function either together in acetyl-CoA formation or independently from each other, e.g. in the synthesis of acetolactate via hydroxy-ethyl-thiamine pyrophosphate or dihydrolipoyl dehydrogenase activities.

N-terminal sequence determination and secondary structure analysis of extrinsic membrane proteins in the water-splitting complex of spinach

The N-terminal sequences of the 16 and 23 kDa extrinsic membrane proteins in photosystem II of spinach have been determined by solid-phase sequencing. The sequence of the 23 kDa protein has been corroborated by a small 3 kDa CNBr fragment confirming partial N-terminal processing by a protease of unknown nature. A secondary structure analysis of the 23 kDa protein by circular dichroism has been performed indicating a β-structure content of 61 % with only 14% α-helix conformation.

Trimeric structure and other properties of the chloroplast reductase binding protein

A complex between the ferredoxin-NADP + reductase and a 17.5 kDa polypeptide was isolated from lettuce chloroplasts by the same procedure used previously in spinach [(1984) J. Biol. Chem. 259, 8048-8051]. The stoichiometry of the complex from both sources was determined by quantification of stained protein bands after gel electrophoresis and, in the case of the spinach complex, by rocket inmunolectrophoresis. The results suggest that the binding protein is a trimer of the 17.5 kDa polypeptide. The amino arid composition of the spinach reductase binding protein and partial sequencing of its tryptic peptides clearly show that it is not identical to the 17 kDa polypeptide of the cytochrome b 6-ƒ complex.