Relationship between the structural and functional changes of the photosynthetic apparatus during chloroplast-chromoplast transition in flower bud of Lilium longiflorum

The relationship between the structural and functional changes of the photosynthetic apparatus in the flower bud of Lilium longiflorum during chloroplast-chromoplast transition was examined. Compared with green petals, there was a five-fold increase of the carotenoid content in yellow petals, whereas the chlorophyll content decreased five-fold. Absorption and emission fluorescence spectra of chromoplasts indicated that newly synthesized carotenoids were not associated with photosystem II (PSII) photochemistry. The maximum quantum yield in the remaining PSII reaction centers remained constant during the chromoplast formation, whereas the photosynthetic electron transport beyond PSII became inhibited, as indicated by a marked decrease of the O2 evolution capacity, of the photochemical quenching of chlorophyll-alpha fluorescence and of the operational quantum yield of photosynthetic electron transport. Deconvoluted fluorescence emission spectra indicated a more rapid degradation of photosystem I (PSI) complexes than of PSII during chromoplast formation. Compared with green petals, the spillover between PSII and PSI decreased by approximately 40% in yellow petals. Our results indicate that during chloroplast-chromoplast transition in the flower bud of L. longiflorum, PSII integrity was preserved longer than the rest of the photosynthetic apparatus.     

Electrochemical oxidation and cleavage of proteins with on-line mass spectrometric detection: Development of an instrumental alternative to enzymatic protein digestion

An electrochemical flow cell coupled on-line to a mass spectrometer is used to oxidize a range of proteins. Oxidation of tyrosine and tryptophan can give rise to peptide bond cleavage at their C-terminal side. This suggests the possible use of electrochemistry as an alternative protein digestion method. For the small proteins insulin and alpha-lactalbumin (6 and 14 kD) almost all potential sites are cleaved, while for the largest successfully tested protein (carbonic anhydrase, 29 kD) 7 of the 15 available sites were specifically cleaved. Several proteins did not produce peptides upon electrochemical oxidation, possibly due to problems with accessibility of tyrosine and tryptophan residues, or to competing oxidation reactions. Peptides were generally not the major oxidation products: non-cleavage oxidation products observed as protein mass + n x 16 Da, presumably by oxidation of tyrosine, tryptophan, cysteine and methionine, account for the major fraction of protein oxidation products. Nevertheless the amount and variety of cleavage products at the present conditions shows good prospects for further improvement of the system. The efficient protein oxidation also allows the use of the EC-MS system as a tool to study protein oxidation reactions in general. The preconditioning and life history and/or age of the electrochemical cell was relevant to the solvent and sample conditions needed for efficient oxidative cleavage as opposed to other oxidation reactions.     

Oxidation of bovine serum albumin: identification of oxidation products and structural modifications

Albumin is an important plasma antioxidant protein, contributing to protecting mechanisms of cellular and regulatory long‐lived proteins. The metal‐catalyzed oxidation (MCO) of proteins plays an important role during oxidative stress. In this study, we examine the oxidative modification of albumin using an MCO in vitro system. Mass spectrometry, combined with off‐line nano‐liquid chromatography, was used to identify modifications in amino acid residues. We have found 106 different residues oxidatively damaged, being the main oxidized residues lysines, cysteines, arginines, prolines, histidines and tyrosines. Besides protein hydroxyl derivatives and oxygen additions, we detected other modifications such as deamidations, carbamylations and specific amino acid oxidative modifications. The oxidative damage preferentially affects particular subdomains of the protein at different time‐points. Results suggest the oxidative damage occurs first in exposed regions near cysteine disulfide bridges with residues like methionine, tryptophan, lysine, arginine, tyrosine and proline appearing as oxidatively modified. The damage extended afterwards with further oxidation of cysteine residues involved in disulfide bridges and other residues like histidine, phenylalanine and aspartic acid. The time‐course evaluation also shows the number of oxidized residues does not increase linearly, suggesting that oxidative unfolding of albumin occurs through a step‐ladder mechanism. Copyright © 2009 John Wiley & Sons, Ltd.     

Bioinspired Artificial Photosynthetic Systems

Mimicking photosynthesis using artificial systems, as a means for solar energy conversion and green fuel generation, is one of the holy grails of modern science. This perspective presents recent advances towards developing artificial photosynthetic systems. In one approach, native photosystems are interfaced with electrodes to yield photobioelectrochemical cells that transform light energy into electrical power. This is exemplified by interfacing photosystem I (PSI) and photosystem II (PSII) as an electrically contacted assembly mimicking the native Z-scheme, and by the assembly of an electrically wired PSI/glucose oxidase biocatalytic conjugate on an electrode support. Illumination of the functionalized electrodes led to light-induced generation of electrical power, or to the generation of photocurrents using glucose as the fuel. The second approach introduces supramolecular photosensitizer nucleic acid/electron acceptor complexes as functional modules for effective photoinduced electron transfer stimulating the subsequent biocatalyzed generation of NADPH or the Pt-nanoparticle-catalyzed evolution of molecular hydrogen. Application of the DNA machineries for scaling-up the photosystems is demonstrated. A third approach presents the integration of artificial photosynthetic modules into dynamic nucleic acid networks undergoing reversible reconfiguration or dissipative transient operation in the presence of auxiliary triggers. Control over photoinduced electron transfer reactions and photosynthetic transformations by means of the dynamic networks is demonstrated.     

Dual selective nitration in Arabidopsis: Almost exclusive nitration of PsbO and PsbP,and highly susceptible nitration of four non‐PSII proteins,including peroxiredoxin II E

Protein tyrosine nitration is a selective process, as revealed in studies of animals. However, evidence for selective protein nitration in plants is scarce. In this study, Arabidopsis plants were exposed to air with or without nitrogen dioxide at 40 ppm for 8 h in light. Proteins extracted from whole leaves or isolated chloroplasts were subjected to 2D PAGE followed by SYPRO Ruby staining and immunoblotting using an anti‐3‐nitrotyrosine antibody. We determined the relative intensity of a spot on an immunoblot (designated RISI), and relative intensity of the corresponding spot on SYPRO Ruby gel (designated RISS). Proteins that exhibited a high RISI value and/or a high RISI/RISS ratio were considered selectively nitrated. In whole leaf proteins from exposed plants, all immunopositive spots were identified as PsbO1, PsbO2 or PsbP1 by PMF. Thus, nitration was exclusive to PsbO and PsbP, extrinsic proteins of photosystem II (PSII). Their RISI/RISS ratio was ≤1.5. Non‐exposed plants showed very faint nitration. In purified chloroplast proteins, PsbO and PsbP accounted for >80% of the total RISI values, while four non‐PSII proteins, including peroxiredoxin II E, exhibited high RISI/RISS ratios (2.5～6.6). Tyr⁹ of PsbO1 was identified as a nitration site. Thus, nitration is selective for two PSII and four non‐PSII proteins in Arabidopsis.     

When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer??s disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8?% and 15?% for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar K_D values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.     

Identification of Nitrotyrosine Containing Peptides using Combined Fractional Diagonal Chromatography (COFRADIC) and Off-Line Nano-LC-MALDI

Protein nitration take place on tyrosine residues under oxidative stress conditions and may influence a number of processes including enzyme activity, protein-protein interactions and phospho-tyrosine signalling pathways. Nitrated proteins have been identified in a number of diseases, however, the study of these proteins has been compromised by the lack of good methods for identifying nitrated proteins, their nitration sites and the level of nitration. Here, we present a method for identification of nitrated peptides that allows the site specific assignment of nitration, is easy to use and reproducible, and opens up for the possibility to quantify the level of nitration of specific peptides as function of different oxidative conditions, namely combined fractional diagonal chromatography (COFRADIC) in combination with off-line nano-LC-MALDI. We identify six nitrated peptides from in vitro nitrated bovine serum albumin and propose that automated COFRADIC using nano-LC and off-line MALDI-MS might be a possibility for identification of tyrosine nitrated proteins and the nitration sites in complex samples.     

ON THE FORMATION OF PHOTOSYNTHETIC MEMBRANES IN BEAN PLANTS*

Abstract— The formation of lamellar chlorophyll-protein complexes I and II, solubilized by sodium dodecyl sulfate, was studied by hydroxylapatite column chromatography during greening of etiolated Phaseohis vulgaris leaves.
The protein moiety of both complexes preexists in the prolamellar body of etiolated tissue. The complex II to complex I protein ratio is of the order of 0.5. During greening in intermittent illumination the 'proto'-chloroplast is agranal, and contains 'primary' thylakoids and chlorophyll a (Chl a ). At this stage the complex II to complex I protein ratio increases only slightly. Further greening of the plant tissue in continuous illumination results in grana, Chi b (chlorophyll b ) and more Chl a formation. The complex II to complex I protein ratio in unfractionated thylakoids is now of the order of 2.5, while in grana it is of the order of 4.0.
The binding of chlorophyll formed during greening to the protein moiety of the two complexes is found to be selective. The Chi a selectively formed under intermittent illumination is more strongly bound to the complex I protein. The Chi b and Chl a formed in continuous illunination are found bound to both complex I and complex II proteins.
Analysis by hydroxylapatite column chromatography of subchloroplast fractions obtained by different fractionation procedures have shown that these two chlorophyll-protein complexes are most probably derived from the PSI (photosystem I) and PSII (photosystem II) particles of the photosynthetic membrane. These findings suggest that PSI units are assembled ahead of PSII units. Moreover, they indicate that the complex I protein is the main protein component in the prolamellar body membranes, the 'primary' thylakoids. and the stroma lamellae, while in the grana membranes the major protein is the complex II protein. Finally our results show that formation of the photosynthetic membranes is a multi-step process.     

Mass spectrometry‐based proteomics of oxidative stress: Identification of 4‐hydroxy‐2‐nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins

Modification of proteins by 4‐hydroxy‐2‐nonenal (HNE), a reactive by‐product of ω6 polyunsaturated fatty acid oxidation, on specific amino acid residues is considered a biomarker for oxidative stress, as occurs in many metabolic, hereditary, and age‐related diseases. HNE modification of amino acids can occur either via Michael addition or by formation of Schiff‐base adducts. These modifications typically occur on cysteine (Cys), histidine (His), and/or lysine (Lys) residues, resulting in an increase of 156 Da (Michael addition) or 138 Da (Schiff‐base adducts), respectively, in the mass of the residue. Here, we employed biochemical and mass spectrometry (MS) approaches to determine the MS “signatures” of HNE‐modified amino acids, using lysozyme and BSA as model proteins. Using direct infusion of unmodified and HNE‐modified lysozyme into an electrospray quadrupole time‐of‐flight mass spectrometer, we were able to detect up to seven HNE modifications per molecule of lysozyme. Using nanoLC‐MS/MS, we found that, in addition to N‐terminal amino acids, Cys, His, and Lys residues, HNE modification of arginine (Arg), threonine (Thr), tryptophan (Trp), and histidine (His) residues can also occur. These sensitive and specific methods can be applied to the study of oxidative stress to evaluate HNE modification of proteins in complex mixtures from cells and tissues under diseased versus normal conditions.     

Hydroxyl radical probe of the surface of lysozyme by synchrotron radiolysis and mass spectrometry.

A new approach is reported that combines synchrotron radiolysis and mass spectrometry to probe the surface of proteins. Hydroxyl radicals produced upon the radiolysis of protein solutions with synchrotron light for several milliseconds result in the reaction of amino acid side chains. This results in the formation of stable oxidation products where the level of oxidation at the reactive residues is influenced by the accessibility of their side chains to the bulk solvent. The aromatic and sulfur-containing residues have been found to react preferentially in accord with previous peptide studies. The sites of oxidation have been determined by tandem mass spectrometry. The rate of oxidation at these reactive markers has been measured for each of the proteolytic peptides as a function of exposure time based on the relative proportion of modified and unmodified peptide ions detected by mass spectrometry. Oxidation rates have been found to correlate closely with a theoretical measure of the accessibility of residue side chains to the bulk solvent in the native protein structure. The synchrotron-based approach is able to distinguish the relative accessibility of the tryptophan residue side chains of lysozyme at positions 62 and 123 from each other and all other tryptophan residues based on their rates of oxidation.     

Electrospray-assisted modification of proteins: a radical probe of protein structure

A new approach is described to probe the structure of proteins through their reactivity with oxygen-containing radicals. Radical-induced oxidative modification of proteins is achieved within an electrospray ion source using oxygen as a reactive nebulizer gas at high needle voltages. This method facilitates the rapid oxidation of proteins as the molecules emerge from the electrospray needle tip. Electrospray mass spectra of both ubiquitin and lysozyme reveal that over 50% of the protein can be modified under these conditions. The radical-induced oxidative modification of amino acid side chains is correlated with their solvent accessibility to obtain information on a protein's higher-order structure. The oxidation sites in hen lysozyme have been identified by proteolysis of the condensed protein solution and tandem mass spectrometry (MS/MS). Oxidation of tryptophan at positions 62 and 123 occurs exclusively over all other tryptophan residues, consistent with the relative solvent accessibilities of the residue side chains based on the NMR structure of the protein. Radical-induced oxidative modification of cysteine (Cys), methionine (Met), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), proline (Pro), histidine (His), and leucine (Leu) residues is also reported, providing sufficient reactive markers to span a protein sequence. This facile oxidation process could be applied to investigate the molecular mechanism by which reactive oxygen species interact with a particular protein domain as a means to investigate the onset of certain diseases.     

Identification of the tyrosine nitration sites in human endothelial nitric oxide synthase by liquid chromatography-mass spectrometry

The formation of nitric oxide (NO) in biological systems has led to the discovery of a number of post- translational protein modifications that can affect biological conditions such as vasodilation. Studies both from our laboratory and others have shown that beside its effect on cGMP generation from soluble guanylate cylcase, NO can produce protein modifications through both S-nitrosylation of cysteine residues. Previously, we have identified the potential S-nitrosylation sites on endothelial NO synthase (eNOS). Thus, the goal of this study was to further increase our understanding of reactive nitrogen protein modifications of eNOS by identifing tyrosine residues within eNOS that are susceptible to nitration in vitro. To accomplish this, nitration was carried out using tetranitromethane followed by tryptic digest of the protein. The resulting tryptic peptides were analyzed by liquid chromatography/mass spectrometry (LC/MS) and the position of nitrated tyrosines in eNOS were identified. The eNOS sequence contains 30 tyrosine residues and our data indicate that multiple tyrosine residues are capable of being nitrated. We could identify 25 of the 30 residues in our tryptic digests and 19 of these were susceptible to nitration. Interstingly, our data identified four tyrosine residues that can be modified by nitration that are located in the region of eNOS responsible for the binding to heat shock protein 90 (Hsp90), which is responsible for ensuring efficient coupling of eNOS.     

Electrochemically Gated Long‐Distance Charge Transport in Photosystem I

The transport of electrons along photosynthetic and respiratory chains involves a series of enzymatic reactions that are coupled through redox mediators, including proteins and small molecules. The use of native and synthetic redox probes is key to understanding charge transport mechanisms and to the design of bioelectronic sensors and solar energy conversion devices. However, redox probes have limited tunability to exchange charge at the desired electrochemical potentials (energy levels) and at different protein sites. Herein, we take advantage of electrochemical scanning tunneling microscopy (ECSTM) to control the Fermi level and nanometric position of the ECSTM probe in order to study electron transport in individual photosystem I (PSI) complexes. Current–distance measurements at different potentiostatic conditions indicate that PSI supports long‐distance transport that is electrochemically gated near the redox potential of P700, with current extending farther under hole injection conditions.     

Ultraviolet resonance Raman microprobe spectroscopy of photosystem II

Photosystem II (PSII) carries out photosynthetic oxygen production and is responsible for the maintenance of aerobic, heterotrophic life. In PSII, protein amino acid residues play an important role in the light-driven electron transfer reactions. Here, we describe an approach to enhancing vibrational signals from PSII proteins through ultraviolet resonance Raman (UVRR) and a microprobe jet flow technique. Our work shows that pump-probe UVRR can be used to monitor intermediates during photosynthetic oxygen evolution.     

Analysis of Redox Activity of Proteins on the Carbon Screen Printed Electrodes

Direct redox activity of different proteins was investigated on the surface of carbon screen printed electrodes (SPE). The signal attributed to the electrochemical oxidation of amino acid residues (cysteine (Cys), tryptophan (Trp) and tyrosine (Tyr)) was registered at E_max from 0.6 to 0.7 V (vs. Ag/AgCl). Based on the difference in the redox behavior of L ‐tyrosine and 3‐nitro‐L ‐tyrosine, the selective electrochemical detection of native and nitrated albumins was demonstrated. It was shown that the electrochemical signal correlated with the surface density of electroactive amino acid residues on the protein molecule. A simple electrochemical method for the total protein analysis was proposed.     

Immobilization of photosystem I or II complexes on electrodes for preparation of photoenergy-conversion devices

Photosystems, PSI and PSII isolated from Thermosynechococcus elongatus were successfully immobilized on a TiO₂ nanostructured film for use in dye-sensitized biosolar cells (DSBCs). The photosystem complexes were also immobilized on an ITO electrode modified with 3-aminopropyltriethoxysilane by utilizing the interactions between the electrode and the surface of the PSI or PSII polypeptide. Illumination of the PSI and PSII complexes immobilized on the ITO electrode resulted in action spectra in the presence of methyl viologen, which corresponded to the absorption spectra of the complexes. Compared with the ITO electrode, PSI or PSII complexes assembled on the TiO₂ electrode had much higher energy-conversion efficiency in the presence of an iodide/triiodide redox system of an ionic-liquid-based electrolyte. This could have interesting applications in the development of DSBCs.     

Evidence for a post-translational modification, aspartyl aldehyde, in a photosynthetic membrane protein

In oxygenic photosynthesis, photosystem II (PSII) carries out the oxidation of water and reduction of plastoquinone. Three PSII subunits contain reactive groups that covalently bind amines and phenylhydrazine. It has been proposed that these reactive groups are carbonyl-containing, co- or post-translationally modified amino acids. To identify modified amino acid residues in one of the PSII subunits (CP47), tandem mass spectrometry was performed. Modified residues were affinity-tagged with either biotin-LC-hydrazide or biocytin hydrazide, which are known to label carbonyl groups. The affinity-tagged subunit was isolated by denaturing gel electrophoresis, and tryptic peptides were then subjected to affinity purification and tandem mass spectrometry. This procedure identified a hydrazide-labeled peptide, which has the sequence XKEGR. This result is supported by quantitative results acquired from peptide mapping and methylamine labeling. The gene sequence and these tandem data predict that the first amino acid, X, which is labeled with the hydrazide reagent, is a modified form of aspartic acid. On the basis of these data, we propose that D348 of the CP47 subunit is post- or co-translationally modified to give a novel amino acid side chain, aspartyl aldehyde.     

Comparison by PAM fluorometry of photosynthetic activity of nine marine phytoplankton grown under identical conditions

The photosynthetic activity of marine phytoplankton from five algal classes (Phaeodactylum tricornutum, Skeletonema costatum, Thalassiosira oceanica, Thalassiosira weissflogii, Dunaliella tertiolecta, Mantoniella squamata, Emiliania huxleyi, Pavlova lutheri and Heterosigma akashiwo) was investigated under identical growth conditions to determine interspecies differences. Primary photochemistry and electron transport capacity of individual species were examined by pulse amplitude-modulated (PAM) fluorescence. Although few differences were found in maximal photosystem II (PSII) photochemical efficiency between various species, large differences were noticed in their PSII-photosystem I (PSI) electron transport activity. We found that species such as T. oceanica and M. squamata have much lower photochemical activity than H. akashiwo. It appeared that processes involved in electron transport activity were more susceptible to change during algal evolution compared with the primary photochemical act close to PSII. Large variations in the nonphotochemical energy dissipation event among species were also observed. Light energy required to saturate photosynthesis was very different between species. We have shown that M. squamata and H. akashiwo required higher light energy (>1300 micromol m(-2) s(-1)) to saturate photosynthesis compared with S. costatum and E. huxleyi (ca 280 micromol m(-2) s(-1)). These differences were interpreted to be the result of variations in the size of light-harvesting complexes associated with PSII. These disparities in photosynthetic activity might modulate algal community structure in the natural environment where light energy is highly variable. Our results suggest that for an accurate evaluation of primary productivity from fluorescence measurements, it is essential to know the species composition of the algal community and the individual photosynthetic capacity related to the major phytoplankton species present in the natural phytoplankton assemblage.     

TRIPLET STATE STUDIES OF FLAVlNS BY ELECTRON PARAMAGNETIC RESONANCE—II

Abstract— The triplet states of proteins, bovine serum albumin, ovalbumin and d-amino acid oxidase, were observed by electron paramagnetic resonance at 77°K.
The triplet state of aromatic amino acids, tryptophan, tyrosine and phenylalanine was also detected.
The protein triplet originates from the tryptophan residues of these proteins.
It is suggested that an energy transfer takes place between tyrosine and tryptophan.     

LIGHT-ABSORPTION AND ELECTRON-TRANSPORT BALANCE BETWEEN PHOTOSYSTEM II AND PHOTOSYSTEM I IN SPINACH CHLOROPLASTS

Abstract— The distribution of absorbed light and the turnover of electrons by the two photosystems in spinach chloroplasts was investigated. This was implemented upon quantitation of photochemical reaction centers, chlorophyll antenna size and composition of each photosystem (PS), and rate of light absorption in situ. In spinach chloroplasts, the photosystem stoichiometry was PSIIJPSII_α/PSII_β/PSI= 1.3/0.4/1.0. The number (N) of chlorophyll (a+b) molecules associated with each PS was N(PSII_α)/N(PSII_β)/N(PSI)=230/100/200, i.e. about 65% of all Chl is associated with PSII and about 35% with PSI. Light absorption by PSII in vivo is selectively attenuated at the molecular, membrane and leaf levels, (a) The rate of light absorption by PSII was only 0.85 that of PSI because of the lower rate of light absorption by Chl b as compared to Chl a (approximately 80% of all Chl b in the chloroplast is associated with PSII). (b) The exclusive localization of PSII_α in the membrane of the grana partition regions and of PSI in intergrana lamellae resulted in a differential “sieve effect” or “flattening of absorbance” by the photosystems in the two membrane regions. Due to this phenomenon, the rate of light absorption by PSII was lower than that of PSI by 15-20%. (c) Selective filtering of sunlight through the spinach leaf results in a substantial distortion of the effective absorbance spectra and concomitant attenuation of light absorption by the two photosystems. Such attenuation was greater for PSII than for PSI because the latter benefits from light absorption in the 700-730 nm region. It is concluded that, in spite of its stoichiometric excess in spinach chloroplasts, light absorption by PSII is not greater than that by PSI due to the different molecular composition of the two light-harvesting antenna systems, due to the localization of PSII in the grana, and also because of the light transmission properties through the leaf. The elevated PSII/PSI reaction center ratio of 1.7 and the association of 65% of all Chl with PSII help to counter the multilevel attenuation of light absorption by PSII and ensure a balanced PSII/PSI electron turnover ratio of about 1:1.