Parag R. Chitnis

PsaL subunit is required for the formation of photosystem I trimers in the cyanobacterium Synechocystis sp. PCC 6803

When membranes of the wild type strain of the cyanobacterium Synechocystis sp. PCC 6803 were solubilized with detergents and fractionated by sucrose‐gradient ultracentrifugation, photosystem I could be obtained as trimers and monomers. We could not obtain trimers from the membranes of any mutant strain that lacked PsaL subunit. In contrast, absence of PsaE, PsaD, PsaF, or PsaJ did not completely abolish the ability of photosystem I to form trimers. Furthermore, PsaL is accessible to digestion by thermolysin in the monomers but not in the trimers of photosystem I purified from wild type membranes. Therefore, PsaL is necessary for trimerization of photosystem I and may constitute the trimer‐forming domain in the structure of photosystem I.

The proteome of maize leaves: use of gene sequences and expressed sequence tag data for identification of proteins with peptide mass fingerprints.

As a first step in establishing a proteome database for maize, we have embarked on the identification of the leaf proteins resolved on two‐dimensional (2‐D) gels. We detected nearly 900 spots on the gels with a pH 4–7 gradient and over 200 spots on the gels with a pH 6–11 gradient when the proteins were visualized with colloidal Coomassie blue. Peptide mass fingerprints for 300 protein spots were obtained with matrix assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometer and 149 protein spots were identified using the protein databases. We also searched the pdbEST databases to identify the leaf proteins and verified 66% of the protein spots that had been identified using the protein databases. Sixty‐seven additional protein spots were identified from expressed sequence tags (ESTs). Many abundant leaf proteins are present in multiple spots. Functions of over 50% of the abundant leaf proteins are either unknown or hypothetical. Our results show that EST databases in conjunction with peptide mass fingerprints can be used for identifying proteins from organisms with incomplete genome sequence information.

Recruitment of a Foreign Quinone into the A1 Site of Photosystem I I. GENETIC AND PHYSIOLOGICAL CHARACTERIZATION OF PHYLLOQUINONE BIOSYNTHETIC PATHWAY MUTANTS IN SYNECHOCYSTIS SP. PCC 6803

Genes encoding enzymes of the biosynthetic pathway leading to phylloquinone, the secondary electron acceptor of photosystem (PS) I, were identified inSynechocystis sp. PCC 6803 by comparison with genes encoding enzymes of the menaquinone biosynthetic pathway inEscherichia coli. Targeted inactivation of themenA and menB genes, which code for phytyl transferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, prevented the synthesis of phylloquinone, thereby confirming the participation of these two gene products in the biosynthetic pathway. The menA and menB mutants grow photoautotrophically under low light conditions (20 μE m−2 s−1), with doubling times twice that of the wild type, but they are unable to grow under high light conditions (120 μE m−2 s−1). The menA andmenB mutants grow photoheterotrophically on media supplemented with glucose under low light conditions, with doubling times similar to that of the wild type, but they are unable to grow under high light conditions unless atrazine is present to inhibit PS II activity. The level of active PS II per cell in the menAand menB mutant strains is identical to that of the wild type, but the level of active PS I is about 50–60% that of the wild type as assayed by low temperature fluorescence, P700 photoactivity, and electron transfer rates. PS I complexes isolated from themenA and menB mutant strains contain the full complement of polypeptides, show photoreduction of FA and FB at 15 K, and support 82–84% of the wild type rate of electron transfer from cytochrome c 6 to flavodoxin. HPLC analyses show high levels of plastoquinone-9 in PS I complexes from the menA and menB mutants but not from the wild type. We propose that in the absence of phylloquinone, PS I recruits plastoquinone-9 into the A1site, where it functions as an efficient cofactor in electron transfer from A0 to the iron-sulfur clusters.

Function and organization of Photosystem I polypeptides.

Photosystem I functions as a plastocyanin:ferredoxin oxidoreductase in the thylakoid membranes of chloroplasts and cyanobacteria. The PS I complex contains the photosynthetic pigments, the reaction center P700, and five electron transfer centers (A0, A1, FX, FA, and FB) that are bound to the PsaA, PsaB, and PsaC proteins. In addition, PS I complex contains at least eight other polypeptides that are accessory in their functions. Recent use of cyanobacterial molecular genetics has revealed functions of the accessory subunits of PS I. Site-directed mutagenesis is now being used to explore structure-function relations in PS I. The overall architecture of PSI complex has been revealed by X-ray crystallography, electron microscopy, and biochemical methods. The information obtained by different techniques can be used to propose a model for the organization of PS I. Spectroscopic and molecular genetic techniques have deciphered interaction of PS I proteins with the soluble electron transfer partners. This review focuses on the recent structural, biochemical and molecular genetic studies that decipher topology and functions of PS I proteins, and their interactions with soluble electron carriers.

Recruitment of a Foreign Quinone into the A1 Site of Photosystem I II. STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF PHYLLOQUINONE BIOSYNTHETIC PATHWAY MUTANTS BY ELECTRON PARAMAGNETIC RESONANCE AND ELECTRON-NUCLEAR DOUBLE RESONANCE SPECTROSCOPY

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance studies of the photosystem (PS) I quinone acceptor, A1, in phylloquinone biosynthetic pathway mutants are described. Room temperature continuous wave EPR measurements at X-band of whole cells of menA andmenB interruption mutants show a transient reduction and oxidation of an organic radical with a g-value and anisotropy characteristic of a quinone. In PS I complexes, the continuous wave EPR spectrum of the photoaccumulated Q−radical, measured at Q-band, and the electron spin-polarized transient EPR spectra of the radical pair P700+ Q−, measured at X-, Q-, and W-bands, show three prominent features: (i) Q− has a larger g-anisotropy than native phylloquinone, (ii) Q− does not display the prominent methyl hyperfine couplings attributed to the 2-methyl group of phylloquinone, and (iii) the orientation of Q− in the A1 site as derived from the spin polarization is that of native phylloquinone in the wild type. Electron spin echo modulation experiments on P700+ Q− show that the dipolar coupling in the radical pair is the same as in native PS I,i.e. the distance between P700+ and Q− (25.3 ± 0.3 Å) is the same as between P700+ and A1 − in the wild type. Pulsed electron-nuclear double resonance studies show two sets of resolved spectral features with nearly axially symmetric hyperfine couplings. They are tentatively assigned to the two methyl groups of the recruited plastoquinone-9, and their difference indicates a strong inequivalence among the two groups when in the A1site. These results show that Q (i) functions in accepting an electron from A0 − and in passing the electron forward to the iron-sulfur clusters, (ii) occupies the A1 site with an orientation similar to that of phylloquinone in the wild type, and (iii) has spectroscopic properties consistent with its identity as plastoquinone-9.

Identification of Surface-Exposed Domains on the Reducing Side of Photosystem I

Photosystem I (PSI) is a multisubunit enzyme that catalyzes the light-driven oxidation of plastocyanin or cytochrome c6 and the concomitant photoreduction of ferredoxin or flavodoxin. To identify the surface-exposed domains in PSI of the cyanobacterium Synechocystis sp. PCC 6803, we mapped the regions in PsaE, PsaD, and PsaF that are accessible to proteases and N-hydroxysuccinimidobiotin (NHS-biotin). Upon exposure of PSI complexes to a low concentration of endoproteinase glutamic acid (Glu)-C, PsaE was cleaved to 7.1- and 6.6-kD N-terminal fragments without significant cleavage of other subunits. Glu63 and Glu67, located near the C terminus of PsaE, were the most likely cleavage sites. At higher protease concentrations, the PsaE fragments were further cleaved and an N-terminal 9.8-kD PsaD fragment accumulated, demonstrating the accessibility of Glu residue(s) in the C-terminal domain of PsaD to the protease. Besides these major, primary cleavage products, several secondary cleavage sites on PsaD, PsaE, and PsaF were also identified. PsaF resisted proteolysis when PsaD and PsaE were intact. Glu88 and Glu124 of PsaF became susceptible to endoproteinase Glu-C upon extensive cleavage of PsaD and PsaE. Modification of PSI proteins with NHS-biotin and subsequent cleavage by endoproteinase Glu-C or thermolysin showed that the intact PsaE and PsaD, but not their major degradation products lacking C-terminal domains, were heavily biotinylated. Therefore, lysine-74 at the C terminus of PsaE was accessible for biotinylation. Similarly, lysine-107, or lysine-118, or both in PsaD could be modified by NHS-biotin.

Recruitment of a Foreign Quinone into the A1 Site of Photosystem I ALTERED KINETICS OF ELECTRON TRANSFER IN PHYLLOQUINONE BIOSYNTHETIC PATHWAY MUTANTS STUDIED BY TIME-RESOLVED OPTICAL, EPR, AND ELECTROMETRIC TECHNIQUES

Interruption of the menAor menB gene in Synechocystis sp. PCC 6803 results in the incorporation of a foreign quinone, termed Q, into the A1 site of photosystem I with a number of experimental indicators identifying Q as plastoquinone-9. A global multiexponential analysis of time-resolved optical spectra in the blue region shows the following three kinetic components: 1) a 3-ms lifetime in the absence of methyl viologen that represents charge recombination between P700+ and an FeS− cluster; 2) a 750-μs lifetime that represents electron donation from an FeS−cluster to methyl viologen; and 3) an ∼15-μs lifetime that represents an electrochromic shift of a carotenoid pigment. Room temperature direct detection transient EPR studies of forward electron transfer show a spectrum of P700+ Q− during the lifetime of the spin polarization and give no evidence of a significant population of P700+ FeS− fort ≤ 2–3 μs. The UV difference spectrum measured 5 μs after a flash shows a maximum at 315 nm, a crossover at 280 nm, and a minimum at 255 nm as well as a shoulder at 290–295 nm, all of which are characteristic of the plastoquinone-9 anion radical. Kinetic measurements that monitor Q at 315 nm show a major phase of forward electron transfer to the FeS clusters with a lifetime of ∼15 μs, which matches the electrochromic shift at 485 nm of the carotenoid, as well as an minor phase with a lifetime of ∼250 μs. Electrometric measurements show similar biphasic kinetics. The slower kinetic phase can be detected using time-resolved EPR spectroscopy and has a spectrum characteristic of a semiquinone anion radical. We estimate the redox potential of plastoquinone-9 in the A1site to be more oxidizing than phylloquinone so that electron transfer from Q− to F X is thermodynamically unfavorable in the menA and menB mutants.

Proteomic study of the peripheral proteins from thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803.

Thylakoid membranes of cyanobacteria and plants contain enzymes that function in diverse metabolic reactions. Many of these enzymes and regulatory proteins are associated with the membranes as peripheral proteins. To identify these proteins, we separated and identified the peripheral proteins of thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Trichloroacetic acid (TCA)‐acetone extraction was used to enrich samples with peripheral proteins and to remove integral membrane proteins. The proteins were separated by two‐dimensional electrophoresis (2‐DE) and identified by peptide mass fingerprinting. More than 200 proteins were detected on the sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gel that was stained with colloidal Coomassie blue. We analyzed 116 spots by peptide mass fingerprinting and identified 78 spots that were derived from 51 genes. Some proteins were found in multiple spots, indicating differential modifications resulting in charge differences. Therefore, a significant fraction of the peripheral proteins in thylakoid membranes is modified post‐translationally. In our analysis, products of 17 hypothetical genes could be identified in the peripheral protein fraction. Therefore, proteomic analysis is a powerful tool to identify location of the products of hypothetical genes and to characterize complexity in gene expression due to post‐translational modifications.

Associating protein activities with their genes: rapid identification of a gene encoding a methylglyoxal reductase in the yeast Saccharomyces cerevisiae

Methylglyoxal is associated with a broad spectrum of biological effects, including cytostatic and cytotoxic activities. It is detoxified by the glyoxylase system or by its reduction to lactaldehyde by methylglyoxal reductase. We show that methylglyoxal reductase (NADPH‐dependent) is encoded by GRE2 (YOL151w). We associated this activity with its gene by partially purifying the enzyme and identifying by MALDI–TOF the proteins in candidate bands on SDS–PAGE gels whose relative intensities correlated with specific activity through three purification steps. The candidate proteins were then purified using a glutathione‐S‐transferase tag that was fused to them, and tested for methylglyoxal reductase activity. The advantage of this approach is that only modest protein purification is required. Our approach should be useful for identifying many of the genes that encode the metabolic pathway enzymes that have not been associated with a gene (about 275 in S. cerevisiae, by our estimate). Copyright

Isolation and functional study of photosystem I subunits in the cyanobacterium Synechocystis sp. PCC 6803.

Publisher Summary This chapter describes biochemical and molecular genetic methods and resources to study photosystem I (PSI) of Synechocystis sp. PCC 6803. In recent years, cyanobacteria have been used increasingly to study structure-function relations in photosynthetic proteins, including PSI. The mesophilic cyanobacterium, Synechocystis sp. PCC 6803 is a model system for using molecular genetic approaches to study functions of PSI proteins. Its genome is completely sequenced and the protein components of its photosystem are identified. Targeted mutations in all genes for PSI proteins are available. To study the topography of PSI, it is important to probe the exposed protein surface by modification of specific residues. Two biotin derivatives can be used to modify surface-exposed residue. N-hydroxysuccinimidobiotin (NHS-biotin) specifically reacts with the N terminus and the e-amino group of lysyl residues, while biotin-maleimide (M-biotin) modifies the sulfhydryl groups of cysteinyl residues. Both reagents are used to study surface-exposed residues in the PSI complex.