A model for the organization of chlorophyll-protein complex of photosystem II and analysis of its photochemical efficiency and excitation migration.

A model is proposed for the organization of chlorophyll-protein complex in photosystem II (PS II) of higher plants. The rates of exciton migration and exciton trapping have been computed using stochastic method to find out the photochemical efficiency of the dimeric PS II. Three dimeric PS II units are assumed to form a group, as transfer of the exciton to the light harvesting bed of the nearest neighbour on either side may only be effective. A relationship has been deduced between the fractions of the reaction centre traps closed and the number of jumps (J) required by the exciton for trapping. The photochemical efficiency and fluorescence quantum yield are computed using J as the parameter in an empirical equation.

An insight into the assembly and organization of photosystem I complex in the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus.

The present study characterizes the assembly and organization of Photosystem I (PSI) complex, and its individual subunits into the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus. PSI is a multiprotein complex that contains peripheral as well as integral subunits. Hence, it serves as a suitable model system for understanding the formation and organization of membrane protein complexes. In the present study, two peripheral cytosol facing subunits of PSI, namely, PsaD and PsaE were overexpressed in E. coli and used for assembly studies. The gene encoding PsaK, an integral membrane spanning subunit of PSI, was cloned and the deduced amino acid sequence revealed PsaK to have two transmembrane alpha-helices. The characterization of the in vitro assembly of the peripheral subunits, PsaD and PsaE, as well as of the integral subunit, PsaK, was performed by incubating each subunit with thylakoids isolated from Mastigocladus laminosus. All three subunits studied were found to assemble into the thylakoids in a spontaneous mechanism, showing no requirement for cytosolic factors or NTP's (nucleotide 5'-triphosphate). Nevertheless, further characterization of the assembly of PsaK revealed its membrane integration to be most efficient at 55 degrees C. The associations and protein-protein interactions between different subunits within the assembled PSI complex were directly quantified by measurements performed using the BIACORE technology. The preliminary results indicated the existence of specific interaction between PsaD and PsaE, and revealed a very high binding affinity between PsaD and the PSI electron acceptor ferridoxin (Kd = 5.8 x 10(-11) M). PsaE has exhibited a much lower binding affinity for ferridoxin (Kd = 3.1 x 10(-5) M), thereby supporting the possibility of PsaE being one of the subunits responsible for the dissociation of ferridoxin from the PSI complex.

A set-up to study photochemically induced dynamic nuclear polarization in photosynthetic reaction centres by solid-state NMR.

Recently, solid-state NMR spectroscopy became a viable method to investigate photosynthetic reaction centres (RCs) on the atomic level. To study the electronic structure of the radical cation state of the RC, occurring after the electron emission, solid-state NMR using an illumination set-up can be exploited. This paper describes the illumination set-up we designed for a standard Bruker wide-bore MAS NMR probe. In addition we demonstrate its application to get information from the active site in photosynthetic reaction centres of Rhodobacter sphaeroides R-26 by photochemically induced dynamic nuclear polarization (photo-CIDNP). Solid-state NMR spectra of natural abundance 13C in detergent solubilized quinone depleted photosynthetic reaction centres under continuous illumination showed exceptionally strong nuclear spin polarization in NMR lines. Both enhanced-absorptive and emissive polarization were seen in the carbon spectrum which could be assigned to a bacteriochlorophyll a (BChl a) cofactor, presumably the special pair BChl a. The sign and intensities of the 13C NMR signals provide information about the electron spin density distribution of the transiently formed radical P.+ on the atomic level.

Addition of C-terminal histidyl tags to PsaL and PsaK1 proteins of cyanobacterial photosystem I.

In vitro mutagenesis was used to produce two photosystem I mutants of the cyanobacterium Synechocystis sp. PCC 6803. The mutant HK and HL contained hexahistidyl tags at the C-termini of the PsaK1 and PsaL subunits, respectively. The HK mutant contained wild-type amounts of trimeric PS I complexes, but the level of hexahistidine-tagged PsaK1 was found only ten per cent in the PS I complexes and membranes of the wild type level. Therefore, attachment of a tag at the C-terminus interferes with the expression or assembly of PsaK1. In contrast, the HL mutant contained a similar level of tagged PsaL as that in the wild type. However, trimeric PS I complexes could not be obtained from this strain, indicating that the C-terminus of PsaL is involved in the formation of PS I trimers. Hexahistidine-tagged complexes of the HL and HK strains could not be purified with Nickel-affinity chromatography, unless photosystem I was denatured with urea, demonstrating that tagged C-termini of PsaK1 and PsaL were embedded inside of the PS I complex. Protection of the C-terminus from trypsin cleavage further supported this conclusion. Thus, histidine tagging allowed us to demonstrate role of C-termini of two proteins of photosystem I.

Prediction of structure of antenna proteins of photosystem II.

Membrane spanning regions of 43 kDa and 47 kDa antenna proteins of photosystem II of thylakoid membranes are theoretically predicted. Prediction of topology of chlorophyll-a and beta-carotene molecules in the proteins and interaction of the proteins with 33 kDa extrinsic protein on the lumenal side of thylakoid membrane is based on the findings reported earlier. Each antenna protein is predicted to have six transmembrane alpha-helices with twelve chlorophyll-a and five beta-carotene molecules binding to it. Both N- and C- terminal ends are proposed to be on the stromal side of thylakoid membrane. The proposed structural model conforms to the reported experimental results from the literature.

Prediction of structure and organisation of chlorophyll a/b binding polypeptides in PS I and II.

Secondary structures, functionally important residues, antigenic sites, membrane spanning segments and hydropathicity of light harvesting chlorophyll a/b binding polypeptides (LHC) are predicted by theoretical methods from the amino acid sequence of the polypeptides. The reported structural features of the Pea LHC (Lhcb 1 gene product) from electron crystallographic studies have been compared by alignment with other types of chlorophyll a/b binding polypeptides for structural prediction. Fifteen conserved residues D85, D89, E113, H116, E/Q133, E/Q181, E189, D/N233, E252, N/H255, Q/E269, E/D/Q280, N281, H285, D288 (number indicates position in the aligned sequence), are identified which are potential ligands to Mg2+ of chlorophylls. Three amino acid residues D89, E/Q131 and D/N 233 are proposed as ligands to chlorophylls b2, a7 and b2 respectively, for which ligands are not identified in electron crystallographic study.

Action of K-crown ether on photosystem II electron transport: characterization of the site of action.

We have investigated the inhibitory effect of K-crown (18-crown-6 potassium picrate) on photosystem II (PSII)-enriched membrane fragments and O2-evolving core complexes. K-crown at 2-4 microM inhibits about half the control level of O2-evolution activity in both types of PSII samples. Oxygen-evolution studies demonstrated that the ether works by inactivating the centres and not by interfering with antenna function or energy transfer to the reaction centre. K-crown does not disrupt binding of the extrinsic proteins associated with O2 evolution nor complex with bound Ca2+ or Cl- cofactors, but rather it directly inhibits electron transfer after the tetrameric Mn cluster. Fluorescence studies on active and Tris-treated samples showed that K-crown does not prevent artificial donors from transferring electrons to PSII but like DCMU inhibits on the acceptor side after QA, the primary quinone acceptor. However, the ether is a leaky inhibitor and may also act as a weak donor when the Mn cluster is not present. Oxygen-production experiments using silicomolybdate as an artificial acceptor (which accepts from both pheophytin and QB in PSII membranes) demonstrated that the inhibition is at or near the DCMU site.