Trimeric Photosystem I Research Articles

Fluorescence spectroscopy of the longwave chlorophylls in trimeric and monomeric photosystem I core complexes from the cyanobacterium Spirulina platensis.

The organization and interaction of chlorophylls (Chl) and the kinetics of the energy transfer in the core antenna of photosystem I (PSI) trimeric and monomeric complexes, isolated from Spirulina platensis with Triton X-100 have been studied by stationary and time-resolved fluorescence. At 295 K both complexes show an unusually intense long-wavelength emission band with prominent peaks at 730 nm (trimers) or 715 nm (monomers), whose intensity is independent of the redox state of P700. A broad band extending from 710 to 740 nm in the absorption and fluorescence excitation spectra of trimers also indicates the existence of the longwave Chls at 295 K. The 77 K fluorescence emission of PSI trimers frozen after addition of dithionite under illumination (P700 and the PSI acceptor side reduced) shows an intense band at 760 (F760) and a smaller one at 725 nm (F725); when P700 is oxidized, the intensity of F760 decreases about 15 times. In the 77 K spectrum of monomers only F725 is present in the longwave region, and its intensity does not depend on the redox state of P700. Bands of Chls with maxima near 680, 710, and 738 nm were found in the 77 K excitation spectrum of trimers, and bands near 680 and 710 nm were seen in the spectrum of monomers. Five spectrally different red Chl forms in PSI trimers and three red Chl in monomers have been resolved by deconvolution of their 77 K absorption spectra. The difference absorption spectrum, trimers-minus-monomers, shows that the appearance of the 735 nm band in trimers is accompanied by a decrease of 708, 698, and 688 nm bands present in monomers. The reversible changes of F760 intensity of Spirulina membranes as a result of their salt treatment confirm the idea that the most longwave Chl form originates from an interaction of Chls bound to different monomeric PSI subunits forming the trimer. The time-resolved fluorescence spectra of PSI trimers and monomers, measured at 287 K in the region 680-770 nm, are substantially different, although a set of similar lifetimes (9, approximately 30, approximately 66, and 1400-2200 ps) was necessary for a good fit. No effect of P700 redox state was observed on the fluorescence kinetics of both complexes at 287 K.

Efficient energy transfer from the long-wavelength antenna chlorophylls to P700 in photosystem I complexes from Spirulina platensis

To study the role of the long-wavelength chlorophylls (Chl) in photosystem I (PSI), the action spectra of P700 photooxidation at 293 and 77 K have been measured for PSI trimeric and monomeric complexes isolated from Spirulina platensis. The long-wavelength Chls which absorb in the region 710dash740 nm transfer excitation energy to the reduced P700 with the same efficiency as bulk antenna Chls, causing the oxidation of P700. The relative quantum yield of P700 photooxidation is about unity (293-77 K) even under the direct excitation of Chl absorbing at 735 nm (Chl735). At 77 K Chl735 exhibits a fluorescence band at 760 nm (F760) whose intensity is quenched under illumination of the PSI trimeric complexes from Spirulina. The relative quantum yield of F760 quenching is not dependent on the wavelength of excitation in the region 620–750 nm. Since the value of the overlap integral between the band of F760 and the absorption band of the cation radical of P700 (P700 +) is higher than that of the P700 band, it is suggested that Chl735 transfers energy to P700 + more efficiently than to reduced P700; energy transfer to P700 + causes the quenching of F760. A linear relationship between the photooxidation rate of P700 and the fraction of P700 + at 293 K indicates that the energy exchange between PSI subunits of the trimer is negligible. Thus, the antenna of PSI trimers of Spirulina is organized in separate photosynthetic units.

Isolation and structural characterization of trimeric cyanobacterial photosystem I complex with the help of recombinant antibody fragments.

A monoclonal antibody was derived from mice immunized with the native trimeric, photosystem I (PSI) complex from the cyanobacterium Synechococcus PCC 7002 which reacts with a conformational epitope of the PSI complex. As seen by immunoelectron microscopy, the mAb bound to the stromal side of the thylakoid membranes. The DNA sequence encoding variable regions of the mAb was cloned into recombinant plasmids, sequenced and expressed in Escherichia coli. ELISA, Western blots and immunoelectron microscopy provided evidence that the expressed paired variable domain (Fv) fragments bind to the antigen in the same way as the parent mAb. A one-step purification was applied to purify the trimeric PSI complex using an affinity tag attached to the Fv fragment. Analysis by gel electrophoresis and N-terminal sequencing revealed the presence of the psaA, psaB, psaC, psaD, psaE, psaF and psaL gene products. The antenna size of the isolated PSI/Fv was 139 +/- 9 chlorophyll a/primary electron donor. Flash-induced absorption-change measurements showed that the complex exhibited electron transfer from the primary electron donor, P700, to the Fe-S center, FA/FB. The position of the bound Fv fragment on the trimeric PSI surface was determined by high-resolution electron microscopy and digital image processing.

Isolation and Structural Characterization of Trimeric Cyanobacterial Photosystem I Complex with the Help of Recombinant Antibody Fragments

A monoclonal antibody was derived from mice immunized with the native trimeric, photosystem I (PSI) complex from the cyanobacterium Synechococcus PCC 7002 which reacts with a conformational epitope of the PSI complex. As seen by immunoelectron microscopy, the mAb bound to the stromal side of the thylakoid membranes. The DNA sequence encoding variable regions of the mAb was cloned into recombinant plasmids, sequenced and expressed in Escherichia coli. ELISA, Western blots and immunoelectron microscopy provided evidence that the expressed paired variable domain (Fv) fragments bind to the antigen in the same way as the parent mAb.A one‐step purification was applied to purify the trimeric PSI complex using an affinity tag attached to the Fv fragment. Analysis by gel electrophoresis and N‐terminal sequencing revealed the presence of the psaA, psaB, psaC, psaD, psaE, psaF and psaL gene products. The antenna size of the isolated PSI/Fv was 139±9 chlorophyll a/primary electron donor. Flash‐induced absorption‐change measurements showed that the complex exhibited electron transfer from the primary electron donor, P700, to the Fe‐S center, FA/FB. The position of the bound Fv fragment on the trimeric PSI surface was determined by high‐resolution electron microscopy and digital image processing.

Laser flash absorption spectroscopy study of ferredoxin reduction by photosystem I in Synechocystis sp. PCC 6803: evidence for submicrosecond and microsecond kinetics.

The kinetics of reduction of soluble ferredoxin by photosystem I (PSI), both purified from the cyanobacterium Synechocystis sp. PCC 6803, were investigated by flash-absorption spectroscopy between 460 and 600 nm. Most experiments were made with isolated monomeric PSI reaction centers prepared with the detergent beta-dodecyl maltoside. Analysis of absorption transients, in parallel at 480 and 580 nm and under several conditions, shows the existence of three different first-order components in the presence of ferredoxin (t1/2 approximately 500 ns, 20 microseconds, and 100 microseconds). A second-order phase of ferredoxin reduction is also present [k = (2-5) x 10(8) s-1 at pH 8 and at moderate ionic strength]. Similar first-order kinetic components were found with membranes from Synechocystis, with dissolved crystals of trimeric PSI reaction centers from Synechococcus, and also when ferredoxin from Synechocystis is replaced by ferredoxin from Chlamydomonas reinhardtii. The three first-order phases exhibit similar, though not identical, spectra which are consistent with electron transfer from the [4Fe-4S] centers of PSI to the [2Fe-2S] center of ferredoxin and are all attributed to reduction of ferredoxin bound to PSI. At pH 8 and at moderate ionic strength, the dissociation constants associated with each of these components are also similar, with a global value varying between 0.2 and 0.8 microM in different cyanobacterial preparations. The presence of three exponential components is discussed assuming homogeneity of the two partners and using the estimated values for the shortest possible distance of approach of soluble ferredoxin from the different iron-sulfur centers of PSI. It is concluded that the 500-ns phase corresponds to electron transfer from either FA- or FB-, the terminal iron-sulfur acceptors of PSI, to ferredoxin and that the immediate electron donor to ferredoxin is reduced within less than 500 ns. The presence of at least two different types of PSI-ferredoxin complex, all competent in electron transfer, is also deduced from the kinetic behavior.

Trimeric forms of the photosystem I reaction center complex pre-exist in the membranes of the cyanobacterium Spirulina platensis

Oligomeric and monomeric forms of chlorophyll—protein complexes of photosystem I (PSI) have been isolated from the mesophilic cyanobacterium Spirulina [(1992) FEBS Lett. 309, 340–342]. Electron microscopic analysis of the complexes showed that the oligomeric form is a trimer of the shape and dimensions similar to those of the trimer from thermophilic cyanobacteria. The chlorophyl ratio in the isolated trimer and monomer was found to be 7:3. The trimeric form of PSI complex in contrast to the monomeric one contains the chlorophyll emitting at 760 nm (77K), which is also found in Spirulina membranes and therefore could be used as an intrinsic probe for the trimeric complex. The 77K circular dichroism spectrum of the trimeric form is much more similar to that of Spirulina membranes than the spectrum of the monomer. Thus, the trimeric PSI complexes exist and dominate in the Spirulina membranes.

Monomeric and trimeric forms of photosystem I reaction center of Mastigocladus laminosus: crystallization and preliminary characterization.

Photosystem I (PSI) reaction centers (RCs) of the thermophilic cyanobacterium Mastigocladus laminosus were purified and characterized. The PSI RC was obtained in two forms, monomeric and trimeric. The two forms contained the same number of pigments per P700 and displayed similar photochemical activities. The two forms had nearly identical polypeptide subunit compositions; the only observed difference was an additional subunit of about 12 kDa observed in the trimeric form. The purified preparations of both the monomeric and the trimeric forms were used for crystallization and preliminary crystallographic analysis. The trimeric PSI RC preparations produced several three-dimensional crystal forms, one of which, the "hexagonal needle" form (THN), had a hexagonal unit cell with dimensions of 300 x 300 x 160 A, containing four PSI RC trimers. The monomeric preparations also produced single crystals of several forms under various crystallization conditions. One of these crystal forms, the "hexagonal plate" (MHP), diffracted to a resolution of about 5.5 A. It had a hexagonal unit cell with dimensions of 192 x 192 x 163 A, containing six PSI RC monomers. Comparison of the PSI RCs in the crystals with those in the precrystallization preparations demonstrated that neither the monomeric nor the trimeric form of PSI RC was altered by the crystallization process. Both forms retained their original polypeptide subunit composition and their pigment content.