A photosystem II reaction center complex consisting of D-1 and D-2 polypeptides and cytochrome b-559 was isolated from spinach grana thylakoids, treated with 4% (wt/vol) Triton X-100, by ion-exchange chromatography using DEAE-Toyopearl 650S. The isolated complex appears to contain five chlorophyll a, two pheophytin a, one (3-carotene, and one or two cytochrome b-559 heme(s) (molar ratio) and exhibits a reversible absorbance change attributable to the photochemical accumulation of reduced pheophytin typical for the intermediary electron acceptor of photosystem H reaction center. These results strongly suggest that the site of primary charge separation in photosystemIH is located on the heterodimer composed of D-1 and D-2 subunits.It has been well established that the photosystem II reaction center of oxygenic photosynthetic organisms is contained in a chlorophyll-protein complex consisting of the following six polypeptide subunits: 47-and There is a controversy concerning the localization of the site of primary charge separation in the photosystem II reaction center complex (5-10). Some evidence supports the proposal that the 47-kDa subunit is the site of primary photochemistry (5-8). However, the amino acid sequence homology between the D-1 and D-2 subunits and the L and M subunits of reaction center from purple photosynthetic bacteria has led to a proposal (9, 10) that the D-1 and D-2 polypeptides play a role in the photosystem II reaction center similar to that of L and M subunits in the bacterial reaction center whose structure has been determined by x-ray crystallographic analysis (11).In this paper we provide experimental evidence supporting the latter proposition. We have succeeded in isolating a pigment-protein complex consisting of D-1 and D-2 polypeptides and cytochrome b-559 that is capable of reversible photochemical accumulation of reduced pheophytin. A preliminary account of this work has been presented (12). MATERIALS AND METHODSThe membrane preparation of grana thylakoids (Triton/ photosystem II particles) was prepared from spinach as described by Kuwabara and Murata (13).Pigments and quinones were extracted with 80% (vol/vol) acetone and quantitatively determined by HPLC using a reverse-phase column (ZORBAX-ODS). Following the procedure described (14), methanol/water, 49:1 (vol/vol) and methanol/isopropanol, 3:1 (vol/vol) were used for chromatographic development, and the elution of components was monitored by the absorption either at 440 nm (chlorophylls and carotenoids) or at 255 nm (pheophytin a and plastoquinone-9). The spectrophotometry of the components was based on the absorption coefficients described by Eskins et al. (14) for chlorophylls and carotenoids, by Vernon (15) for pheophytins, and by Barr and Crane (16) for plastoquinones. The amounts of cytochrome b-559 with different midpotentials were determined by the method of Hind and Nakatani (17) using a difference millimolar absorption coefficient (559-570 nm) of 15 (18). NaDodSO4/polyacrylamide gel electrophoresis was carrie...

No abstract

No abstract

No abstract

Genes for -like factors of bacterial-type RNA polymerase have not been characterized from any multicellular eukaryotes, although they probably play a crucial role in the expression of plastid photosynthesis genes. We have cloned three distinct cDNAs, designated SIG1, SIG2, and SIG3, for polypeptides possessing amino acid sequences for domains conserved in 70 factors of bacterial RNA polymerases from the higher plant Arabidopsis thaliana. Each gene is present as one copy per haploid genome without any additional sequences hybridized in the genome. Transient expression assays using green f luorescent protein demonstrated that N-terminal regions of the SIG2 and SIG3 ORFs could function as transit peptides for import into chloroplasts. Transcripts for all three SIG genes were detected in leaves but not in roots, and were induced in leaves of dark-adapted plants in rapid response to light illumination. Together with results of our previous analysis of tissue-specific regulation of transcription of plastid photosynthesis genes, these results indicate that expressed levels of the genes may inf luence transcription by regulating RNA polymerase activity in a green tissue-specific manner.The chloroplast is a semi-autonomous organelle whose genetic information is encoded in the nuclear and plastid genomes. The plastid genome encodes genes for photosynthesis, as well as genes for housekeeping functions such as protein synthesis. There is evidence that photosynthesis genes are transcribed by a multimeric Escherichia coli-type RNA polymerase (RNAP), and that housekeeping genes are transcribed by a monomeric T7 or T3 bacteriophage-type RNAP (1). The Ϫ10 and Ϫ35 sequences, 5Ј-TATAAT-3Ј and 5Ј-TTGACA-3Ј, respectively, in promoters of many plastid genes (2) are recognized by the E. coli-type RNAP (3). The E. coli RNAP is composed of a core complex of ␣, ␤, and ␤Ј subunits and one of a variety of factors, the principal one being 70, which is capable of binding to the Ϫ10 and Ϫ35 sequences (4-6). Determination of the complete nucleotide sequences of plastid genomes from liverwort (7), tobacco (8), rice (9), and other plants has resulted in finding of genes, rpoA, rpoB, and rpoC, probably encoding ␣, ␤, and ␤Ј subunits, respectively, of a plastid RNAP. In higher plants, rpoC is duplicated, rpoC1 for ␤Ј subunit and rpoC2 for ␤Љ subunit. Amino acid sequences deduced from maize plastid genes, rpoC2, rpoB, rpoC1, and rpoA, have proved to correspond to those determined chemically of 180-, 120-, 78-, and 38-kDa polypeptides, respectively, of highly purified maize plastid RNAP (10,11).No homolog for the bacterial-like factor of RNAP has been detected in the plastid genomes so far sequenced. However, some reports indicate that plastid -like factors (SLFs) exist in higher plants. Antibodies against an E. coli 70 homolog from the cyanobacterium Anabaena sp. PCC7120 have cross-reacted with polypeptides in purified plastid RNAPs from maize and rice (12). In spinach 90-and 33-kDa polypeptides have been identified in a plastid RNAP that a...

The structure and molecular interactions of the primary donor (P680) in the reaction center (D1-D2-cytochrome b-559 complex) of photosystem II (PS II) have been investigated by detecting light-induced FT-IR difference spectra upon the formation of its triplet state (3P680). The 3P680/P680 spectrum obtained was analyzed by comparing it with difference spectra between the ground and lowest triplet states of purified chlorophyll a (Chl) in organic solvents. The negative peaks at 1669 and 1707 cm-1 accompanied by the positive peaks at 1627 and 1659 cm-1 in the 3P680/P680 spectrum were assigned to the keto C = O stretching mode, and the appearance of these two pairs of bands indicated that P680 has a dimeric structure analogous to that of the bacterial primary donor. From the band positions of the keto and carbomethoxy C = O stretches, the hydrogen-bonding properties of these two Chl molecules were found to be asymmetrical; in one Chl molecule both the keto and carbomethoxy C = O groups form hydrogen bonds, while in the other Chl molecule the keto C = O is not hydrogen-bonded whereas the carbomethoxy C = O probably is hydrogen-bonded. The temperature dependence of the intensity ratios of the keto C = O bands revealed that the triplet state is equilibrated between the two Chl molecules with an energy gap of 8.4 +/- 0.7 meV. Most of the triplet population was found to be localized on one Chl molecule (86% at 80 K), in which both of the two C = O groups are hydrogen-bonded, that is probably attached to the D1 subunit. Considering the structure of the bacterial reaction center determined by X-ray crystallography and the sequence homology between the D1 and D2 subunits of PS II and the L and M subunits of bacteria, a model of the P680 structure and its interactions with apoproteins has been proposed.

Spinach (Spmacis okracea L.) chloroplasts solubilized by digitonin were separated into five fractions by sucrose density gradient centrifugation. Three of the fractions, FI, F,,, and F,,,, corresponding to photosystem I, photosystem II, and the chlorophyll a/b complex, were purified further by two steps of diethylaminoethyl-cellulose chromatography followed by electrofocusing on an Ampoine column. The polypeptide patterns of the fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the spectral properties of the fractions at -196 C determined by absorption spectra, fourth derivative curves of the absorption spectra, fluorescence emission spectra, and fluorescence excitation spectra.The activity of purified Fii (photosystem II) was also assayed by the photoreduction of dichlorophenol-indophenol at room temperature using 1,5-diphenylcarbohydrazine as the electron donor and by the photoreduction of C-550 at -196 C. The different fractions showed unique polypeptide patterns and unique sets of low temperature-absorbing forms ofchlorophyll.The fluorescence emission spectra of F1, F11, present work was to extract and purify the three types of Chl complexes and to measure their fluorescence and absorption properties at -196 C. In the course of this work which involved fairly extensive purification procedures, the photochemical properties and polypeptide profiles of the fractions were also examined at different stages of purity. MATERIALS AND METHODSThe initial purification procedures followed those of Wessels et al. (19) and Huzisige et al. (10) After 1 hr at 0 C with stirring, the suspension was centrifuged at 1,000g for 2 min to remove debris and the supernatant centrifuged again at 30,000g for 1 hr at 0 C. The supernatant containing about 30%o of the Chl with an a/b ratio of 5:8 was discarded and the pellet, now somewhat enriched in PSII, was resuspended in a medium containing 0.05 M Tris-HCI (pH 7.8) with 5 mM MgC12, 0.35 M NaCl, and 1.25% digitonin. After 18 to 20 hr at 0 C with stirring, the suspension was centrifuged at 100,000g for 1 hr at 0 C. The pellet was discarded and the supernatant (1-to 1.5-ml aliquots containing about 700 ,ug of Chl) was placed in each of six centrifuge tubes on a sucrose density gradient, 10 to 30%o sucrose in 0.05 M Tris-HCI (pH 7.8), 5 mM MgCl2, and 0.5% digitonin, and centrifuged 40 to 48 hr at 90,000g at 0 C in an SW 27 rotor. After equilibration had been reached four or five green bands were apparent in the centrifuge tube depending on whether or not the top band could be resolved visually into two bands. Fractions (0.4 ml) were removed from the top of the centrifuge tube with an Instrumentation Specialties Co. model 640 density gradient fractionator which continuously monitored the A at 280 nm. Absorption spectra were measured at room temperature and at -196 C on the 0.4-ml fractions. Fractions from a parallel experiment were assayed for PSII activity as the rate of photoreduction of DPIP2 with DPC as the electron donor. The P700 cont...

Pigment stoichiometries in the D,/D&ytochrome 6-559 reaction center complex from spinach have been studied by spectrophotometry and normalphase silica HPLC of the solvent extracts. Based on the well-accepted molar ratio Pheo a/P680=2, the results are summarized by Chl a/P680 = 6.01 k 0.37 (n = 24) and b-carotene/P680 = 1.84 f 0.11 (n = 4). These stoichiometries are significantly different from those of bacterial reaction center complexes.