Modification of photosystem I reaction center by the extraction and exchange of chlorophylls and quinones.

The photosystem (PS) I photosynthetic reaction center was modified thorough the selective extraction and exchange of chlorophylls and quinones. Extraction of lyophilized photosystem I complex with diethyl ether depleted more than 90% chlorophyll (Chl) molecules bound to the complex, preserving the photochemical electron transfer activity from the primary electron donor P700 to the acceptor chlorophyll A(0). The treatment extracted all the carotenoids and the secondary acceptor phylloquinone (A(1)), and produced a PS I reaction center that contains nine molecules of Chls including P700 and A(0), and three Fe-S clusters (F(X), F(A) and F(B)). The ether-extracted PS I complex showed fast electron transfer from P700 to A(0) as it is, and to FeS clusters if phylloquinone or an appropriate artificial quinone was reconstituted as A(1). The ether-extracted PS I enabled accurate detection of the primary photoreactions with little disturbance from the absorbance changes of the bulk pigments. The quinone reconstitution created the new reactions between the artificial cofactors and the intrinsic components with altered energy gaps. We review the studies done in the ether-extracted PS I complex including chlorophyll forms of the core moiety of PS I, fluorescence of P700, reaction rate between A(0) and reconstituted A(1), and the fast electron transfer from P700 to A(0). Natural exchange of chlorophyll a to 710-740 nm absorbing chlorophyll d in PS I of the newly found cyanobacteria-like organism Acaryochloris marina was also reviewed. Based on the results of exchange studies in different systems, designs of photosynthetic reaction centers are discussed.

Selective extraction of antenna chlorophylls, carotenoids and quinones from photosystem I reaction center.

By the ether treatment of lyophilized PSI pigment-protein complexes, all the carotenoids and the secondary acceptor phylloquinone (A1), and more than 90% of the Chl were removed to yield the PSI complex with 9-11 molecules of Chl per reaction-center unit. The complexes retained the primary electron donor and acceptor (P700 and A0), in addition to three FeS clusters (F(X), F(A) and F(B)), and showed an activity of highly efficient electron transfer when phylloquinone was reconstituted. The methods for the preparation and the characterization of the ether-extracted PSI complexes are reviewed in this article. We also review the studies done with this PSI preparation on (1) the identification of the absorption and fluorescence spectra of P700, (2) the nano- and picosecond reaction of A0 and A1, (3) the energy-gap dependency of the reaction rate between A0 and the artificial quinones reconstituted at the A1 site, (4) the direct excitation of P700 followed by the ultra-fast electron transfer from P700 to A0, and (5) the de- and re-stabilization of the PSI structure by the removal and reconstitution, respectively, of antenna Chl in the presence of certain lipids.

[A preliminary study to determine the activity of topoisomerase II in human kidney cancer cells with DNA unknotting method].

For the purpose of examining the Topoisomerase II (Topo II) activity in human kidney cancer cells, we performed experiments with using DNA unknotting method. This method check relative Topo II activity with its conversion of knotted form P4 phage DNA to unknotted form. Our preliminary results demonstrate remarkable activity of Topo II with specific conversion of knotted form P4 DNA to unknotted form in human kidney cancer cells, YCR and ACHN. Moreover, addition of etoposide to the same experiment suppressed Topo II activity in a dose dependent manner. Our results suggest that kidney cancer has a certain amount of Topo II, a target for etoposide. We believe this method is useful to measure Topo II activity in cancer cells and to estimate chemotherapeutic potential of Topo II inhibitors including etoposide in human kidney cancers.

Enrichment of bacteriochlorophyll g' in membranes of Heliobacterium chlorum by ether extraction. Unequivocal evidence for its existence in vivo.

Treatment of H. chlorum membrane preparations with diethyl ether of high degrees of water saturation raised the bacteriochlorophyll (BChl) g' mole fraction, as determined by HPLC analysis of their acetone extracts, toward a level of 40% of total BChl g or higher. Starting from pure BChl g, the BChl g' mole fraction should never exceed 24.6% which is the equilibrium value in diethyl ether. The existence (and possible functioning) of BChl g' in vivo is thus unequivocally demonstrated.

Structure of the primary electron donor in photosystem I: a resonance Raman study.

Low-temperature resonance Raman (RR) spectra have been obtained at resonance with the Soret transition of chlorophyll a in photosystem I particles containing large amounts either of the triplet state of P700 or of its radical cation state. Subtracting these spectra from those of resting reaction centers yielded RR spectra of P700 in its neutral, ground state. These spectra arise from two distinct chlorophyll a molecules differing by the strengths of the bonding interactions assumed by their keto carbonyl groups, the stretching frequencies of which are found at 1655 and 1675 cm-1. The present results rule out previous hypotheses that P700 might have consisted of a single, chemically modified chlorophyll a molecule. Neither of the bonding interactions assumed by the keto carbonyls of the P700 chlorophylls most probably involves chlorophyll-chlorophyll bridging through water molecules, as surmised in the so-called special pair models, but likely consists of H bonds with distinct protein sites. The magnesium atoms of the two P700 chlorophylls are 5-coordinated. Hence, the structural model of P700 provided by the present data is qualitatively the same, in terms of bonding interactions, as that currently accepted for the bacterial primary donor.

Orientation of Photosystem-I pigments: low temperature linear dichroism spectroscopy of a highly-enriched P700 particle isolated from spinach.

The linear dichroism of Photosystem I particles containing 10 chlorophylls per P700 has been investigated at 10 K. The particles were oriented by uniaxial squeezing of polyacrylamide gels. The oxidation state of P700 was altered either by incubation of the gels with redox mediators or by low temperature illumination. The QY transitions of the primary electron donor P700, of the remaining unoxidized chlorophyll in P700(+) and of a chlorophyll molecule absorbing at 686 nm, which presumably corresponds to the primary electron acceptor A0, are all preferentially oriented perpendicular to the gel squeezing direction. The QY transition of the chlorophyll forms absorbing at 670 and 675 nm appear tilted at 40 ± 5° from this orientation axis. This orientation of the various chlorophylls is compared to that previously reported for more native Photosystem I particles.

Nanosecond flash studies of the absorption spectrum of the Photosystem I primary acceptor Ao.

Photosystem I particles devoid of the secondary electron acceptor A1 were studied by nanosecond flash absorption. The primary radical pair (P-700(+), A0 (-)) decays with a half-time of 35 ns. The difference spectrum was measured (400-870 nm). After subtraction of the P-700(+)/P-700 difference spectrum, the A0 (-)/A0 was obtained. It includes bleachings centered at 690 and 430 nm, and broad positive bands in the near infra-red and the blue-green. This spectrum is consistent with A0 being chlorophyll a absorbing at 690 nm.

Light-induced charge separation in Photosystem I at low temperature is not influenced by vitamin K-1.

The photoreduction of iron-sulfur centers was studied at low temperature in Photosystem I particles from spinach and the cyanobacterium Synechocystis 6803, which contain various amounts of vitamin K-1 (recently tentatively identified as the acceptor A1). The irreversible charge separation that was progressively induced at low temperature between P-700 and FA (or FB) by successive laser flashes was studied at 15 K. Its maximum amount after a large number of flashes was shown to be fairly independent of the number (0, 1 or 2) of vitamins K-1 per reaction center. Moreover, the first flash yield of this charge separation was diminished by only about 50% when vitamin K-1 was completely absent from the particles by comparison with particles containing one or two vitamin K-1 per reaction center. When FA and FB were prereduced, the iron-sulfur center FX was also reversibly photoreduced at 9 K in the absence of vitamin K-1. The implications of these results for the electron pathways of Photosystem I are discussed and it is proposed that a direct electron transfer from A0- to the iron-sulfur centers is highly efficient at low temperature.

Fluorescence changes related in the primary photochemical reaction in the P-700-enriched particles isolated from spinach chloroplasts.

The light-induced changes in the yield of chlorophyll alpha fluorescence and photooxidation of P-700 in the P-700-enriched particles isolated from spinach chloroplasts were studied. 1. Fluorescence emitted from the particles was found to show light-induced transient changes in the yield. In the presence of ascorbate, illumination induced quenching of fluorescence in parallel to the photooxidation of P-700. The time course of dark reduction of photooxidized P-700 agreed well with that of dark recovery of variable fluorescence yield in the presence of ascorbate. When illuminated in the presence of dithionite, the emission yield increased, whereas no photooxidation of P-700 was observed. 2. The yield of variable fluorescence and redox state of P-700 depended similarly upon the redox potential. 3. At liquid nitrogen temperature, illumination induced a rise of the fluorescence yield and a complete photooxidation of P-700 in the ascorbate-treated sample. When the particles had been preincubated with dithionite in the light before cooling, light-induced rise in the fluorescence yield was accompanied by only a small extent of P-700 photooxidation. It is suggested that both the oxidized form of P-700 and the primary electron acceptor act as quenchers for the variable fluorescence. 4. The emission spectrum for the constant part of fluorescence (F679) has a peak at 679 nm, and that for the variable part of fluorescence (F694) has a peak at 694 nm at room temperature. The emission maxima were slightly shifted and the yield of variable fluorescence was markedly enhanced at liquid nitrogen temperature. 5. Excitation spectra determined show a peak at 672 nm for F679, and a peak at 672 nm and a shoulder at 685 nm for F694. Action spectrum for P-700 photooxidation was similar to the excitation spectrum for F694.

Enrichment of photosystem I reaction center chlorophyll from spinach chloroplasts.

The reaction center chlorophyll of Photosystem I in spinach chloroplasts was highly enriched. Preparations having 5-9 chlorophylls per 1 P700 were obtained by treating the Photosystem I particles prepared by digitonin treatment of chloroplasts with wet diethyl ether. All P700 present in the extracted particles was found to be photoactive, undergoind oxidation upon illumination.