Isamu Ikegami

Bacteriochlorophyll g epimer as a possible reaction center component of heliobacteria

Normal-phase HPLC analysis of acetone extracts of cells, membranes and antenna-reaction center complexes of Heliobacterium chlorum and Heliobacillus mobilis showed the presence of bacteriochlorophyll (BChl) g′, the 132-epimer of BChl g. The molar ratio of BChl g:BChl g′ in these preparations was approx. 18 in cells and membranes of both species. This value, when combined with a molar ratio of BChl g to the primary electron donor, P-798, of 35–40, yields a BChl g′:P-798 ratio of 2, suggesting that P-798 may be a dimer of BChl g′. The amount of BChl g′ in the antenna-reaction center complex of H. chlorum was slightly higher, suggesting that some epimerization may have occurred during the isolation of the complex. In contrast, bacteriopheophytins (BPhe) g and g′ were present in too small amouts to be essential components of the photosynthetic apparatus. This confirms the idea that the reaction center of heliobacteria is basically different from those of purple bacteria and Photosystem II. A detailed interpretation, based on Correlated Spectroscopy and Double Resonance experiments is given of the 1H-NMR spectra of BChl g and BChl g′, confirming the structure and identity of both pigments. Absorption and circular dichroism spectra of BChl g and BChl g′ are also presented.

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.

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.

Chlorophyll a′/P-700 and pheophytin a/P-680 stoichiometries in higher plants and cyanobacteria determined by HPLC analysis

Abstract The chlorophyll (CHl) a ′ and pheophytin (Pheo) a contents in photosynthetic organs have been reinvestigated by means of silica HPLC. Previously reported data (Watanabe, T. et al. (1985) BBA 807, 110–117 and FEBS Lett. 191, 252–256) were found to contain errors from two sources: slight epimerization and pheophytinization of Chl a during pigment extraction and PS I particle preparation, and an accidental overlap of a Chl a alteration product ( meso -chlorinated Chl a ) with Chl a ′ on the HPLC trace. The pigment molar ratios determined with the improved extraction/HPLC procedure were Chl a /Chl a ′ ÷ 460 and Chl a /Pheo a ÷ 120 in more than 100 samples from 13 different higher plants, and Chl a /Chl a ′ ÷ 125 and Chl a /Pheo a ÷ 100 in three cyanobacteria. These values, when combined with the PS I particle composition (Chl a ′/P-700 = 1) and the widely accepted molar ratio Pheo a /P-680 of 2, yield P-680/P-700 = 1.9 ± 0.5 and 0.65 ± 0.20 as reaction center stoichiometries in higher plants and cyanobacteria, respectively. The present results correspond well with the recently published Chl a /reaction center stoichiometry data acquired by (photo-)redox titrations of key components.

Extraction of vitamin K-1 from Photosystem I particles by treatment with diethyl ether and its effects on the A−1 EPR signal and System I photochemistry

Treatment of Photosystem I particles, prepared by digitonin treatment of spinach chloroplasts, with dry diethyl ether extracted all the vitamin K-1 (2.2 molecules/P-700 in the original Photosystem I particles) and carotenoids as well as 60% of antenna chlorophylls. P-700 and FeS centers (FX, FA and FB) were not extracted, and were photochemically oxidized and reduced, respectively, upon continuous illumination. This preparation showed the A−0 EPR signal but no A−1 signal even after long illumination at 210 or 230 K, in which condition A−1 was accumulated in the untreated Photosystem I particles. Laser flash excitation induced only a small absorption change, corresponding to 20% of the total P-700, at 429 nm in the extracted preparation, suggesting that charge recombination took place on a submicrosecond time scale. Addition of vitamin K-1 or K-3 but not that of benzyl viologen increased the extent of the slow decay phase of P-700+. The extraction also induced a new fluorescence band peaking at 692 nm, the intensity of which increased upon addition of dithionite. This fluroescence band is possibly related to the rapid charge recombination between A−0 and P-700+. These results strongly suggest that A1 is vitamin K-1 and that it functions to mediate electron transfer between A0 and FeS centers. Similar results were also obtained with the ‘P-700 enriched particles’ with 7–16 antenna chlorophyll aP-700 which can be obtained by extraction of Photosystem I particles with diethyl ether containing (80% saturated) water.

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.

Electron transfer in menaquinone-depleted membranes of Heliobacterium chlorum

Abstract Treatment of membranes of Heliobacterium chlorum with diethyl ether at various levels of water saturation resulted in extraction of bacteriochlorophyll (BChl) g and menaquinones. Only a minor enrichment of P-798 with respect to antenna BChl g could be achieved, whereas menaquinone (vitamin K-2), the only quinone found in this species (Hiraishi, A. (1989) Arch. Microbiol. 151, 378–379), was essentially completely removed. Extraction of menaquinone, however, did not result in significant changes in electron transport. Electron transport to secondary electron acceptors was not impaired by wet ether extraction, either at room temperature or at low temperature. As in intact membranes, radical pair recombination and subsequent triplet formation were only observed under strongly reducing conditions. These results suggest that menaquinone is not an essential participant in the electron acceptor chain of heliobacteria.

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.

Absorption studies of Photosystem I photochemistry in the absence of vitamin K-1

Abstract Flash-induced absorption changes were studied on different timescales (nanosecond to millisecond) and at different temperatures (10 to 278 K) in highly enriched spinach PS I particles lacking vitamin K-1 and in which the electron transfer from the primary acceptor to the secondary acceptors was blocked. At all temperatures, the initial absorption change at 820 nm was followed by a fast decay ( t 1 2 ≈ 47 ns at 278 K and ≈ 82 ns at 10 K) which is attributed to the decay of the primary radical pair (P-700+-A−0). A slower phase of absorption decay is attributed to the P-700 triplet state, which was formed as a result of the biradical recombination, with a yield of about 30% at 278 K and about 75% at 10 K. Under air, the 3P-700 state decayed with a t 1 2 of about 50 μs at 278 K, whereas in the absence of oxygen it decayed with t 1 2 ≈ 560 μs . At 278 K, this yield was shown to depend on the presence of a magnetic field, with a maximum around 60 G. The 3P-700 decay halftime was nearly independent of temperature in the absence of oxygen ( t 1 2 ≈ 1 ms at 10 K ). The implications for the mechanisms involved in this decay are discussed. Addition of vitamin K-1 to these particles resulted in a decrease in the amplitude of the fast submicrosecond decay and a concomitant increase in the amplitude of a slow phase, indicating an efficient transfer from A−0 to vitamin K-1. However, most functional properties of the acceptor A1 were not reconstituted under these conditions.

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.