Spectral, Photophysical, and Stability Properties of Isolated Photosystem II Reaction Center

Abstract

Photosystem II reaction center (RC) preparations isolated from spinach (Spinacea oleracea) by the Nanba-Satoh procedure (O Nanba, K Satoh 1987 Proc Nati Acad Sci USA 84: 109-112) are quite labile, even at 4°C in the dark. Simple spectroscopic criteria were developed to characterize the native state of the material. Degradation of the RC results in (a) blueshifting of the red-most absorption maximum, (b) a shift of the 77 K fluorescence maximum from -682 nm to -670 nm, and (c) a shift of fluorescence lifetime components from 1.3-4 nanoseconds and >25 nanoseconds to -6-7 nanoseconds. Fluorescence properties at 77 K seem to be a more sensitive spectral indicator of the integrity of the material. The >25 nanosecond lifetime component is assigned to P680+ Pheophytinrecombination luminescence, which suggests a correlation between the observed spectral shifts and the photochemical competence of the preparation. Substitution of lauryl maltoside for Triton X-100 immediately after RC isolation stabilizes the RCs and suggests that Triton may be responsible for the instability.RC4 complexes found in photosynthetic membranes contain specialized pigment components that carry out the primary photochemical processes of photosynthesis. These light reactions provide the chemical potential to drive dark electron transport that leads to the storage of energy required by the organism. The first RC fractions were isolated from the photosynthetic bacteria Rhodobacter sphaeroides R-26 (25) and Rhodospirillum rubrum S1 (14) in the late 1960s. This accomplishment has led to a great deal of progress in understanding of the function of the RC. Perhaps the most far-reaching effect of this work, however, culminated in the crystallization of the Rhodopseudomonas viridis RC, which was the first integral membrane protein complex to be crystallized (22 (8,10,18) have suggested that the Dl and D2 proteins constitute the reaction center of PSII. The first experimental evidence suggesting that this might be true was the observation that the Dl protein functions or affects function on both sides of the PSII RC (21). More recently, Nanba and Satoh (24) reported the isolation of a PSII complex composed of Dl, D2, and Cyt b-559 which they identified as the RC. Danielius et al. (9) confirmed this identification by showing that the material could transfer an electron from P680 to endogenous pheophytin. In this paper we confirm the isolation of the Nanba-Satoh preparation and report on several spectral properties that appear to relate to the stability of the isolated PSII reaction center.MATERIALS AND METHODS PSII RC fractions were prepared from market spinach (Spinacia oleracea) by the procedure of Nanba and Satoh (24). All steps were carried out at 4°C in the dark. Appressed thylakoid membrane fragments devoid of PSI (13) were obtained from market spinach using Triton X-100 (17). PSII membranes were pelleted and then resuspended in 50 mm Tris-HCl (pH 7.2). The material (30 ml of 1 mg Chl/ml final concentration) was then stirred for 1 h with 4%...

Characterization of a D1‐D2‐cyt b‐559 complex containing 4 chlorophyll a/2 pheophytin a isolated with the use of MgSO4

“…A selective bleaching around 680 nm has been observed upon oxidation of the primary electron donor P680 [24,25], 3P680 formation [26] and reaction center inactivation [3,6].…”

“…Corrected fluorescence emission and excitation spectra were carried out with a SPEX Fluorolog spectrometer. Absorption difference spectra upon continuous illumination were measured under the sameconditions as in [6]. The Dl-DZZcyt b-559 complex (OD,,, 5 = 0.7) was treated with 1 PM methyl viologen and 10 mM sodium dithlonite.…”

“…Basically, the method developed by Nanba and Satoh [l] yields, after a long-term Triton X-100 exposure, a PS II RC stoichiometry of 4-5 chlorophyll a, 1 B-carotene and 1 cytochrome b-559 per 2 pheophytin a. Several reports have shown that the Dl-D2cyt b-559 complex is fairly unstable in the presence of Triton X-100 [3,6] and some alternative methods have been described to obtain more stable preparations [7-lo] which contain 6 chlorophyll a, 2 p-carotene, 1 cytochrome b-559 per 2 pheophytin a. At present, many research groups work with such prepara-tions assuming that the 6 Chl a/2 Pheo a stoichiometry represents the intact Dl-D2-cyt b-559 complex [l l-131.…”

Wavelength and intensity dependent primary photochemistry of isolated Photosystem II reaction centers at 5°C

“…Di-Dz-cytochrome b-559 PSII RC complex was isolated from PSII-enriched appressed membrane fragments obtained from market spinach [4]. PSII membranes (120 mg Chl a) were pelleted; resuspended in 50 mM Tris-HCl (pH 7.2), 30 mM NaCI, and 4% Triton X-100 at 1 mg Chl a/ml, and incubated with stirring for I h. All manipulations in this procedure were done at 4°C in the dark.…”

The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes

“…The emission peak from band 2 from plasma membrane was blue-shifted to 680 nm. It is noteworthy that emission spectrum of purified D1-D2-cyt b559 reaction center preparations from higher plants often exhibit such a blue-shifted peak (34). Next, the excised band 1 and band 2 material from plasma membranes were fractionated by denaturing SDS͞PAGE and analyzed by Western blotting for the presence of various PSI and PSII proteins.…”