The light-induced cation radical of the primary electron donor, Ps70, in photosynthetic reaction centers from Rhodospirillum rubrum G-9, has been investigated by electron-nuclear double resonance (ENDOR) in liquid aqueous solution. The measured hyperfine coupling constants are assigned to specific molecular positions by partial deuteration.Comparison with the bacteriochlorophyll a cation radical shows different reduction factors of the individual coupling constants deviating from the value 2.0 reported in earlier investigations in frozen solutions. The average of the coupling constants is, however, reduced by a factor very close to 2.0. EPR simulations using the ENDOR coupling constants support a dimer model for PsO with C2 symmetry, where the two macrocycles are close enough to form a supermolecular orbital resulting in a different distribution of the unpaired electron, compared with the monomeric bacteriochlorophyll a cation radical. Molecular orbital calculations were used to obtain structural information about this dimer.In bacterial photosynthesis, the light-induced charge separation starts with the fast donation of an electron from an excited singlet primary donor P to an electron-transport chain in the reaction center protein (RC) (1-5). RCs offer a convenient system for the investigation of the cation and anion radicals formed in this process. Much of our present knowledge about the various species and their interactions in RCs has evolved from the application of paramagnetic resonance methods (3, 6). The dimeric nature of the primary donor cation radical P8t,0 in some bacteria was originally proposed by Norris et al. (7) to explain the observed narrowing of the EPR line by a reduction factor (RF) of \/2 compared with the monomeric bacteriochlorophyll a cation radical (BChl-a+ ) (Fig. 1). Further support for this model came from electron-nuclear double resonance (ENDOR) at low temperatures in which a reduction of the hyperfine coupling constants (hfcs) by a factor of two was deduced when going from BChl-a+ to P8+70 (8)(9)(10)(11)(12)(13)(14). The detailed structure of the suggested dimer is still controversial (3,4,14), but its geometry is of prime importance for basic understanding of the primary act of light-induced charge separation in photosynthesis.ENDOR in solution has been used to elucidate the electronic structure of the various isolated pigment radicals and has provided an almost complete set of isotropic hfcs (15)(16)(17)(18)(19)(20). Liquid-state ENDOR is superior to frozen-solution EN-DOR (10, 21, 22) because the linewidths are smaller due to the absence of anisotropic broadening so that hfcs from all magnetic nuclei in the radical can often be obtained (23,24). However, most of the ENDOR studies of photosynthesis thus far were carried out in frozen matrices (8)(9)(10)(11)(12)(13)). An EN-DOR in solution study on RC [Rhodopseudomonas (Rp.) sphaeriodes R-26] in water at room temperature has been reported only recently (25). In those experiments, the additional application of electron...

At least three forms of Triton X-100-solubilized bacteriochlorophyll a (BChI a) have been characterized by UV/ visible/near-IR absorption and CD spectra. One, absorbing at 770 nm, is similar to a monomeric solution in methanol. The two others have strongly red-shifted absorption peaks (860 nm and 930, 835 nm) and intense and complex CD bands in this region, indicative of strong interaction of at least two and three molecules of BChl a, respectively.Triton X-100 (II, m = 9 or 10) is next to dodecyldimethylamineoxide the most commonly used detergent for the isolation of bacterial reaction center and light-harvesting complexes.The understanding of bacterial photosynthesis has greatly advanced with the isolation and characterization of well-defined complexes of bacteriochlorophyll a (BChl a, I) with proteins ( (Fig. 2). The red shifts of these BCh1 a-protein complexes (as well as similar shifts in chlorophyll a-protein complexes) are as yet only partly understood and subject to much theoretical, spectroscopic, and synthetic work. They have been related to aggregation (5-14) or hydration (or both) (5,6,(12)(13)(14)(15), enolization of the chlorophyll groups (16)(17)(18)(19), point charges in their environment (20), and charge-transfer complexes (21). In particular, the primary donor P870 in BChl a-containing reaction centers has been suggested to be a "special pair" of BChl a, with their exact relationship still under discussion (22-27). IIWe noticed, during the isolation of reaction centers from Rhodospirillum rubrum G9 and Rhodopseudmownas spheroides R26 with Triton X-100, fractions that looked superficially like reaction centers contaminated with solubilized BChl a and bacteriopheophytin a. The 805-nm band was, however, small or even absent, and the 870-nm band was somewhat blue shifted and did not show any reversible bleaching. By systematic solubilization studies of BCh1 a with detergents, we have now shown that Triton X-100 induces strongly red-shifted absorption peaks in solubilized BChl a, with maxima around 830, 860, and 930 nm, and we here present our results on the BChl a species present in these solutions. EXPERIMENTALRs. rubrum G9 and Rp. spheroides R26 were grown in Hutner's medium (28) at 270C. BChl a was isolated by a modification of the method of Sato and Murata (29), which principally involves solvent extraction, precipitation of BChl a as a dioxane complex (30), and chromatography on DEAE-cellulose (29). The chromatography was alternatively also done on sugar columns (30). Triton X-100 was purchased from Serva (Heidelberg, Federal Republic of Germany) and used as received. All solvents were distilled or purified over alumina (Woelm, Eschwege, Federal Republic of Germany) prior to use. UV/visible/near-IR absorption spectra were recorded on a DMR 22 (Zeiss, Toberkochen, Federal Republic of Germany) spectrophotometer equipped for cross-illumination with suitable interference and cutoff filters. CD spectra were recorded with a dichrograph Mark V (Yvon-Jobin, Longjumeau, France) equipped w...

Micellar aggregates have been prepared from chloro­phyll a. Whereas most detergents including non-ionic, zwitterionic and anionic ones, produce aggregates with long-wavelength shifted and enhanced QY absorption, the cationic detergents hexadecyl-trimethylammonium-bromide and hexadecyl-pyridiniumchloride produce complexes with opposite traits: The QY absorption is shifted to shorter wavelengths, and it is hypochromic. The optical activity is increased as in the long-wave­ length shifted complexes, but the cd bands are non-conservative and coincide with the absorption maxima. The complexes are most likely small aggregates of chlorophyll with a changed geometry as compared to the “standard” ones.

Micellar complexes were prepared from bacteriochlorophyll a and bacteriopheophytin a with the cationic detergents, cetyltrimethyl ammonium bromide and cetylpyridinium chloride. These complexes have spectroscopic properties (absorption, circular dichroism) which are very different from the ones formed with non-ionic detergents like Triton X-100, and also with anionic detergents. Bacteriochlorophyll a forms two complexes: One is blue-shifted and has excitonically coupled Qy transitions. The second one is extremely red-shifted. The unusual properties are suggested to result from interactions of the positively charged head-group of the detergent with the tetrapyrrole.

SummaryThe (Y-and &subunits of C-phycocyanin from Mastigocladus laminosus were prepared according to revised procedures. Both subunits are isolated as dimers, which can be dissociated into monomers with detergent mixtures. The fluorescence decay kinetics are similar for the respective monomers and dimers. In no case could they be fitted by only one (o-subunit) or two exponentials (P-subunit) which are predicted by theory for samples with a unique chromophore-protein arrangement containing one and two chromophores, respectively. It is suggested that there exists a heterogeneity among the chromophores of the subunits, which may persist in the highly aggregated complexes present in cyanobacterial antennas.