Reconstitution of Okenone into light harvesting complexes from Allochromatium minutissimum.

Okenone was reconstituted into light harvesting (LH) complexes of the purple photosynthetic bacterium Allochromatium minutissimum possessing the spirilloxanthin pathway for carotenoid biosynthesis. Suppression of this pathway by diphenylamine, an inhibitor of carotenogenesis, yielded nearly carotenoidless complexes preserving their native spectral properties. Using a previously developed technique, okenone was readily reconstituted into LH1 complex (>90%) whereas its reconstitution into LH2 complex was of low efficacy (10-20%). The absorption band of the reconstituted okenone was shifted to shorter wavelength compared with its position in vivo. This is typical for other reconstituted carotenoids. The reconstitution of okenone was confirmed by Li-DS electrophoresis (in contrast to free okenone the reconstituted okenone migrated with complexes), circular dichroism spectra (reconstituted okenone exhibited optical activity), and fluorescence excitation spectrum (energy transfer from okenone to bacteriochlorophyll was at the control level).

Formation of bacteriochlorophyll form B820 in light harvesting 2 complexes from purple sulfur bacteria treated with dioxane.

Treatment of some sulfur bacteria (Allochromatium minutissimum, Thiorhodospira sibirica, and Ectothiorhodospira halovacuolata WN22) with dioxane results in formation of the bacteriochlorophyll form B820 in the light harvesting complex LH2. This form characterized by absorption maximum at 820 nm has the same absorption spectrum as B820 subcomplex from LH1 complex. Appearance of the B820 form was accompanied by a sharp decrease in absorption in the carotenoid region. This phenomenon observed in all LH2 complexes investigated may be attributed to formation of colorless carotenoid aggregates. This is very similar to the previously reported dissociation of the LH1 complex with carotenoids into B820 subcomplexes. Although the B820 form corresponded the bacteriochlorophyll dimer, its circular dichroism spectrum showed that pigment molecules in this dimer exhibit different interaction than those in the B820 subcomplex. The dioxane treatment of LH2 complexes isolated from Rhodopseudomonas palustris bacteria grown under normal or low intensity illumination did not result in formation of such dimers. It is suggested that bacteriochlorophyll B820 formation is related to unique structure of LH2 complexes from the sulfur bacteria.

Reconstitution of carotenoids into the light-harvesting complex B800-850 of Chromatium minutissimum.

Chromatophores and peripheral light-harvesting complexes B800-850 with a trace of carotenoids were isolated from Chromatium minutissimum cells in which carotenoid biosynthesis was inhibited by diphenylamine. Three methods previously used for the reconstitution of carotenoids into either the light-harvesting (LH1) type complexes or reaction centers (RC) of carotenoidless mutants were examined for the possibility of carotenoid reconstitution into the carotenoid depleted chromatophores. All these methods were found to be unsuitable because carotenoid depleted complex B800-850 from Chr. minutissimum is characterized by high lability. We have developed a novel method maintaining the native structure of the complexes and allowing reconstitution of up to 80% of the carotenoids as compared to the control. The reconstituted complex has a similar CD spectrum in the carotenoid region as the control, and its structure restores its stability. These data give direct proof for the structural role of carotenoids in bacterial photosynthesis.

[Two-photon excitation fluorescence spectrum of the light-harvesting complex LH2 from Chromatium minutissimum within 650-745 nm range is determined by two-photon absorption of bacteriochlorophyll rather than of carotenoids]. / Spektr dvukhfotonnogo vozbuzhdeniia fluorestsentsii svetosobiraiushchego kompleksa LH2 iz Chromatium minutissimum v oblasti 650-745 nm opredeliaetsia dvukhfotonnym pogloshcheniem bakteriokhlorofilla, a ne karotinoidov.

Two-photon fluorescence excitation spectra of the peripheral light-harvesting complex LH2 from the purple photosynthetic bacterium Chromatium minutissimum were examined within the expected spectral range of the optically forbidden S1 singlet state of carotenoids. LH2 preparations isolated from wild-type and carotenoid-depleted cells were used. 100-fs laser pulses in the range of 1300-1490 nm with an energy of 7-9 mW (corresponding to one-photon absorption between 650 and 745 nm) were used for two-photon fluorescence excitation. It was shown that two-photon fluorescence excitation spectra of LH2 complex from wild and carotenoid-depleted cells are very similar to each other and to the two-photon fluorescence excitation spectrum of bacteriochlorophyll a in acetone. It was concluded that direct two-photon excitation of bacteriochlorophyll a determines the fluorescence of both samples within the 650-745 nm spectral range.

Chemical crosslinking studies of the isolated light-harvesting B800-850 complex of Chromatium minutissimum.

The spatial relationship of polypeptides comprising the light-harvesting B800-850 complex of Chromatium minutissimum has been studied by means of chemical crosslinking of the isolated complex with cleavable, 1.2 nm-long dithiobis(succinimidyl propionate). The samples were analyzed by different types of electrophoresis and spectrophotometrically. No difference was shown between crosslinking of the B800-850 complex either solubilized or incorporated into proteoliposomes. It was found that two main polypeptides form only one type of heterodimer. The crosslinked complex was more thermostable. This crosslinkage restricted the conformational transitions causing the shift of the long wavelength band in the near infrared region. A structure of the complex is discussed.

[Detection by the use of silver ions of additional protein zones in gradient polyacrylamide gels stained with Coomassie]. / Vyiavlenie s pomoshch'iu ionov serebra dopolnitel'nykh zon belkov v gradientnykh poliakrilamidnykh geliakh, okrashennykh kumassi.

A simple method of revealing the additional zones of proteins in gradient polyacrylamide gels, preliminary dyed Coomassie by means of silver ions is described. The dyeing of Coomassie allows to avoid the time-consuming stages of preliminary treatment of gels as well to reveal more sensitive zones in gels. On the second stage of dyeing silver minor zones appear there which were not seen while Coomassie was dyed. The suggested method preserves high sensitivity characteristic of the methods of gel dyeing with silver.