Isolation and characterization of a subunit form of the light-harvesting complex of Rhodospirillum rubrum

Abstract

A new method is described for the isolation of subunits of the light-harvesting complex from Rhodospirillum rubrum (wild type and the G-9 mutant) in yields that approach 100%. The procedure involved treating membrane vesicles with ethylenediaminetetraacetic acid-Triton X-100 to remove components other than the light-harvesting complex and reaction center. In the preparation from wild-type cells, a benzene extraction was then employed to remove carotenoid and ubiquinone. The next step involved a careful addition of the detergent n-octyl beta-D-glucopyranoside, which resulted in a quantitative shift of the long-wavelength absorbance maximum from 873 to 820 nm. This latter complex was then separated from reaction centers by gel filtration on Sephadex G-100. The pigment-protein complex, now absorbing at 820 nm, contained two polypeptides of about 6-kilodalton molecular mass (referred to as alpha and beta) in a 1:1 ratio and two molecules of bacteriochlorophyll (BChl) for each alpha beta pair. This complex is much smaller in size than the original complex absorbing at 873 nm but probably is an associated form such as alpha 2 beta 2 X 4BChl or alpha 3 beta 3 X 6BChl. The 820-nm form could be completely shifted back to a form once again having a longer wavelength lambda max near 873 nm by decreasing the octyl glucoside concentration. Thus, the complex absorbing at 820 nm appears to be a subunit form of the original 873-nm complex.

Oligomerization of Light-Harvesting I Antenna Peptides of Rhodospirillum rubrum

“…This agrees with the studies of Miller et al who found a stoichiometry of two for the BChl to R and polypeptides (13). For calculation of the association constants, we assumed that such a dimerization reaction is indeed leading to the formation of B820.…”

“…They observed a fast (within seconds) red shift of 820 nm toward 860 nm upon reassociation, followed by a slow final 12-15 nm shift, taking hours to days. Further, Miller et al measured spectra slightly blue-shifted from the B873 spectrum in the reassociation process (13). The forms absorbing around 860 nm are probably the intermediate spectra that we also observed.…”

“…Here we observed a rapid collapse of the B873 peak already at temperatures above 290 K. The appearance of monomeric B777 together with a decreasing and blue-shifting B873 peak could be explained by an opening of the LH1 ring via the loss of some dissociated subunits, giving a combination of incomplete (blue-shifted) rings and monomeric polypeptides as reaction products. In the studies of Miller et al (13), it was found that reassociation of B873 from B820 subunits yielded an absorption spectrum that was slightly blue-shifted from the original B873 spectrum; this could be the same phenomenon having uncompleted rings, appearing in the reassociation process.…”

“…rubrum a mutant strain, called G9+, exists that produces LH1 complexes lacking carotenoids. Isolated LH1 antenna complexes of this strain can be reversibly dissociated into B820 subunit pigment-protein complexes (12)(13)(14), which can be further dissociated into monomeric † …”

“…rubrum dissolved in this detergent can reversibly be dissociated into its B820 subunits (13,32), whereas after treatment with other detergents dissociation directly into B777 monomers was observed (13,14). We used the fact that the reversible dissociation of B873 into B820 subunits can be obtained not only by treatment with detergent, but also by varying the temperature in the range 278-300 K. This temperature dependence was observed already by Miller et al (13). The latter finding provided us with a controlable method to modulate the reversible association reaction without changing sample.…”