Substitution of Isoleucine L177 by Histidine Affects the Pigment Composition and Properties of the Reaction Center of the Purple Bacterium Rhodobacter sphaeroides

Abstract

Using site-directed mutagenesis, we obtained the mutant of the purple bacterium Rhodobacter sphaeroides with Ile to His substitution at position 177 in the L-subunit of the photosynthetic reaction center (RC). The mutant strain forms stable and photochemically active RC complexes. Relative to the wild type RCs, the spectral and photochemical properties of the mutant RC differ significantly in the absorption regions corresponding to the primary donor P and the monomer bacteriochlorophyll (BChl) absorption. It is shown that the RC I(L177)H contains only three BChl molecules compared to four BChl molecules in the wild type RC. Considering the fact that the properties of both isolated and membrane-associated mutant RCs are similar, we conclude that the loss of a BChl molecule from the mutant RC is caused by the introduced mutation but not by the protein purification procedure. The new mutant missing one BChl molecule but still able to perform light-induced reactions forming the charge-separated state P+QA- appears to be an interesting object to study the mechanisms of the first steps of the primary electron transfer in photosynthesis.

Substitution of isoleucine L177 by histidine in Rhodobacter sphaeroides reaction center results in the covalent binding of PA bacteriochlorophyll to the L subunit

“…In particular, in the room temperature absorption spectrum of I(L177)H RC a strong blue shift of the long‐wavelength P band was accompanied by a substantial decrease of its dipole strength. Besides, it was found that in acetone–methanol (7:2, v/v) extract of the I(L177)H RCs the BChl:BPhe ratio was dramatically altered [4]. In the present work, we report new data on the I(L177)H RC which suggest that Ile‐His substitution in this position cause covalent attachment of a BChl molecule, namely P A , to the L‐subunit.…”

“…Cell growth, preparation of intracytoplasmic membranes and construction of the antenna deficient strains of Rb. sphaeroides containing reaction centers with I(L177)H mutation were described previously [4]. For all experiments, wild‐type (WT) reaction centers were those isolated from the puf ‐deficient strain DD13 [5] complemented with the wild type genes.…”

Different effects of identical symmetry-related mutations near the bacteriochlorophyll dimer in the photosynthetic reaction center of Rhodobacter sphaeroides

“…Rhodobacter sphaeroides recombinant strains were grown on Huttner medium in the presence of tetracycline (1 μg/ml), kanamycin (5 μg/ml), and streptomycin (5 μg/ml) as described earlier [16]. The procedure of preparation of chromatophores and purification of RCs from "no antenna" strains of Rba.…”

“…The procedure of preparation of chromatophores and purification of RCs from "no antenna" strains of Rba. sphaeroides by ion exchange chromatography was described previously [16,17]. The yield of reaction centers after their purification from membranes was estimated using absorption near 800 nm (Q Y B band) measured in 1 cm cuvette (ODV 800 ) calculated per liter of 3 day culture.…”

Physical stage of photosynthesis charge separation

“…±ÑÕÇÓâ BChl Ä P ÒÓÑËÔØÑAEËÕ Ä ÏÖÕÂÐÕÇ IL177H Rba. sphaeroides Ä ÓÇÊÖÎßÕÂÕÇ ÔÕÓÖÍÕÖÓ-ÐÞØ Ë àÎÇÍÕÓÑÔÕÂÕËÚÇÔÍËØ ËÊÏÇÐÇÐËÌ ÑÔÕÂÕÍÂ, ÐÂØÑAEâÜÇ-ÅÑÔâ ÏÇÉAEÖ B B Ë P [204].…”