Modification of a hydrogen bond to a bacteriochlorophyll a molecule in the light-harvesting 1 antenna of Rhodobacter sphaeroides.

Abstract

Site-directed mutagenesis has been used to examine the function of a highly conserved aromatic residue, darp43, in the light-harvesting 1 antenna ofthe photosynthetic bacterium Rhodobacter sphaeroides. In this antenna dfrp43 is thought to be located near the putative binding site for bacteriochlorophyll; in this work it was changed to both Tyr and The photosynthetic apparatus of the purple bacterium Rhodobacter sphaeroides consists of a membrane protein complex, the reaction center (RC), surrounded by a core light-harvesting complex, LH1, also referred to as B875, in a fixed stoichiometry of -12 LH1 a(3 dimers per RC. An additional peripheral light-harvesting complex, LH2, also referred to as B800-850, occurs in variable amounts in response to environmental levels of light and oxygen (1, 2). The structure of the RC of Rb. sphaeroides is known to atomic resolution (3, 4); however, the structures of the LH1 and LH2 complexes are not known, although diffracting crystals ofvarious LH complexes have been reported (5-10). As yet, these structures have not been solved, so it is necessary to employ other methods such as mutagenesis and spectroscopy to obtain relevant data. As a result of these approaches, more information is becoming available on the interaction of the chromophores of LH1 and LH2 with nearby residues of the antenna polypeptides (11,12). These data can be used to test existing model structures derived from spectroscopic and electrophoretic mobility studies of wild-type (WT) complexes. The models that have been proposed for bacterial LH complexes all follow a similar pattern in that the af8 heterodimer, which in the case of LH1 would bind 2 bacteriochlorophyll (bchl) molecules, is considered to be the minimum possible building block. The topology and conformation of these subunits are thought to be similar, each with a cytoplasmically exposed N terminus, a single transmembrane helix, and a periplasmic C terminus. There are indications of turns at or near the membrane interfaces (13 (20), and Rhodocyclus gelatinosus (21), leading to the speculation that this bchl dimer is the building block of the LH1 complex. Characterization of the B820 form (22) supports the idea that it is an excitonically coupled bchl a dimer. Moreover, resonance Raman measurements of the B820 showed that it differed from the B873 form in the interactions of the C2 acetyl carbonyl; the loss of a H bond was one possible explanation of the Raman data. A more detailed Raman study of the LH1 ofRs. rubrum G9 has shown that upon formation of the B820, the strength of one H bond was increased (23 FT, Fourier transform; WT, wild type; P-OG, n-octyl P-Dglucopyranoside; ODPS, n-octyldipropyl sulfoxide. 7124The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Photoprotection in a purple phototrophic bacterium mediated by oxygen-dependent alteration of carotenoid excited-state properties

“…The biosynthetic incorporation of a single keto oxygen into the structure of spheroidene changes the energy transfer pathways, and the S 1 ∕ICT route becomes dominant. Because the ICT state is not active in LH2 complexes binding the same carotenoid, spheroidenone, an LH1-specific interaction must exist between the keto group of spheroidenone and amino acid residues such as tryptophan and tyrosine, as found for interactions of these residues with LH1 BChls (43,44). There is evidence that the Trp residue of the highly conserved Lys-Ile-Trp (KIW) motif found in the N-terminal domain of the LH1α polypeptide plays a role in binding spheroidenone in the LH1 complex of Rba.…”

“…The LH2-minus crtA mutant DBCΩ ΔcrtA was constructed by in-frame deletion of the crtA gene using an LH2-minus background, DBCΩ (51), so spheroidene became the dominant carotenoid in the core complexes; this mutant was grown photosynthetically. Intracytoplasmic membrane vesicles were then prepared from both LH2-minus mutants according to published methods (43). PufX-minus (see SI Text) membranes were prepared from a PufX-minus mutant with monomeric core complexes (52).…”

The Organization of LH2 Complexes in Membranes from Rhodobacter sphaeroides

“…The R. sphaeroides deletion strain DPF2G (16) was grown and the membranes were prepared according to the methods in Olsen et al (26) and further treated with the detergent ␤-DDM (Glycon Biochemistry, GmbH Biotechnology, Germany), at 0.005 and 0.01% (w/v) concentration, following the protocol of Bahatyrova et al (9). The membrane patches were adsorbed onto freshly cleaved mica (Agar Scientific) in 20 mM HEPES, pH 7.5, 150 mM KCl, 25 mM MgCl 2 , 0.5 mM NiCl 2 for an hour at room temperature.…”

Spectral properties of LH2 exhibit very similar even when heterologously express LH2 with <i>β</i>-subunit fusion protein in <i>Rhodobacter</i> <i>sphaeroides</i>

“…sphaeroides [17,18]. Similarly, the LH1 αTrp43 was proposed to be located near the putative binding site for BChl molecule [19]. Dissimilar to the purple non-sulfur bacteria, calcium ions were demonstrated to be involved in the unusual red shift of the LH1 Q y transition of the core complex in Thermochromatium tepidum [20].…”