Abstract:Photosynthesis in some phototrophic bacteria requires the PufX component of the reaction centre–light‐harvesting 1–PufX (RC‐LH1‐PufX) complex, which creates a pore for quinone/quinol (Q/QH
2) exchange across the LH1 barrier surrounding the RC. However, photosynthetic bacteria such as Thermochromatium (T.) tepidum do not require PufX because there are fewer carotenoid binding sites, which creates multiple pores in the LH1 ring for Q/QH
2 exchange. We show that an αTrp‐24→Phe alteration of the Rhodobacter (Rba.)… Show more
“…Deletion of pufX from Rba. species prevents photosynthetic growth [37] , [38] unless LH1 is also deleted [39] , or LH1 is modified by suppressor mutants or mutagenesis to prevent ring closure [3] , [40] , [41] , [42] or to have lowered carotenoid content [43] . Here, deletion of rpa4402 did not have any effect on the photosynthetic growth of Rps.…”
The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13 years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes.
“…Deletion of pufX from Rba. species prevents photosynthetic growth [37] , [38] unless LH1 is also deleted [39] , or LH1 is modified by suppressor mutants or mutagenesis to prevent ring closure [3] , [40] , [41] , [42] or to have lowered carotenoid content [43] . Here, deletion of rpa4402 did not have any effect on the photosynthetic growth of Rps.…”
The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13 years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes.
“…Rba. sphaeroides RCLH1(X) complexes contain two carotenoids per LH1 subunit, 38 but these were not resolved in the available X-ray crystal structure. 22 These rings of BChl and carotenoid pigments harvest light energy and “feed” the resulting excited state to the RC electron transfer chain.…”
We address concerns that PufX-deficient RCLH1 complexes from photosynthetically-incompetent bacteria may not be suitable as photovoltaic materials for incorporation into biohybrid photoelectrochemical cells.
“…Suppressor mutations that relieve this blockage do so by impairing LH1 assembly and opening gaps in the LH1 ring [24] , [25] . Other LH1 mutations that prevent encirclement of the RC or impair carotenoid binding also compensate for the loss of PufX [26] , [27] . Fig.…”
In bacterial photosynthesis reaction center-light-harvesting 1 (RC-LH1) complexes trap absorbed solar energy by generating a charge separated state. Subsequent electron and proton transfers form a quinol, destined to diffuse to the cytochrome bc1 complex. In bacteria such as Rhodobacter (Rba.) sphaeroides and Rba. capsulatus the PufX polypeptide creates a channel for quinone/quinol traffic across the LH1 complex that surrounds the RC, and it is therefore essential for photosynthetic growth. PufX also plays a key role in dimerization of the RC-LH1-PufX core complex, and the structure of the Rba. sphaeroides complex shows that the PufX C-terminus, particularly the region from X49-X53, likely mediates association of core monomers. To investigate this putative interaction we analysed mutations PufX R49L, PufX R53L, PufX R49/53L and PufX G52L by measuring photosynthetic growth, fractionation of detergent-solubilised membranes, formation of 2-D crystals and electron microscopy. We show that these mutations do not affect assembly of PufX within the core or photosynthetic growth but they do prevent dimerization, consistent with predictions from the RC-LH1-PufX structure. We obtained low resolution structures of monomeric core complexes with and without PufX, using electron microscopy of negatively stained single particles and 3D reconstruction; the monomeric complex with PufX corresponds to one half of the dimer structure whereas LH1 completely encloses the RC if the gene encoding PufX is deleted. On the basis of the insights gained from these mutagenesis and structural analyses we propose a sequence for assembly of the dimeric RC-LH1-PufX complex.
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