Carotenoid assembly regulates quinone diffusion and the Roseiflexus castenholzii reaction center-light harvesting complex architecture
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eLife assessment
This is a valuable analysis of the structure of Roseiflexus castenholzii native and carotenoid-depleted light harvesting complexes. The authors have investigated the relationship between Carotenoid pigment depletion in the photosynthesis-related light harvesting complex, the assembly of the prokaryotic reaction center LH complex, and quinone exchange in Roseiflexus castenholzii, a chlorosome-less filamentous anoxygenic phototroph that forms the deepest branch of photosynthetic bacteria. The evidence supporting the claims is solid, with application of rigorous biochemical and biophysical techniques, including cryo-electron microscopy of the purified of the RC-LH complexes with or depleted of carotenoids. This study will be of interest to biologists working on the evolution and diversity of prokaryotic photosynthetic apparatus.
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Abstract
Carotenoid (Car) pigments perform central roles in photosynthesis-related light harvesting (LH), photoprotection, and assembly of functional pigment-protein complexes. However, the relationships between Car depletion in the LH, assembly of the prokaryotic reaction center (RC)-LH complex, and quinone exchange are not fully understood. Here, we analyzed native RC-LH (nRC-LH) and Car-depleted RC-LH (dRC-LH) complexes in Roseiflexus castenholzii , a chlorosome-less filamentous anoxygenic phototroph that forms the deepest branch of photosynthetic bacteria. Newly identified exterior Cars functioned with the bacteriochlorophyll B800 to block the proposed quinone channel between LHαβ subunits in the nRC-LH, forming a sealed LH ring that was disrupted by transmembrane helices from cytochrome c and subunit X to allow quinone shuttling. dRC-LH lacked subunit X, leading to an exposed LH ring with a larger opening, which together accelerated the quinone exchange rate. We also assigned amino acid sequences of subunit X and two hypothetical proteins Y and Z that functioned in forming the quinone channel and stabilizing the RC-LH interactions. This study reveals the structural basis by which Cars assembly regulates the architecture and quinone exchange of bacterial RC-LH complexes. These findings mark an important step forward in understanding the evolution and diversity of prokaryotic photosynthetic apparatus.
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eLife assessment
This is a valuable analysis of the structure of Roseiflexus castenholzii native and carotenoid-depleted light harvesting complexes. The authors have investigated the relationship between Carotenoid pigment depletion in the photosynthesis-related light harvesting complex, the assembly of the prokaryotic reaction center LH complex, and quinone exchange in Roseiflexus castenholzii, a chlorosome-less filamentous anoxygenic phototroph that forms the deepest branch of photosynthetic bacteria. The evidence supporting the claims is solid, with application of rigorous biochemical and biophysical techniques, including cryo-electron microscopy of the purified of the RC-LH complexes with or depleted of carotenoids. This study will be of interest to biologists working on the evolution and diversity of prokaryotic photosynthetic …
eLife assessment
This is a valuable analysis of the structure of Roseiflexus castenholzii native and carotenoid-depleted light harvesting complexes. The authors have investigated the relationship between Carotenoid pigment depletion in the photosynthesis-related light harvesting complex, the assembly of the prokaryotic reaction center LH complex, and quinone exchange in Roseiflexus castenholzii, a chlorosome-less filamentous anoxygenic phototroph that forms the deepest branch of photosynthetic bacteria. The evidence supporting the claims is solid, with application of rigorous biochemical and biophysical techniques, including cryo-electron microscopy of the purified of the RC-LH complexes with or depleted of carotenoids. This study will be of interest to biologists working on the evolution and diversity of prokaryotic photosynthetic apparatus.
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Reviewer #1 (Public Review):
In this work, the authors have investigated the relationship between Carotenoid pigment depletion in the photosynthesis-related light harvesting complex, the assembly of the prokaryotic reaction center LH complex, and quinone exchange in Roseiflexus castenholzii, a chlorosome-less filamentous anoxygenic phototroph that forms the deepest branch of photosynthetic bacteria. By means of different biochemical and biophysical techniques, including cryo-electron microscopy of the purified RC-LH complexes with or depleted of carotenoids, the authors provide evidence of the structural basis by which Carotenoid assembly regulates the architecture and quinone exchange of bacterial RC-LH 40 complexes. Although most of the experiments described in this manuscript are structural, by analyzing Cryo-MS results, the authors …
Reviewer #1 (Public Review):
In this work, the authors have investigated the relationship between Carotenoid pigment depletion in the photosynthesis-related light harvesting complex, the assembly of the prokaryotic reaction center LH complex, and quinone exchange in Roseiflexus castenholzii, a chlorosome-less filamentous anoxygenic phototroph that forms the deepest branch of photosynthetic bacteria. By means of different biochemical and biophysical techniques, including cryo-electron microscopy of the purified RC-LH complexes with or depleted of carotenoids, the authors provide evidence of the structural basis by which Carotenoid assembly regulates the architecture and quinone exchange of bacterial RC-LH 40 complexes. Although most of the experiments described in this manuscript are structural, by analyzing Cryo-MS results, the authors also propose some predictions about the functional roles of proteins/pigments in LH complex, such as the role of the gap in the ring that persists without a canonical subunit X. Together, the results presented are important to understand the evolution and diversity of prokaryotic photosynthetic apparatus.
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Reviewer #2 (Public Review):
In this work, Xin et al. describe cryo-EM structures of the native and carotenoid-depleted forms of RC-LH from R. castenholzii, attempting to reveal how differences in the carotenoid composition may result in the structural and functional differences in the RC-LH complex. Previously, the authors obtained the nRC-LH structure at 4.1 angstrom resolution. The current work extends the earlier moderate-resolution to a higher resolution (2.8 angstrom), which allowed them to identify 14 additional carotenoid molecules located at the external positions between adjacent LHs. These external carotenoids, together with bacteriochlorophylls, result in an impenetrable LH ring surrounding the RC, leaving only the LH opening shaped by subunit X and c-TM as the pathway for quinone exchange. They further solve the dRC-LH …
Reviewer #2 (Public Review):
In this work, Xin et al. describe cryo-EM structures of the native and carotenoid-depleted forms of RC-LH from R. castenholzii, attempting to reveal how differences in the carotenoid composition may result in the structural and functional differences in the RC-LH complex. Previously, the authors obtained the nRC-LH structure at 4.1 angstrom resolution. The current work extends the earlier moderate-resolution to a higher resolution (2.8 angstrom), which allowed them to identify 14 additional carotenoid molecules located at the external positions between adjacent LHs. These external carotenoids, together with bacteriochlorophylls, result in an impenetrable LH ring surrounding the RC, leaving only the LH opening shaped by subunit X and c-TM as the pathway for quinone exchange. They further solve the dRC-LH structure at 3.1 angstrom resolution, and find that while nRC-LH binds 15 internal and 14 external carotenoids, dRC-LH contains only five internal carotenoids, as well as a highly mobile c-TM, but no subunit X. Comparing the two types of complexes at both structural and biochemical levels, they show that these structural changes may result in the accelerated quinone exchange in dRC-LH than that in nRC-LH.
The structural data in this work are solid. The cryo-EM structures are well discussed and presented by the authors to highlight the structural features that may arise from carotenoid depletion. The authors also measured the oxidation rate of the auracyanin to characterize the quinone exchange rate. The work carried out by the authors is useful in the understanding of the regulatory role of carotenoids in complex assembly and quinone exchange. -
Reviewer #3 (Public Review):
Light harvesting (LH) associated with photosynthesis, photoprotection, and the formation of useful pigment-protein complexes are all major functions of carotenoid (Car) pigments. However, the connections between quinone exchange, prokaryotic reaction center (RC)-LH complex formation, and Car depletion in the LH are not entirely understood. This article examined the native RC-LH (nRC-LH) and Car-depleted RC-LH (dRC-LH) complexes in the filamentous anoxygenic phototroph Roseiflexus castenholzii. The authors show with a high degree of detail using crystallography and Cryo-EM complemented with biophysical techniques important results of a new conformation of a LH. They could assigned the amino acid sequences of subunit X and two hypothetical proteins, Y and Z, that formed the quinone channel and maintained the …
Reviewer #3 (Public Review):
Light harvesting (LH) associated with photosynthesis, photoprotection, and the formation of useful pigment-protein complexes are all major functions of carotenoid (Car) pigments. However, the connections between quinone exchange, prokaryotic reaction center (RC)-LH complex formation, and Car depletion in the LH are not entirely understood. This article examined the native RC-LH (nRC-LH) and Car-depleted RC-LH (dRC-LH) complexes in the filamentous anoxygenic phototroph Roseiflexus castenholzii. The authors show with a high degree of detail using crystallography and Cryo-EM complemented with biophysical techniques important results of a new conformation of a LH. They could assigned the amino acid sequences of subunit X and two hypothetical proteins, Y and Z, that formed the quinone channel and maintained the RC-LH connections. This study identifies a new architectural basis for the regulation of bacterial RC-LH complex and quinone exchange by Cars assembly, which is distinct from the well known purple bacteria. These findings represent a significant advancement of diversity and development of bacterial photosynthetic machinery.
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