Philip J. Jackson

Three-Dimensional Structure of the Rhodobacter sphaeroides RC- LH1-PufX Complex: Dimerization and Quinone Channels Promoted by PufX

Reaction center-light harvesting 1 (RC-LH1) complexes are the fundamental units of bacterial photosynthesis, which use solar energy to power the reduction of quinone to quinol prior to the formation of the proton gradient that drives ATP synthesis. The dimeric RC-LH1-PufX complex of Rhodobacter sphaeroides is composed of 64 polypeptides and 128 cofactors, including 56 LH1 bacteriochlorophyll a (BChl a) molecules that surround and donate energy to the two RCs. The 3D structure was determined to 8 Å by X-ray crystallography, and a model was built with constraints provided by electron microscopy (EM), nuclear magnetic resonance (NMR), mass spectrometry (MS), and site-directed mutagenesis. Each half of the dimer complex consists of a RC surrounded by an array of 14 LH1 αβ subunits, with two BChls sandwiched between each αβ pair of transmembrane helices. The N- and C-terminal extrinsic domains of PufX promote dimerization by interacting with the corresponding domains of an LH1 β polypeptide from the other half of the RC-LH1-PufX complex. Close contacts between PufX, an LH1 αβ subunit, and the cytoplasmic domain of the RC-H subunit prevent the LH1 complex from encircling the RC and create a channel connecting the RC QB site to an opening in the LH1 ring, allowing Q/QH₂ exchange with the external quinone pool. We also identified a channel that connects the two halves of the dimer, potentially forming a long-range pathway for quinone migration along rows of RC-LH1-PufX complexes in the membrane. The structure of the RC-LH1-PufX complex explains the crucial role played by PufX in dimer formation, and it shows how quinone traffic traverses the LH1 complex as it shuttles between the RC and the cytochrome bc₁ complex.

A Cyanobacterial Chlorophyll Synthase-HliD Complex Associates with the Ycf39 Protein and the YidC/Alb3 Insertase

Chlorophyll synthase attaches geranylgeraniol to the chlorophyll macrocycle and was used as bait to retrieve an enzymatically active complex comprising the synthase, the high-light-inducible protein HliD, Ycf39, and the YidC/Alb3 insertase. These data reveal a link between chlorophyll biosynthesis and Sec/YidC-dependent cotranslational insertion of nascent photosystem polypeptides into membranes. Macromolecular membrane assemblies of chlorophyll-protein complexes efficiently harvest and trap light energy for photosynthesis. To investigate the delivery of chlorophylls to the newly synthesized photosystem apoproteins, a terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), was tagged in the cyanobacterium Synechocystis PCC 6803 (Synechocystis) and used as bait in pull-down experiments. We retrieved an enzymatically active complex comprising ChlG and the high-light-inducible protein HliD, which associates with the Ycf39 protein, a putative assembly factor for photosystem II, and with the YidC/Alb3 insertase. 2D electrophoresis and immunoblotting also provided evidence for the presence of SecY and ribosome subunits. The isolated complex contained chlorophyll, chlorophyllide, and carotenoid pigments. Deletion of hliD elevated the level of the ChlG substrate, chlorophyllide, more than 6-fold; HliD is apparently required for assembly of FLAG-ChlG into larger complexes with other proteins such as Ycf39. These data reveal a link between chlorophyll biosynthesis and the Sec/YidC-dependent cotranslational insertion of nascent photosystem polypeptides into membranes. We expect that this close physical linkage coordinates the arrival of pigments and nascent apoproteins to produce photosynthetic pigment-protein complexes with minimal risk of accumulating phototoxic unbound chlorophylls.

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides.

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll-protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc1 whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc1-RC-LH1-PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.

Rapid resonance Raman microspectroscopy to probe carbon dioxide fixation by single cells in microbial communities

Photosynthetic microorganisms play crucial roles in aquatic ecosystems and are the major primary producers in global marine ecosystems. The discovery of new bacteria and microalgae that play key roles in CO2 fixation is hampered by the lack of methods to identify hitherto-unculturable microorganisms. To overcome this problem we studied single microbial cells using stable-isotope probing (SIP) together with resonance Raman (RR) microspectroscopy of carotenoids, the light-absorbing pigments present in most photosynthetic microorganisms. We show that fixation of 13CO2 into carotenoids produces a red shift in single-cell RR (SCRR) spectra and that this SCRR–SIP technique is sufficiently sensitive to detect as little as 10% of 13C incorporation. Mass spectrometry (MS) analysis of labelled cellular proteins verifies that the red shift in carotenoid SCRR spectra acts as a reporter of the 13C content of single cells. Millisecond Raman imaging of cells in mixed cultures and natural seawater samples was used to identify cells actively fixing CO2, demonstrating that the SCRR–SIP is a noninvasive method for the rapid and quantitative detection of CO2 fixation at the single cell level in a microbial community. The SCRR–SIP technique may provide a direct method for screening environmental samples, and could help to reveal the ecophysiology of hitherto-unculturable microorganisms, linking microbial species to their ecological function in the natural environment.

Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium-protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803

Background: The cyclase step in chlorophyll biosynthesis remains uncharacterized. Results: Ycf54 forms a complex with other oxidative cyclase components, and a ycf54 mutant accumulates the cyclase substrate. Conclusion: Ycf54 is essential for cyclase function. Significance: Identification of all of the components of chlorophyll biosynthesis is a step closer. The cyclase step in chlorophyll (Chl) biosynthesis has not been characterized biochemically, although there are some plausible candidates for cyclase subunits. Two of these, Sll1214 and Sll1874 from the cyanobacterium Synechocystis 6803, were FLAG-tagged in vivo and used as bait in separate pulldown experiments. Mass spectrometry identified Ycf54 as an interaction partner in each case, and this interaction was confirmed by a reciprocal pulldown using FLAG-tagged Ycf54 as bait. Inactivation of the ycf54 gene (slr1780) in Synechocystis 6803 resulted in a strain that exhibited significantly reduced Chl levels. A detailed analysis of Chl precursors in the ycf54 mutant revealed accumulation of very high levels of Mg-protoporphyrin IX methyl ester and only traces of protochlorophyllide, the product of the cyclase, were detected. Western blotting demonstrated that levels of the cyclase component Sll1214 and the Chl biosynthesis enzymes Mg-protoporphyrin IX methyltransferase and protochlorophyllide reductase are significantly impaired in the ycf54 mutant. Ycf54 is, therefore, essential for the activity and stability of the oxidative cyclase. We discuss a possible role of Ycf54 as an auxiliary factor essential for the assembly of a cyclase complex or even a large multienzyme catalytic center.

Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway

Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon–carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N = 10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoea agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2′-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC–LH1–PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2′-diketo-spirilloxanthin (15 conjugated C 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 C bonds; N = 15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N = 9; 94%), spheroidene (N = 10; 96%) and spheroidenone (N = 11; 95%), whereas intermediate values were measured for lycopene (N = 11; 64%), rhodopin (N = 11; 62%) and spirilloxanthin (N = 13; 39%). The variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the energy transfer dynamics of carotenoids in bacterial photosynthesis.

Identification of an 8-vinyl reductase involved in bacteriochlorophyll biosynthesis in Rhodobacter sphaeroides and evidence for the existence of a third distinct class of the enzyme

The purple phototrophic bacterium Rhodobacter sphaeroides utilises bacteriochlorophyll a for light harvesting and photochemistry. The synthesis of this photopigment includes the reduction of a vinyl group at the C8 position to an ethyl group, catalysed by a C8-vinyl reductase. An active form of this enzyme has not been identified in R. sphaeroides, but its genome contains two candidate ORFs (open reading frames) similar to those reported to encode C8-vinyl reductases in the closely related Rhodobacter capsulatus (bchJ), and in plants and green sulfur bacteria (rsp_3070). To determine which gene encodes the active enzyme, knock-out mutants in both genes were constructed. Surprisingly, mutants in which one or both genes were deleted still retained the ability to synthesize C8-ethyl bacteriochlorophyll. These genes were subsequently expressed in a cyanobacterial mutant that cannot synthesize C8-ethyl chlorophyll a. R. sphaeroides rsp_3070 was able to restore synthesis of the WT (wild-type) C8-ethyl chlorophyll a in the mutant, whereas bchJ did not. The results of the present study demonstrate that Rsp_3070 is a functional C8-vinyl reductase and that R. sphaeroides utilises at least two enzymes to catalyse this reaction, indicating the existence of a third class, while there remains no direct evidence for the activity of BchJ as a C8-vinyl reductase.

PucC and LhaA direct efficient assembly of the light‐harvesting complexes in Rhodobacter sphaeroides

The mature architecture of the photosynthetic membrane of the purple phototroph Rhodobacter sphaeroides has been characterised to a level where an atomic‐level membrane model is available, but the roles of the putative assembly proteins LhaA and PucC in establishing this architecture are unknown. Here we investigate the assembly of light‐harvesting LH2 and reaction centre‐light‐harvesting1‐PufX (RC‐LH1‐PufX) photosystem complexes using spectroscopy, pull‐downs, native gel electrophoresis, quantitative mass spectrometry and fluorescence lifetime microscopy to characterise a series of lhaA and pucC mutants. LhaA and PucC are important for specific assembly of LH1 or LH2 complexes, respectively, but they are not essential; the few LH1 subunits found in ΔlhaA mutants assemble to form normal RC‐LH1‐PufX core complexes showing that, once initiated, LH1 assembly round the RC is cooperative and proceeds to completion. LhaA and PucC form oligomers at sites of initiation of membrane invagination; LhaA associates with RCs, bacteriochlorophyll synthase (BchG), the protein translocase subunit YajC and the YidC membrane protein insertase. These associations within membrane nanodomains likely maximise interactions between pigments newly arriving from BchG and nascent proteins within the SecYEG‐SecDF‐YajC‐YidC assembly machinery, thereby co‐ordinating pigment delivery, the co‐translational insertion of LH polypeptides and their folding and assembly to form photosynthetic complexes.

Quantitative proteomic analysis of intracytoplasmic membrane development in Rhodobacter sphaeroides.

The purple phototrophic bacteria elaborate a specialized intracytoplasmic membrane (ICM) system for the conversion of solar energy to ATP. Previous radiolabelling and ultrastructural experiments have shown that ICM assembly in Rhodobacter sphaeroides is initiated at indentations of the cytoplasmic membrane, termed UPB. Here, we report proteomic analyses of precursor (UPB) and mature (ICM) fractions. Qualitative data identified 387 proteins, only 43 of which were found in the ICM, reflecting its specialized role within the cell, the conversion of light into chemical energy; 236 proteins were found in the significantly more complex UPB proteome. Metabolic labelling was used to quantify the relative distribution of 173 proteins between the UPB and ICM fractions. Quantification reveals new information on assembly of the RC‐LH1‐PufX, ATP synthase and NAD(P)H transhydrogenase complexes, as well as showing that the UPB is enriched in enzymes for lipid, carbohydrate and amino acid metabolism, tetrapyrrole biosynthesis and proteins representing a wide range of other metabolic and biosynthetic functions. Proteins involved in light harvesting, photochemistry, electron transport and ATP synthesis are all enriched in ICM, consistent with the spatial proximity of energy capturing and transducing functions. These data provide further support to the developmental precursor–product relationship between UPB and ICM.

Aberrant Assembly Complexes of the Reaction Center Light Harvesting 1 PufX (RC-LH1-PufX) Core Complex of Rhodobacter sphaeroides Imaged by Atomic Force Microscopy

Background: Photosynthesis relies upon connected pigment protein complexes to capture, transfer, and utilize light energy. Results: Aberrant forms of light-harvesting 1 (LH1) complex occur without the reaction center (RC); complete RC-LH1 complexes and isolated RCs were found when LH1 levels were reduced. Conclusion: The subunits of the RC-LH1 complex of Rhodobacter sphaeroides are assembled cooperatively. Significance: This represents a paradigm for assembly of other purple bacterial photosystems. In the purple phototrophic bacterium Rhodobacter sphaeroides, many protein complexes congregate within the membrane to form operational photosynthetic units consisting of arrays of light-harvesting LH2 complexes and monomeric and dimeric reaction center (RC)-light-harvesting 1 (LH1)-PufX “core” complexes. Each half of a dimer complex consists of a RC surrounded by 14 LH1 αβ subunits, with two bacteriochlorophylls (Bchls) sandwiched between each αβ pair of transmembrane helices. We used atomic force microscopy (AFM) to investigate the assembly of single molecules of the RC-LH1-PufX complex using membranes prepared from LH2-minus mutants. When the RC and PufX components were also absent, AFM revealed a series of LH1 variants where the repeating α1β1(Bchl)2 units had formed rings of variable size, ellipses, and spirals and also arcs that could be assembly products. The spiral complexes occur when the LH1 ring has failed to close, and short arcs are suggestive of prematurely terminated LH1 complex assembly. In the absence of RCs, we occasionally observed captive proteins enclosed by the LH1 ring. When production of LH1 units was restricted by lowering the relative levels of the cognate pufBA transcript, we imaged a mixture of complete RC-LH1 core complexes, empty LH1 rings, and isolated RCs, leading us to conclude that once a RC associates with the first α1β1(Bchl)2 subunit, cooperative associations between subsequent subunits and the RC tend to drive LH1 ring assembly to completion.