The Type 1 Homodimeric Reaction Center in Heliobacterium modesticaldum

Project: Research project

Description

Compared to Photosystem I, our understanding of homodimeric, Type I (Fe/S-cluster) reaction centers is primitive. The electron transfer cofactors are still being characterized, the protein composition is just coming into focus, the ability to make mutants is not yet possible, and there are few, if any, structural details available. This lack of progress is due partly to the fact that heliobacteria and green sulfur bacteria are strict anaerobes, with all of the attendant problems of growing the organisms and purifying the components in an oxygen-free environment. In the case of the heliobacterial reaction center (HbRC), the oxygen sensitivity is exacerbated by rapid destruction of the FA/FB clusters and the facile conversion of the BChl g pigments to a Chl a-like molecule. In this proposal, two experienced photosynthesis researchers, Kevin Redding (Arizona State University) and John Golbeck (Penn State University) propose to continue their collaborative research program aimed at understanding the process of charge separation and stabilization in the Type I homodimeric reaction center in Heliobacterium modesticaldum. What we know from the current funding period is that (i) a reaction center core consisting of the (PshA)2 homodimer can be purified to homogeniety; (ii) the FA and FB clusters are located on two loosely-bound proteins, PshB1 and PshB2; (iii) the genes for these proteins, HM1-1461 and HM1-1462 form an operon from which a single message is transcribed; (iv) the FX cluster is present in a ground spin state of S = 3/2 and has a surprisingly high reduction potential; (v) the HbRC core lacking PshB polypeptides can photo-reduce several different electron acceptors; (v) the enigmatic fluorescence emission from whole heliobacterial cells correlates with charge recombination between A0 and P800+, and (vi) the partial conversion of Bchl g to Chl aF is accompanied by a change in the CIDNP spectrum that is best explained if the intermediate accessory pigment, rather than the primary donor (P8

Description

Compared to Photosystem I, our understanding of homodimeric, Type I (Fe/S-cluster) reaction centers has been primitive. This lack of progress was due partly to the fact that heliobacteria are strict anaerobes, with all of the attendant problems of growing the organisms and purifying the components in an oxygen-free environment. In the case of the heliobacterial reaction center (HbRC), the oxygen sensitivity is exacerbated by the facile conversion of the bacteriochlorophyll (BChl) g pigments to a Chl a-like molecule, which ultimately renders the reaction center incapable of carrying out long-lived charge separation.
We have made the following major accomplishments in the current funding period. (1) We have determined the structure of the HbRC from Heliobacterium modesticaldum by X-ray crystallography to a resolution of 2.2 . (2) We have demonstrated that the FX cluster, which is the terminal acceptor, is present in a ground spin state of S = 3/2 and has a reduction potential of -0.50 V. (3) We have measured the reduction potentials of the 3 small, dicluster ferredoxins predicted to be present based on the genome. (4) We have discovered that the HbRC can reduce menaquinone to menaquinol, thus breaking the Type I/II reaction center paradigm. (5) We have created a genetic system for deleting genes for the first time in any heliobacterium. (6) We have created a system for expressing the HbRC in a new host by engineering the biosynthesis of BChl g in a purple proteobacterium (Rhodobacter sphaeroides). We have thus shown that the HbRC is not only the simplest of all known photosynthetic reaction centers in terms of its polypeptide composition, it also possesses unique activities among RCs in that it uses its FX cluster as the reductant of multiple soluble acceptor proteins (e.g. ferredoxins) but can reduce membrane-soluble quinones, like a Type II RC, in the absence of soluble acceptors. The advances that we have made in key infrastructural aspects (e.g. genetic transformation and X-ray crystallography) position us to take this project to the next level. Our long-term goal is ultimately to bring knowledge of this reaction center to the same level of sophistication as that of the bacterial reaction center and of Photosystems I and II.
With our new capabilities in hand, we propose to expand this project, while retaining a focus on light-driven electron transfer, both the reactions occurring within the HbRC as well as electron transport in the heliobacterial cell driven by the HbRC. Our goals for the next three years are to: (1) genetically test our model of light-driven cyclic electron flow pathways within heliobacterial cells by deleting genes for each protein implicated in the pathways and measuring the activities of the mutants; (2) measure the in vivo abundance of each ferredoxin by mass spectrometry and the affinity of each for the HbRC by isothermal calorimetry; (3) investigate key structural features of the electron transfer chain within the HbRC by a combination of site-directed mutagenesis and X-ray crystallography, including identification of the quinone-binding site; (4) explore the reduction of quinone to quinol in a newly-created proteoliposome system using biochemical techniques and probe reduction of bound quinone to a semiquinone in a mutant lacking the FX cluster with time-resolved optical and EPR (electron paramagnetic resonance) spectroscopy; (5) elucidate the electronic structure of the primary electron donor and acceptor using advanced pulsed EPR techniques and quantum mechanical computational chemistry, as well as determine the mechanism of primary charge separation using a combination of site-directed mutagenesis and ultra-fast pump-probe spectroscopy; (6) determine the requirements for assembling an active HbRC by isolating heliobacterial mutants unable to do so and by co-expressing heliobacterial proteins in R. sphaeroides cells that synthesize BChl g and the HbRC subunits.

Description

Compared to Photosystem I, our understanding of homodimeric, Type I (Fe/S-cluster) reaction centers is primitive. The electron transfer cofactors are still being characterized, the protein composition has just come into focus, the ability to make mutants is not yet possible, and there are few, if any, structural details available. This lack of progress is due partly to the fact that heliobacteria are strict anaerobes, with all of the attendant problems of growing the organisms and purifying the components in an oxygen-free environment. In the case of the heliobacterial reaction center (HbRC), the oxygen sensitivity is exacerbated by the facile conversion of the bacteriochlorophyll (BChl) g pigments to a Chl a-like molecule, which ultimately renders the reaction center incapable of carrying out long-lived charge separation. What we have learned from the current funding period is that (i) a reaction center core consisting of the (PshA)2 homodimer can be purified and crystallized, and the crystals can be obtained that diffract to ~2.4 resolution; (ii) the FX cluster, which is the terminal acceptor, is present in a ground spin state of S = 3/2 and has a reduction potential of -0.50 V); (iii) the heterodimeric BChl g'/Chl a'F special pair formed by partial conversion is photochemically active; (iv) the HbRC can reduce its loosely-bound quinone to quinol; (v) heliobacteria can be stably transformed with replicating plasmids, allowing genetic engineering of this group of organisms for the first time. We have thus shown that the HbRC is not only the simplest of all known photosynthetic reaction centers, in terms of its polypeptide composition, it also possesses unique activities among RCs in that it uses its FX cluster as the reductant of multiple soluble acceptor proteins (e.g. ferredoxins) but can reduce loosely bound quinones, like a Type II RC, in the absence of soluble acceptors. Furthermore, the ability to convert in a controlled fashion the BChl g bound within the RC into a functionally distinct pigment (Chl aF) in situ provides a powerful system to analyze the effects of pigment conversion upon reaction center photochemistry without requiring solvent extraction and reconstitution.
In this proposal, we build on these important advances. Our goals for the next three years are to: (i) Devise promoter sequences (constitutive or inducible) with predictable transcription levels to drive expression of introduced genes and to develop methodologies (e.g. CRISPR/CAS9 system) for the generation of gene deletion and site-directed mutants. (ii) Study the optical, EPR and redox properties of the heterodimeric BChl g'/Chl a'F special pair and characterize energy transfer to the trap in partially converted reaction centers. (iii) Determine the properties and function of the four low molecular mass ferredoxins by studying their interactions with the HbRC core, their X-ray crystal structures, and by characterizing the phenotype of the cells in selective knockout mutants. (iv) Characterize menaquinone reduction by creating an in vitro system to study the process in isolation, by spectroscopically examining menaquinone reduction in artificial liposomes, and by genetic abrogation of the quinone biosynthetic pathway in cells. (v) Overcome the phasing problem to solve the 3-dimensional structure of the (PshA)2 homodimer by producing highly diffracting crystals and derivatives, with the expectation that we should be able to obtain a high-resolution 3-dimensional structure in the next grant period. Our long-term goal is to ultimately bring knowledge of this reaction center to the same level of sophistication as that of the bacterial reaction center and of Photosystems I and II.

StatusActive
Effective start/end date9/1/138/31/20

Funding

  • US Department of Energy (DOE): $2,160,337.00

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Bacteriochlorophylls
Photosystem I Protein Complex
Ferredoxins
Pigments
Oxygen
Electrons
Vitamin K 2
X ray crystallography
Proteins
Genes
Hydroquinones
Photosynthetic Reaction Center Complex Proteins
Paramagnetic resonance
Quinones
Mutagenesis
Photosystem II Protein Complex
Reducing Agents
Chemical analysis
Peptides
Molecules