The Type I Homodimeric Reaction Center in Heliobacterium modesticaldum

Project: Research project

Project Details


The Type I Homodimeric Reaction Center in Heliobacterium modesticaldum The Type I Homodimeric Reaction Center in Heliobacterium modesticaldum 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.
Effective start/end date9/1/199/2/19


  • DOE: Office of Science (OS): $1.00


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