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DE-SC0010575: The Type I Homodimer Reaction Center in Heliobacterium Modesticaldum

Award Status: Active
  • Institution: Arizona Board of Regents for Arizona State University, Tempe, AZ
  • UEI: NTLHJXM55KZ6
  • DUNS: 943360412
  • Most Recent Award Date: 08/17/2023
  • Number of Support Periods: 11
  • PM: Herbert, Stephen
  • Current Budget Period: 09/01/2023 - 08/31/2024
  • Current Project Period: 09/01/2022 - 08/31/2025
  • PI: Redding, Kevin
  • Supplement Budget Period: N/A
 

Public Abstract

Photochemical reaction centers (RCs), the protein-pigment complexes that allow biological organisms to harvest light energy and convert it into a biologically useful form of chemical energy, first appeared on this planet over 3 billion years ago. Since then they have diversified and are now present in very different forms of life. The RCs are divided into two groups based on their composition and types of electron acceptor: type I RCs (like Photosystem I in plants, algae and cyanobacteria) contain iron-sulfur clusters and reduce soluble low-potential proteins, such as ferredoxins, while type II RCs (like Photosystem II) contain pheophytin and reduce membrane-soluble quinones. Photosystem I (PSI) is heterodimeric and has been the exemplar of Type I RCs, but the other members of this group are all homodimeric. Our understanding of these homodimeric RCs had been rudimentary – but what we have discovered in the last decade indicates that they function quite differently from PSI. The heliobacteria are a group of phototrophic anaerobic Gram-positive bacteria that use a homodimeric Type I RC and bacteriochlorophyll (BChl) g as their major pigment. Our DOE-funded work has confirmed previous findings that the embedded quinone is not required for forward electron transfer in the heliobacterial RC (HbRC), in contrast to PSI; our work indicates that the HbRC can reduce membrane-soluble quinones in the absence of water-soluble protein acceptors. These results blur the distinction between Type I and II RCs. 

During the course of this project, we have made two major advances: (1) determination of the structure of the HbRC from Heliobacterium modesticaldum and (2) creation of a genetic system for this species. During the process, we discovered an additional HbRC subunit: PshX, a 31-residue polypeptide consisting of a single transmembrane helix. Leveraging the organism's own CRISPR/Cas system, we have developed techniques to delete genes cleanly from the chromosome in H. modesticaldum. We have deleted the genes for both subunits of the HbRC; loss of the major subunit (PshA) resulted in no HbRC, while loss of the minor subunit (PshX) had little effect. Expression of mutant versions of PshA on a plasmid in the strain lacking the endogenous gene now allow us to study the effects of modifications of key amino acid residues identified in the structure. 

Our overall goal is to understand how the HbRC functions as well as the light-dependent electron transport pathways that it drives. This will entail an integrated computational and experimental research program. We have two major specific aims for the 3 years of this grant. Aim 1 is an in-depth study of the HbRC and the electron transfer events within it. We propose to (1) test the role of low-energy pigments in light harvesting, (2) test the role of a postulated intermediate in charge separation, (3) test mechanistic hypotheses for the postulated long-range (~13 Å) electron transfer step between the chlorophyll and FeS cluster, and (4) identify the mechanism used to reduce quinone to quinol. We focus heavily on the last goal, as it is the biggest challenge.

Aim 2 focuses on understanding the light-driven electron transport pathways in heliobacteria. There are two possible electron transport cycles driven by the HbRC, depending on which molecule it reduces. Reduction of quinone results in a short cycle, with the cytochrome bc complex reoxidizing quinol and reducing the membrane-bound cytochrome c553, which returns electrons to the HbRC. Reduction of ferredoxin results in a long cycle, with electrons transiting from the ferredoxin pool to the NAD(P)H pool and thence to the quinone pool via Complex I; it would thus result in more protons pumped (and ATP made) per electron cycled. We have started to test this hypothesis genetically by deleting genes of components. As expected, loss of all 4 genes encoding the major subunits of the cytochrome bc complex resulted in viable mutants that were non-phototrophic. We propose to computationally model the function of the short cycle using detailed structural models based on experimental data. This will enable computation of the ATP synthesis rate and cellular doubling times, which will be tested by quantifying growth rates of cells under different conditions. We will also gather the data required to model the long cyclic pathway. This includes identifying the ferredoxins reduced by the HbRC, their affinity to the HbRC, and their cellular abundance, as well as the role of the other components required to recycle electrons back to the quinone pool.

The end result of these studies will be a profound understanding of, and the consequent ability to manipulate, a relatively minimal biological energy conversion system that has the capability of using alternate pathways based on light-driven electron transfer reactions occurring within a simple photochemical reaction center.



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