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Identification and characterization of the low molecular mass ferredoxins involved in central metabolism in Heliomicrobium modesticaldum

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Abstract

The homodimeric Type I reaction center (RC) from Heliomicrobium modesticaldum lacks the PsaC subunit found in Photosystem I and instead uses the interpolypeptide [4Fe–4S] cluster FX as the terminal electron acceptor. Our goal was to identify which of the small mobile dicluster ferredoxins encoded by the H. modesticaldum genome are capable of accepting electrons from the heliobacterial RC (HbRC) and pyruvate:ferredoxin oxidoreductase (PFOR), a key metabolic enzyme. Analysis of the genome revealed seven candidates: HM1_1462 (PshB1), HM1_1461 (PshB2), HM1_2505 (Fdx3), HM1_0869 (FdxB), HM1_1043, HM1_0357, and HM1_2767. Heterologous expression in Escherichia coli and studies using time-resolved optical spectroscopy revealed that only PshB1, PshB2, and Fdx3 are capable of accepting electrons from the HbRC and PFOR. Modeling studies using AlphaFold show that only PshB1, PshB2, and Fdx3 should be capable of docking on PFOR at a positively charged patch that overlays a surface-proximal [4Fe–4S] cluster. Proteomic analysis of wild-type and gene deletion strains ΔpshB1, ΔpshB2, ΔpshB1pshB2, and Δfdx3 grown under nitrogen-replete conditions revealed that Fdx3 is undetectable in the wild-type, ΔpshB1, and Δfdx3 strains, but it is present in the ΔpshB2 and ΔpshB1pshB2 strains, implying that Fdx3 may substitute for PshB2. When grown under nitrogen-deplete conditions, Fdx3 is present in the wild-type and all deletion strains except for Δfdx3. None of the knockout strains demonstrated significant impairment during chemotrophic dark growth on pyruvate, photoheterotrophic light growth on pyruvate, or phototrophic growth on acetate+CO2, indicating a high degree of redundancy among these three electron transfer proteins. Loss of both PshB1 and PshB2, but not FdxB, resulted in poor growth under N2-fixing conditions.

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Acknowledgements

We wish to thank Patricia Baker for technical assistance with construction of the gene knockouts and growth studies.

Funding

This work was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0010575.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 16802, USA

    Karim A. Walters & John H. Golbeck

  2. School of Molecular Sciences, Arizona State University, 1711 S Rural Rd, Box 871604, Tempe, AZ, 85287-1604, USA

    Kevin E. Redding

  3. Center for Bioenergy & Photosynthesis, Arizona State University, Tempe, AZ, 85287, USA

    Kevin E. Redding

  4. Department of Chemistry, The Pennsylvania State University, University Park, PA, 16802, USA

    John H. Golbeck

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  1. Karim A. Walters
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JG, KM, and KR wrote the main manuscript text and jointly prepared the figures. All authors reviewed the manuscript.

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Correspondence to Kevin E. Redding or John H. Golbeck.

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Walters, K.A., Redding, K.E. & Golbeck, J.H. Identification and characterization of the low molecular mass ferredoxins involved in central metabolism in Heliomicrobium modesticaldum. Photosynth Res (2024). https://doi.org/10.1007/s11120-023-01069-z

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