Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase

  1. Chris Furlan
  2. Nipa Chongdar
  3. Pooja Gupta
  4. Wolfgang Lubitz
  5. Hideaki Ogata
  6. James N Blaza  Is a corresponding author
  7. James A Birrell  Is a corresponding author
  1. University of York, United Kingdom
  2. Max Planck Institute for Chemical Energy Conversion, Germany
  3. Nara Institute of Science and Technology, Japan

Abstract

Electron-bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate potential electron donor are split so that one is sent along a high potential pathway to a high potential acceptor and the other is sent along a low potential pathway to a low potential acceptor. This process allows endergonic reactions to be driven by exergonic ones and is an alternative, less recognised, mechanism of energy coupling to the well-known chemiosmotic principle. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron-bifurcation in HydABC remains enigmatic in spite of intense research efforts over the last few years. Structural information may provide the basis for a better understanding of spectroscopic and functional information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure shows a heterododecamer composed of two independent 'halves' each made of two strongly interacting HydABC heterotrimers connected via a [4Fe-4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and for proton reduction. We identified two conformations of a flexible iron-sulfur cluster domain: a 'closed bridge' and an 'open bridge' conformation, where a Zn2+ site may act as a 'hinge' allowing domain movement. Based on these structural revelations, we propose a possible mechanism of electron-bifurcation in HydABC where the flavin mononucleotide serves a dual role as both the electron bifurcation center and as the NAD+ reduction/NADH oxidation site.

Data availability

Protein databank (PDB) files for the four model presented in this manuscript are available at https://www.rcsb.org/ under PDB ID 7P5H (D2 tetramer, 7P8N (Bridge closed forward), 7P91 (Bridge closed reverse), and 7P92 (Open bridge). Cryo-EM maps are available at https://www.ebi.ac.uk/pdbe/emdb/. All other data are available in the main text or the supplementary materials.

Article and author information

Author details

  1. Chris Furlan

    Department of Chemistry, University of York, York, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Nipa Chongdar

    Max Planck Institute for Chemical Energy Conversion, Muelheim an der Ruhr, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Pooja Gupta

    Department of Chemistry, University of York, York, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Wolfgang Lubitz

    Max Planck Institute for Chemical Energy Conversion, Muelheim an der Ruhr, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Hideaki Ogata

    Division of Materials Science, Nara Institute of Science and Technology, Nara, Japan
    Competing interests
    The authors declare that no competing interests exist.
  6. James N Blaza

    Department of Chemistry, University of York, York, United Kingdom
    For correspondence
    jamie.blaza@york.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5420-2116
  7. James A Birrell

    Max Planck Institute for Chemical Energy Conversion, Muelheim an der Ruhr, Germany
    For correspondence
    James.Birrell@cec.mpg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0939-0573

Funding

Deutsche Forschungsgemeinschaft (BI 2198/1-1)

  • Nipa Chongdar
  • James A Birrell

UK Research and Innovation (MR/T040742/1)

  • James N Blaza

Japan Society for the Promotion of Science (JP20H03215)

  • Hideaki Ogata

Max-Planck-Gesellschaft (n/a)

  • Nipa Chongdar
  • Wolfgang Lubitz
  • James A Birrell

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Amie K Boal, Pennsylvania State University, United States

Publication history

  1. Preprint posted: September 13, 2021 (view preprint)
  2. Received: April 8, 2022
  3. Accepted: August 25, 2022
  4. Accepted Manuscript published: August 26, 2022 (version 1)
  5. Version of Record published: September 21, 2022 (version 2)

Copyright

© 2022, Furlan et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 839
    Page views
  • 327
    Downloads
  • 2
    Citations

Article citation count generated by polling the highest count across the following sources: PubMed Central, Crossref, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Chris Furlan
  2. Nipa Chongdar
  3. Pooja Gupta
  4. Wolfgang Lubitz
  5. Hideaki Ogata
  6. James N Blaza
  7. James A Birrell
(2022)
Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase
eLife 11:e79361.
https://doi.org/10.7554/eLife.79361
  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    Lu Hu, Yang Sun ... Xu Wu
    Short Report Updated

    The TEA domain (TEAD) transcription factor forms a transcription co-activation complex with the key downstream effector of the Hippo pathway, YAP/TAZ. TEAD-YAP controls the expression of Hippo-responsive genes involved in cell proliferation, development, and tumorigenesis. Hyperactivation of TEAD-YAP activities is observed in many human cancers and is associated with cancer cell proliferation, survival, and immune evasion. Therefore, targeting the TEAD-YAP complex has emerged as an attractive therapeutic approach. We previously reported that the mammalian TEAD transcription factors (TEAD1–4) possess auto-palmitoylation activities and contain an evolutionarily conserved palmitate-binding pocket (PBP), which allows small-molecule modulation. Since then, several reversible and irreversible inhibitors have been reported by binding to PBP. Here, we report a new class of TEAD inhibitors with a novel binding mode. Representative analog TM2 shows potent inhibition of TEAD auto-palmitoylation both in vitro and in cells. Surprisingly, the co-crystal structure of the human TEAD2 YAP-binding domain (YBD) in complex with TM2 reveals that TM2 adopts an unexpected binding mode by occupying not only the hydrophobic PBP, but also a new side binding pocket formed by hydrophilic residues. RNA-seq analysis shows that TM2 potently and specifically suppresses TEAD-YAP transcriptional activities. Consistently, TM2 exhibits strong antiproliferation effects as a single agent or in combination with a MEK inhibitor in YAP-dependent cancer cells. These findings establish TM2 as a promising small-molecule inhibitor against TEAD-YAP activities and provide new insights for designing novel TEAD inhibitors with enhanced selectivity and potency.

    1. Biochemistry and Chemical Biology
    2. Cancer Biology
    Luca Costantino, Stefania Ferrari ... Maria Paola Costi
    Research Article

    Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anti-cancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, E7, we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.