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

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 recognized, 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.

[1]  Huilin Li,et al.  Structure and electron transfer pathways of an electron-bifurcating NiFe-hydrogenase , 2022, Science advances.

[2]  F. Baymann,et al.  The dyad of the Y-junction- and a flavin module unites diverse redox enzymes. , 2021, Biochimica et biophysica acta. Bioenergetics.

[3]  G. Gilardi,et al.  A safety cap protects hydrogenase from oxygen attack , 2021, Nature Communications.

[4]  M. Maldonado,et al.  Atomic structures of respiratory complex III2, complex IV, and supercomplex III2-IV from vascular plants. , 2021, eLife.

[5]  J. Artz,et al.  The role of thermodynamic features on the functional activity of electron bifurcating enzymes. , 2021, Biochimica et biophysica acta. Bioenergetics.

[6]  Conrad C. Huang,et al.  UCSF ChimeraX: Structure visualization for researchers, educators, and developers , 2020, Protein science : a publication of the Protein Society.

[7]  J. A. Letts,et al.  Atomic structures of respiratory complex III2, complex IV and supercomplex III2-IV from vascular plants , 2020, bioRxiv.

[8]  D. Gallagher,et al.  Key role of quinone in the mechanism of respiratory complex I , 2020, Nature Communications.

[9]  E. Boyd,et al.  The Beta Subunit of Non-bifurcating NADH-Dependent [FeFe]-Hydrogenases Differs From Those of Multimeric Electron-Bifurcating [FeFe]-Hydrogenases , 2020, Frontiers in Microbiology.

[10]  T. Mielke,et al.  Cryo-EM structures reveal intricate Fe-S cluster arrangement and charging in Rhodobacter capsulatus formate dehydrogenase , 2020, Nature Communications.

[11]  W. Lubitz,et al.  Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase , 2019, JBIC Journal of Biological Inorganic Chemistry.

[12]  J. W. Peters,et al.  Tuning catalytic bias of hydrogen gas producing hydrogenases. , 2019, Journal of the American Chemical Society.

[13]  Jasenko Zivanov,et al.  Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1 , 2019, bioRxiv.

[14]  Christopher J. Williams,et al.  Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix , 2019, Acta crystallographica. Section D, Structural biology.

[15]  T. Ikegami,et al.  Structural adaptations of photosynthetic complex I enable ferredoxin-dependent electron transfer , 2019, Science.

[16]  Paul D Adams,et al.  Iron–sulfur clusters have no right angles , 2019, Acta crystallographica. Section D, Structural biology.

[17]  Andrew C. R. Martin,et al.  ZincBind—the database of zinc binding sites , 2019, Database J. Biol. Databases Curation.

[18]  Erik Lindahl,et al.  New tools for automated high-resolution cryo-EM structure determination in RELION-3 , 2018, eLife.

[19]  Volker Müller,et al.  Electron Bifurcation: A Long-Hidden Energy-Coupling Mechanism. , 2018, Annual review of microbiology.

[20]  Jasenko Zivanov,et al.  A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis , 2018, bioRxiv.

[21]  David N Beratan,et al.  On the nature of organic and inorganic centers that bifurcate electrons, coupling exergonic and endergonic oxidation-reduction reactions. , 2018, Chemical communications.

[22]  R. Thauer,et al.  Flavin-Based Electron Bifurcation, A New Mechanism of Biological Energy Coupling. , 2018, Chemical reviews.

[23]  R. Thauer,et al.  Flavin-Based Electron Bifurcation, Ferredoxin, Flavodoxin, and Anaerobic Respiration With Protons (Ech) or NAD+ (Rnf) as Electron Acceptors: A Historical Review , 2018, Front. Microbiol..

[24]  B. Guigliarelli,et al.  Roles of the F-domain in [FeFe] hydrogenase. , 2018, Biochimica et biophysica acta. Bioenergetics.

[25]  Yong Zi Tan,et al.  Routine single particle CryoEM sample and grid characterization by tomography , 2017, bioRxiv.

[26]  T. Friedrich,et al.  Reduction of the off-pathway iron-sulphur cluster N1a of Escherichia coli respiratory complex I restrains NAD+ dissociation , 2017, Scientific Reports.

[27]  J. W. Peters,et al.  Defining Electron Bifurcation in the Electron-Transferring Flavoprotein Family , 2017, Journal of bacteriology.

[28]  J. W. Peters,et al.  Syntrophomonas wolfei Uses an NADH-Dependent, Ferredoxin-Independent [FeFe]-Hydrogenase To Reoxidize NADH , 2017, Applied and Environmental Microbiology.

[29]  J. W. Peters,et al.  Reduction Potentials of [FeFe]-Hydrogenase Accessory Iron-Sulfur Clusters Provide Insights into the Energetics of Proton Reduction Catalysis. , 2017, Journal of the American Chemical Society.

[30]  L. Casalot,et al.  Hydrogen production by the hyperthermophilic bacterium Thermotoga maritima part I: effects of sulfured nutriments, with thiosulfate as model, on hydrogen production and growth , 2016, Biotechnology for Biofuels.

[31]  David I Stuart,et al.  Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes , 2015, Nature Communications.

[32]  N. Grigorieff,et al.  CTFFIND4: Fast and accurate defocus estimation from electron micrographs , 2015, bioRxiv.

[33]  K. Klein,et al.  A structural view of synthetic cofactor integration into [FeFe]-hydrogenases† †Electronic supplementary information (ESI) available: Tables listing and comparing the RMSD of the structures, distances and angles of the 2FeH-subclusters, the distances from 2FeH-subcluster atoms to selected amino acids , 2015, Chemical science.

[34]  Michael J E Sternberg,et al.  The Phyre2 web portal for protein modeling, prediction and analysis , 2015, Nature Protocols.

[35]  J. W. Peters,et al.  [FeFe]-hydrogenase oxygen inactivation is initiated at the H cluster 2Fe subcluster. , 2015, Journal of the American Chemical Society.

[36]  W. Lubitz,et al.  Spontaneous activation of [FeFe]-hydrogenases by an inorganic [2Fe] active site mimic. , 2013, Nature chemical biology.

[37]  J. Hirst,et al.  Investigating the function of [2Fe–2S] cluster N1a, the off-pathway cluster in complex I, by manipulating its reduction potential , 2013, The Biochemical journal.

[38]  W. Lubitz,et al.  Biomimetic assembly and activation of [FeFe]-hydrogenases , 2013, Nature.

[39]  J. W. Peters,et al.  The modular respiratory complexes involved in hydrogen and sulfur metabolism by heterotrophic hyperthermophilic archaea and their evolutionary implications. , 2013, FEMS microbiology reviews.

[40]  R. Thauer,et al.  Energy conservation via electron bifurcating ferredoxin reduction and proton/Na(+) translocating ferredoxin oxidation. , 2013, Biochimica et biophysica acta.

[41]  John M. Berrisford,et al.  Crystal structure of the entire respiratory complex I , 2013, Nature.

[42]  J. Swartz,et al.  High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli , 2010, PloS one.

[43]  P. Dutton,et al.  An Electronic Bus Bar Lies in the Core of Cytochrome bc1 , 2010, Science.

[44]  J. W. Peters,et al.  Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydAΔEFG , 2010, Nature.

[45]  M. Adams,et al.  The Iron-Hydrogenase of Thermotoga maritima Utilizes Ferredoxin and NADH Synergistically: a New Perspective on Anaerobic Hydrogen Production , 2009, Journal of bacteriology.

[46]  M. Adams,et al.  Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels. , 2008, Metabolic engineering.

[47]  F. Guerlesquin,et al.  Solution structure of HndAc: A thioredoxin‐like domain involved in the NADP‐reducing hydrogenase complex , 2006, Protein science : a publication of the Protein Society.

[48]  T. Ikegami,et al.  Identification of the N- and C-terminal substrate binding segments of ferredoxin-NADP+ reductase by NMR. , 2005, Biochemistry.

[49]  P. Dutton,et al.  Large scale domain movement in cytochrome bc(1): a new device for electron transfer in proteins. , 2001, Trends in biochemical sciences.

[50]  Christopher C. Moser,et al.  Natural engineering principles of electron tunnelling in biological oxidation–reduction , 1999, Nature.

[51]  M. Adams,et al.  The hyperthermophilic bacterium, Thermotoga maritima, contains an unusually complex iron-hydrogenase: amino acid sequence analyses versus biochemical characterization. , 1999, Biochimica et biophysica acta.

[52]  J. W. Peters,et al.  Electron bifurcation. , 2016, Current opinion in chemical biology.

[53]  Tanja Sušec,et al.  Historical Review , 1917, Acta Cytologica.

[54]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[55]  Arthur Schweiger,et al.  EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. , 2006, Journal of magnetic resonance.

[56]  B. Lagoutte,et al.  Ferredoxin-NADP (cid:1) Reductase KINETICS OF ELECTRON TRANSFER, TRANSIENT INTERMEDIATES, AND CATALYTIC ACTIVITIES STUDIED BY FLASH-ABSORPTION SPECTROSCOPY WITH ISOLATED PHOTOSYSTEM I AND FERREDOXIN* , 2005 .

[57]  M. Adams,et al.  Fe-only hydrogenase from Thermotoga maritima. , 2001, Methods in enzymology.

[58]  N. B. Zhadanovskii Hydrogen production , 1972 .

[59]  Michael Davis Cost of Living , 1969, Nature.