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Open Dataset XSDUNFBU

Correlating conformational equilibria with catalysis in the electron bifurcating Fix/EtfABCX of Thermotoga maritima

Abstract

Electron bifurcation (BF) is an evolutionarily ancient means of energy conservation in anaerobes whose enzymatic mechanism remains enigmantic. In BF-flavoenzymes, a chemically high-potential electron forms in a thermodynamically favorable fashion by simultaneously dropping the potential of a second electron before their donation to physiological acceptors. The cryo-EM and spectroscopic analyses of the BF-enzyme Fix/EtfABCX from Thermotoga maritima suggests that the BF-site contains a special FAD and, upon its reduction with NADH, a low-potential electron transfers to ferredoxin and a high-potential electron reduces menaquinone. The transfer of energy from high-energy intermediates must be carefully orchestrated conformationally to avoid equilibration. Herein, anaerobic size exclusion-coupled small-angle X-ray scattering (SEC-SAXS) shows the Fix/EtfAB heterodimer subcomplex, which houses BF- and electron transfer (ET)-flavins, exists in a conformational equilibrium of compacted and extended states between flavin-binding domains, the abundance of which is impacted by reduction and NAD(H) binding. The conformations identified provide novel snapshots of the T. maritima enzyme and also recapitulate states identified in static structures of homologous BF-flavoenzymes. Reduction of Fix/EtfABCX’s flavins alone is insufficient to elicit domain movements conducive to ET, but requires a structural “trigger” induced by NAD(H) binding. Models show Fix/EtfABCX’s superdimer exists in a combination of states with respect to its BF-subcomplexes, suggesting a cooperative mechanism between supermonomers for optimizing catalysis. Correlation of conformational states with pathway steps suggest a structural means with which Fix/EtfABCX may progress through its catalytic cycle. Collectively, these observations provide a structural framework for tracing Fix/EtfABCX’s catalysis and enable future studies of BF-enzymes.

Experimental description

The SIBYLS beamline was adapted to an anaerobic modality prior to performing SEC-MALS-SAXS studies.

File description

Merged I(q) versus q (A^-1) data files are uploaded as .dat files as generated in Scatter or BioXTAS RAW softwares following their processing from SEC-SAXS datasets.

Created

2022-07-16

Updated

2023-07-26

Data collection technique

SEC-SAXS

Journal DOI

Source

ALS

Beamline

BL 12.3.1

Wavelength

1.23984 Å

Sample to Detector Distance

2.075 m

Submitting Author

Dan Murray

Lawrence Berkeley National Laboratory, The SIBYLS Beamline

United States of America

[email protected]

Collaborators

  • Michal Hammel, LBNL
  • Russ Hille, UC Riverside
  • Michael W. W. Adams, University of Georgia
  • Xiaoxuan Ge, University of Georgia
  • Gerrit J. Schut, University of Georgia
  • Jan C. Bierma, LBNL
  • Daniel J. Rosenberg, LBNL / SLAC

Project Leader

Greg Hura

[email protected]


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Complete Set of SAS Data Files

The complete SAS dataset is downloadable as a zip file.

Individual SAS Data Files (total 0)

These are individual SAS data files that may be raw or processed (merged, etc.). These may or not be included in a zip file containing a larger dataset.

Supplemental Data and Supporting Materials (total 0)

These data and materials may include X-ray crystal structure coordinates, multi-angle light scattering data, .etc. It may also include additional details or methods pertaining to the SAXS experiments, or the researcher's interpretations of the results.

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SAXS Similarity SAXS FrameSlice

Sample:

  • Macromolecule 1: EtfABCX
    • Sample Full Name: 9x His-tagged Thermotoga maritima electron-transferring flavoprotein-menaquinone oxidoreductase ABCX
    • Sample Type: Protein
    • Source Organism: Thermotoga maritima
    • Source Organism NCBI Taxonomy ID: 2336
    • Expression System: E. coli
    • Expression NCBI Taxonomy ID: 562
    • Uniprot ID:
    • Sequence or Chemical Formula:
    • >7KOE_1|Chains A, E|Electron transfer flavoprotein, beta subunit|Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099) (243274) MAHHHHHHHHHANVVVCIKQVPDTTNVRIDRKTNNLVREGVPSIINPDDERALELASQLKEKFGATVYVITMGPPQAKEALKDAIAFGLDEAVHLSDRTFAGADTLATTYTLYWGIKKIEERIGKIDLILTGKQAVDGDTGQVGPGLATRFGYALGAYVVRIEEIDPEKKEMVIVRRLDQGFEKIRLKLPAVLTITDELNKPRYADLPNLIRAIRYEPIVWTHKDLGLDPKKCGFFGSPTRVVSTNIPPARKGGDIISKNEDPEVAAEKLIEALKKFEAVRLVEALKPVLEGEKDE >7KOE_2|Chains B, F|Electron transfer flavoprotein, alpha subunit|Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099) (243274) MSEKKIIFVLIEHHGGKAHPVSWELIGKARDLASKLENSEVWGVLLGEGLESVAKEAIQRGADKVLYVKNREFNTYVNYLYKKALVDMVRKYRPEIFLIGATLEGRELAGMVATELETGLTADCTGLDIIPDKKLLAMTRPTFGGNLMATIMCPDHRPQMATVRPGVMKELPPDPERTGEIIEEEYDLGTFDKLIEILETIPLQTQVNLEYAPVVVAGGKGVGGPEGFKKLKELADLLGGEVGASRAAVKAGWISPEHQVGQTGKTVRPVLYFACGISGAIQHVVGIKESEIIVAINIDEKAPIFDIADIGIVGDLHKVVPALTAKLRELLNKSGVKK >7KOE_3|Chains C, G|Electron transfer flavoprotein-quinone oxidoreductase FixC|Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099) (243274) MKIEFDVVVVGAGPSGLSCAYVLAKNGLKVAVVEKGEYPGSKNVMGGVLYVHPLKEIMPDFLEKAANSKALERNVIEQNLWLLGNEGVIKIGHRNVEWKENPNAFTVLRANFDRWFAQEVEKAGALIIPKTKVEDFLRNEKGEIAGVVTSRPKGEIHSKAVVIAEGVNPILTMKAGLRKEDLKPHMVAVAVKEVISVPEDVVNRVFGVEGNDGATIELLGSWSEGMFGMGFLYANRSSVSLGCGVLLEDLRKKKIKPYQLLENLKNHPVISDMLGEYRNNTMEYLAHLIPEGGYYAMPKVYGDRVLVCGDAAMLVNSIHREGSNHAITSGRLAAETLLEAFEKGDFSEKILKNYYLRLKESFILKDLEKYKDLMPTMEKNHQFVEIYPDLANDALKRFLQVDGTPKWDVQKQIADMVLSRRSLIGISLDLLRFWRAVR >7KOE_4|Chains D, H|Ferredoxin-like protein|Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099) (243274) MRIEDKLYLNRYRTDEENPHLKIKDESICAEKCSDRPCVSCCPADVYEWTESGMEVKFEGCLECGTCRIVCPFGNIEWNYPRGNYGVLYKFG