Extended conformations of bifurcating electron transfer flavoprotein constitute up to half the population, possibly mediating conformational change

Electron transfer bifurcation allows biological systems to facilitate unfavorable (endergonic) electron transfers by pairing them with favorable (exergonic) transfers of a second electron. In electron transfer flavoproteins (ETFs), it is thought that a large-scale conformational change within the protein disrupts the favorable pathway after the first electron transfer, thus preventing energy loss from the exergonic transfer of the second electron. To investigate the conformational changes involved during this process, we used small-angle neutron scattering (SANS) and computational methods to study the bifurcating ETF from Acidaminococcus fermentans (AfeETF). SANS results showed an overall radius of gyration (Rg) of 30.1 ± 0.2 Å and a maximum dimension (Dmax) of 100 Å for oxidized AfeETF, which are 4 Å and 30 Å larger, respectively, than any previously reported bifurcating ETF structures. These findings indicate that none of the existing ETF structures can account for the observed scattering data, nor can any single conformation generated by our molecular dynamics simulations. To refine ensembles that best match the SANS data, we adapted a genetic algorithm. The successful ensembles featured a compact conformation similar to one seen in crystallographic studies, along with a much more extended conformation, and these two states were sufficient to explain the data. The extended conformations identified had Rgs at least 4 Å larger than any currently known ETF structures, yet they were strongly represented, making up 20% of the reduced ETF population and over 50% of the oxidized AfeETF population. Consequently, the previously published (compact) structures offer an incomplete view of AfeETF's conformation in solution. Additionally, since the conformational ensemble changes upon the reduction of AfeETF's flavins, the interconversion between these conformations may play a role in turnover. We suggest that the extended conformations could provide energetically favorable pathways for the rapid switching between the open and closed compact conformations, which are believed to be crucial at different stages of turnover.

SAXS measurements were performed on the ETF sample at Brookhaven National Laboratory (BNL). SANS datasets were also collected on ETF, see dataset XSMELGAC

Data in the file SAXS_ETF.dat represents ETF SAXS data with the buffer subtracted

2024-09-23

2024-10-03

SEC-SAXS

National Synchrotron Light Source II

Life Sciences X-ray Scattering Beamline (LIX)

Sample:

• Macromolecule 1: ETF
• Sample Full Name: Electron transfer flavoprotein
• Sample Type: Protein
• Source Organism: Acidaminococcus fermentans
• Source Organism NCBI Taxonomy ID: 905
• Expression System: Escherichia coli
• Expression NCBI Taxonomy ID: 562
• Uniprot ID's: D2RIQ2, D2RIQ3
• Sequence or Chemical Formula:
• >4KPU_1|Chain A|Electron transfer flavoprotein alpha subunit|Acidaminococcus fermentans (591001) MANTKGLKTGNEKDLWVYVEHYKGEPVHVVYELLGECRKLADKCNQKLAAVLITDDAKDVPSKLIARGADLVYVCQDPAFKYYSTDEYTNAFCEMIDEYQPSSVFIGATNDGRDLGPRIAARVNTGLCADCTILDAEEDGLIEWTRPAAGGNIMATILCKEHRPQMGTVRPKTFKAMEPDASRTGEVINYTLKNHVDDRVTCIRREEVVSEGEMAIDDAPFVCSGGRGMKAKENFSLLYDLAHALGGAVGGSRAAVDEGFIEHPRQVGQSGKTVTPKIYFACGISGSVQHKAGMSKSDTIVCINKDPDAPMFEISKYGIVGDALKILPLLTAKIKAFKESHHHHHH >4KPU_2|Chain B|Electron transfer flavoprotein alpha/beta-subunit|Acidaminococcus fermentans (591001) MNIVVCVKQVPDTAEMKIDPVTNNLVRDGVTNIMNPYDQYALETALQLKDELGAHVTVITMGPPHAESVLRDCLAVGADEAKLVSDRAFGGADTLATSAAMANTIKHFGVPDLILCGRQAIDGDTAQVGPEIAEHLGLPQVTAALKVQVKDDTVVVDRDNEQMSMTFTMKMPCVVTVMRSKDLRFASIRGKMKARKAEIPVYTAAALEIPLDIIGKAGSPTQVMKSFTPKVTQVHGEIFDDEDPAVAVDKLVNKLIEDKIITK