Electrochemical Observation and pH Dependence of All Three Expected Redox Couples in an Extremophilic Bifurcating Electron Transfer Flavoprotein with Fused Subunits

Authors

• Debarati Das, University of KentuckyFollow
• Wassim El Housseini, Missouri University of Science and Technology
• Monica Brachi, Missouri University of Science and Technology
• Shelley D. Minteer, Missouri University of Science and TechnologyFollow
• Anne-Frances Miller, University of KentuckyFollow

Abstract

Bifurcating enzymes employ energy from a favorable electron transfer to drive unfavorable transfer of a second electron, thereby generating a more reactive product. They are therefore highly desirable in catalytic systems, for example, to drive challenging reactions such as nitrogen fixation. While most bifurcating enzymes contain air-sensitive metal centers, bifurcating electron transfer flavoproteins (bETFs) employ flavins. However, they have not been successfully deployed on electrodes. Herein, we demonstrate immobilization and expected thermodynamic reactivity of a bETF from a hyperthermophilic archaeon, Sulfolobus acidocaldarius (SaETF). SaETF differs from previously biochemically characterized bETFs in being a single protein, representing a concatenation of the two subunits of known ETFs. However, SaETF retains the chemical properties of heterodimeric bETFs, including possession of two FADs: one that undergoes sequential 1-electron (1e) reductions at high E° and forms an anionic semiquinone, and another that is amenable to lower-E° 2e reduction, including by NADH. We found homologous monomeric ETF genes in archaeal and bacterial genomes, accompanied by genes that also commonly flank heterodimeric ETFs, and SaETF’s sequence conservation is 50% higher with bETFs than with canonical ETFs. Thus, SaETF is best described as a bETF. Our direct electrochemical trials capture reversible redox couples for all three thermodynamically expected redox events. We document electrochemical activity over a range of pH values and reveal a conformational change coupled to proton acquisition that affects the electrochemical activity of the higher-E° FAD. Thus, this well-behaved monomeric bETF opens the door to bioinspired bifurcating devices or bifurcation on a chip.

Document Type

Article

Publication Date

2025

Notes/Citation Information

© 2025 The Authors. Published by American Chemical Society.

Digital Object Identifier (DOI)

https://doi.org/10.1021/jacsau.4c01219

Funding Information

D.D. and A.F.M. gratefully acknowledge the support of the National Science Foundation Award CHE-2108134, and A.F.M. acknowledges partial support from DOE BES under DE- SC0021283. W.E.H., M.B., and S.D.M. were supported by the National Science Foundation Grant 2406605.

Repository Citation

Das, Debarati; El Housseini, Wassim; Brachi, Monica; Minteer, Shelley D.; and Miller, Anne-Frances, "Electrochemical Observation and pH Dependence of All Three Expected Redox Couples in an Extremophilic Bifurcating Electron Transfer Flavoprotein with Fused Subunits" (2025). Chemistry Faculty Publications. 205.
https://uknowledge.uky.edu/chemistry_facpub/205