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.
Recommended Citation
D. Das et al., "Electrochemical Observation and PH Dependence of all Three Expected Redox Couples in an Extremophilic Bifurcating Electron Transfer Flavoprotein with Fused Subunits," JACS Au, American Chemical Society, Jan 2025.
The definitive version is available at https://doi.org/10.1021/jacsau.4c01219
Department(s)
Chemistry
Publication Status
Open Access
Keywords and Phrases
bifurcating electron transfer flavoprotein; bifurcation; direct electrochemistry; fusion enzyme; hyperthermophilic; proton coupled electron transfer; spectroelectrochemistry
International Standard Serial Number (ISSN)
2691-3704
Document Type
Article - Journal
Document Version
Final Version
File Type
text
Language(s)
English
Rights
© 2025 American Chemical Society, All rights reserved.
Creative Commons Licensing
This work is licensed under a Creative Commons Attribution 4.0 License.
Publication Date
01 Jan 2025
Comments
National Science Foundation, Grant CHE-2108134